EA045900B1 - COMBINATION OF ANTI-PD-1 ANTIBODIES AND RADIATION FOR THE TREATMENT OF MALIGNANT TUMOR - Google Patents

Description

This application was filed on May 12, 2017 as a PCT international patent application and claims priority to U.S. Provisional Application Nos. 62/335,743, filed May 13, 2016; 62/340142, filed May 23, 2016; 62/348546, filed June 10, 2016; 62/350305, filed June 15, 2016; 62/364920, filed July 21, 2016; 62/374020, filed August 12, 2016; and 62/451274, filed January 27, 2017, the description of each is incorporated herein by reference in its entirety.

List of sequences

This application contains a list of sequences in electronic format in the form of a txt file called Sequence Lists. The contents of the txt file Sequence Lists are incorporated herein by reference.

Field of technology to which the invention relates

The present invention relates to methods of treating cancer, comprising administering to a patient a therapeutically effective amount of an antibody that specifically binds to the programmed cell death protein 1 (PD-1) receptor.

State of the art

Radiation therapy uses the DNA-damaging properties of ionizing radiation to control tumor growth and/or kill tumor cells and has become widespread in the last six decades for the treatment of localized tumors. Radiation therapy may contribute to stimulatory effects on the immune system, for example due to the release of tumor antigens. However, radiation can also lead to immunosuppressive effects, such as stimulation of inhibitory cells of the immune system such as regulatory T cells (Le et al 2015, Clin. Cancer Res. 21: 3393-3401).

The development of immune checkpoint inhibitors for cancer therapy has led to interest in combining them with radiotherapy (Tang et al 2014, Cancer Immunol. Res. 2: 831-838; Teng et al 2015, Cancer Lett. 365: 23-29) . One such immune checkpoint inhibitor is programmed cell death protein 1 (PD-1). PD-1 receptor signaling in the tumor microenvironment plays a key role in mediating tumor cell evasion from host immune surveillance. Blocking the PD-1/PD-L1 T cell checkpoint pathway is an effective and well-tolerated approach to stimulating an immune response that has achieved significant objective responses in advanced melanoma, renal cell carcinoma (RCC), and non-small cell lung cancer (NSCLC) (Topalian et al 2012, New Engl J Med 366: 2443-2454). However, optimal therapy likely requires combining anti-PD-1 monoclonal antibody treatment with standard therapy and new immunotherapeutic approaches. Combination approaches to stimulate the converging aspects of host immunity using complementary immunomodulators, as well as the immunostimulatory aspects of standard modalities such as radiation and chemotherapy, may lead to the development of more effective cancer therapies.

Disclosure of the invention

In accordance with certain embodiments, the present invention provides methods for treating or improving at least one symptom or indication or inhibiting the growth of a cancer in a patient. Methods in accordance with this aspect of the invention include administering to a patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to programmed cell death protein 1 (PD-1), optionally in combination with radiation therapy.

In accordance with certain embodiments, the present invention provides methods for treating a cancer, including a solid tumor, the methods comprising selecting a patient with the cancer and administering one or more doses of an anti-PD-1 antibody in combination with one or more doses of radiation therapy. In certain embodiments, administration of the combination results in an enhanced therapeutic effect or antitumor effect compared to administration of the antibody or radiation alone.

In certain embodiments of the present invention, methods are provided for treating or improving at least one symptom or indication or inhibiting the growth of a cancer in a patient. In certain embodiments, the present invention provides methods for delaying tumor growth or preventing tumor recurrence. In certain embodiments of the present invention, methods are provided for improving overall survival or progression-free survival in a patient with a cancer. Methods in accordance with this aspect of the invention include sequential administration of one or more doses of a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds PD-1. In one embodiment, the administration of an anti-PD-1 antibody is administered in combination with radiation therapy.

In certain embodiments, the malignancy or tumor is a solid tumor or cancer. In certain embodiments, the solid tumor is selected from

- 1 045900 group consisting of colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer , lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.

In certain embodiments, an anti-PD-1 antibody is administered as a first-line treatment to a patient with a cancer where the patient has not received prior systemic treatment for the cancer. In certain embodiments, an anti-PD-1 antibody is administered as a second-line treatment to a patient with a cancer (eg, metastatic cancer) where the patient has previously been treated with standard therapy, including, but not limited to, chemotherapy, surgery, and radiation.

One embodiment of the invention relates to an anti-PD-1 antibody for use in the treatment of skin cancer. In certain embodiments, the skin cancer is a non-melanoma skin cancer, including, but not limited to, squamous cell carcinoma of the skin and basal cell carcinoma. An anti-PD-1 antibody can be administered as disclosed herein to a patient with metastatic or locally advanced squamous cell carcinoma of the skin. In certain embodiments, an anti-PD-1 antibody is administered, as disclosed herein, to a patient with advanced basal cell carcinoma, where the patient is intolerant to a Hedgehog pathway inhibitor (e.g., vismodegib, sonedegib) or has been treated with a Hedgehog pathway inhibitor and is experiencing progressive disease.

In certain embodiments, each dose of anti-PD-1 antibody contains 0.1-20 mg/kg of patient body weight. In certain embodiments, each dose of anti-PD-1 antibody contains 0.3, 1, 3, 5, or 10 mg/kg of the patient's body weight. In certain embodiments, each dose of anti-PD-1 antibody contains 20-600 mg. In one embodiment, each dose of anti-PD-1 antibody contains approximately 200 mg. In one embodiment, each dose of anti-PD-1 antibody contains approximately 250 mg. In one embodiment, each dose of anti-PD-1 antibody contains approximately 350 mg.

In certain embodiments, radiation therapy is administered in one or more doses. In certain embodiments, each radiation therapy dose contains 2-100 Grays (Gy). In certain embodiments, the radiation therapy is hypofractionated radiation therapy. In certain embodiments, the radiation therapy provides 2-12 fractions.

In certain embodiments, the methods of the present invention include administering a therapeutically effective amount of an anti-PD-1 antibody before, concurrently with, or after radiation therapy. In one embodiment, the methods of the present invention include administering an anti-PD-1 antibody prior to administering a dose of radiation therapy.

In certain embodiments, the methods of the present invention include administering 0-50 therapeutic doses, each of which is an anti-PD-1 antibody, wherein each dose is administered 0.5-12 weeks after the immediately preceding dose. In one embodiment, each dose is administered 1 week after the immediately preceding dose. In one embodiment, each dose is administered 2 weeks after the immediately preceding dose. In one embodiment, each dose is administered 3 weeks after the immediately preceding dose.

In certain embodiments, one or more doses of an anti-PD-1 antibody and optionally radiation therapy are included in the treatment cycle. Methods in accordance with this aspect of the invention include subjecting a patient to at least one cycle of treatment, where at least one cycle of treatment includes one or more doses of an anti-PD-1 antibody. In certain embodiments, up to 12 cycles of treatment are administered to a patient. In certain embodiments, the at least one treatment cycle further comprises one or more doses of radiation therapy. In certain embodiments, radiation therapy is administered in only one treatment cycle. In certain embodiments, the radiation therapy is hypofractionated radiation therapy. In certain embodiments, the anti-PD-1 antibody is administered prior to radiation therapy.

In certain embodiments, the anti-PD-1 antibody and radiation therapy are administered in combination with an additional therapeutic agent or therapy (eg, cyclophosphamide, or any agent or therapy disclosed herein).

In certain embodiments, the treatment produces one or more therapeutic effects selected from the group consisting of tumor regression, abscopal effect, inhibition of tumor metastasis, reduction of metastatic lesions over a period of time, reduced use of chemotherapeutic or cytotoxic agents, reduced tumor burden, increased survival without progression, improved overall survival, complete response, partial response and stable disease.

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In certain embodiments, the anti-PD-1 antibody or antigen binding protein comprises heavy chain complementarity determining regions (HCDRs) from a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1, and light chain CDRs from a light chain variable region chain (LCVR) containing the amino acid sequence of SEQ ID NO:2. One such type of antigen binding protein that can be used in the context of the methods of the present invention is an anti-PD-1 antibody, such as REGN2810.

In certain embodiments, the present invention provides the use of an anti-PD-1 antibody or an antigen-binding fragment thereof for the production of a medicament for treating or inhibiting the growth of a cancer in a patient, including a human. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer gland cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer or myeloma.

In certain embodiments, the present invention provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof to produce a medicament in combination with radiation therapy to treat or inhibit the growth of a cancer in a patient, including a human. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer gland cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer or myeloma.

In one aspect, the present invention provides a kit for treating a patient suffering from cancer, the kit containing: (a) a dose of an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-1; and (b) instructions for using an anti-PD-1 antibody to treat a patient in accordance with the methods disclosed herein. In certain embodiments, the cancer is selected from the group consisting of colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, cancer liver, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.

Other embodiments of the present invention will become apparent upon consideration of the following detailed description.

Brief description of drawings

In fig. 1 shows a study design involving anti-PD-1 antibody dosing and radiation (XRT) for mice implanted with MC38 tumors (the study described in Example 1 herein).

In fig. Figure 2 shows average tumor growth in mice treated with isotype control antibody (·), anti-PD-1 antibody (), isotype control+radiation (XRT) (A), or anti-PD-1+XRT (▼) in the study described in example 1 in the present description.

In fig. 3 shows the overall survival of mice treated with isotype control antibody (·), anti-PD-1 antibody (), isotype control+irradiation (XRT) ( ^A ), or anti-PD-1+XRT (▼) in the study described in the example. 1 in this specification.

In fig. 4 shows a study design involving anti-PD-1 antibody dosing and radiation (XRT) for mice implanted with B16F10.9 tumors (the study described in Example 2 herein).

In fig. Figure 5 shows average tumor growth in mice treated with isotype control antibody (·), anti-PD-1 antibody (), isotype control+irradiation (XRT) (♦), or anti-PD-1+XRT (O) in the study described in example 2 in the present description.

In fig. 6 shows the overall survival of mice treated with isotype control antibody (·), anti-PD-1 antibody (), isotype control+irradiation (XRT) (♦), or anti-PD-1+XRT antibody (O) in the study described in the example. 2 in the present description.

In fig. 7 shows a study design including anti-PD-1 antibody dosing and radiation (XRT) for mice implanted with MC38 tumors (study described in Example 4 herein)

In fig. 8 shows average primary tumor growth in mice treated with isotype control antibody (·), anti-PD-1 antibody (), isotype control+radiation (XRT) (A), or anti-PD-1+XRT (▼) in the study. described in example 4 in the present description.

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In fig. 9 shows the overall survival of mice treated with isotype control antibody (·), anti-PD-1 antibody (), isotype control+irradiation (XRT) ( ^A ), or anti-PD-1+XRT (▼) in the study described in the example. 4 in the present description.

In fig. 10 shows second tumor growth in mice treated with isotype control antibody (·), anti-PD-1 antibody (), isotype control+radiation (XRT) ( ^A ), or anti-PD-1+XRT (▼) in the study described in example 4 in the present description.

In fig. 11 shows a study design including dosing of anti-PD-1 antibody, anti-GITR antibody, and radiation (XRT) for mice implanted with MC38 tumors (the study described in Example 5 herein).

In fig. 12 shows average tumor growth in mice treated with isotype control antibody (·), anti-PD-1 antibody (), anti-GITR antibody (^), combination of anti-PD-1 antibody and anti-GITR antibody (▼), isotype control + irradiation (XRT) (♦), anti-PD-1+XRT (O), anti-GITR+XRT (□), or a combination of anti-PD-1, anti-GITR+XRT (A) in the study described in Example 5 in this description.

In fig. 13 shows the overall survival of mice treated with isotype control antibody (·), anti-PD-1 antibody (), anti-GITR antibody ( ^A ), combination of anti-PD-1 antibody and anti-GITR antibody (▼), isotype control+radiation (XRT ) (♦), anti-PD-1+XRT (O), anti-GITR+XRT ( ^P ), or a combination of anti-PD-1, anti-GITR+XRT (A) in the study described in Example 5 herein .

In fig. 14A shows radiographic images of pulmonary metastases in a patient with basal cell carcinoma (BCC), indicated by arrows, at baseline, left, and at week 24, right.

In fig. 14B shows radiographic images of a cervical mass in a patient with cutaneous squamous cell carcinoma (CSCC) at baseline, left, and at week 16, right.

Carrying out the invention

Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used in the present description serves only the purpose of describing specific embodiments and is not intended as limitation, since the scope of the present invention is limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. As used herein, the term approximately, when used in relation to a specific specified numerical value, means that the value may vary from the specified value by no more than 1%. For example, as used herein, the expression approximately 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in order to be described in their entirety.

Methods for treating or inhibiting the growth of a malignant tumor

The present invention includes methods of treating, improving or reducing the severity of at least one symptom or indication, or inhibiting the growth of a cancer in a patient. Methods in accordance with this aspect of the invention include administering to a patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds PD-1. In certain embodiments, an anti-PD-1 antibody is administered in combination with antitumor therapy (described elsewhere herein). In one embodiment, the antitumor therapy is radiation therapy. As used herein, the terms treat, cure or the like mean to alleviate symptoms, eliminate the cause of symptoms on a temporary or permanent basis, delay or inhibit tumor growth to reduce tumor cell burden or tumor burden, promote tumor regression, cause tumor shrinkage, necrosis and/or disappearance to prevent tumor recurrence, to prevent or inhibit metastasis, to inhibit metastatic tumor growth and/or to increase the survival time of an individual.

As used herein, the term patient refers to a human or non-human mammal that exhibits one or more symptoms or indications of a cancer, and/or has been diagnosed with a cancer, including a solid tumor, and is in need of treatment for it. In many embodiments, the term individual may be used interchangeably with the term patient. For example, in a human patient

- 4 045900 can diagnose a primary or metastatic tumor and/or one or more symptoms or indications, including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, bloating abdomen, chest pain/pressure, enlarged spleen, and elevated levels of a biological marker associated with cancer (eg, CA125). The expression includes individuals with primary or advanced tumors. In specific embodiments, the term includes a person who suffers from and/or needs treatment for a solid tumor, for example, colon cancer, breast cancer, lung cancer, prostate cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, cancer cervical cancer, pancreatic cancer, head and neck cancer and brain cancer. The term includes individuals with primary or metastatic tumors (advanced malignancies). In certain embodiments, the expression patient includes patients with a solid tumor that is resistant or refractory to or insufficiently controlled by prior therapy (eg, treatment with an anti-cancer agent). For example, the expression includes individuals who have been treated with one or more lines of prior therapy, such as treatment with chemotherapy (eg, carboplatin or docetaxel). In certain embodiments, the expression patient includes patients with a solid tumor who were treated with one or more lines of prior therapy, but who subsequently experienced recurrence or metastasis. For example, patients with a solid tumor who may have received treatment with one or more anti-malignancy agents leading to tumor regression; however, have subsequently had a recurrence of a cancer that is resistant to one or more anti-cancer agents (eg, chemotherapy-resistant cancer) and are treated with the methods of the present invention. The expression also includes individuals with a solid tumor in whom standard therapy against malignancy is inappropriate, for example, due to toxic side effects. For example, the expression includes patients who have received one or more cycles of chemotherapy with toxic side effects.

In certain embodiments, the methods of the present invention can be used to treat patients who have elevated levels of one or more cancer-associated biological markers [e.g., programmed death protein 1 ligand (PD-L1), CA125, CA19-9, specific antigen prostate cancer (PSA), lactate dehydrogenase, KIT, embryonal tumor antigen, epidermal growth factor receptor (EGFR), ALK gene rearrangement]. For example, the methods of the present invention include administering a therapeutically effective amount of an anti-PD-1 antibody in combination with radiation therapy to a patient with elevated levels of PD-L1 and/or EGFR. In a preferred embodiment, the methods of the present invention are used in patients with cancer who are selected based on the expression of PD-L1 in the cancer tissue. In certain embodiments, the methods of the present invention are used to treat patients with cancer, where patients are selected based on at least 1%, at least 2%, at least 5%, at least 10%, at least at least 20%, at least 30%, at least 40%, or at least 50% of PD-L1 expression in cancer tissue and/or immune system cells. Methods for determining PD-L1 expression in cancer tissue and/or immune system cells are well known in the art. In certain embodiments, PD-L1 expression in tumor tissue is determined using any assay known in the art, for example, using an ELISA assay or an immunohistochemical (IHC) assay as described in PCT publications WO2016124558 or WO2016191751 or patent application publication US20160305947. In certain embodiments, PD-L1 expression is determined by quantifying RNA expression, such as by in situ hybridization or RT-PCR. In certain embodiments, PD-L1 expression is determined by imaging using a labeled anti-PD-L1 antibody, such as immune positron emission tomography or iPET [see, for example, The Oncologist, 12: 1379 (2007); Journal of Nuclear Medicine, 52(8): 1171 (2011); US Provisional Patent Application No. 62/428,672, filed December 1, 2016].

In certain embodiments, the methods of the present invention are used in a patient with a solid tumor. The terms tumor, cancer and malignancy are used interchangeably herein.

As used herein, the term solid tumor refers to an abnormal mass of tissue that does not typically contain cysts or fluid areas. Solid tumors can be benign (non-cancerous tumor) or metastatic (malignant tumor). For purposes of the present invention, the term solid tumor refers to metastatic solid tumors. The term includes various types of solid tumors, named after the types of cells that form them, such as sarcomas, carcinomas and lymphomas. However, the term does not include leukemias. In various embodiments, the term solid tumor includes malignant tumors arising from

- 5 045900 connective or supporting tissue (for example, bone or muscle) (called sarcomas), malignant tumors arising from somatic glandular cells and epithelial cells that line the tissues of the body (called carcinomas), and malignant tumors of lymphoid organs such as lymph nodes , spleen and thymus (referred to as lymphomas). Lymphoid cells are found in almost all tissues of the body and, therefore, lymphomas can develop in a wide range of organs. In certain embodiments, the term solid tumor includes malignant tumors, including, but not limited to, colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer , colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma. In certain embodiments, the term solid tumor includes malignant tumors, including, but not limited to, hepatocellular carcinoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, basal cell carcinoma, breast carcinoma, squamous cell carcinoma of the skin, chondrosarcoma, angiosarcoma, cholangiocarcinoma, soft tissue sarcoma , colon cancer, melanoma, Merkel cell carcinoma and glioblastoma multiforme. In certain embodiments, the term solid tumor includes more than one solid tumor lesion that is located separately from each other, for example, 2, greater than 2, greater than 5, greater than 10, greater than 15, greater than 20, or greater than 25 lesions in a patient in need of treatment. In certain embodiments, more than one lesion is located distal to each other in the same organ. In certain other embodiments, the tumor lesions may be located in various organs.

In certain embodiments, the present invention includes methods of treating or inhibiting the growth of a cancer, including, but not limited to, colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma. In certain embodiments, the present invention includes methods of treating or inhibiting the growth of a malignant tumor, including, but not limited to, hepatocellular carcinoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, basal cell carcinoma, squamous cell carcinoma of the skin, chondrosarcoma, angiosarcoma, cholangiocarcinoma, soft tissue sarcoma, colon cancer, melanoma, Merkel cell carcinoma and glioblastoma multiforme. In certain embodiments, the present invention includes methods for treating advanced solid tumors, including, but not limited to, metastatic cutaneous squamous cell carcinoma (CSCC), unresectable locally advanced CSCC, metastatic colon cancer, advanced or metastatic hepatocellular cancer, advanced non-small cell lung cancer, basal cell carcinoma, recurrent glioblastoma multiforme, castration recurrent prostate cancer and any advanced solid tumor refractory to first-line therapy. Methods in accordance with this aspect include administering a therapeutically effective amount of an anti-PD-1 antibody. In certain embodiments, the methods include administering a therapeutically effective amount of an anti-PD-1 antibody in combination with an antitumor therapy.

Antitumor therapy includes, but is not limited to, standard anticancer therapy such as chemotherapy, radiation, surgery. Other antineoplastic therapies are described elsewhere herein. In one embodiment, the antitumor therapy includes radiation therapy. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to a patient, wherein each dose is administered 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the immediately preceding dose. In certain embodiments, each dose contains 0.1-10 mg/kg (eg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg) of the patient's body weight. In certain other embodiments, each dose contains 20-600 mg of anti-PD-1 antibody, such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg of anti-PD-1 antibody.

In certain embodiments, the present invention includes methods for treating a cancer or inhibiting the growth of a cancer with microsatellite instability (MSI). As used herein, the term microsatellite instability, also known as MSI, refers to an alteration in microsatellite repeats in tumor cells or genetic hypermutability caused by deficient DNA mismatch repair. Microsatellites, also known as simple sequence repeats, are repeated sequences of DNA containing repeating units 1-6 base pairs in length. Although the length of microsatellites varies greatly from person to person and contributes to DNA fingerprinting, every individual has microsatellites of a given length. MSI is

- 6 045900 result from the inability of mismatch repair (MMR) proteins to correct DNA replication errors. MSI contains DNA polymorphisms in which replication errors vary in length rather than in sequence. MSI includes frameshift mutations, through insertions or deletions, or hypermethylation leading to gene silencing. It is known in the art that microsatellite instability can lead to colon cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer and skin malignancies. The present invention includes methods for treating MSI cancers, the methods comprising administering to a patient in need thereof a therapeutically effective amount of an anti-PD-1 antibody, optionally in combination with radiation therapy.

One embodiment of the invention provides an anti-PD-1 antibody (eg, REGN2810) for use in the treatment of advanced solid tumors with MSI, including, but not limited to, metastatic colon cancer with MSI, metastatic endometrial cancer with MSI, and castration-recurrent prostate cancer with MSI. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with an advanced solid tumor with MSI, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered over 0.5 to 4 weeks after the immediately preceding dose. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with an advanced solid tumor with MSI, wherein each dose contains 20-600 mg of an anti-PD-1 antibody and wherein each dose is administered 0.5 to 4 weeks after the immediate previous dose.

As used herein, the term radiation therapy, also referred to as XRT, refers to the use of ionizing radiation to kill cancer cells generally as part of therapy against cancer. X-rays, gamma rays, or charged particles (such as protons or electrons) are used to create ionizing radiation. Radiation therapy can be delivered from a device located outside the patient's body (external beam radiation therapy), or from a source located inside the patient's body (internal beam therapy or brachytherapy), or through systemic radioisotopes delivered intravenously or orally (systemic radioisotope therapy). . Radiation therapy can be planned and administered in combination with imaging-based modalities such as computed tomography (CT) and magnetic resonance imaging (MRI) to accurately determine the dose and location of radiation to be delivered. In various embodiments, the radiation therapy is selected from the group consisting of whole body radiation therapy, standard external beam radiation therapy, stereotactic radiosurgery, stereotactic body radiation therapy, three-dimensional conformal radiation therapy, intensity modulated radiation therapy, image-guided radiation therapy , tomotherapy, brachytherapy and systemic radiation therapy. Depending on the purpose, in certain embodiments, the radiation therapy is curative, adjuvant, or palliative. In specific embodiments, the term radiation therapy refers to hypofractionated radiation therapy. Hypofractionated radiation therapy refers to radiation therapy in which the radiation dose is contained in 2 or more fractions. In various embodiments, each fraction contains 2-20 Gy. For example, a radiation dose of 50 Gy can be divided into 10 fractions, each containing 5 Gy. In certain embodiments, 2 or more fractions are administered on the next or consecutive days. In certain other embodiments, 2 or more fractions are administered every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or a combination thereof.

In accordance with certain embodiments, the present invention includes methods of treating or delaying or inhibiting tumor growth. In certain embodiments, the present invention includes methods for promoting tumor regression. In certain embodiments, the present invention includes methods for reducing tumor cell burden or reducing tumor burden. In certain embodiments, the present invention includes methods for preventing tumor recurrence. Methods in accordance with this aspect of the invention involve sequentially administering a therapeutically effective amount of an anti-PD-1 antibody in combination with radiation therapy to a patient, wherein the antibody is administered to the individual in multiple doses, for example, as part of a specific therapeutic dosing regimen. For example, a therapeutic dosing regimen may include administering one or more doses of an anti-PD-1 antibody to an individual at a frequency of approximately once daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 2 months, once every 3 months, once every 4 months or less often. In certain embodiments, one or more doses of anti-PD-1 antibody are administered in combination with one or more doses of radiation therapy, wherein one or more doses of radiation are administered to the individual at a frequency of approximately once per day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, once every 2 months, once every 3 months, once every 4 months or less often.

In certain embodiments, one or more doses are included in a treatment cycle. Methods in accordance with this aspect include administering to a patient at least one cycle of treatment,

- 7 045900 where at least one treatment cycle includes 1-10 doses of an anti-PD-1 antibody and optionally one or more doses of radiation therapy. In certain embodiments, 2-12 cycles of treatment are administered to the patient.

In specific embodiments, the present invention provides methods for increased antitumor effect or increased tumor inhibition. Methods in accordance with this aspect of the invention include administering to an individual with a solid tumor a therapeutically effective amount of an anti-PD-1 antibody prior to a dose of radiation, wherein the anti-PD-1 antibody may be administered over about 1 day, more than 1 day, more than 2 days , more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days or more than 8 days before radiation therapy. In certain embodiments, the methods provide increased tumor inhibition, e.g., about 20%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70% or greater than 80% compared to an individual dosed before the anti-PD-1 antibody. In certain embodiments, the radiation therapy includes hypofractionated radiation therapy.

In certain embodiments, the present invention provides methods for treating a malignant tumor, the methods comprising selecting a patient with a first tumor lesion and at least a second tumor lesion and administering one or more doses of an anti-PD-1 antibody in combination with radiation therapy such that both lesions are treated. In specific embodiments, the methods include administering radiation therapy to a first tumor lesion but not to a second tumor lesion, where administration results in tumor regression in both tumor lesions (abscopal effect). In certain embodiments, the methods include selecting a patient with a first tumor lesion and at least a second tumor lesion and administering one or more doses of an anti-PD-1 antibody in combination with hypofractionated radiation therapy, wherein the hypofractionated radiation therapy is administered to the first lesion but not the second damage, and where both injuries are treated with this administration. In certain embodiments, the anti-PD-1 antibody is administered prior to radiation therapy.

In certain embodiments, the present invention includes methods for treating cancer, the methods comprising administering to an individual in need thereof one or more subtherapeutic doses of an anti-PD-1 antibody in combination with one or more anticancer therapies, such as radiation therapy. As defined elsewhere herein, the term subtherapeutic dose refers to a dose less than the therapeutic dose that can be used to reduce the toxicity of the administered therapy. In certain embodiments, administration of a subtherapeutic dose of an anti-PD-1 antibody in combination with radiation therapy results in a therapeutic antitumor effect compared to administration of a subtherapeutic dose of an anti-PD-1 antibody alone. In certain other embodiments, the methods of the present invention include administering a therapeutically effective amount of an anti-PD-1 antibody in combination with a subtherapeutic dose of an anticancer therapy, such as chemotherapy or radiation. For example, a therapeutically effective amount of an anti-PD-1 antibody can be administered in combination with a subtherapeutic dose of cyclophosphamide for increased effect compared to either monotherapy.

In certain embodiments, the present invention includes methods for inhibiting, slowing, or stopping tumor metastasis or tumor infiltration into peripheral organs. Methods in accordance with this aspect include administering a therapeutically effective amount of an anti-PD-1 antibody to a patient. In certain embodiments, the anti-PD-1 antibody is administered in combination with radiation. In one embodiment, the radiation is hypofractionated radiation. In one embodiment, radiation is administered after administration of one or more doses of anti-PD-1 antibody.

In certain embodiments, the methods of the present invention include administering a therapeutically effective amount of an anti-PD-1 antibody to an individual with advanced solid tumors. In specific embodiments, the advanced solid tumor is a metastatic lung cancer, a head and neck cancer, a hepatocellular cancer, or a breast cancer. In certain other embodiments, the advanced solid tumor is a squamous cell malignant tumor of the skin. In certain embodiments, the advanced solid tumor is indolent or aggressive. In certain embodiments, the individual does not respond to prior therapy or has relapsed after prior therapy (eg, carboplatin). In certain embodiments, the individual has an advanced solid tumor that is refractory to first-line chemotherapy. In certain additional embodiments, the methods of the present invention further comprise administering radiation and/or cyclophosphamide to an individual with an advanced solid tumor.

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In certain embodiments, the present invention includes methods of treating or inhibiting the growth of a cancer, including, but not limited to, colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma. In certain embodiments, the present invention includes methods of treating or inhibiting the growth of a malignant tumor, including, but not limited to, hepatocellular carcinoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, basal cell carcinoma, squamous cell carcinoma of the skin, chondrosarcoma, angiosarcoma, cholangiocarcinoma, soft tissue sarcoma, colon cancer, melanoma, Merkel cell carcinoma and glioblastoma multiforme. In certain embodiments, the present invention includes methods for treating common solid tumors, including, but not limited to, metastatic cutaneous squamous cell carcinoma (CSCC), unresectable locally advanced CSCC, metastatic colon cancer, advanced or metastatic hepatocellular cancer, advanced non-small cell lung cancer, recurrent multiforme glioblastoma, newly diagnosed glioblastoma multiforme, castration-recurrent prostate cancer, and any advanced solid tumor refractory to first-line therapy.

In one aspect, the present invention includes methods for treating or inhibiting tumor growth, the methods comprising: (a) selecting a patient with cutaneous squamous cell carcinoma (CSCC), wherein the patient is selected based on a feature selected from the group consisting of: (i) the patient has locally advanced CSCC; (ii) the patient has metastatic CSCC; (iii) the tumor is unresectable; (iv) the patient had previously been treated with at least one anticancer therapy; (v) the patient suffers from a disease that is considered inoperable; (vi) surgery and/or radiation are contraindicated; (vii) the patient has previously been treated with radiation and the tumor is resistant or does not respond to radiation; (viii) the patient has locally advanced CSCC and is not eligible for curative surgery; (ix) the tumor contains UV-induced DNA damage; and (x) the patient has >1%, >5%, or >10% PD-L1 expression in tumor cells; and (b) administering a therapeutically effective amount of an anti-PD-1 antibody to the patient. In certain embodiments, one or more doses of anti-PD-1 antibody are administered 1-12 weeks after the immediately preceding dose, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the immediately preceding dose. In certain embodiments, each dose of anti-PD-1 antibody contains 0.1, 1, 0.3, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg of patient body weight. In certain embodiments, each dose contains 50-500 mg of anti-PD-1 antibody, such as 200 mg, 250 mg, or 350 mg of anti-PD-1 antibody, wherein each dose is administered over 0.5, 1, 2, 3, or 4 weeks after the immediately preceding dose. In one embodiment, the anti-PD-1 antibody is REGN2810.

In accordance with one aspect, the present invention includes methods for treating or inhibiting tumor growth, the methods comprising: (a) selecting a patient with basal cell carcinoma (BCC), wherein the patient is selected based on a trait selected from the group consisting of: (i) the patient has locally advanced SCD; (ii) the patient has metastatic SCD; (iii) the tumor is unresectable; (iv) the patient had previously been treated with at least one anticancer therapy; (v) the patient was previously treated and progressed with a Hedgehog pathway inhibitor (eg, vismodegib, sonedegib); (vi) the patient is intolerant to a Hedgehog pathway inhibitor; (vii) the patient suffers from a disease that is considered inoperable or is not amenable to surgical intervention with a curative chain; (viii) surgery and/or radiation is contraindicated; (ix) the patient has previously received radiation treatment and the tumor is resistant or does not respond to radiation; (viii) the patient has >1%, >5%, or >10% PD-L1 expression in tumor cells; and (ix) the tumor contains UV-induced DNA damage; and (b) administering a therapeutically effective amount of an anti-PD-1 antibody to the patient. In certain embodiments, one or more doses of anti-PD-1 antibody are administered 1-12 weeks after the immediately preceding dose, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the immediately preceding dose. In certain embodiments, each dose of anti-PD-1 antibody contains 0.1, 1, 0.3, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg of patient body weight. In certain embodiments, each dose contains 50-500 mg of anti-PD-1 antibody, such as 200 mg, 250 mg, or 350 mg of anti-PD-1 antibody, wherein each dose is administered over 0.5, 1, 2, 3, or 4 weeks after the immediately preceding dose. In one embodiment, the anti-PD-1 antibody is REGN2810.

In certain embodiments, each dose of anti-PD-1 antibody is administered 1 week, 2 weeks, 3 weeks, or 4 weeks after the immediately preceding dose, where each dose contains 50-600 mg of anti-PD-1 antibody. In one embodiment, each dose contains 200, 250, 300, or 350 mg of anti-PD-1 antibody.

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One embodiment of the invention relates to an anti-PD-1 antibody (eg, REGN2810) for use in the treatment of cholangiocarcinoma. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with cholangiocarcinoma, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered 0.5 to 4 weeks after the immediately preceding doses. In certain embodiments, each dose contains 50-500 mg of anti-PD-1 antibody, such as 200 mg, 250 mg, or 350 mg of anti-PD-1 antibody, wherein each dose is administered over 0.5, 1, 2, 3, or 4 weeks after the immediately preceding dose.

One embodiment of the invention provides an anti-PD-1 antibody (eg, REGN2810) for use in the treatment of advanced hepatocellular malignancy (HCC). In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with SUD, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered 0.5 to 4 weeks after the immediately preceding doses. In certain embodiments, each dose contains 50-500 mg of anti-PD-1 antibody, such as 200 mg, 250 mg, or 350 mg of anti-PD-1 antibody, wherein each dose is administered over 0.5, 1, 2, 3, or 4 weeks after the immediately preceding dose.

One embodiment of the invention relates to an anti-PD-1 antibody (eg, REGN2810) for use in the treatment of soft tissue sarcoma. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with soft tissue sarcoma, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered 0.5 to 4 weeks after the immediately preceding dose. In certain embodiments, each dose contains 50-500 mg of anti-PD-1 antibody, such as 200 mg, 250 mg, or 350 mg of anti-PD1 antibody, wherein each dose is administered 0.5, 1, 2, 3, or 4 weeks after the immediate previous dose.

One embodiment of the invention provides an anti-PD-1 antibody (eg, REGN2810) for use in the treatment of non-small cell lung cancer (NSCLC). In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with NSCLC, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered 0.5 to 4 weeks after the immediately preceding doses. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with NSCLC, wherein each dose contains 50-600 mg of an anti-PD-1 antibody, and wherein each dose is administered 0.5 to 4 weeks after the immediately preceding dose.

In one aspect, the present invention includes methods of treating or inhibiting tumor growth, the methods comprising selecting a patient with brain cancer and administering a therapeutically effective amount of an anti-PD-1 antibody or antigen binding fragment thereof to an individual in need thereof. In certain embodiments, the malignant brain tumor is glioblastoma multiforme. In one embodiment, the individual has a newly diagnosed glioblastoma multiforme. In one embodiment, the individual is >65 years of age. In one embodiment, the anti-PD-1 antibody is administered in one or more doses, with each dose administered 0.5 to 4 weeks after the immediately preceding dose. In one embodiment, each dose of anti-PD-1 antibody contains 1, 3, or 10 mg/kg of the patient's body weight. In certain embodiments, an anti-PD-1 antibody is administered in combination with radiation therapy. In one embodiment, the radiation therapy is hypofractionated radiation therapy. In one embodiment, the individual is administered 20-60 Gy in 2-20 fractions. In certain embodiments, one or more doses of an anti-PD-1 antibody are contained in one or more treatment cycles, where each treatment cycle includes 1-6 doses of an anti-PD-1 antibody. In one embodiment, at least one treatment cycle further comprises radiation therapy. In a further embodiment, the radiation therapy is hypofractionated radiation therapy. In certain embodiments, the individual is administered hypofractionated radiation therapy in the first cycle of treatment, wherein the hypofractionated radiation therapy contains 20-60 Gy in 2-20 fractions. In one embodiment, the individual receives hypofractionated radiation therapy one week after administration of the anti-PD-1 antibody in the first cycle of treatment. In certain embodiments, the methods of the present invention further comprise administering an antiangiogenic agent to the individual if the patient develops intracranial edema following administration of an anti-PD-1 antibody. In one embodiment, the antiangiogenic agent is a vascular endothelial growth factor (VEGF) inhibitor. In one embodiment, the anti-angiogenic agent is an angiopoietin-2 (Ang-2) inhibitor (eg, an anti-Ang-2 antibody such as nesvacumab). In certain embodiments, the VEGF inhibitor is selected from the group consisting of a VEGF inhibitory fusion protein (e.g., VEGF-Trap, such as aflibercept or another VEGF inhibitory fusion protein as set forth in US 7,087,411), an anti-VEGF antibody (e.g., bevacizumab), and a small molecule kinase inhibitor VEGF receptor (eg sunitinib, sorafenib or pazopanib).

In certain embodiments, the methods of the present invention include introducing

- 10 045900 anti-PD-1 antibody in combination with radiation therapy to the patient as first-line treatment (eg, initial treatment). In other embodiments, an anti-PD-1 antibody in combination with radiation therapy is administered as second-line treatment (eg, after prior therapy). For example, an anti-PD-1 antibody in combination with radiation therapy is administered as a second-line treatment to an individual who has relapsed after prior therapy, such as chemotherapy.

The methods of the present invention, in accordance with certain embodiments, comprise administering to an individual a therapeutically effective amount of an anti-PD-1 antibody and radiation in combination with an additional therapeutic agent or therapeutic regimen or procedure. An additional therapeutic agent or therapeutic regimen or procedure may be performed to enhance the antitumor effect, to reduce the toxic effects of one or more therapies, and/or to reduce the dose of one or more therapies. In various embodiments, the additional therapeutic agent or therapeutic regimen or procedure is selected from the group consisting of, for example, chemotherapy, cyclophosphamide, surgery, cancer vaccine, programmed death ligand 1 (PD-L1) inhibitor (for example, anti-PD antibodies -L1), lymphocyte activation gene 3 (LAG3) inhibitor (eg, anti-LAG3 antibodies), cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitor (eg, ipilimumab), glucocorticoid-induced tumor necrosis factor receptor (GITR) inhibitor ( e.g., anti-GITR antibodies), T-cell immunoglobulin and mucin-3 (TIM3) inhibitor, B-T-lymphocyte attenuator (BTLA) inhibitor, T-cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitor, CD47 inhibitor, indoleamine 2,3-dioxygenase (IDO) inhibitor, vascular endothelial growth factor (VEGF) antagonist, angiopoietin-2 (Ang2) inhibitor, transforming growth factor β (TGFe) inhibitor, epidermal growth factor receptor (EGFR) inhibitor, tumor-specific antibody antigen [eg, CA9, CA125, melanoma associated antigen 3 (MAGE3), embryonal tumor antigen (CEA), vimentin, M2-PK tumors, prostate specific antigen (PSA), mucin-1, MART-1 and CA19-9 ], anti-CD3/anti-CD20 bispecific antibody, vaccine (eg, bacillus Calmette-Guérin), granulocyte-macrophage colony-stimulating factor, cytotoxin, chemotherapeutic agent, IL-6R inhibitor, IL-4R inhibitor, IL-10 inhibitor, cytokine such as IL-2, IL-7, IL-21 and IL-15, an anti-inflammatory drug such as corticosteroids and non-steroidal anti-inflammatory drugs, and a nutritional supplement such as antioxidants. In certain embodiments, an anti-PD-1 antibody may be administered in combination with therapy, including a chemotherapy agent, and surgery. As used herein, the phrase in combination with means that the anti-PD-1 antibody is administered to an individual simultaneously, immediately before or immediately after radiation therapy and an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is co-formulated with an anti-PD-1 antibody.

One embodiment of the invention relates to a combination of an anti-PD-1 antibody (eg, REGN2810), radiation therapy, cyclophosphamide and GM-CSF for use in the treatment of head and neck squamous cell carcinoma (HNSCC). In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with HNSCC, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered 0.5 to 4 weeks after the immediately preceding doses. In certain embodiments, each dose contains 50-500 mg of anti-PD-1 antibody, such as 200 mg, 250 mg, or 350 mg of anti-PD-1 antibody, wherein each dose is administered over 0.5, 1, 2, 3, or 4 weeks after the immediately preceding dose.

One embodiment of the invention relates to a combination of an anti-PD-1 antibody (eg, REGN2810), radiation therapy and cyclophosphamide for use in the treatment of breast cancer. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to an individual with breast cancer, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered over 0.5 to 4 weeks after the immediately preceding dose.

One embodiment of the invention provides a combination of an anti-PD-1 antibody (eg, REGN2810), radiation therapy, cyclophosphamide, and GM-CSF for use in the treatment of advanced solid tumors in patients who have been previously treated with an anti-PD-1 antibody or an anti-PD-1 antibody. L1. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to a patient in need thereof, wherein each dose contains from 0.1 to 20 mg/kg of the patient's body weight and wherein each dose is administered 0.5 to 4 weeks after the immediately preceding dose.

One embodiment of the invention provides a combination of an anti-PD-1 antibody (eg, REGN2810), docetaxel, and optionally carboplatin for use in the treatment of advanced solid tumors that are refractory to first-line chemotherapy. In certain

- 11 045900 embodiments, docetaxel is administered at a low dose. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to a patient, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered 0.5 to 4 weeks after the immediately preceding dose.

One embodiment of the invention relates to a combination of an anti-PD-1 antibody (eg, REGN2810) and radiation therapy for use in the treatment of newly diagnosed or recurrent glioblastoma multiforme (GBM). In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to a patient, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered 0.5 to 4 weeks after the immediately preceding dose. In certain embodiments, the radiation treatment is hypofractionated radiation therapy, as disclosed herein.

Certain embodiments of the invention relate to the combination of an anti-PD1 antibody (eg, REGN2810) and radiation therapy for use in the treatment of squamous cell carcinoma of the cervix, squamous cell carcinoma of the anus, Merkel cell carcinoma, adenocarcinoma of the small intestine, or serous carcinoma of the ovary. In certain embodiments, one or more doses of an anti-PD-1 antibody are administered to a patient, where each dose contains from 0.1 to 20 mg/kg of the patient's body weight and where each dose is administered 0.5 to 4 weeks after the immediately preceding dose. In certain embodiments, the radiation treatment is hypofractionated radiation therapy as disclosed herein.

In certain embodiments, the present invention includes methods for treating large tumors or advanced malignancies, the methods comprising administering to an individual an anti-PD-1 antibody in combination with radiation therapy and an additional therapeutic agent, wherein the additional therapeutic agent is administered to overcome immunosuppression mediated by regulatory T cells ( Treg). In certain embodiments, the additional therapeutic agent is selected from the group consisting of an anti-GITR antibody, an anti-LAG3 antibody, cyclophosphamide, and GM-CSF.

As used herein, the term large tumor refers to the size of the tumor. It usually correlates with a higher tumor load or tumor burden. In certain embodiments, it correlates with the stage of the disease, such as advanced malignancy. In certain embodiments, it correlates with an increased likelihood of metastasis.

In certain embodiments, the present invention includes methods comprising administering one or more doses of an anti-PD-1 antibody in combination with radiation therapy and a subtherapeutic dose of cyclophosphamide. As used herein, a subtherapeutic dose of cyclophosphamide (also referred to herein as low dose cyclophosphamide) refers to an amount of cyclophosphamide that does not, by itself, produce a therapeutic effect and preferably does not cause toxicity. Exemplary doses of cyclophosphamide that are considered subtherapeutic in the context of the present invention include 100 mg/m ² , 90 mg/m ² , 80 mg/m ² or less.

In one aspect, the present invention includes methods comprising administering a therapeutically effective amount of an anti-PD-1 antibody in combination with radiation to an individual who is on a background therapeutic regimen against cancer. The background therapeutic regimen against a malignant tumor may include a course of administration, for example, of a chemotherapeutic agent. An anti-PD-1 antibody in combination with radiation therapy can be added on top of a background therapeutic regimen against the malignancy. In some embodiments, the anti-PD-1 antibody is added as part of a background tapering regimen in which background anti-cancer therapy is gradually withdrawn from the patient over time (e.g., in a stepwise manner) while the anti-PD-1 antibody is administered to the individual on a continuous basis. dose or in an increasing dose or in a decreasing dose over time. For example, background therapy against cancer may include a chemotherapeutic agent, which can be administered at a low dose or at a subtherapeutic dose. In certain embodiments, the present invention includes methods for treating cancer, the methods comprising administering one or more doses of an anti-PD-1 antibody in combination with radiation therapy and one or more doses of a chemotherapeutic agent, wherein the chemotherapeutic agent is administered at a subtherapeutic dose.

In certain embodiments, radiation therapy is administered to the first tumor lesion, but not to the second tumor lesion, where administration in combination with an anti-PD-1 antibody results in tumor regression in both the first and second tumor lesions (abscopal effect). In certain embodiments, the methods of the present invention include administering an anti-PD-1 antibody in combination with radiation therapy to produce a prolonged abscopal effect.

In certain embodiments, the methods of the present invention include administering to a patient a therapeutically effective amount of an anti-PD-1 antibody, optionally in a combination

- 12 045900 nations with radiation therapy, where administration of the combination leads to increased inhibition of tumor growth. In certain embodiments, tumor growth is inhibited by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% by compared with an individual without treatment or an individual who received either the antibody or radiation as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and/or radiation therapy results in increased tumor regression, tumor shrinkage, and/or disappearance. In certain embodiments, administration of an anti-PD-1 antibody and/or radiation therapy results in a delay in tumor growth and development, for example, tumor growth can be delayed by about 3 days, more than 3 days, about 7 days, more than 7 days , more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years or more than 3 years compared with an individual without treatment or an individual , which was treated with antibody or radiation as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody in combination with radiation therapy prevents tumor recurrence and/or increases the duration of survival of the patient, for example, increases the duration of survival by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months than a patient without treatment or a patient receiving antibody or radiation in as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody in combination with radiation therapy improves progression-free survival or overall survival. In certain embodiments, administration of an anti-PD-1 antibody in combination with radiation therapy increases the response and duration of response in a patient, e.g., greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater by more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40% or more than 50% relative to a patient without treatment or a patient who received antibody or radiation as monotherapy. In certain embodiments, administration of an anti-PD-1 antibody and/or radiation therapy to an individual with a cancer results in complete resolution of all signs of tumor cells (complete response). In certain embodiments, administration of an anti-PD-1 antibody and/or radiation therapy to an individual with a cancer results in at least a 30% or greater reduction in tumor cells or tumor size (partial response). In certain embodiments, administration of an anti-PD-1 antibody and/or radiation therapy to an individual with a cancer results in complete or partial clearance of tumor cells/lesions, including new measurable lesions. Tumor shrinkage can be measured by any of the methods known in the art, for example, x-rays, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology or molecular genetic analyses.

In certain embodiments, the methods of the present invention include administering to a patient a therapeutically effective amount of an anti-PD-1 antibody, wherein administration of the anti-PD-1 antibody results in increased overall survival (OS) or progression-free survival (PFS) in the patient compared to a patient who standard of care (SOC) therapy (eg, chemotherapy, surgery, or radiation) was administered. In certain embodiments, the PFS is increased by at least one month, by at least 2 months, by at least 3 months, by at least 4 months, by at least 5 months, by at least 6 months, by at least for at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years or at least for 3 years compared with a patient treated with any one or more SOC therapies. In certain embodiments, the OS is increased by at least one month, by at least 2 months, by at least 3 months, by at least 4 months, by at least 5 months, by at least 6 months, by at least for at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years or at least for 3 years compared with a patient treated with any one or more SOC therapies.

The present invention also provides kits containing an anti-PD-1 antibody for therapeutic use. Kits typically contain a label indicating the intended use of the kit's contents and instructions for use. The term label includes any written or recorded material supplied on or with the kit, or which otherwise accompanies the kit. Accordingly, this disclosure provides a kit for the treatment of a patient suffering from a cancer, the kit containing: (a) a dose of an antibody or antigen-binding portion thereof that specifically binds to PD-1 and inhibits the activity of PD-1; and (b) instructions for use of an anti-PD-1 antibody in any of the therapies disclosed herein. In certain embodiments, for the treatment of human patients, the kit contains an antibody against

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Human PD-1 as disclosed herein, for example, REGN2810. In other embodiments, the anti-PD-1 antibody may be any one of nivolumab, pembrolizumab, or any of the anti-PD-1 antibodies disclosed herein. In certain embodiments, the dose of the anti-PD-1 antibody is in the range of 0.1 to 10 mg/kg body weight. In certain embodiments, the dose of the anti-PD-1 antibody comprises from 50 to 600 mg.

Methods for suppressing regulatory T cells.

In accordance with certain aspects, the present invention provides methods for suppressing or inhibiting the activation and/or proliferation of regulatory T cells (Treg). In certain embodiments, the present invention provides methods for suppressing Treg cell activity. Methods in accordance with these aspects include selecting a patient with a solid tumor and administering to the individual an anti-PD-1 antibody or an antigen-binding fragment thereof in combination with at least one of (i) radiation therapy and (ii) a glucocorticoid-induced tumor necrosis factor receptor antagonist. (GITR). In certain embodiments, the methods include administering to a patient an anti-PD-1 antibody or an antigen-binding fragment thereof in combination with radiation therapy and a GITR antagonist.

In certain embodiments, the GITR antagonist is an anti-GITR antibody or an antigen-binding fragment thereof. In accordance with certain exemplary embodiments of the present invention, an anti-GITR antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and/or complementarity determining regions (CDRs) comprising the amino acid sequences of any of the anti-GITR antibodies. GITR as set forth in USSN 62/256,922 (filed November 18, 2015), the contents of which are incorporated herein in their entirety. Other anti-GITR antibodies that may be used in the context of the methods of the present invention include any of the anti-GITR antibodies as set forth, for example, in US Patent Nos. 9228016, 8709424, 8591886, 7812135 or US Patent Publication No. 20150368349.

In certain embodiments, the present invention provides methods for suppressing or eliminating Treg activity, the methods comprising administering to an individual in need thereof an anti-PD-1 antibody or an antigen-binding fragment thereof in combination with one or more doses of radiation and a cytotoxic T lymphocyte antigen 4 antagonist (CTLA ). In certain embodiments, the CTLA antagonist is an anti-CTLA antibody (eg, ipilimumab).

In certain embodiments, the present invention provides methods for suppressing or eliminating Treg activity, the methods comprising administering to an individual in need thereof an anti-PD-1 antibody or an antigen-binding fragment thereof in combination with one or more doses of radiation and a lymphocyte activation gene 3 (LAG-3) antagonist ). In certain embodiments, the LAG-3 antagonist is an anti-LAG-3 antibody. Antibodies against LAG-3 that can be used in the context of the methods of the present invention are disclosed in USSN 15/289,032 (filed October 7, 2016), the contents of which are incorporated herein in their entirety.

In certain embodiments, the present invention provides methods for suppressing or eliminating Treg activity, the methods comprising administering to an individual in need thereof an anti-PD-1 antibody or antigen binding fragment thereof in combination with one or more doses of radiation and cyclophosphamide.

In one aspect, the methods of the present invention include administering an anti-PD-1 antibody in combination with radiation therapy and an additional therapeutic agent selected from the group consisting of a GITR antagonist, an anti-LAG-3 antibody, and cyclophosphamide to an individual with a solid tumor, wherein administration leads to an effect selected from the group consisting of inhibition of tumor growth, reduction of tumor size, delay of tumor growth, inhibition of tumor metastasis, reduction of metastatic lesions over time, reduced use of chemotherapeutic or cytotoxic agents, prolonged survival, complete response, partial response and stable diseases. In certain embodiments, administration results in a reduction in the patient's tumor burden. In certain embodiments, the individual has a large tumor. As defined elsewhere herein, the term large tumor refers to the size of the tumor and correlates with an increased tumor burden and an increased likelihood of metastasis. In certain embodiments, the term refers to a common malignancy. Anti-PD-1 Antibodies and Antigen-Binding Fragments Thereof In accordance with certain exemplary embodiments of the present invention, methods include administering a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding fragment thereof. The term antibody as used herein includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as multimers thereof (eg, IgM). In a typical antibody, each heavy chain contains a heavy chain variable region (abbreviated as

- 14 045900 herein as HCVR or _VH ) and the heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or V _L ) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and _VL regions can be further subdivided into regions of hypervariability, designated complementarity determining regions (CDRs), which alternate with regions that are more conserved, called framework regions (FRs). Each VH and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, FRs from an anti-IL-4R antibody (or an antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. The consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs.

The term antibody, as used herein, also includes antigen-binding fragments of entire antibody molecules. The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding antibody fragments can be recovered, for example, from whole antibody molecules using any suitable standard methods, such as proteolytic digestion or recombinant genetic engineering techniques, including manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biological techniques, for example, to arrange one or more variable and/or constant domains in a suitable configuration or introduce codons, create cysteine residues, modify, add or remove amino acids, etc.

Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) fragments F(ab') ₂ ; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as the CDR3 peptide) or the FR3-CDR3-FR4 restricted peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-transplanted antibodies, diabodies, tri-bodies, tetrabodies, mini-bodies, nanobodies (e.g. monovalent nanobodies, divalent nanobodies, etc.) , small modular immunopharmaceuticals (SMIPs) and shark IgNAR variable domains are also included in the expression of the antigen binding fragment as used herein.

The antigen binding fragment of an antibody typically contains at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR that is adjacent or in frame with one or more framework sequences. In antigen binding fragments having a VH domain linked to a VL domain, the VH and _VL domains can be arranged relative to each other in any suitable arrangement. For example, the variable region may be dimeric and the dimers comprise _VH - _VH , VH-VL or VL-VL. Alternatively, the antigen binding antibody fragment may comprise a monomeric VH or _VL domain.

In certain embodiments, an antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary variable and constant domain configurations that may be found in an antigen binding fragment of an antibody of the present invention include: (i) Vh-Ch1; (ii) Vh-Ch2; (iii) Vh-Ch3; (iv) Vh-Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) Vl-Ch2-Ch3; and (xiv) V _l -C _l . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be directly linked to each other or can be linked through a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen binding fragment of an antibody of the present invention may contain a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V _H or V _L domains (eg via disulfide bond(s)).

The term antibody, as used herein, also includes multispecific

- 15 045900 (for example, bispecific) antibodies. A polyspecific antibody or antigen-binding antibody fragment typically contains at least two different variable domains, where each variable domain is capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format can be adapted for use in the context of an antibody or antigen-binding antibody fragment of the present invention using standard methods available in the art. For example, the present invention includes methods involving the use of bispecific antibodies in which one immunoglobulin arm has specificity for PD-1 or a fragment thereof and the other immunoglobulin arm has specificity for a second therapeutic target or is conjugated to a therapeutic fragment. Exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, for example, scFv-based formats or bispecific diabody formats, IgG-scFv fusions, double variable domain (DVD) Ig, quad, peak-to-valley, common light chain (e.g. common knob-in-valley light chain, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, IgG with Dual Action Fab (DAF) and bispecifics Mab2 formats (for a review of the above formats, see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, for example, where unnatural amino acids with orthogonal chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes of defined composition, valency and geometry ( see, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Antibodies used in the methods of the present invention may be human antibodies. The term human antibody, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may, however, contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo), for example, in the CDR and, in particular , CDR3. However, the term human antibody, as used herein, is not intended to include antibodies in which CDR sequences derived from the mammalian germ line of other species, such as mouse, are transplanted into human framework sequences.

Antibodies used in the methods of the present invention may be recombinant human antibodies. The term recombinant human antibody, as used herein, is intended to include all human antibodies that are produced, expressed, created or isolated by recombinant means, for example, antibodies expressed using a recombinant expression vector transferred into a host cell (further described below ), antibodies isolated from libraries of recombinant human combination antibodies (further described below), antibodies isolated from an animal (for example, a mouse) that are transgenic for human immunoglobulin genes [see for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295], or antibodies produced, expressed, generated or released by any other means that involve splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subject to in vitro mutagenesis (or, when using an animal transgenic for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline V _H and V _L sequences, may not naturally exist in the human germline antibody repertoire in vivo.

In accordance with certain embodiments, the antibodies used in the methods of the present invention specifically bind to PD-1. The term specifically bind or the like means that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, an antibody that specifically binds PD-1, as used in the context of the present invention, includes antibodies that bind to PD-1 or a portion thereof with a KD of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about

- 16 045900 specifically 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured in a surface assay plasmon resonance. However, an isolated antibody that specifically binds human PD-1 may be cross-reactive with other antigens, such as PD-1 molecules from other (non-human) species.

In accordance with certain exemplary embodiments of the present invention, an anti-PD-1 antibody or antigen binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR), and/or complementarity determining regions (CDRs) comprising amino acid sequences of any of anti-PD-1 antibodies as set forth in US Patent Publication No. 20150203579, hereby incorporated in its entirety. In certain exemplary embodiments, an anti-PD-1 antibody or antigen binding fragment thereof that can be used in the context of the methods of the present invention comprises heavy chain complementarity determining regions (HCDRs) from a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO :1, and a light chain complementarity determining region (LCDR) from a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:2. In certain embodiments, an anti-PD-1 antibody or antigen binding fragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:3; HCDR2 contains the amino acid sequence SEQ ID NO:4; HCDR3 contains the amino acid sequence SEQ ID NO:5; LCDR1 contains the amino acid sequence SEQ ID NO:6; LCDR2 contains the amino acid sequence SEQ ID NO:7; and LCDR3 contains the amino acid sequence of SEQ ID NO:8. In other embodiments, the anti-PD-1 antibody or antigen binding fragment thereof comprises an HCVR comprising SEQ ID NO:1 and an LCVR comprising SEQ ID NO:2. In certain embodiments, the methods of the present invention include the use of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:9. In some embodiments, the anti-PD-1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:10. An exemplary antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO:9 and a light chain containing the amino acid sequence of SEQ ID NO:10 is a fully human anti-PD-1 antibody known as REGN2810 and also known as emplumab. In accordance with certain exemplary embodiments, the methods of the present invention include the use of REGN2810 or a bioequivalent thereof. The term bioequivalent, as used herein, refers to anti-PD-1 antibodies or PD-1 binding proteins or fragments thereof that are pharmaceutical equivalents or pharmaceutical alternatives in which the rate and/or extent of absorption does not show significant differences from those of REGN2810 when administered at the same molar dose under similar experimental conditions, either as a single dose or in multiple doses. As used herein, the term refers to antigen binding proteins that bind to PD-1 that are not clinically significantly different from REGN2810 with respect to their safety, purity and/or potency.

In accordance with certain embodiments of the present invention, the anti-human PD-1 agent or antigen binding fragment thereof comprises an HCVR that is 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO:1.

In accordance with certain embodiments of the present invention, the anti-human PD-1 agent or antigen binding fragment thereof comprises an LCVR that is 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO:2.

In accordance with certain embodiments of the present invention, the anti-human PD-1 agent or antigen binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO:1 that has no more than 5 amino acid substitutions. In accordance with certain embodiments of the present invention, the anti-human PD-1 agent or antigen binding fragment thereof comprises an LCVR comprising the amino acid sequence of SEQ ID NO:2 that has no more than 2 amino acid substitutions.

Sequence identity can be measured by any method known in the art (eg, GAP, BESTFIT and BLAST).

The present invention also relates to the use of anti-PD-1 antibodies in methods of treating cancer, wherein the anti-PD-1 antibodies contain variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein that have one or more conservative amino acid substitutions . For example, the present invention includes the use of anti-PD-1 antibodies having amino acid sequences HCVR, LCVR and/or CDR, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

- 17 045900

Other anti-PD-1 antibodies that can be used in the context of the methods of the present invention include, for example, antibodies designated and known in the art as nivolumab (US Pat. No. 8,008,449), pembrolizumab (U.S. Pat. No. 8,354,509), MEDI0608 (U.S. Pat. No. 8609089), pidilizumab (U.S. Patent No. 8686119), or any of the anti-PD-1 antibodies as set forth in U.S. Patent Nos. 6808710, 7488802, 8168757, 8354509, 8779105 or 8900587.

Anti-PD-1 antibodies used in the context of the methods of the present invention may have pH-dependent binding characteristics. For example, an anti-PD-1 antibody for use in the methods of the present invention may exhibit reduced binding to PD-1 at acidic pH compared to neutral pH. Alternatively, the anti-PD-1 antibody of the invention may exhibit enhanced binding to its antigen at acidic pH compared to neutral pH. The expression acidic pH includes pH values less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6 , 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less . As used herein, the expression neutral pH means a pH of about 7.0 to about 7.4. The expression neutral pH includes pH values of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

In certain cases, reduced binding to PD-1 at acidic pH compared to neutral pH is expressed in terms of the ratio of the KD value of antibody binding to PD-1 at acidic pH to the KD value of antibody binding to PD-1 at neutral pH (or vice versa). For example, an antibody or antigen binding fragment thereof may be considered to exhibit reduced binding to PD-1 at acidic pH compared to neutral pH for purposes of the present invention if the antibody or antigen binding fragment thereof exhibits an acidic/neutral KD ratio of approximately 3.0 or greater. In certain exemplary embodiments, the acidic/neutral KD ratio for an antibody or antigen binding fragment of the present invention may be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6 ,5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5 , 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 7 0.0, 100.0 or more.

Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at an acidic pH compared to a neutral pH. Additionally, modifications of the antigen binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, by replacing one or more amino acids of the antigen binding domain (eg, in the CDR) with a histidine residue, it is possible to produce an antibody with reduced antigen binding at an acidic pH relative to a neutral pH. As used herein, the expression acidic pH means a pH of 6.0 or less.

Combination therapy.

The methods of the present invention, in accordance with certain embodiments, comprise administering to an individual a therapeutically effective amount of an anti-PD-1 antibody. In certain embodiments, the methods of the present invention include administering radiation therapy in combination with an anti-PD-1 antibody for additive or synergistic activity to treat the cancer. As used herein, the expression in combination with means that radiation therapy is administered before, after, or in parallel with the anti-PD-1 antibody. The term in combination with also includes sequential or concomitant administration of an anti-PD-1 antibody and radiation therapy. For example, when administered before radiation therapy, the anti-PD-1 antibody may be administered more than 150 hours before, about 150 hours before, about 100 hours before, about 72 hours before, about 60 hours before, about 48 hours before, about 36 hours before , approximately 24 hours, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, approximately 1 hour or approximately 30 minutes, approximately 15 minutes or approximately 10 minutes before radiation therapy. When administered after radiation therapy, the anti-PD-1 antibody can be administered after about 10 minutes, after about 15 minutes, after about 30 minutes, after about 1 hour, after about 2 hours, after about 4 hours, after about 6 hours, after about 8 hours, approximately 10 hours, approximately 12 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 60 hours, approximately 72 hours, or more than 72 hours after radiation therapy. Administration concurrent with radiation therapy means that the anti-PD-1 antibody is administered to an individual within less than 10 minutes (before, after, or simultaneously) of radiation therapy.

In certain embodiments, the methods of the present invention include administering an additional therapeutic agent, wherein the additional therapeutic agent is an anti-cancer drug. As used herein, an anticancer drug means any agent that can be used to treat a cancer, including but not limited to cytotoxins and agents such as antimetabolites, alkylating agents,

- 18 045900 tracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), biological agents (for example, antibodies and interferons) and radioactive agents. As used herein, a cytotoxin or cytotoxic agent also refers to a chemotherapeutic agent and refers to any agent that is harmful to cells. Examples include, but are not limited to, Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin , actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues.

In certain embodiments, the methods of the present invention include administering an additional therapeutic agent or therapeutic regimen or procedure selected from the group consisting of surgery, radiation, a programmed death ligand 1 (PD-L1) inhibitor (e.g., anti-PD-L1 antibodies, such as disclosed in US Patent Publication 2015/0203580, or atezolizumab), lymphocyte activation gene 3 (LAG-3) inhibitor (e.g., anti-LAG-3 antibodies), cytotoxic T lymphocyte associated protein 4 (CTLA4) inhibitor (e.g., ipilimumab ), glucocorticoid-induced tumor necrosis factor receptor (GITR) inhibitor (eg, anti-GITR antibodies), T-cell immunoglobulin and mucin-3 (TIM3) inhibitor, B and T-lymphocyte attenuator (BTLA) inhibitor, T-cell immunoglobulin and mucin-3 inhibitor ( Ig and ITIM domains (TIGIT), a CD47 inhibitor, an antagonist of another T cell coinhibitor or ligand (for example, antibodies to CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA), a CD20 inhibitor (for example, antibodies against CD20 or bispecific CD3 /CD20 antibody), an indoleamine 2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., VEGF-Trap such as aflibercept or other VEGF inhibitory fusion protein as set forth in US 7,087,411, or antibodies against VEGF or its antigen-binding fragment (eg, bevacizumab or ranibizumab) or a small molecule inhibitor of VEGF receptor kinase (eg, sunitinib, sorafenib, or pazopanib)], angiopoietin 2 (Ang2) inhibitor (eg, nesvacumab), transforming growth factor β (TGFe) inhibitor ), CD38 inhibitor, epidermal growth factor receptor (EGFR) inhibitor (eg, erlotinib, cetuximab), costimulatory receptor agonist (eg, glucocorticoid-inducible TNFR-related protein agonist), tumor-specific antigen antibody [eg, CA9, CA125, melanoma-associated protein antigen 3 (MAGE3), embryonal tumor antigen (CEA), vimentin, M2-RK tumors, prostate specific antigen (PSA), mucin-1, MART-1 and CA19-9], vaccines (for example, bacillus Calmette-Guerin, cancer vaccine), cyclophosphamide, adjuvant to enhance antigen presentation (eg, granulocyte-macrophage colony-stimulating factor), cytotoxin, chemotherapeutic agent (eg, dacarbazine, temozolomide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel and vincristine), interleukin-6 receptor (IL-6R) inhibitor (eg, sarilumab), IL-4R inhibitor (eg, dupilumab), IL-10 inhibitor, cytokine such as IL-2, IL-7, IL-21 and IL-15, antibody-drug conjugate (ADC) (eg, anti-CD19-DM4 ADC and anti-DS6-DM4 ADC), chimeric antigen receptor T cells (eg, CD19-targeted T cells ), an anti-inflammatory drug (eg, corticosteroids and non-steroidal anti-inflammatory drugs) and a dietary supplement such as antioxidants.

In certain embodiments, the methods of the invention include administering an anti-PD-1 antibody in combination with radiation therapy and optionally an anti-GITR antibody in order to generate long-term sustained antitumor responses and/or enhance survival of patients with cancer. In some embodiments, the methods of the invention include administering radiation therapy before, concomitantly, or after administering an anti-PD-1 antibody and an anti-GITR antibody to a patient with a cancer. For example, radiation therapy can be administered in one or more doses to tumor lesions after administration of one or more doses of antibodies. In some embodiments, radiation therapy can be administered locally to a tumor lesion to enhance the local immunogenicity of a patient's tumor (auxiliary radiation) and/or kill tumor cells (ablative radiation) following systemic administration of an anti-PD-1 antibody and/or an anti-GITR antibody. In certain embodiments, radiation therapy is administered to the first tumor lesion but not to the second tumor lesion, where administration in combination with an anti-PD-1 antibody results in tumor regression in both the first and second tumor lesions (abscopal effect). In certain embodiments, the methods of the present invention include administering an anti-PD-1 antibody in combination with radiation therapy and optionally an anti-GITR antibody to produce a prolonged abscopal effect.

In certain embodiments, an anti-PD-1 antibody may be administered in combination with

- 19 045900 radiation therapy and a chemotherapeutic agent (eg temozolomide or cyclophosphamide), a VEGF antagonist (eg aflibercept) or granulocyte-macrophage colony-stimulating factor.

Pharmaceutical compositions and administration.

The present invention includes methods that involve administering an anti-PD-1 antibody in combination with irradiation to an individual, wherein the anti-PD-1 antibody is included in a pharmaceutical composition. The pharmaceutical compositions of the invention can be formulated with suitable carriers, excipients and other means that provide suitable transport, delivery, tolerability, etc. Many suitable formulations can be found in a formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions and water-in-water emulsions. in-oil, Carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax. See also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238–311.

Various delivery systems are known that can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987 , J Biol Chem 262: 4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, through absorption through epithelial or mucocutaneous membranes (eg, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biological active means.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, with respect to subcutaneous delivery, a pen-shaped delivery device can easily be used for delivering the pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. A reusable pen delivery device typically uses a replaceable cartridge that contains the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily removed and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. The disposable pen-style delivery device does not have a replaceable cartridge. In contrast, a disposable pen-style delivery device arrives pre-filled with a pharmaceutical composition contained in a reservoir within the device. When the pharmaceutical composition reservoir is empty, the entire device is discarded.

In certain situations, the pharmaceutical composition can be delivered in controlled release systems. In one embodiment, a pump may be used. In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed close to the target of the composition, thereby requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, volume 2, pp. 115-138). Other controlled release systems are reviewed in Langer, 1990, Science 249:1527-1533.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, there are, for example, physiological saline solution, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with a suitable solubilizing agent such as alcohol (eg ethanol), polyhydric alcohol (eg propylene glycol, polyethylene glycol), non-ionic surfactant [eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) hydrogenated castor oil adduct)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into a suitable ampoule.

Advantageously, pharmaceutical compositions for oral or parenteral use as described above are prepared in dosage forms at a unit dose adjusted to

- 20 045900 correspond to the dose of active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

In certain embodiments, the present invention provides a pharmaceutical composition comprising a therapeutic amount of an anti-PD-1 antibody and a pharmaceutical carrier. In certain embodiments, the present invention provides an anti-PD-1 antibody formulated in a pharmaceutical composition for use for intravenous administration.

Administration schemes.

The present invention includes methods comprising administering an anti-PD-1 antibody to an individual at a dosing frequency of approximately four times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks , once every 6 weeks, once every 8 weeks, once every 12 weeks, or less often, provided that a therapeutic response is achieved. In certain embodiments, the present invention includes methods comprising administering radiation therapy to an individual at a dosing frequency of about seven times per week, about four times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, every 5 weeks, every 6 weeks, every 8 weeks, every 12 weeks or less often, provided that a therapeutic response is achieved. In certain embodiments, the methods include administering an anti-PD-1 antibody in combination with radiation therapy at a dosing frequency of about seven times per week, about four times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks , once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 9 weeks, once every 12 weeks or less often, provided that a therapeutic response is achieved.

In certain embodiments, the methods of the present invention include administering radiation therapy, wherein the radiation therapy is hypofractionated radiation therapy. In certain embodiments, hypofractionated radiation therapy contains 2-12 fractions. In certain embodiments, 2-12 fractions are administered on consecutive days. In certain embodiments, radiation therapy is administered following administration of one or more doses of an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is administered 0.5 to 2 weeks before the administration of one or more fractions of radiation therapy.

In accordance with certain embodiments of the present invention, multiple doses of an anti-PD-1 antibody in combination with radiation therapy can be administered to an individual over a period of time. Methods in accordance with this aspect of the invention include sequentially administering to an individual one or more doses of an anti-PD-1 antibody in combination with one or more doses of radiation. As used herein, sequential administration means that each dose of antibody is administered to an individual at a different point in time, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). In certain embodiments, the methods of the present invention include sequential administration of one or more doses of an anti-PD-1 antibody, with each dose administered 0.5 to 12 weeks after the immediately preceding dose. In certain additional embodiments, the methods further include administering radiation therapy. Radiation therapy may be hypofractionated radiation therapy. In certain embodiments, the radiation therapy contains 2-12 fractions. In some embodiments, irradiation fractions are administered on consecutive days or every other day. In certain embodiments, irradiation fractions are administered once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, or a combination thereof.

In certain embodiments, the present invention includes methods that include sequentially administering to a patient one initial dose of an anti-PD-1 antibody, followed by one or more second doses of an anti-PD-1 antibody, and optionally followed by one or more third doses of an anti-PD-1 antibody. 1. In certain embodiments, the methods further include sequentially administering to the patient one initial dose of radiation therapy, followed by one or more second doses of radiation therapy, and optionally followed by one or more third doses of radiation therapy. In alternative embodiments, the methods further include sequential administration of one or more fractions of hypofractionated radiation therapy.

In accordance with certain embodiments of the present invention, multiple doses of anti-PD-1 antibody and radiation therapy can be administered to an individual over a period of time. Methods in accordance with this aspect of the invention involve sequentially administering multiple doses of an anti-PD-1 antibody and radiation to an individual. As used herein, sequential administration means that each dose of anti-PD-1 antibody in combination with radiation therapy is administered to an individual at a different point in time, for example, on different days separated by a predetermined interval (for example, hours, days, weeks or months ).

The terms initial dose, second doses and third doses refer to the time sequence of administration. Thus, the initial dose is the dose that is administered at the beginning

- 21 045900 treatment regimens (also referred to as the base dose); second doses are doses that are administered after the initial dose; and third doses are doses that are administered after the second doses. The initial, second, and third doses may all contain the same amount of antibody (anti-PD-1 antibody). However, in certain embodiments, the amount included in the initial, second and/or third doses varies among themselves (eg, it is adjusted up or down depending on the situation) during the course of treatment. In certain embodiments, one or more (eg, 1, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses, followed by subsequent doses that are administered on a less frequent basis (eg, maintenance doses). For example, an anti-PD-1 antibody may be administered to a cancer patient at a loading dose of about 1-3 mg/kg, followed by one or more maintenance doses of about 0.1 to about 20 mg/kg of the patient's body weight.

In one exemplary embodiment of the present invention, every second and/or tertiary dose is administered every 0.5-14 (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4, 5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or more) weeks after the immediately preceding dose. The phrase immediately preceding dose, as used herein, means, in a sequence of multiple administrations, the dose of anti-PD-1 antibody (and/or radiation) that is administered to a patient before the administration of the next next dose in the sequence, with no intervening doses.

Methods in accordance with the present aspect of the invention may include administering to the patient any number of second and/or third doses of anti-PD-1 antibody (and/or radiation therapy). For example, in certain embodiments, only one second dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) second doses. Likewise, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8, or more) third doses.

In embodiments involving multiple second doses, each second dose may be administered at the same frequency as the other second doses. For example, every second dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Likewise, in embodiments involving multiple third doses, each tertiary dose may be administered at the same frequency as the other third doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which second and/or third doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by the physician, depending on the needs of the individual patient, after a clinical examination.

In certain embodiments, one or more doses of anti-PD-1 antibody and/or radiation are administered at the beginning of the treatment regimen as induction doses on a more frequent basis (twice weekly, weekly, or biweekly), followed by subsequent doses (consolidation doses or maintenance doses) that are administered on a less frequent basis (eg, every 2 to 12 weeks). In certain embodiments, one or more doses of anti-PD-1 antibody and/or radiation are administered early in the treatment regimen as induction doses on a more frequent basis (twice weekly, weekly, or biweekly), followed by subsequent doses of anti-PD-1 antibody.

The present invention includes methods that include sequential administration of one or more doses of an anti-PD-1 antibody in combination with one or more doses of radiation therapy, where one or more doses are included in one or more treatment cycles.

In accordance with certain embodiments of the present invention, the methods comprise administering at least one cycle of treatment, wherein the at least one cycle of treatment comprises administering one or more doses of an anti-PD-1 antibody and optionally one or more doses of radiation therapy. In certain embodiments, a treatment cycle includes 1-10 doses of an anti-PD-1 antibody, wherein each dose of an anti-PD-1 antibody is administered 0.5-8 weeks after the immediately preceding dose. In certain embodiments, the methods of the present invention include administering up to 6 or 8 treatment cycles. In certain other embodiments, the methods of the present invention include administering up to 12 treatment cycles or more, if necessary, for therapeutic effect. In certain embodiments, at least one treatment cycle further comprises radiation therapy. In some embodiments, the radiation therapy is hypofractionated radiation therapy, where the hypofractionated radiation therapy contains 2-12 fractions. In certain embodiments, 2-12 fractions are administered on consecutive days.

The present invention includes methods involving sequential administration of an anti-PD-1 antibody in combination with radiation therapy to a patient in order to treat a malignant tumor (eg, a solid tumor). In some embodiments, these methods include administering one or more doses of an anti-PD-1 antibody followed by radiation therapy. In the definition

- 22 045900 In additional embodiments, radiation therapy is administered in fractions (hypofractionated irradiation). In certain embodiments, these methods include administering a single dose of an anti-PD-1 antibody, followed by 2-10 fractions of radiation therapy, followed by one or more doses of an anti-PD-1 antibody. In some embodiments, one or more doses of about 0.1 mg/kg to about 20 mg/kg of anti-PD-1 antibody may be administered, followed by radiation therapy to inhibit tumor growth and/or prevent tumor recurrence in the patient with a malignant tumor (for example, a solid tumor). In some embodiments, the anti-PD-1 antibody is administered in one or more doses, followed by radiation therapy leading to increased antitumor effect (e.g., greater inhibition of tumor growth, increased prevention of tumor recurrence compared to an untreated individual or an individual treated with antibody or radiation as monotherapy). Alternative embodiments of the invention relate to the concomitant administration of an anti-PD-1 antibody and radiation, which are administered at a similar or different frequency relative to the anti-PD-1 antibody. In some embodiments, radiation therapy is administered before, after, or simultaneously with the anti-PD-1 antibody.

Dose.

The amount of anti-PD-1 antibody administered to an individual in accordance with the methods of the present invention is generally a therapeutically effective amount. As used herein, the phrase a therapeutically effective amount refers to an amount of an antibody (an anti-PD-1 antibody) that results in one or more of: (a) a reduction in the severity or duration of a symptom or indication related to a malignant tumor, e.g., a solid tumor; ( b) inhibiting tumor growth or increasing tumor necrosis, reducing tumor and/or disappearing tumor; (c) delaying tumor growth and development; (d) inhibiting tumor metastasis; (e) preventing relapse of tumor growth; (f) increasing survival of a patient with malignant tumor; and/or (g) reducing the use or need for standard cancer therapy (eg, reduced or avoided use of chemotherapeutic or cytotoxic agents) compared to an untreated individual or an individual administered the antibody as monotherapy.

In the case of an anti-PD-1 antibody, a therapeutically effective amount may be from about 0.05 mg to about 600 mg, from about 1 mg to about 500 mg, from about 10 mg to about 450 mg, from about 50 mg to about 400 mg , from about 75 mg to about 350 mg or from about 100 mg to about 300 mg of antibody. For example, in various embodiments, the amount of anti-PD-1 antibody is about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 110 mg, approximately 120 mg, approximately 130 mg, approximately 140 mg, approximately 150 mg, approximately 160 mg, approximately 170 mg, approximately 180 mg, approximately 190 mg, approximately 200 mg, approximately 210 mg, approximately 220 mg, approximately 230 mg, approximately 240 mg, approximately 250 mg, approximately 260 mg, approximately 270 mg, approximately 280 mg, approximately 290 mg, approximately 300 mg, approximately 310 mg, approximately 320 mg, approximately 330 mg, approximately 340 mg, approximately 350 mg, approximately 360 mg, approximately 370 mg, approximately 380 mg, approximately 390 mg, approximately 400 mg, approximately 410 mg, approximately 420 mg, approximately 430 mg, approximately 440 mg, approximately 450 mg, approximately 460 mg, approximately 470 mg, approximately 480 mg, approximately 490 mg, approximately 500 mg, approximately 510 mg, approximately 520 mg, approximately 530 mg, approximately 540 mg, approximately 550 mg, approximately 560 mg, approximately 570 mg, approximately 580 mg, approximately 590 mg, or approximately 600 mg of anti-PD-1 antibody. In one embodiment, 250 mg of anti-PD-1 antibody is administered in accordance with the methods of the present invention. In one embodiment, 200 mg of anti-PD-1 antibody is administered in accordance with the methods of the present invention. In one embodiment, 350 mg of anti-PD-1 antibody is administered in accordance with the methods of the present invention.

The amount of any anti-PD-1 antibody included in individual doses can be expressed in units of mg of antibody per kg of patient body weight (i.e., mg/kg). In certain embodiments, the anti-PD-1 antibody used in the methods of the present invention can be administered to an individual at a dose of from about 0.0001 to about 100 mg/kg of the patient's body weight. In certain embodiments, the anti-PD-1 antibody can be administered at a dose of from about 0.1 mg/kg to about 20 mg/kg of the patient's body weight. In certain embodiments, the methods of the present invention include administering an anti-PD-1 antibody at a dose of about 1 mg/kg, 3 mg/kg, 5 mg/kg, or 10 mg/kg of the patient's body weight.

In certain embodiments, the amount of anti-PD-1 antibody administered to a patient is

- 23 045900 may be less than a therapeutically effective amount, i.e. a subtherapeutic dose. For example, if the therapeutically effective amount of anti-PD-1 antibody contains 3 mg/kg, the subtherapeutic dose contains an amount less than 3 mg/kg, e.g., 2 mg/kg, 1.5 mg/kg, 1 mg/kg, 0.5 mg /kg or 0.3 mg/kg. As defined herein, a subtherapeutic dose refers to an amount of anti-PD-1 antibody that does not lead to a therapeutic effect by itself. However, in certain embodiments, a subtherapeutic dose of an anti-PD-1 antibody is administered with a second and optionally third therapeutic agent to promote a therapeutic effect.

In certain embodiments, the radiation therapy administered to the patient contains 2-100 Grays (Gy). In certain embodiments, the radiation therapy contains 5, 7, 8, 9, 10, 11, 12, 15, 20, 23, 25, 27, 30, 35, 40, or 45 Gy. In certain other embodiments, the radiation therapy comprises 50-100, 60-90, or 70-80 Gy. In certain embodiments, radiation therapy is administered in 2-12 fractions (hypofractionated radiation therapy), where each fraction contains 2-10 Gy. For example, 30 Gy of radiation is administered, which is contained in 5 fractions, each fraction is 6 Gy.

Selected Embodiments

Selected embodiments of the present invention include the following.

In some embodiments, the present disclosure provides a method of treating or inhibiting the growth of a tumor in a patient, which includes:

(a) selection of an individual with a malignant tumor; and (b) administering to an individual in need thereof one or more doses of radiation therapy in combination with one or more doses of a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds programmed death 1 (PD-1), wherein administration of the combination leads to enhanced therapeutic effect compared to the introduction of a separate antibody or radiation.

In one embodiment, each dose of anti-PD-1 antibody contains between 0.1-20 mg/kg of the patient's body weight.

In another embodiment, each dose of anti-PD-1 antibody contains 0.3, 1, 3, 5, or 10 mg/kg of the patient's body weight.

In other embodiments, each dose of anti-PD-1 antibody contains 20-400 mg.

In some embodiments, each dose of anti-PD-1 antibody contains 200 mg.

In one embodiment, each radiation dose contains 2-80 Grays (Gy).

In another embodiment, each dose of anti-PD-1 antibody contains 1, 3, or 10 mg/kg of the patient's body weight and each dose of radiation therapy contains 20-50 Gy.

In other embodiments, the radiation therapy is fractionated radiation therapy.

In some embodiments, fractionated radiation therapy contains 2-10 fractions.

In one embodiment, fractionated radiation therapy contains 30 Gy in 5 fractions.

In another embodiment, fractionated radiation therapy contains 27 Gy in 3 fractions.

In other embodiments, 4-50 doses of anti-PD-1 antibody are administered, and each dose is administered 0.5-4 weeks after the immediately preceding dose.

In some embodiments, each dose of anti-PD-1 antibody is administered 2 weeks after the immediately preceding dose.

In one embodiment, the anti-PD-1 antibody is administered before, concurrently with, or after radiation therapy.

In another embodiment, the anti-PD-1 antibody is administered prior to radiation therapy.

In other embodiments, the anti-PD-1 antibody is administered 1 week before radiation therapy.

In some embodiments, the enhanced therapeutic effect includes an effect selected from the group consisting of tumor regression, abscopal effect, inhibition of tumor metastasis, reduction of metastatic lesions over time, reduced use of chemotherapeutic or cytotoxic agents, reduced tumor burden, increased progression-free survival, increased overall survival, complete response, partial response, and stable disease.

In one embodiment, the enhanced therapeutic effect includes tumor regression in a tumor that is distant from the irradiated tumor.

In another embodiment, tumor growth is inhibited by at least 50% compared to an individual treated with antibody or radiation alone.

In other embodiments, tumor growth is inhibited by at least 50% compared to an individual dosed prior to the anti-PD-1 antibody.

In some embodiments, the present disclosure provides a method of treating...

- 24 045900 holi, which includes: (a) selection of a patient with a malignant tumor; and (b) administering to the individual at least one cycle of treatment, wherein the at least one cycle of treatment comprises 1-6 doses of the anti-PD-1 antibody and wherein each dose is administered 2 weeks after the immediately preceding dose.

In one embodiment, each dose of anti-PD-1 antibody contains 1, 3, 5, or 10 mg/kg of the patient's body weight.

In another embodiment, at least one treatment cycle further comprises radiation therapy.

In other embodiments, the radiation therapy contains approximately 20-50 Gy.

In some embodiments, the radiation therapy contains approximately 27 Gy.

In one embodiment, the radiation therapy contains approximately 30 Gy.

In another embodiment, the radiation therapy is fractionated radiation therapy.

In other embodiments, fractionated radiation therapy contains 2-6 fractions.

In some embodiments, fractionated radiation therapy contains 3 fractions.

In one embodiment, fractionated radiation therapy contains 5 fractions.

In another embodiment, the radiation therapy contains approximately 27 Gy in 3 fractions.

In other embodiments, the radiation therapy contains approximately 30 Gy in 5 fractions.

In some embodiments, the fractions are administered on consecutive days.

In one embodiment, the anti-PD-1 antibody is administered 1 week before radiation therapy.

In another embodiment, up to 10 cycles of treatment are administered to an individual in need thereof.

In other embodiments, 6 cycles of treatment are administered to an individual in need thereof.

In some embodiments, radiation therapy is administered in the first treatment cycle.

In one embodiment, the radiation therapy contains approximately 20-50 Gy.

In another embodiment, the radiation therapy comprises hypofractionated radiation therapy.

In other embodiments, fractionated radiation therapy contains 2-6 fractions.

In some embodiments, the radiation therapy contains approximately 27 Gy in 3 fractions.

In one embodiment, the radiation therapy contains approximately 30 Gy in 5 fractions.

In another embodiment, fractions are administered on consecutive days.

In other embodiments, the anti-PD-1 antibody is administered 1 week before radiation therapy.

In some embodiments, each treatment cycle includes 4 doses of an anti-PD-1 antibody.

In one embodiment, the treatment produces a therapeutic effect selected from the group consisting of inhibition of tumor growth, tumor regression, reduction in tumor size, reduction in the number of tumor cells, inhibition of tumor growth, abscopal effect, inhibition of tumor metastasis, reduction of metastatic lesions over time, decreased use of chemotherapeutic or cytotoxic agents, decreased tumor burden, increased progression-free survival, increased overall survival, complete response, partial response, and stable disease.

In another embodiment, the treatment effect includes tumor regression in a tumor that is distant from the irradiated tumor in the patient.

In other embodiments, tumor growth is delayed by at least 10 days compared to an individual without treatment.

In some embodiments, tumor growth is inhibited by at least 50% compared to an individual without treatment.

In one embodiment, tumor growth is inhibited by at least 50% compared to an individual treated with antibody or radiation alone.

In some embodiments, the present disclosure provides a method of treating a tumor that includes: (a) selecting a patient with a first solid tumor lesion and a second solid tumor lesion, wherein the second solid tumor lesion is located remote from the first solid tumor lesion; and (b) administration of an anti-PD-1 antibody or antigen-binding fragment thereof in combination with radiation therapy.

In other embodiments, radiation therapy is administered to the first tumor lesion but not to the second tumor lesion, where administration results in tumor regression in both the first and second tumor lesions.

In one embodiment, the anti-PD-1 antibody is administered prior to radiation therapy.

In another embodiment, the individual is resistant to or does not respond sufficiently to

- 25 045900 or relapsed after previous therapy.

In other embodiments, the cancer is a recurrent or metastatic cancer.

In some embodiments, the method further includes administering to the individual an additional therapeutic agent or therapy, wherein the additional therapeutic agent or therapy is selected from the group consisting of surgery, a chemotherapy agent, a cancer vaccine, a programmed death ligand 1 (PD-L1) inhibitor, an lymphocyte activation gene 3 (LAG3), cytotoxic T lymphocyte associated protein 4 (CTLA-4) inhibitor, glucocorticoid-induced tumor necrosis factor receptor (GITR) inhibitor, T-cell immunoglobulin and mucin-3 (TIM3) inhibitor, inhibitor B and T lymphocyte attenuator (BTLA), T cell immunoreceptor inhibitor with Ig and ITIM domains (TIGIT), CD47 inhibitor, indoleamine 2,3-dioxygenase (IDO) inhibitor, anti-CD3/anti-CD20 bispecific antibody, factor antagonist vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang2) inhibitor, transforming growth factor β (TGFe) inhibitor, epidermal growth factor receptor (EGFR) inhibitor, granulocyte-macrophage colony-stimulating factor (GM-CSF), cyclophosphamide, tumor-specific antigen antibody , bacillus Calmette-Guérin vaccine, cytotoxin, interleukin-6 receptor (IL6R) inhibitor, interleukin-4 receptor (IL-4R) inhibitor, IL-10, IL-2, IL-7, IL-21, IL-15 inhibitor , an antibody-drug conjugate, an anti-inflammatory drug and a dietary supplement.

In one embodiment, the additional therapeutic agent is an anti-GITR antibody.

In another embodiment, the additional therapeutic agent is cyclophosphamide.

In other embodiments, the additional therapeutic agent is GM-CSF.

In some embodiments, the additional therapeutic agent is selected from the group consisting of docetaxel, carboplatin, paclitaxel, cisplatin, gemcitabine, and pemetrexed.

In one embodiment, the anti-PD-1 antibody is administered intravenously, subcutaneously, or intraperitoneally.

In another embodiment, the malignant tumor includes a solid tumor.

In other embodiments, the solid tumor is selected from the group consisting of colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, cancer liver, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.

In some embodiments, the solid tumor is selected from the group consisting of hepatocellular carcinoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, basal cell carcinoma, breast carcinoma, squamous cell carcinoma of the skin, chondrosarcoma, angiosarcoma, cholangiocarcinoma, soft tissue sarcoma, colon cancer, melanoma , Merkel cell carcinoma and glioblastoma multiforme.

In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof comprises heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1, and three light chain complementarity determining regions chains (LCDR1, LCDR2 and LCDR3) of the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:2.

In another embodiment, an anti-PD-1 antibody or antigen binding fragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:3; HCDR2 contains the amino acid sequence SEQ ID NO:4; HCDR3 contains the amino acid sequence SEQ ID NO:5; LCDR1 contains the amino acid sequence SEQ ID NO:6; LCDR2 contains the amino acid sequence SEQ ID NO:7; and LCDR3 contains the amino acid sequence of SEQ ID NO:8.

In other embodiments, HCVR contains the amino acid sequence of SEQ ID NO:1 and LCVR contains the amino acid sequence of SEQ ID NO:2.

In another embodiment, an anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the present disclosure provides a method of inhibiting the activation and/or proliferation of regulatory T cells (Tregs), which comprises: (a) selecting a patient with a solid tumor; and (b) administering to the individual (i) an anti-PD-1 antibody or antigen-binding antibody thereof

- 26 045900 fragment, (ii) administering radiation therapy and (iii) administering at least one of an antibody or an antigen-binding fragment thereof that specifically binds to the glucocorticoid-induced tumor necrosis factor receptor (GITR), cyclophosphamide, GM-CSF, anti-LAG3 antibody , docetaxel or carboplatin.

In one embodiment, the individual has a large tumor.

In another embodiment, the radiation dose is 2-50 Gy.

In other embodiments, administration results in at least one effect selected from the group consisting of inhibition of tumor growth, tumor regression, tumor size reduction, tumor cell number reduction, tumor growth inhibition, abscopal effect, tumor metastasis inhibition, reduction of metastatic lesions with over time, decreased use of chemotherapeutic or cytotoxic agents, decreased tumor burden, increased progression-free survival, increased overall survival, complete response, partial response, and stable disease.

In some embodiments, the solid tumor is selected from the group consisting of colon cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, malignant head and neck tumors, salivary gland malignancy and myeloma.

In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof comprises heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1, and three light chain complementarity determining regions chains (LCDR1, LCDR2 and LCDR3) of the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:2.

In another embodiment, an anti-PD-1 antibody or antigen binding fragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:3; HCDR2 contains the amino acid sequence SEQ ID NO:4; HCDR3 contains the amino acid sequence SEQ ID NO:5; LCDR1 contains the amino acid sequence SEQ ID NO:6; LCDR2 contains the amino acid sequence SEQ ID NO:7; and LCDR3 contains the amino acid sequence of SEQ ID NO:8.

In other embodiments, HCVR contains the amino acid sequence of SEQ ID NO:1, and LCVR contains the amino acid sequence of SEQ ID NO:2.

In one embodiment, an anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the present disclosure provides a method of treating or inhibiting tumor growth, which includes:

(a) selection of a patient with a malignant skin tumor; and (b) administering to the individual a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds PD-1.

In one embodiment, said antibody or antigen-binding fragment thereof that specifically binds PD-1 is administered as monotherapy.

In another embodiment, said skin cancer is a UV-associated skin cancer.

In other embodiments, said skin cancer is selected from the group consisting of cutaneous squamous cell carcinoma (CSCC), basal cell carcinoma (BCC), Merkel cell carcinoma, and melanoma.

In some embodiments, it is provided that said skin cancer is not squamous cell carcinoma of the head and neck.

In one embodiment, said skin cancer is a metastatic, unresectable and/or locally advanced malignancy.

In another embodiment, said skin cancer is SCD, wherein the patient is intolerant to or progresses after treatment with a hedgehog pathway inhibitor.

In other embodiments, the antibody or antigen-binding fragment thereof is administered in one or more doses, where each dose is administered 0.5 to 4 weeks after the immediately preceding dose.

In some embodiments, each dose is administered 2 weeks after the immediate

- 27 045900 previous dose.

In one embodiment, each dose contains 1, 3, or 10 mg/kg of the patient's body weight.

In another embodiment, said antibody or antigen binding fragment thereof that specifically binds PD-1 is an antibody as defined in any one of the preceding embodiments.

In some embodiments, the present disclosure provides a method of treating or inhibiting the growth of a tumor in a patient, the method comprising: selecting a patient with a malignant brain tumor; and administering to an individual in need thereof a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds PD-1.

In one embodiment, the individual has glioblastoma multiforme (GBM).

In another embodiment, the individual has newly diagnosed GBM.

In other embodiments, the age of the individual is >65 years.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered in one or more doses, with each dose administered 0.5 to 4 weeks after the immediately preceding dose.

In one embodiment, each dose is administered 2 weeks after the immediately preceding dose.

In another embodiment, each dose contains 1, 3 or 10 mg/kg of the patient's body weight.

In other embodiments, the method further includes administering radiation therapy to an individual in need thereof.

In some embodiments, the radiation therapy is hypofractionated radiation therapy.

In one embodiment, the individual is administered 20-50 Gy of radiation in 2-20 fractions.

In another embodiment, the individual is administered radiation therapy 1 week after the first dose of anti-PD-1 antibody.

In other embodiments, one or more doses of an anti-PD-1 antibody are contained in one or more cycles of treatment, where each cycle contains 1-6 doses of an anti-PD-1 antibody.

In some embodiments, each treatment cycle contains 4 doses of an anti-PD1 antibody, with each dose administered 2 weeks after the immediately preceding dose.

In one embodiment, each dose contains 1, 3, or 10 mg/kg of the patient's body weight.

In another embodiment, the first cycle of treatment further comprises radiation therapy.

In other embodiments, the radiation therapy is hypofractionated radiation therapy.

In some embodiments, the individual is administered 20-50 Gy of radiation in 2-20 fractions.

In one embodiment, the individual is administered 30 Gy in 5 daily fractions.

In another embodiment, radiation therapy is administered one week after administration of the anti-PD-1 antibody.

In other embodiments, the method further comprises administering an antiangiogenic agent to the individual if the patient develops intracranial edema following administration of the anti-PD-1 antibody.

In some embodiments, the antiangiogenic agent is selected from the group consisting of a vascular endothelial growth factor (VEGF) inhibitor and an angiopoietin-2 (Ang-2) inhibitor.

In one embodiment, the antiangiogenic agent is bevacizumab or aflibercept.

In other embodiments, said antibody or antigen-binding fragment thereof that specifically binds PD-1 is an antibody as defined in any one of the preceding embodiments.

Examples

The following examples are provided to provide those of ordinary skill in the art with complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider their invention to be. Efforts have been made to ensure accuracy in terms of numbers used (eg quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1: Effect of anti-PD-1 antibody in vivo in combination with radiation therapy against MC38 tumors.

In this example, the effect of blocking PD-1 in combination with radiation therapy was examined in established MC38 tumors in mice.

- 28 045900

5x105 MC38 colon carcinoma cells were implanted subcutaneously in the right flank of female C57BL/6 mice (Jackson Laboratory). Treatment was initiated on day 9 after implantation, when average tumor volumes reached approximately 100 mm ³ . Mice were randomly assigned to receive either isotype control (2A3, BioXcell) or a PD-1 blocking antibody (RMP1-14, BioXcell) at 5 mg/kg, twice weekly, for a total of 5 intraperitoneal injections. One day after the start of antibody treatment, mice were assigned to radiation therapy groups that received 12 Gy of radiation to their right flank tumors. Radiation therapy was delivered using an RS 2000 Biological Research Irradiator (Rad Source) to anesthetized mice (ketamine/xylazine) that were shielded using a partial irradiation body mount (Precision X-ray) and lead shielding (Images Scientific Instruments). Tumor growth was assessed 3 times a week until days 70-80, when all mice were sacrificed. In fig. Figure 1 shows the study design for the experiment, which includes anti-PD-1 antibody dosing and radiation.

In fig. 2 and in table. Figure 1 shows average tumor volumes in mice treated with anti-PD-1 antibody alone or in combination with radiation.

Table 1

Average tumor volumes in mice treated with anti-PD-1 antibody alone or in combination with radiation

Days after implantation Average tumor volume (mm ³ ±ZEM) Isotypic control Antibody against PD-1 Isotype control + irradiation Antibody against PD- 1+irradiation 4 15.39±3.70 8.62±3.02 13.28±3.44 10.78±3.01 7 41.11±8.81 38.90±7.09 49.86±11.38 39.36±6.32 8 68.64±10.01 72.03±12.13 74.03±14.83 73.70±14.86 10 85.82±4.10 94.98±22.68 100.88±11.46 122.05±15.05 14 725.87±68.45 834.37±206.70 320.10±58.80 300.67±60.74 16 1023.61±191.4 1 1123.51±310.0 4 276.17±82.81 219.29±45.94 18 1573.64±263.6 5 1710.30±424.3 0 353.45±121.47 250.17±74.70 21 2688.69±502.3 9 2569.65±633.3 5 494.53±211.90 188.98±105.8 0 23 597.70±267.02 141.37±73.76 25 671.93±347.76 134.87±75.67 28 879.64±403.70 147.82±70.88 thirty 1081.39±426.80 133.13±88.88 32 177.73±112.8 1

- 29 045900

36 233.44±152.9 1 39 258.23±158.6 7 42 316.58±160.9 1 44 332.73±152.4 3 46 456.13±209.4 5 49 564.05±262.3 2 51 925.92±434.2 9 53 747.14±350.9 0 56 1290.10±584, 62

Blockade of PD-1 (RMP1-14) was synergistic with local irradiation (XRT) and significantly reduced tumor regression (4/6 mice) in tumor-bearing MC38 mice compared to mice treated with XRT + isotype control (2/6 mice ). Tumor growth was inhibited or delayed in mice treated with anti-PD-1 antibody in combination with radiation. Mice treated with anti-PD-1 antibody and radiation took more than 40 days to reach a tumor volume of 500 mm ³ , compared with mice on monotherapy, which took less than 20 days to achieve a tumor volume of 500 mm ³ . Tumor regression continued for up to 4 weeks for the combination treatment group (XRT + anti-PD-1 antibody) (1 of 4 rejected tumors recurred at this time point) compared to 1.5 weeks for the XRT + isotype treatment group (1 of 4 rejected tumors recurred). 2 rejected tumors). In this tumor model, blocking PD-1 as monotherapy had no effect on primary tumor growth.

- 30 045900

table 2

Percentage of survival of mice treated with anti-PD-1 antibody alone or in combination with radiation

Days after implantation Isotype Against PD-1 Irradiation+isotype Irradiation + against PD-1 4 100 100 100 100 7 100 100 100 100 8 100 100 100 100 10 100 100 100 100 14 100 100 100 100 16 100 100 100 100 18 100 100 100 100 21 100 100 100 100 23 100 83 100 100 25 67 50 100 100 28 33 50 100 100 thirty 17 17 100 100 32 0 17 67 100 36 0 0 67 100 44 0 0 50 100 46 0 0 50 100 49 0 0 50 100 51 0 0 33 100 53 0 0 17 100 56 0 0 17 100 58 0 0 17 83 60 0 0 17 50 63 0 0 17 50 65 0 0 17 50 81 0 0 17 50 85 0 0 17 50

The therapeutic effect of combination treatment (XRT+anti-PD-1 antibody) was demonstrated by the statistically increased overall survival of this group (50% alive at 70 days after tumor implantation) compared to all other treatment groups: isotype control (0% alive at d70) , anti-PD-1 antibody treatment (0% alive at d70) and XRT+ isotype-treated mice (17% alive at d70) (Fig. 3; Table 2).

Example 2: Effect of anti-PD-1 antibody in vivo and radiotherapy against B16 tumors.

In this example, the antitumor effect of a mouse anti-PD-1 antibody in combination with radiation therapy against established B16 tumors in mice was examined.

^2x105 B16F10.9 melanoma cells were implanted subcutaneously in the right flank of female C57BL/6 mice (Jackson Laboratory). Treatment was initiated when the average tumor volume reached approximately 150 mm ³ . Mice were randomly assigned to receive either isotype control (2A3, BioXcell) or a PD-1 blocking antibody (RMP1-14, BioXcell) at 5 mg/kg, twice weekly, for a total of 5 intraperitoneal injections. One day after the start of antibody treatment, mice were assigned to radiation therapy groups that received 8 Gy of radiation to their right flank tumors. Radiation therapy was delivered using an RS 2000 Biological Research Irradiator (Rad Source) to anesthetized mice (ketamine/xylazine) that were shielded using a partial irradiation body mount (Precision Xray) and lead shielding (Images Scientific Instruments). Tumor growth was assessed 3 times a week until days 70-80, when all mice were sacrificed. In fig. 4 shows the study design for the experiment, which includes anti-PD-1 antibody dosing and radiation.

- 31 045900

Treatment with a PD-1 (RMP1-14) blocking antibody in combination with local irradiation (XRT) delayed primary B16 tumor growth compared with XRT or anti-PD-1 antibody alone (Fig. 5; Table 3).

Table 3

Average tumor volumes in mice treated with anti-PD-1 antibody alone or in combination with radiation

Days after implantation Average tumor volume mm ³ ±ZEM Isotypic control Antibody against PD-1 Isotype control + irradiation Antibody vs PD- Noirradiation 6 5.7515.75 8.32±8.32 13.79±13.79 1.1410.85 8 55.98127.15 62.66±15.80 57.18±37.79 50.57138.33 12 157.34137.88 144.36±37.81 237.84±71.27 177.91159.17 15 334.71±61.71 193.32±35.53 510.95±171.15 372.531147.5 0 18 621.43±136.09 363.80±45.72 739.62±244.10 440.331194.9 0 20 939.69±158.50 561.64±49.44 677.48±175.75 486.351207.6 5 22 1329.77±202.0 1 772.16±118.26 759.15±235.94 512.671220.3 0 25 2602.08±434.0 8 1343.42±120.6 5 1182.271336.32 789.801299.2 4 27 1533.03±179.8 8 1321.131400.18 877.821296.5 1 29 2104.46±350.4 8 944.671333.1 6 33 1024.711321, 20 36 1237.681340, 52

- 32 045900

Table 4

Percentage of survival of mice treated with anti-PD-1 antibody alone or in combination with radiation

Days after implantation Isotype Against PD-1 Irradiation+isotype Irradiation + against PD-1 0 100 100 100 100 6 100 100 100 100 8 100 100 100 100 12 100 100 100 100 15 100 100 100 100 18 100 100 100 100 20 100 100 100 100 22 100 100 100 100 25 100 100 100 100 27 40 100 100 100 29 0 100 80 100 33 0 80 80 100 36 0 20 60 100 39 0 0 60 83 41 0 0 60 83 43 0 0 20 67 46 0 0 20 67 48 0 0 20 50 50 0 0 0 50 53 0 0 0 33 55 0 0 0 33 57 0 0 0 33 60 0 0 0 17 62 0 0 0 17 64 0 0 0 17 66 0 0 0 0

The combination of XRT+anti-PD-1 treatment increased overall survival (50% alive at d50 after implantation) compared with XRT alone (0% alive at d50), anti-PD-1 alone (0% alive at d40) and isotype alone (0% alive by d30) (Fig. 6; Table 4).

Example 3: Effect of anti-PD-1 antibody in vivo in combination with radiation therapy against metastatic lung tumors.

In this example, the effect of blocking PD-1 in combination with radiation therapy was tested against established and metastatic tumors in mice.

1.5 x 105 4T1 mammary carcinoma cells were implanted subcutaneously in the right flank of female Balb/c mice (Jackson Laboratory). Treatment was initiated on day 12 after implantation, when average tumor volumes reached approximately 100 mm ³ . Mice were randomly assigned to receive either isotype control (2A3, BioXcell) or a PD-1 blocking antibody (RMP114, BioXcell) at 5 mg/kg, twice weekly, for a total of 5 intraperitoneal injections. One day after the start of antibody treatment, mice were assigned to radiation therapy groups that received 8 Gy of radiation to their right flank tumors. Radiation therapy was delivered using an RS 2000 Biological Research Irradiator (Rad Source) to anesthetized mice (ketamine/xylazine)

- 33 045900 was shielded using a partial irradiation body mount (Precision X-ray) and lead shielding (Images Scientific Instruments). Tumor growth was assessed 3 times per week until day 28, when all mice were sacrificed to assess lung metastatic burden using a clonogenic assay. Briefly, lung tissue was dissociated using DNAase/Liberase TL (Roche) and cultured in media supplemented with 60 μM 6-thioguanine. After two weeks in culture, the plates were counterstained with methylene blue and the number of colonies was determined (one colony represents one metastatic 4T1 cell).

Anti-PD-1 antibody treatment in combination with radiation is expected to promote tumor regression and also mediate suppression of metastatic growth.

Example 4: In vivo effect of anti-human PD-1 antibody in combination with radiation therapy promotes abscopal effect against distant tumors.

In this example, the effect of blocking PD-1 in combination with radiation therapy was examined against primary and distant MC38 tumors in mice humanized for PD-1 using anti-human PD-1 antibodies.

The exemplary anti-PD-1 antibody used in this example is REGN2810 (also known as H4H7798N, as disclosed in US20150203579), a fully human anti-PD-1 monoclonal antibody containing a heavy chain containing the amino acid sequence of SEQ ID NO:9, and a light chain containing the amino acid sequence of SEQ ID NO:10; the amino acid sequence pair HCVR/LCVR contains SEQ ID NO:1/2; and the CDR sequences of the heavy and light chains contain SEQ ID NO:3-8.

PD-1 humanized mice were constructed using VelociGene® technology (Valenzuela et al 2003, Nat. Biotechnol. 21: 652-659; US patent application publication 2015/0366174).

5x105 MC38 colon carcinoma cells were implanted subcutaneously into female humanized PD-1/C57BL/6 mice on day 0 (right flank primary tumor) and day 3 (left flank tumor; distant tumor). Treatment was initiated when the average volumes of the primary tumors reached approximately 150 mm ³ . Mice were randomly assigned to receive isotype control or PD-1 blocking antibody (REGN2810) at 5 mg/kg, twice weekly, for a total of 8 intraperitoneal injections. One day after the start of antibody treatment, mice were assigned to radiation therapy groups that received 8 Gy of radiation to their right flank tumors. Radiation therapy was delivered using an RS 2000 Biological Research Irradiator (Rad Source) to anesthetized mice (ketamine/xylazine) that were shielded using a partial irradiation body mount (Precision X-ray) and lead shielding (Images Scientific Instruments). Primary and second tumor growth was assessed 3 times a week until days 70-80, when all mice were sacrificed. In fig. 7 shows the study design for the experiment, which includes anti-PD-1 antibody dosing and radiation.

Results.

Primary tumor: PD-1 blockade treatment (REGN2810) was synergistic with local irradiation (XRT) in the rejection of primary MC38 tumors (4 of 6 tumor-free mice) compared to mice treated with XRT+isotype control (1/6 tumor-free mice ). Tumor regression continued in the combination treatment group for 8 weeks until the end of the experiment, compared with three weeks for the XRT+isotype treatment group (the rejected tumor recurred at this time point) (Fig. 8; Table 5).

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Table 5

Average volumes of primary tumors in mice treated with REGN2810 alone or in combination with radiation

24 hours after Average tumor volume (mm ³ ±ZEM) implantation Isotypic control REGN2810 Isotype control + irradiation REGN2810+ irradiation 4 8.47±5.22 13.86±7.13 9.02±3.07 3.75±3.75 5 14.32±4.76 22.08±2.69 27.54±4.90 10.00±3.17 7 39.43±5.36 35.47±6.73 42.72±8.00 32.80±10.60 9 62.68±12.03 84.73± 20.91 68.27±11.65 47.26±11.65 10 111.78±24.45 108.15± 27.17 96.18±18.07 75.13±11.56 eleven 147.89±36.11 176.67± 43.99 111.87±10.12 110.27±25.02 12 171.76±41.23 154.97± 44.16 153.69±16.06 121.88±29.86 14 304.95±94.96 221.70± 65.96 147.22±19.77 144.71±34.28 17 609.24±227.64 296.69± 95.14 116.65±27.03 135.26±57.41 19 369.17± 128.37 114.85±38.73 124.59±55.50 21 442.13± 158.80 127.77±36.92 130.00±78.30 24 641.92± 275.23 198.40±67.81 113.25±74.51 26 896.32± 389.54 252.51±98.39 116.90±101.35 28 1200.99± 498.27 331.78±125.55 120.05±89.32 31 477.34±181.97 168.62±151.18 33 189.07±154.91 35 164.70±127.33 38 216.32±159.47 40 219.35±156.39 42 292.37±204.83

Blocking PD-1 as monotherapy mediated rejection in 2 of 5 mice; however, 1 of the mice that rejected their primary tumor underwent a second tumor growth, resulting in only 1 mouse surviving to the end of the experiment. The potent therapeutic effect of the combination treatment (XRT+REGN2810) was demonstrated by statistically increased overall survival (~67% alive at 70 days after tumor implantation) compared with all other groups: isotype control alone or XRT (0% alive at d70) and REGN2810 as monotherapy (20% alive at d70) (Fig. 9; Table 6).

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Table 6

Percentage of survival in mice treated with REGN2810 alone or in combination with radiation

Days after implantation Isotype REGN2810 Irradiation+isotype Irradiation+KEbI2 8U 0 100 100 100 100 4 100 100 100 100 5 100 100 100 100 7 100 100 100 100 9 100 100 100 100 10 100 100 100 100 eleven 100 100 100 100 12 100 100 100 100 14 100 100 100 100 17 100 100 100 100 19 83 100 100 100 21 83 100 100 100 24 83 100 100 100 26 50 100 100 100 28 33 100 100 100 31 33 67 100 100 33 33 33 83 100 35 33 33 67 100 38 33 33 50 100 40 17 17 50 100 42 17 17 50 100 44 17 17 50 100 49 0 17 17 83 54 17 0 83 56 17 0 83 59 17 0 83 61 17 0 67 63 17 0 67

Distant tumor: REGN2810 in combination with XRT significantly promoted abscopal effect (rejection of tumor implanted at a distant site) in 5 of 6 tumor-free mice compared to XRT alone (2/6 without distant tumors), REGN2810 alone (1/6 without distant tumors) ) and mice treated with isotype control (1/6 without distant tumors) (Fig. 10; Table 7).

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Table 7

Average distal tumor volumes in mice treated with REGN2810 alone or in combination with radiation

Days after implantation Average tumor volume (mm ³ ±ZEM) Isotypic control REGN2810 Isotypic control + irradiation REGN2810+ irradiation 1 0 0 0 0 2 0 0 0 0 4 0 0 0 0 6 0 0 0 0 7 11.13±11.13 0 0 0 8 20.01±20.01 0 3.2613.26 0 9 25.43±25.43 7.00±7.00 9.75±9.75 0 eleven 31.93±29.32 42.24±26.88 28.81±17.01 12.13112.13 14 56.20±34.46 59.40±29.41 57.64±29.91 20.93114.07 16 58.64±29.57 95.78±52.87 14.0319.79 18 151.71±7b.86 115.1b±59.43 22.87116.20 21 207.13±128.83 227.22±105.46 17.01117.01 23 333.43±220.57 335.13±148.86 9.5119.51 25 506.55±355.36 503.711211.49 11.45111.45 28 968.921418.57 31.59131.59 thirty 57.40157.40 32 83.94183.94 35 133.891133.89 37 224.651224.65

Example 5: Effect of anti-PD-1 antibody in vivo in combination with radiation therapy and GITR antagonist against MC38 tumors.

In this example, the effect of blocking PD-1 in combination with radiation therapy and a glucocorticoid-induced tumor necrosis factor receptor (GITR) antagonist (anti-GITR antibody) was examined against large established MC38 tumors in mice.

5x105 MC38 colon carcinoma cells were implanted subcutaneously in the right flank of female C57BL/6 mice (Jackson Laboratory). Treatment was initiated when the average tumor volumes reached approximately 150-200 mm ³ (classified as large tumors). Mice were randomized to receive isotype control antibody (2A3 or LTF-2; BioXcell), anti-PD-1 antibody (RMP1-14; BioXcell), anti-GITR antibody (DTA-1; BioXcell), or a combination of anti-PD antibodies -1 and anti-GITR antibodies 5 mg/kg, 2 times a week, for a total of 5 intraperitoneal injections. One day after the start of antibody treatment, mice were assigned to radiation therapy groups that received 8 Gy of radiation to their right flank tumors. Radiation therapy was delivered using an RS 2000 Biological Research Irradiator (Rad Source) to anesthetized mice (ketamine/xylazine) that were shielded using a partial irradiation body mount (Precision X-ray) and lead shielding (Images Scientific Instruments). Tumor growth was assessed 3 times a week until days 70-80, when all mice were sacrificed. In fig. 11 shows the study design for the experiment, which includes dosing of anti-PD-1 antibody, anti-GITR antibody, and radiation.

Treatment with anti-PD-1 antibody (RMP1-14) was synergistic with local irradiation (XRT) and anti-GITR antibody in the rejection of large MC3 8 tumors (4 of 6 tumor-free mice) compared with XRT + anti-GITR antibody (2/6 without tumors), XRT+anti-PD-1 antibody (2/6 rejected) or mice treated with XRT alone (0/6 tumor-free). Monotherapy (using anti-PD-1 antibody or anti-GITR antibody) or combination treatment (anti-PD-1 antibody+anti-GITR antibody) had minimal effect on tumor growth, with treatment with anti-PD-1 antibody or anti-GITR antibody mediating rejection in 1 /5 mice and combination

- 37 045900 two antibodies mediated rejection in 2/5 mice. Tumor regression continued for up to 6.5 weeks after the start of treatment for mice treated with the triple combination versus 2 weeks for mice treated with XRT+anti-GITR antibody (FIG. 12).

Table 8

Percentage of survival of mice treated with anti-PD-1 antibody in combination with radiation and anti-GITR antibody

Days after implantation Iso type Anti PD-1 Anti GITR Anti PD-1 +anti GITR Irradiation + ISOTYPE Irradiation + against PD-1 Radiation + vs GITR Radiation + against PD1 + against GITR 0 100 100 100 100 100 100 100 100 28 100 100 80 100 100 100 100 100 32 80 80 80 80 100 100 100 100 35 60 60 40 60 100 80 83 100 38 20 20 20 60 40 80 50 100 41 0 20 20 60 20 60 50 100 48 0 20 20 60 0 60 50 83 56 0 20 20 40 0 40 33 67 66 0 20 20 40 0 40 17 67 77 0 20 20 40 0 40 17 67

In table 8 and FIG. 13 shows the survival of mice treated with anti-PD-1 antibody in combination with radiation therapy and anti-GITR antibody. In addition, administration of anti-PD1+XRT antibody led to tumor regression in very large tumors (~300 mm ³ ).

Example 6: Effect of anti-PD-1 antibody in vivo in combination with radiation therapy and a GITR antagonist against B16 tumors.

In this example, the effect of blocking PD-1 in combination with radiation therapy and a GITR antagonist (anti-GITR antibody) was tested against established B16 tumors in mice.

2.5x105 B16F10.9 melanoma cells were implanted subcutaneously in the right flank of female C57BL/6 mice (Jackson Laboratory).

Treatment was initiated when average tumor volumes reached approximately 100 mm ³ . Mice were randomly assigned to receive isotype controls (2A3, LTF-2; BioXcell), anti-PD-1 antibody (RMP1-14, BioXcell), anti-GITR antibody (DTA-1; BioXcell), or a combination of anti-PD-1 antibody 1 and anti-GITR antibodies 5 mg/kg, 2 times a week, for a total of 5 intraperitoneal injections. One day after the start of antibody treatment, mice were assigned to radiation therapy groups that received 8 Gy of radiation to their right flank tumors. Radiation therapy was delivered using an RS 2000 Biological Research Irradiator (Rad Source) to anesthetized mice (ketamine/xylazine) that were shielded using a partial irradiation body mount (Precision X-ray) and lead shielding (Images Scientific Instruments). Tumor growth was assessed 3 times a week until days 70-80, when all mice were sacrificed.

Anti-PD-1 antibody in combination with anti-GITR antibody and radiation therapy is expected to promote greater tumor regression and delayed tumor growth than monotherapy or anti-PD-1 antibody in combination with radiation therapy.

Example 7: Clinical study of anti-PD-1 antibody and radiation therapy in patients with advanced solid tumors.

This study is an open-label, multicenter, dose-escalation study with multiple dose escalation and expansion arms to investigate the effect, safety, and tolerability of anti-PD-1 antibody alone and in combination with other cancer therapies (including radiation therapy) in adult patients with common solid tumors.

The exemplary anti-PD-1 antibody used in this study is REGN2810 (also known as H4H7798N, as disclosed in US20150203579), a fully human anti-PD-1 monoclonal antibody containing a heavy chain containing the amino acid sequence of SEQ ID NO:9, and a light chain containing the amino acid sequence of SEQ ID NO:10; a pair of amino acid sequences HCVR/LCVR containing SEQ ID NO:1/2; and heavy and light chain CDR sequences comprising SEQ ID NO:3-8.

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Objectives of the study.

The primary objective of the study is to determine the safety, tolerability, dose-limiting toxicity (DLT) characteristics of REGN2810, which was administered intravenously (IV) as monotherapy or in combination with targeted radiation (with the intention of having it as an immunostimulatory rather than primary ablative tumor therapy), low dose cyclophosphamide (therapy has been shown to inhibit regulatory T cell responses), granulocyte-macrophage colony-stimulating factor, carboplatin, docetaxel, or a combination thereof in patients with advanced malignancies.

The secondary objectives of the study are: (1) to determine the recommended phase 2 dose (RP2D) for REGN2810 as monotherapy and in combination with other cancer therapy (targeted radiation, low dose cyclophosphamide, or both); (2) describe the preliminary antitumor activity of REGN2810, alone and with each combination partner(s); (3) determine the PK characteristics of REGN2810 as monotherapy and in combination with other cancer therapy (targeted radiation, low dose cyclophosphamide, or both); and (4) evaluate the immunogenicity of REGN2810.

Rationale for the research design.

The 3+3 model for the dose escalation phase of this study was designed to allow assessment of the safety of REGN2810, both as monotherapy at different dose levels and in combination with immunoenhancing treatment: cyclophosphamide; limited targeted radiation delivered by 1 of 2 dosing schedules; or combined radiation and cyclophosphamide.

When tolerability of REGN2810 was established alone and in combination with radiation and/or cyclophosphamide, multiple expansion cohorts using various combinations or monotherapy in selected indications [NSCLC, VS, HNSCC, CSCC, tumors with MSI (colon, rectum, endometrial, prostate or tumors of other types), HCC and other common solid tumors] are added to further confirm safety and evaluate the increase in antitumor activity. Granulocyte-macrophage colony-stimulating factor (GM-CSF), carboplatin, and/or docetaxel are added to some of these combinations.

In table 9 lists some of the cohorts that used REGN2810 monotherapy and combinations with other treatment modalities.

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Table 9

List of selected expansion cohorts for REGN2810 monotherapy and combination therapy

Cohort Indication Treatment 1 Non-small cell lung cancer (NSCLC) Constant dose - 200 mg REGN2810 2 NSCLC 3 mg/kg PECN2810+radiation therapy (9 Grx3) 3 Head and neck squamous cell carcinoma (HNSCC) 3 mg/kg PECN2810+radiation therapy (9 Grx3)+cyclophosphamide+SM-SZE 4 Malignant breast tumor (BC) 3 mg/kg PECN2810+radiation therapy (9 Grx3)+cyclophosphamide 5 Advanced solid tumors - previous treatment with anti-PD-1/PD-L1 antibody 3 mg/kg PECN2810+radiation therapy (9 Grx3)+cyclophosphamide+SM-SZE 6 Advanced solid tumors (except NSCLC, HNSCC, and VS) 3 mg/kg PECN2810+radiation therapy (9 Grx3)+cyclophosphamide+SM-SZE 7 Metastatic (Ml) cutaneous squamous cell carcinoma (CSCC) 3 mg/kg REGN2810 8 Locally and/or regionally advanced CSCC (MO) that is unresectable 3 mg/kg REGN2810 9 Metastatic malignant tumor of the colon with 3 mg/kg REGN2810

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microsatellite instability (MSI) 10 Metastatic endometrial malignancy with MSI 3 mg/kg REGN2810 eleven Castration recurrent prostate cancer with MSI 3 mg/kg REGN2810 12 Any other advanced solid tumor with MSI 3 mg/kg REGN2810 13 Common or metastatic hepatocellular malignancy (HCC) 3 mg/kg REGN2810 14 Advanced solid tumor refractory to first-line chemotherapy 3 mg/kg REGN2 810+carboplatin+docetaxel (low dose) 15 Advanced solid tumor refractory to first-line chemotherapy 3 mg/kg REGN2810+floneTaKcen (low dose) 16 Metastatic colon malignancy with MSI that has not been previously treated 3 mg/kg REGN2810 17 Common NSCLC, which not pre-treated 3 mg/kg REGN2 810+carboplatin+docetaxel (low dose) 18 Newly diagnosed glioblastoma multiforme (GBM) REGN2810 (1 or 3 mg/kg) + radiation therapy (6 Gy x 5 days) 19 Recurrent GBM REGN2810 (1 or 3 mg/kg) + radiation therapy (6 Gy x 5 days) 20 HIV and solid tumors 3 mg/kg REGN2810 21 Common NSCLC, which not pre-treated 3 mg/kg REGN2 810+Carboplatin+Paclitaxel (full dose) 22 Advanced non-squamous NSCLC that has not been previously treated 3 mg/kg REGN2 810+cisplatin+pemetrexed 23 Advanced squamous NSCLC that has not been previously treated 3 mg/kg REGN2 810+cisplatin+gemcitabine 24 Malignant tumor of the cervix, recurrent or metastatic 3 mg/kg REGN2810 25 Basal cell carcinoma, 3 mg/kg REGN2810 refractory to hedgehog pathway inhibition 26 Advanced solid tumor 3 mg/kg REGN2810

The initial planned treatment with REGN2810 is every 14 days up to 48 weeks, with a follow-up of 24 weeks. Radiation is administered one week after the first dose of REGN2810. A low dose of cyclophosphamide is administered to patients assigned to cyclophosphamide 1 day before each of the first 4 doses of REGN2810.

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Duration of the study.

Patients receive up to 48 weeks of treatment, followed by an observation period of 24 weeks. The patient remains on treatment until completion of the 48-week treatment period or until disease progression, unacceptable toxicity, withdrawal of consent, or other study termination criteria are met. After a minimum of 24 weeks of treatment, patients with confirmed complete responses (CR) may elect to interrupt treatment and continue with all relevant study evaluations (eg, effect assessments). After a minimum of 24 weeks of treatment, patients with tumor burden assessments for stable disease (SD) or partial response (PR) that have not changed over 3 consecutive tumor assessments may also elect to interrupt treatment and continue with all relevant study assessments (eg, effect assessments ).

Study population.

The target population for this study includes patients with advanced malignancies who are not candidates for standard therapy, are unwilling to undergo standard therapy, or for whom available therapy is not expected to provide clinical benefit; and patients with malignancies that are incurable and have failed to demonstrate a response or have demonstrated tumor progression despite standard therapy.

Inclusion Criteria: A patient must meet the following criteria to be eligible for inclusion in the study: (1) demonstrate progression of a solid tumor in the absence of an available alternative to standard therapy; (2) at least 1 lesion to assess response. Patients assigned to radiation therapy require at least one additional lesion that can be safely irradiated while still sparing the index lesion, and for which radiation at the limited palliative doses considered is deemed appropriate for medical use; (3) patients must have relapsed after or be refractory to first-line therapy (and up to 2 prior lines of therapy) in the setting of recurrent or metastatic disease and must have disease for which palliative radiotherapy is indicated; (4) patients with metastatic malignancy with microsatellite instability (MSI), refractory to up to 2 previous lines of therapy; (5) Eastern Cooperative Oncology Group (ECOG) performance status <1; (6) age over 18 years; (7) liver function: a. total bilirubin <1.5x upper limit of normal (ULN; if liver metastases <3x ULN), b. transaminases <3 x ULN (or <5.0 x ULN if liver metastases), p. alkaline phosphatase (ALP) <2.5x ULN (or 5.0x ULN if liver metastases); (8) renal function: serum creatinine <1.5x ULN; (9) neutrophil count (ANC) >1.5x10 ⁹ /l, s. platelet count >75x10 ⁹ /l; (10) ability to provide signed informed consent; and (11) ability and willingness to perform scheduled visits, treatment plan, laboratory tests, and other study-related procedures.

Study treatment.

REGN2810 was supplied as a liquid in sterile single-use vials. Each vial contains a volume sufficient to contain 10 mL of REGN2810 at a concentration of 25 mg/mL. REGN2810 is administered in an outpatient setting as an IV infusion over 30 minutes. The dosage for each patient depends on the individual's body weight. The REGN2810 dose is adjusted each cycle for changes in body weight >10%. REGN2810 is administered alone or in combination with radiation and/or cyclophosphamide. Cyclophosphamide is administered at 200 mg/ ^m2 or at a low dose (100 mg/ ^m2 ).

Monotherapy.

REGN2810 is administered in an outpatient setting via IV infusion over 30 minutes every 14 days for 48 weeks (i.e., days 1, 15±3, 29±3, and 43±3 cycles of 56 days). Planned monotherapy regimens to be considered may include: (i) IV infusion of 1 mg/kg over 30 minutes every 14 days for 48 weeks; (ii) IV infusion of 3 mg/kg over 30 minutes every 14 days for 48 weeks; (iii) infusion of 10 mg/kg over 30 minutes every 14 days for 48 weeks; (iv) IV infusion of 0.3 mg/kg over 30 minutes every 14 days for 48 weeks (if determined to be less than 1 mg/kg MTD) and (v) IV infusion of a constant dose of 200 mg over 30 minutes every 14 days for 48 weeks.

Combination therapy.

Concomitant radiation therapy, cyclophosphamide, GM-CSF, carboplatin, and docetaxel are delivered via prescription, and their use, dose, dose modifications, reductions or delays, and any potential AEs resulting from their use are monitored along with those for REGN2810.

Coadministration of REGN2810 and radiation: REGN2810 is administered by IV infusion over 30 minutes every 14 days for 48 weeks in combination with radiation treatment from day 8 to day 12. Planned combination regimens of REGN2810 and radiation therapy may include:

mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks + radiation therapy 30 Gy (6 Gyx5 times/week; given 1 week after the first dose of REGN2810,

- 42 045900 preferably on consecutive days);

mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks + radiation therapy 27 Gy (9 Gy x 3 times/week; given 1 week after the first dose of REGN2810, preferably not on consecutive days);

mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks + radiation therapy 30 Gy (6 Gyx5 times/week; given 1 week after the first dose of REGN2810, preferably on consecutive days);

mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks + radiation therapy 27 Gy (9 Gy x 3 times/week; given 1 week after the first dose of REGN2810, preferably not on consecutive days).

Patients receive either 30 Gy, given in 5 fractions of 6 Gy given daily, starting 1 week after the first dose of REGN2810, or 27 Gy, given in 3 fractions of 9 Gy, given every other day, starting 1 week after the first dose of REGN2810. The lesion selected for irradiation must be one that can be safely irradiated with focused irradiation while sparing the index lesion(s), and for which irradiation at the limited palliative doses considered is considered appropriate for medical use.

Coadministration of REGN2810 and cyclophosphamide: REGN2810 is administered by IV infusion over 30 minutes every 14 days (2 weeks) for 48 weeks in combination with low dose cyclophosphamide 100 mg/m ² IV infusion every 14 days for 4 doses. Each of the 4 doses of cyclophosphamide is administered 1 day before each of the first 4 doses of REGN2810 (days -1, 14, 28 and 42 of the first cycle of 56 days).

The planned combination regimen of REGN2810 and cyclophosphamide is:

cyclophosphamide 100 mg/ ^m2 or 200 mg/ ^m2 IV every 14 days (days -1, 14, 28, and 42 of the first cycle of 56 days), a total of 4 doses; + mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks (a monotherapy dose of 3 mg/kg < MTD is provided; if 3 mg/kg > MTD, the dose will be 1 mg/kg.

Co-administration of REGN2810, radiation and cyclophosphamide: The planned combination regimen of REGN2810, radiation and cyclophosphamide includes:

cyclophosphamide 100 mg/ ^m2 (low dose) IV every 14 days (days -1, 14, 28, and 42 of the first cycle of 56 days), 4 doses in total; + radiation therapy 27 Gy (9 Gy x 3 times/week; given 7 or 8 days after the first dose of REGN2810, preferably not on consecutive days) or radiation therapy 30 Gy (6 Gy x 5 times/week; given 7 or 8 days after the first dose REGN2810, preferably on consecutive days); + mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks (a monotherapy dose of 3 mg/kg < MTD is provided; if 3 mg/kg > MTD, the dose will be 1 mg/kg)

Co-administration of REGN2810, radiation and GM-CSF: The planned combination regimen of REGN2810, radiation and GM-CSF includes:

GM-CSF 250 mcg SC daily for 7 days, four 7-day intervals (days 1-7, 15-21, 29-35 and 43-49 of the first cycle of 56 days); + radiation therapy 27 Gy (9 Gy x 3 times/week; given 1 week after the first dose of REGN2810, preferably not on consecutive days); + mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks (a monotherapy dose of 3 mg/kg < MTD is provided; if 3 mg/kg > MTD, the dose will be 1 mg/kg).

Co-administration of REGN2810, radiation, GM-CSF and cyclophosphamide: The planned combination regimen of REGN2810, radiation, GM-CSF and cyclophosphamide includes:

GM-CSF 250 mcg SC daily for 7 days, four 7-day intervals (days 1-7, 15-21, 29-35 and 43-49 of the first cycle of 56 days); + radiation therapy 27 Gy (9 Gy x 3 times/week; given 1 week after the first dose of REGN2810, preferably not on consecutive days); + cyclophosphamide 100 mg/ ^m2 or 200 mg/ ^m2 IV every 14 days (days -1, 14, 28, and 42 of the first cycle of 56 days), a total of 4 doses; + mg/kg REGN2810 infusion over 30 minutes every 14 days for 48 weeks (a monotherapy dose of 3 mg/kg < MTD is provided; if 3 mg/kg > MTD, the dose will be 1 mg/kg).

Coadministration of REGN2810 and docetaxel with or without carboplatin: The proposed drug administration sequence is docetaxel followed by carboplatin (if enrolled in a carboplatin-containing cohort) followed by REGN2810:

docetaxel 30 mg/m ² IV for approximately 1 hour per day on days 1, 8, 29 and 36 of the first cycle of 56 days. Dexamethasone 8 mg IV is given before the first dose of docetaxel. For subsequent docetaxel treatment, premedication with a dose of dexamethasone may be 8 mg or 4 mg, at the discretion of the investigator;

carboplatin AUC 2 IV over approximately 30 minutes on days 1, 8, 29, and 36 of the first cycle 56

- 43 045900 days. When dosing carboplatin, use the Calvert formula on the carboplatin label. Creatinine clearance should be calculated using the Cockfort-Gault equation;

mg/kg REGN2810 infusion over approximately 30 minutes every 14 days for 48 weeks.

Procedures and assessments.

Screening procedures to be performed include serum β-HCG, brain MRI, and chest x-ray.

Safety procedures include medical history, physical examination, vital signs, electrocardiogram (ECG), coagulation, immunological safety tests (for patients treated with REGN2810), B-symptom and functional status assessment, clinical laboratory tests, AEs, and concomitant drug therapy.

Effect procedures to be performed for tumor evaluations include CT or MRI scans, ¹⁸ G-fluorodeoxyglucose positron emission tomography (FDG-PET) scans, and/or tumor biopsies. CT or MRI for tumor assessment is performed at the screening visit (within 28 days before infusion) and during each cycle (approximately every 8 weeks) on days 56±3, and when disease progression is suspected. Additionally, in patients who have not progressed on study, tumor assessments are performed at follow-up visits 3, 5, and 7. When the decision is made to use CT or MRI scanning, subsequent assessments are performed using the same modality. Tumor response assessments are performed in accordance with the Response Evaluation Criteria in Solid Tumors RECIST version 1.1 (Eisenhauer et al 2009, Eur. J. Cancer 45: 228247). Measurable lesions selected as target lesions for RECIST measurements are also included as index lesions for immune-related response criteria (irRC; Nishino et al 2013, Clin. Cancer Res. 19: 3936–3943). The RECIST response takes precedence as a statistical estimate of the response rate. For an individual patient, irRC can inform the decision about whether to continue treatment at the discretion of the investigator due to the possibility of nonstandardized responses.

Blood samples are collected to assess PK and anti-drug antibodies (ADA).

Research Variables.

The main variables in the study are DLT incidence and incidence and severity of TEAE and abnormal laboratory findings after 48 weeks of treatment.

The secondary variables are:

antitumor activities assessed using appropriate criteria for the indication (described elsewhere herein):

Response Evaluation Criteria in Solid Tumors (RECIST; Eisenhauer et al 2009, Eur. J. Cancer 45: 228–247), measured by CT or MRI;

other scoring criteria are also used for specific tumors for which RECIST measurements are not standard;

Immune-related response criteria (irRC; Nishino et al 2013, Clin. Cancer Res. 19: 39363943) applied to RECIST measurements. In all cases, RECIST (or other tumor-specific criteria) is the preferred tool to define PD, SD, CR or PR. irRCs are collected for clinical decision and information purposes.

Incidence for antibody formation against REGN2810.

Antitumor activity measured by PFS and overall survival.

For the purposes of this study, patients are re-evaluated for response every 8 weeks. Confirmatory scans are also obtained 4 weeks after initial documentation of objective response or progressive disease. Response and progression are assessed in this study using the international criteria proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) manual (version 1.1; Eisenhauer et al 2009, Eur. J. Cancer 45: 228–247). Changes in tumor lesions of the largest diameter (unidimensional measurement) and the smallest diameter in the case of metastatic lymph nodes are used in the RECIST criteria.

Selection of damages.

Measurable disease: Measurable lesions are defined as those that, when accurately measured in at least one dimension (longest reportable diameter), are >20 mm (>2 cm) by chest radiography or are >10 mm (> 1 cm) using CT scan, MRI or caliper during clinical examination. All tumor measurements should be recorded in millimeters (or tenths of a centimeter). Note: See below for evaluation of irradiated target lesions.

Metastatic lymph nodes: should be considered pathologically enlarged and measurable, the lymph node should be >15 mm (>1.5 cm) in short axis when assessed by CT scan (recommended slice thickness on CT scan is no more than 5 mm [0 .5 cm]). At baseline and follow-up, only the short axis is measured and monitored.

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Non-measurable disease: all other lesions (or sites of disease), including small lesions (longest diameter <10 mm [<1 cm] or abnormal short-axis lymph nodes >10 to <15 mm [>1 to <1.5 cm]) is considered unmeasurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, cutaneous lymphangitis/pulmonitis, inflammatory breast disease, and abdominal masses (not monitored by CT or MRI) are considered not measurable. Note: Cystic lesions that meet the criteria for radiographic simple cysts should not be considered metastatic lesions (neither measurable nor non-measurable) because they are, by definition, simple cysts. Cystic lesions that are presumed to represent cystic metastases may be considered measurable lesions if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, they are preferred for selection as target lesions.

Target lesions: All measurable lesions, up to a maximum of 2 lesions per organ and 5 lesions overall, that are representative of all organs involved should be designated as target lesions and recorded and measured at a baseline level. Target lesions, which are selected based on their size (the lesion with the longest diameter), are representative of all organs involved but also include those suitable for reproducible, repeatable measurements. Sometimes this may be the case where the largest lesion is not suitable for reproducible measurement, in which case the next largest lesion that can be reproducibly measured is selected. The sum of the diameters (longest for nonnodal lesions, shortest axis for nodal lesions) for all target lesions is calculated and reported as the base sum of diameters. If lymph nodes are to be included in the sum, then only the short axis is added to the sum. The baseline sum of diameters is used as a guide to further characterize any objective tumor regression into measurable disease.

Non-target lesions: All other lesions (or disease sites), including any measurable lesions beyond the 5 target lesions, are identified as non-target lesions and recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or, in rare cases, unambiguous progression of each is noted throughout follow-up.

Methods for assessing measurable disease.

All measurements are taken and recorded in metric notation using a ruler or calipers. All baseline assessments are carried out as close to the start of treatment as possible and never more than 4 weeks before the start of treatment. The same assessment method and techniques should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based assessment is preferred to clinical examination until the observed lesion(s) are not visualized but are assessable by clinical examination.

Clinical lesions: Clinical lesions are considered measurable only when they are superficial (eg, skin nodules and palpable lymph nodes) and have a diameter >10 mm (>1 cm) as assessed using calipers (eg, skin nodules). In case of skin damage, documentation using color photography is recommended, as well as a ruler to assess the size of the damage.

Chest X-ray: Lesions on a chest X-ray are acceptable because the lesions are measurable when they are clearly defined and surrounded by air-filled lung. However, ST is preferred.

Standard CT and MRI: This guideline has a specific capability for measuring damage on CT scans, which is based on the assumption that the CT slice thickness is 5 mm (0.5 cm) or less. If the CT scan has a slice thickness greater than 5 mm (0.5 cm), the minimum measurable lesion size must be twice the slice thickness. MRI is also acceptable in certain situations.

RET-CT: If the CT performed as part of the RET-CT has diagnostic quality identical to diagnostic CT (with IV and oral contrast), then the CT portion of the RET-CT can be used for RECIST measurements and can be used interchangeably with standard CT for accurate measuring malignant tumor lesions over time.

Ultrasound: Ultrasound cannot be used in assessing the size of a lesion and should not be used as a measurement method. If new lesions are identified by ultrasound during examination, confirmation by CT or MRI is recommended. If there are concerns about radiation exposure with CT, MRI may be used instead of CT in selected cases.

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Endoscopy, laparoscopy: the use of these methods for tumor assessment is not recommended. However, such methods can be used to confirm complete pathological response when biopsies are obtained, or to determine relapse in studies where recurrence after complete response (CR) or surgical resection is the end point.

Tumor markers: Tumor markers cannot be used alone to assess response. If markers are initially above the upper limit of normal, they should be normalized in a patient considered to be in complete clinical response.

Cytology, histology: these modalities can be used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (eg, residual lesions in certain tumor types, such as germ cell tumors, where known residual benign tumors may persist) . Cytological confirmation of neoplastic origin of any exudate that occurs or worsens during treatment when the measurable tumor meets criteria for response or stable disease is necessary to differentiate between response or stable disease (exudate may represent a side effect of treatment) and progressive disease.

FDG-PET: Although FDG-PET response assessments require additional investigation, it may sometimes make sense to include the use of FDG-PET scans to complement CT scans when assessing progression (particularly possible new disease). New lesions based on FDGPET imaging can be identified according to the following algorithm: a) negative FDG-PET at baseline, with positive FDG-PET at follow-up indicating PD based on new lesion; b) No FDG-PET at baseline and positive FDG-PET at follow-up: If a positive FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, it is PD. If a positive FDG-PET on surveillance is not confirmed as a new site of disease on CT, additional surveillance CT scans are needed to determine if there is true progression occurring at that site (if so, the PD date will be the date of the initial abnormal FDG-PET). PET scan). If a positive FDG-PET on surveillance corresponds to a pre-existing site of disease on CT that has not progressed based on anatomical imaging, it is not PD; c) FDGPET can be used to update the response to CR in a manner similar to biopsy in cases where the residual radiographic abnormality is thought to represent fibrosis or scarring. The use of FDG-PET in this setting should be described in advance in the protocol and supported by disease-specific medical literature for this indication. However, it must be recognized that both approaches may lead to false-positive CR due to the limitations of FDG-PET and biopsy resolution/sensitivity. Note: A positive lesion on an FDG-PET scan denotes one in which the FDG tropism for uptake is greater than twice that of the surrounding tissue on the attenuation-corrected image. Response criteria for assessing target lesions

Complete response (CR): disappearance of all target lesions. Any abnormal lymph nodes (whether target or non-target) should have a short-axis reduction of <10 mm (<1 cm).

Partial response (PR): At least 30% reduction in the sum of diameters of target lesions, using the base sum of diameters as a reference.

Progressive disease (PD): At least a 20% increase in the sum of target lesion diameters, using the smallest sum in the study as the reference (this includes the base sum if it is the smallest in the study). In addition to the 20% relative increase, the sum must also demonstrate an absolute increase of at least 5 mm (0.5 cm) (note: the occurrence of one or more new lesions is also considered progression).

Stable disease (SD): neither sufficient reduction to qualify as PR nor sufficient increase to qualify as PD, using the smallest sum of diameters in the study as the reference.

Response criteria for assessing non-target lesions.

Complete response (CR): disappearance of all non-target lesions and normalization of tumor marker levels. All lymph nodes should be of normal size (<10 mm [<1 cm] short axis). Note: If tumor markers are initially above the upper limit of normal, they should be normalized for a patient considered to be in complete clinical response.

Non-CR/He PD: persistence of one or more non-target lesions and/or persistence of tumor marker levels above normal limits.

Progressive disease (PD): the occurrence of one or more new lesions and/or clear progression of existing non-target lesions. Unambiguous progression should generally not exceed the state of the target lesion. It should be representative of the overall change in disease status rather than an increase in a single lesion.

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Response criteria related to immunity.

The immunity-related response criteria differ from RECIST (version 1.1) with respect to the sum of the longest diameters of all target lesions and new lesions, if any, are used to define response. The presence of new lesions per se does not determine progression; calculate the total tumor burden.

Assessment of target damage.

Complete response (CR): disappearance of all target lesions. Any abnormal lymph nodes (whether target or non-target) should have a short-axis reduction of <10 mm (<1 cm).

Partial Response (PR): At least a 30% reduction in the sum of diameters of target lesions, including new lesions, using the base sum of diameters as a reference.

Progressive disease (PD): At least a 20% increase in the sum of diameters of target lesions, including new lesions, using the smallest sum in the study as the reference (this includes the base sum if it is the smallest in the study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm (0.5 cm).

Stable disease (SD): neither sufficient reduction to qualify as PR nor sufficient increase to qualify as PD, using the smallest sum of diameters in the study, including measurements of new lesions, as the reference.

Assessment of non-target damage.

Complete response (CR): disappearance of all non-target lesions and normalization of tumor marker levels. All lymph nodes should be of nonpathologic size (<10 mm [<1 cm] short axis). Note: If tumor markers are initially above the upper limit of normal, they should be normalized for a patient considered to be in complete clinical response.

Non-CR/He PD: persistence of one or more non-target lesions and/or persistence of tumor marker levels above normal limits.

Progressive disease (PD): clear progression of existing non-target lesions. Unambiguous progression should generally not exceed the state of the target lesion. It should be representative of the overall change in disease status rather than an increase in a single lesion. Although apparent progression of non-target lesions is an exception, the judgment of the treating physician should prevail in such circumstances and the progression status should be confirmed at a later time.

Evaluation of overall response criteria.

The best overall response is the best response recorded from the start of treatment until disease progression/relapse (using the lowest measurement recorded from the start of treatment as the reference for progressive disease). Assigning the best answer to a patient depends on meeting both the measurement and validation criteria. Revised Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (Eisenhauer et al 2009, Eur. J. Cancer 45: 228–247) and immune-related response criteria (irRC; Nishino et al 2013, Clin. Cancer Res. 19: 3936-3943) are summarized below in table. 10 and 11.

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Table 10

Response according to Revised RECIST (version 1.1)

Target damage Not targeted damage New damage General answer Best general answer when confirmation is needed CR CR No CR Confirmation >4 weeks CR Not CR/He PD No PR Confirmation >4 weeks CR Not rated No PR Confirmation >4 weeks PR Not CR/He PD/not assessed No PR Confirmation >4 weeks SD Not CR/He PD/not assessed No SD Documented and at least at least once every >4 weeks from basic level P.D. Any Yes or no P.D. No previous SD, PR or CR Any P.D. Yes or no P.D. No previous SD, PR or CR Any Any Yes P.D. No previous SD, PR or CR

CR: full answer;

PD: progressive disease;

PR: partial response;

SD: stable disease.

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Table 11

Assessment of immune-related response criteria

Target damage Not targeted damage New damage General answer Best general answer when confirmation is needed CR CR No CR Confirmation >4 weeks CR Not CR/He PD No PR Confirmation >4 weeks CR Not rated No PR Confirmation >4 weeks PR Not CR/He PD/not assessed Yes or no PR Confirmation >4 weeks SD Not CR/He PD/not assessed Yes or no SD Documented and at least once in >4 weeks from basic level P.D. Any Yes or no P.D. No previous SD, PR or CR Any P.D. Yes or no P.D. No previous SD, PR or CR

CR: full answer;

PD: progressive disease;

PR: partial response;

SD: stable disease.

Assessment of irradiated target lesions.

Irradiated target lesions are assessed using a modified version of the international criteria proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) Committee, version 1.1. Additional definitions beyond the RECIST 1.1 guidance specific to this protocol are included to define local control.

The response criteria for irradiated lesions are as follows.

Local increase (LE): at least a 20% increase in the LD of the target lesion, using as a reference the smallest LD recorded since the start of treatment. It is ideal to make this determination based on an assessment of the CT image.

Local failure (LF): refers to the primary treatment of the tumor after protocol therapy and corresponds to the fulfillment of both of the following two criteria: (1) an increase in tumor measurement of 20%, as defined above for local enhancement (LE); (2) a measurable tumor meeting LE criteria should be amenable to positron emission tomography (PET) imaging with similar intensity capture as staging on pretreatment PET, or a measurable tumor biopsy confirming viable carcinoma should be obtained.

Local control (LC): no local failure.

The longest diameter (LD) for the irradiated target lesion calculated from the CT scan during treatment planning, using an appropriate tissue-specific window, is reported as the base LD. The baseline LD is used as a reference against which the characteristics of the target tumor are determined. For follow-up assessment, diagnostic CT

- 49 045900 scans that were performed using a 5 mm solid reconstruction algorithm using a pulmonary window taken as part of the planned protocol follow-up are preferred as a means of assessing response. When CT scanning is not available, MRI or x-ray detection is acceptable as long as the target lesion is clearly visible.

Results.

REGN2810 alone and in combination is safe and well tolerated in patients. Administration of REGN2810 alone or in combination with other treatment modalities inhibits tumor growth and/or promotes tumor regression in patients with advanced solid tumors. The overall response rate is better for combination therapy with radiation compared with monotherapy.

To date, 60 patients with advanced solid malignancies have been treated (47% with four or more prior therapies).

Common solid malignancies include colon cancer, head and neck cancer, breast cancer, soft tissue sarcoma, adrenal cancer, anal cancer, appendix cancer, bladder cancer, cervical cancer, endometrial cancer , malignant tumor of the esophagus, malignant tumor of the liver, non-small cell adenocarcinoma of the lung, malignant ovarian tumor, malignant tumor of the pancreas, malignant tumor of the prostate, renal sarcomatoid, malignant tumor of the salivary gland, non-melanoma malignant tumor of the skin, Merkel cell carcinoma, squamous cell carcinoma, basal cell carcinoma , malignant tumor of the small intestine, malignant tumor of the thyroid gland and malignant tumor of the uterus.

patients (70%) experienced one or more treatment-related adverse events (AEs). The most common treatment-related AEs were fatigue (28.3%), arthralgia (11.7%), and nausea (11.7%). Of the 60 patients assessed for tumor responses, 11 (18.3%) had objective responses (PR/CR), while 31 patients (51.7%) demonstrated disease control (CR/PR/SD). In 36 patients who received combination therapy containing radiation, objective response was observed in 6 patients (16.7%) and disease control in 19 patients (52.8%). In 24 patients who did not receive radiation therapy, objective response was observed in 5 patients (20.8%) and disease control was observed in 12 patients (50%). In table Table 12 provides a summary of patients who responded to treatment.

Table 12

Summary of responders

Individual ID Cohorts by dose Type malignant tumors No. previous lines therapy Best answer Best reduction 41 R2810: 1 mg/kg Cholangiocarcinoma 5 PR -41.2

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50 R2810: 1 mg/kg Squamous cell carcinoma of the skin 2 CR -100.0 43 R2810: 10 mg/kg Soft sarcoma fabrics 5 PR -49.1 37 R2810: 10 mg/kg Basal cell carcinoma 1 PR -36.7 36 R2810: 3 mg/kg+SPA: 200 mg/ ^m2 Soft sarcoma fabrics 5 PR -33.3 47 R2810: 1 mg/kg+XRT: 6 Gy x 5 Squamous cell carcinoma of the cervix uterus 4 PR -66.7 46 R2810: 1 mg/kg+XRT: 9 Gy x 3 Squamous cell carcinoma of the anus 3 PR -57.1 49 R2810: 1 mg/kg+XRT: 9 Gy x 3 Squamous cell carcinoma of the cervix uterus 3 CR -100.0 48 R2810: 3 mg/kg+XRT: 6 Gy x 5 Carcinoma from Merkel cells 1 PR -72.5 42 R2810: 3 mg/kg+XRT: 6 Gy x 5 Small bowel adenocarcinoma 2 PR -46.7 44 R2810: 3 mg/kg+XRT: 9 Gy x 3 Serous carcinoma of the ovary 6 PR -52.4

Among responding patients, the median time to response for monotherapy was 113 days (range, 52–226) and for patients with radiation therapy was 59 days (range, 56–113).

Example 8. Descriptions of cases of blocking PD-1 with the monoclonal antibody REGN2810 with the achievement of durable objective responses in metastatic non-melanoma malignant skin tumors: basal cell carcinoma and squamous cell carcinoma of the skin.

Introduction.

Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (CSCC) have exposure to UV light as a major risk factor, and consequently these tumors are hypermutated (Chalmers et al 2016, AACR Ann. Meeting, Abs 3576). In other malignancies, high mutational load is associated with clinical benefit of antibody therapy directed against the PD-1 immune checkpoint [Le et al 2015, New Engl. J. Med. May 30 (electronic publication ahead of print)]. Highly mutated tumors are more likely to express immunogenic tumor neoantigens, which attract effector T cells that can be unleashed by blocking the PD-1 immune checkpoint (Mandal and Chan 2016, Cancer Discov. 6: 1-12). This example describes a patient with metastatic SCD and a patient with metastatic CSCC who were treated with REGN2810, a fully human anti-PD-1 monoclonal antibody in an ongoing phase 1 study (NCT02383212; described in Example 7 herein).

Description of case 1.

The patient was a 66-year-old female who was diagnosed with stage 1 SCD occurring on the left side of her chin, which was excised in a Mohs procedure. A localized recurrence in the same location was identified 2 years later, and extensive local excision revealed invasion into the left mandible involving one of 18 lymph nodes. The patient received assisted radiation and remained in remission for 4 years, when enlarging lung nodules observed on chest imaging during follow-up were biopsied and confirmed to have metastatic SCD. The patient subsequently received the Hedgehog pathway inhibitor (HHI) vismodegib for 5 months. She initially responded but stopped due to progressive disease.

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After 6 months of vismodegib therapy and with continued slow progression, the patient was enrolled in the phase 1 REGN2810 study in the 10 mg/kg IV every 2 weeks cohort and received her first dose. Two pulmonary metastases were observed as target lesions. Response assessments at the end of 8 weeks (3% increase) and 16 weeks (10% decrease) demonstrated stable disease according to RECIST criteria. Response assessment at the end of 24 weeks demonstrated a 37% reduction in tumor measurements (Figure 14A), and this was confirmed at week 32. The patient tolerated the treatment well and remains on REGN2810, on treatment for 10+ months.

Description of case 2.

The patient was a 52-year-old male who was diagnosed with squamous cell carcinoma of the skin on the left cheek. He underwent Mohs surgery with clean margins. He showed several relapses and underwent at least 9 additional Mohs surgeries. He underwent extensive local excision over the left mandible 4 years later and subsequently a left parotidectomy 20 months later. Adjuvant radiation therapy was also administered to the left cheek, left mandible, left neck (with concurrent cetuximab), and bilateral neck (with concurrent carboplatin). Other systemic therapies were capecitabine and cisplatin+docetaxel. Ten years after initial diagnosis, he underwent excision with clean margins for a 2.2 cm recurrence in a left neck scar. Subsequently, invasive CSCC on the C4-C5 vertebral bodies necessitated cervical spinal cord decompression with anterior C4-C5 corpectomy and posterior C4-C6 laminectomy. He also developed lower extremity muscle weakness, presumably due to nerve sheath involvement, which required the use of a walker for ambulation.

He was enrolled in a phase 1 study in the first cohort receiving 1 mg/kg REGN2810 every two weeks. Within weeks of starting treatment, he gradually regained strength in his lower extremities and no longer required the use of a walker. The answer for week 16 is shown in Fig. 14V. Complete radiological response of the left neck lesion was achieved at week 40. The patient completed the planned 48 weeks of treatment protocol using REGN2810. He continues close active surveillance with his medical oncologist without clinical or radiographic evidence of disease recurrence.

Discussion.

This example discloses the first confirmed partial response in a patient with metastatic SCD treated with a PD-1 inhibitor (REGN2810), as well as the current sustained complete response in a patient with metastatic CSCC. The profound and durable responses of these heavily pretreated patients to anti-PD-1 monotherapy in this phase 1 study are consistent with the hypothesis that the high mutational load in SCD and CSCC will induce antitumor cellular immunity that can be unleashed by blocking the PD1 checkpoint pathway /PD-L1.

This example supports the basic principle that UV-associated skin malignancies other than melanoma are sensitive to PD-1 blockade. The reductionist model predicts that UV-associated tumors with a higher burden of nonsynonymous mutations will be more sensitive to PD-1 blockade than those with a lower mutational load.

Example 9: Safety and effect of anti-PD-1 antibody in patients with unresectable locally advanced or metastatic cutaneous squamous cell carcinoma (CSCC).

Prerequisites.

There is no established standard of care for unresectable locally advanced or metastatic CSCC. Due to UV-induced DNA damage, most CSCC are hypermutated. Therefore, these tumors may respond to PD-1 checkpoint blockade. This example describes patients with locally advanced or metastatic CSCC who were treated with REGN2810, a fully human anti-PD-1 monoclonal antibody, in an ongoing phase 1 study (NCT02383212; described in Example 7 herein).

Ways.

The expansion cohorts (EC) of the phase 1 REGN2810 study included patients with distant metastatic CSCC (EC 7) and locally advanced CSCC (EC8) (Table 9). All patients received 3 mg/kg REGN2810 intravenously every 2 weeks for up to 48 weeks. Biopsy studies were performed at baseline and day 29 (and at progression, if possible). To determine the overall response rate, tumor measurements were performed every 8 weeks according to RECIST 1.1.

Results.

25 patients were enrolled (10 in EU 7 and 15 in EU 8): median age 72.5 L (range 56–88 L); median PS 1 (range 0-1); 20M:5F; median number of prior systemic therapy regimens 1 (range 0-3). Median exposure to REGN2810 was 6 doses (range 1-22). The most common treatment-related adverse events of any grade were fatigue (16.7%), nausea, arthralgia, and rash (8.3% each). Each of the following grade >3 adverse events (AEs) occurred once: increased AST, increased ALT, arthralgia, and rash.

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Overall response rate (uPR+PR+CR) and disease control rate (ORR+SD) were 48% (11/23; 3uPR, 5 PR, 2 CR, 1 uCR) and 70% (16/23, including 5 SD), respectively. Two patients remain evaluable. Median PFS and median OS were calculated, and only one patient experienced PD during REGN2810 treatment after an initial response. Correlative scientific studies are underway, including whole exome sequencing of tumor DNA.

Conclusion.

REGN2810 demonstrates robust antitumor activity in patients with advanced CSCC.

Example 10: Clinical study of anti-PD-1 antibody in combination with hypofractionated radiation therapy compared with standard of care in patients aged >65 years with newly diagnosed glioblastoma.

Introduction.

Glioblastoma is a fatal disease with a median survival of approximately 16 months in newly diagnosed patients (nGBM) and approximately 9 months in relapsed patients (rGBM) (Friedman et al., 2009, J. Clin. Oncol. 27: 4733–4740). The current standard of care for patients with newly diagnosed glioblastoma is radiation (60 Gy over weeks) with concurrent temozolomide (TMZ) followed by adjuvant temozolomide (Stupp et al., 2005, N. Engl. J. Med. 352: 987-996 ), although subgroup analysis suggests that the addition of temozolomide may not improve the effect in older individuals (Laperriere et al., 2013, Cancer Treat. Rev. 39: 350-357).

This example describes a phase 3 study to evaluate the effect of anti-PD-1 antibody in combination with hypofractionated radiation therapy (hfRT) compared with standard of care (SoC) on overall survival in patients aged >65 years with nGBM.

The exemplary anti-PD-1 antibody used in this study is REGN2810 (also known as H4H7798N, as disclosed in US20150203579), a fully human anti-PD-1 monoclonal antibody containing a heavy chain containing the amino acid sequence of SEQ ID NO: 9, and a light chain containing the amino acid sequence SEQ ID NO: 10; a pair of amino acid sequences HCVR/LCVR containing SEQ ID NO: No. 1/2; and heavy and light chain CDR sequences containing SEQ ID NO: 3-8.

Objectives of the study.

The primary objective of the study is to evaluate the overall survival (OS) effect of REGN2810 given in combination with hfRT compared with standard of care in patients aged >65 years with nGBM.

The secondary objective of the study is to determine the improvement in progression-free survival (PFS).

Other study objectives are: (i) improvement in objective response rate (ORR), duration of response, and duration of disease control; (ii) clinical assessment using the Neurological Assessment in Neuro-Oncology (NANO) scale; (iii) safety; (iv) improved quality of life (QoL) and mental status; (v) changes in edema and steroid use; (vi) serum REGN2810 concentration and anti-REGN2810 antibodies; and (vii) investigation of potential pharmacodynamic, predictive or predictive biological markers.

Research plan.

This represents a 2:1 randomized phase 3 trial of REGN2810, a fully human anti-PD-1 antibody, in combination with hypofractionated radiotherapy versus standard of care in patients aged >65 years with newly diagnosed glioblastoma. Patients are randomized to REGN2810 in combination with hypofractionated radiotherapy versus standard of care in a 2:1 ratio using methylation status (methylated vs. unmethylated vs. undetermined) and extent of resection (partial vs. gross total resection) as stratification factors. The effect is assessed by overall survival.

Patients with nGBM who are candidates for radiotherapy are randomized 2:1 to receive one of the following treatments:

Investigational therapy: 3 mg/kg REGN2810 IV (every 2 weeks) + hypofractionated RT (6 Gy x 5, second week only). Radiation therapy is provided in week 2 of cycle 1, but not in subsequent cycles;

comparative therapy: standard of care TMZ (oral, 75 mg/ ^m2 , daily) in combination with standard RT (5 daily radiation fractions/week of 2 Gy) for 6 weeks, followed by adjuvant TMZ (oral, from 150 mg/m m ² to 200 mg/m ² 5 days/28 days) for 6 cycles. Radiation therapy is provided only in the first 6-week cycle.

Duration of the study.

The study consists of a 28-day screening period, after which eligible patients can have up to twelve 56-day (8-week) treatment cycles for up to

- 53,045,900 weeks of treatment. During the screening period (day -28 to day -1), all eligible patients should have available pre-treatment tumor resection (partial or complete resection) or biopsy for central pathology confirmation and determination and confirmation of MGMT methylation.

After day 1/baseline, patients returned to the clinic on cycle 1 on days 8±3, 15±3, 29±3, 43±3, and 56±3. In each subsequent 8-week cycle (cycles 2–12), patients returned to the clinic on days 1, 15±3, 29±3, 43±3, and 56±3. Tumor assessments (brain MRI, iRANO and NANO scores, MMSE and EORTC QLQ-C30/BN20 questionnaires) were performed on day 1/baseline and at the end of each treatment cycle. Extended safety assessments are performed on day 1 of each cycle; Standard safety assessments are performed at each visit. Samples are also collected to evaluate biological markers (cellular and molecular, described herein) that are associated with REGN2810 treatment exposure, clinical activity, or underlying disease.

During the 24-week follow-up period, patients return to the clinic 21 to 42 days after the last study treatment for the first follow-up visit. Subsequent follow-up visits (from follow-up visit 2 to follow-up visit 7) occur every 28 days + 7 days. Tumor assessments (brain MRI, piRANO and NANO scores, MMSE and EORTC QLQ-C30/BN20 questionnaires) are performed at follow-up visit 3, follow-up visit 5, and follow-up visit 7. Extended safety assessments are performed at the first follow-up visit; Standard safety assessments are performed at subsequent follow-up visits. Samples are collected to evaluate biological markers (cellular and molecular as described herein) that are associated with REGN2810 treatment exposure, clinical activity, or underlying disease.

Target population.

The target population includes patients aged >65 years with nGBM.

Inclusion Criteria: The patient must meet the following criteria to be eligible for inclusion in the study: (1) newly diagnosed primary glioblastoma with histological confirmation, maximum diameter <5 cm, undergoing partial or complete surgical resection; (2) Eastern Cooperative Oncology Group (ECOG) performance status 0-2; (3) age >65 years; (4) liver function: (a) total bilirubin <1.5 x upper limit of normal; (b) ALT and AST <3xULN; (c) alkaline phosphatase (ALP) <2.5xULN; (5) kidney function: creatinine cbibopotkuM^xULN; (6) bone marrow function: hemoglobin >9.0 g/dL; absolute neutrophil count (ANC) >1.5x 10 ⁹ /l; platelet count >75x109/l; (7) is able to read, understand, and be willing to sign the ICF; and (8) ability and willingness to perform scheduled visits, treatment plan, laboratory tests, and other study-related procedures.

Exclusion Criteria: Patients who meet any of the following criteria should be excluded from the study:

(1) any previous treatment for GBM (other than surgery);

(2) has known contraindications to Gd-MRI;

(3) current or recent (within 5 years) evidence of significant autoimmune disease that required treatment with systemic immunosuppressive therapy, which may indicate a risk of immune-related adverse events (irAEs). The following are not limitations: vitiligo, childhood asthma that has resolved, residual hypothyroidism that requires only hormone replacement therapy, or psoriasis that does not require systemic treatment;

(4) current treatment with systemic corticosteroids, excluding the use of corticosteroids for other (non-tumor or non-immunosuppressive) indications, up to a maximum of 10 mg/day of prednisone or equivalent;

(5) primary tumors located in the brain stem, spinal cord, or any secondary active infection of a brain tumor requiring therapy, including known human immunodeficiency virus infection, or active hepatitis B or hepatitis C virus infection;

(6) history of pneumonitis within the last 5 years;

(7) any investigational or antineoplastic treatment within 30 days prior to the initial administration of REGN2810;

(8) a history of documented allergic reactions or acute hypersensitivity reactions that were attributed to treatment with antibody therapy in general or to the agents specifically used in the study;

(9) inadequately controlled hypertension (defined as systolic blood pressure >150 mmHg and/or diastolic blood pressure >100 mmHg);

(10) known allergy to doxycycline or tetracycline (caution due to the presence of trace components in REGN2810).

(11) a previous history of hypertensive crisis or hypertensive encephalopathy.

(12) history of an invasive malignancy other than the one in the last 5 years

- 54 045900 which is treated in this study, with the exception of resected/ablated basal or squamous cell carcinoma of the skin or cervical carcinoma in situ or other local tumors that are considered curable with local treatment;

(13) acute or chronic psychiatric problems that, in the investigator's assessment, make the patient ineligible for participation;

(14) use of Novocure Tumor Treating Fields (Optune NovoTTF-lOOA device) in screening. Planned or anticipated use of Novocure Tumor Treating Fields while participating in the study;

(15) previous treatment with carmustine plates;

(16) ongoing sexual activity in men who are unwilling to maintain adequate contraception during the study.

Investigational treatments.

Patients receive one of the following treatment regimens.

Investigational therapy: 3 mg/kg REGN2810 (administered IV infusion over 30 minutes every 2 weeks for up to 96 weeks ¹ per week 2 cycles 1.

Comparative therapy: standard of care TMZ (oral, 75 mg/ ^m2 , daily) in combination with standard RT (5 daily radiation fractions/week of 2 Gy) for 6 weeks, followed by adjuvant TMZ (oral, from 150 mg/m m ² to 200 mg/m ² 5 days/28 days) for 6 cycles. Radiation therapy is provided only in the first cycle.

REGN2810 is supplied as a liquid in sterile, single-use vials. Each vial contains a volume sufficient to contain 10 mL of REGN2810 at a concentration of 25 mg/mL. REGN2810 is administered as an IV infusion over 30 minutes. The dose for each patient depends on individual body weight. The dose of REGN2810 should be adjusted each cycle based on changes in body weight >10%.

Radiation therapy: Patients in the control group receive standard radiation therapy (60 Gy over 6 weeks). Patients in the experimental treatment group received hfRT (6 Gy x 5 daily fractions) administered 1 week after the first dose of REGN2810.

REGN2810+radiation (investigational treatment): REGN2810 is administered by IV infusion over 30 minutes every 14 days for 96 weeks in combination with hfRT from day 8 to day 12.

Schedule for the planned combination of REGN2810 and hfRT: infusion of REGN2810 3 mg/kg over 30 minutes every 14 days for 96 weeks + radiation therapy (hfRT in 6 Gyx5 daily fractions; given 1 week after the first dose of REGN2810, preferably on consecutive days).

Description of Radiation Therapy: Patients receive 30 Gy in 5 fractions of 6 Gy administered daily, beginning 1 week after the first dose of REGN2810.

Comparative group: standard of care: TMZ (oral, 75 mg/ ^m2 , daily) in combination with standard RT (5 daily radiation fractions/week of 2 Gy) for 6 weeks, followed by adjuvant oral TMZ. The dose of TMZ is 150 mg/m ² for the first 5 days of the first booster cycle, and is increased to 200 mg/m ² for 5 days/28 days starting from the second cycle if there is no unacceptable hematological toxicity in the first booster cycle.

If during the first booster cycle all observed non-hematologic toxicities are grade <2 (excluding alopecia, nausea and vomiting) and platelets are >100 x 10 ⁹ /L and ANC >1.5 x 10 ⁹ /L, then the TMZ dose should be increased to dose 1 (200 mg/ ^m2 ) and this dose should be used as the initial dose for subsequent cycles. If, after cycle 1, TMZ should be withheld due to current grade >2 non-hematologic toxicity, then upgrading is not possible. If the dose was not increased in the second cycle, then the dose should not be increased in subsequent cycles.

Treatment of CNS Edema: For any patient who develops symptomatic intracranial edema during the study, maintain REGN2810 dosing and radiation therapy until the edema subsides.

In patients who develop intracranial edema, bevacizumab is administered IV, as needed (PRN), at a reduced dose relative to the standard (suggested dose of 5 mg/kg every 2 weeks for up to 3 doses, not more than 10 mg/kg per dose every 2 weeks) unless contraindicated (for example, if the patient has not had surgery within the last 28 days).

If bevacizumab does not relieve intracranial edema, systemic corticosteroids, in addition to or as a substitute for bevacizumab, can be administered at the lowest dose appropriate to control symptoms. In patients who cannot tolerate bevacizumab, use corticosteroids at a dose appropriate to control symptoms.

Research Variables.

The primary outcome measure is overall survival (OS), which is defined as the time interval from the date of randomization to the date of death from any cause.

The key secondary endpoint is progression-free survival (PFS), which is defined as the time interval from the date of randomization to the date of first observation of disease progression or date of death (from any cause). Disease progression is determined using the iRANO criteria.

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Other secondary effect endpoints are:

Objective response rate (ORR): defined as the proportion of patients with a confirmed complete response (CR) or confirmed partial response (PR), as determined by Immunotherapy Response Assessment in Neuro-Oncology (iRANO) criteria, relative to the total number of patients in the study population.

Duration of response: determined for patients with best overall response for CR or PR. Duration of response is measured by measuring the time first to meet the CR/PR (whichever is recorded first) to the first date of recurrent or progressive disease (radiographically) or death from any cause.

Duration of disease control: determined for patients with best overall response for SD, CR or PR.

Duration of disease control is measured from the start of treatment to the first date of recurrent or progressive disease (radiographic) or death from any cause.

Quality of life and symptom control variables: Quality of life and symptom control variables are:

five functional scales, three symptom scales, one global health measure, and six single-item scales that assess symptoms using the EORTC QLQ-C30 questionnaires during the study;

four scales and 7 individual dimensions using the EORTC QLQ-BN20 questionnaires during the study;

clinical evaluation using NANO;

overall MMSE score during the study;

corticosteroid use at baseline, cumulative corticosteroid use during the study, and steroid-free duration or low-dose steroid use during the progression-free period in the study;

bevacizumab PRN use at baseline, cumulative bevacizumab PRN during the study, and bevacizumab-free duration during the progression-free period of the study.

Exploratory Biological Marker Variables: Another endpoint includes pharmacodynamic, prognostic and predictive biological markers associated with clinical response, mechanism of action and possible REGN2810-related AEs following treatment. Biological marker variables include:

expression levels of immune checkpoint receptors PD-L1, GITR and LAG3, as well as other potential biological markers (eg, EGFRvIII, Ki67, etc.) in tumor samples;

number and distribution of TILs in tumor samples;

IDH1 mutational status, microsatellite instability (MSI), and mutational load in tumor samples;

circulating biological markers, including cytokines and angiogenic factors;

Cell subpopulations and expression levels of biological markers of interest in PBMCs;

MGMT promoter methylation status (also used for stratification).

Other variables include serum REGN2810 concentration (pharmacokinetic variables) and the formation of anti-REGN2810 antibodies.

Procedures and assessments.

After a screening period of up to 28 days, patients receive up to twelve 56-day treatment cycles for up to a total of 96 weeks of treatment, followed by an observation period of 24 weeks. Analyze effect, safety, PK, ADA and research biological markers.

Effect procedures.

MRI: MRI for tumor assessment is performed 72 hours after surgery at the screening visit (within 28 days before infusion), on day 56±3 of each cycle (approximately every 8 weeks), and when PD is suspected. In patients whose disease has not progressed, additional tumor assessments are performed at follow-up visits 3, 5, and 7. Note: If PD has been confirmed, additional scans are not required at follow-up visits. If pre- and post-surgery MRI was performed before study entry, these scans should also be included in the study to assist in determining tumor volume and tumor progression.

Tumor response assessment is carried out in accordance with iRANO; and clinical neurological assessment is performed using NANO. RANO assessments are also carried out as a supporting study; however, the initial determination of disease progression for an individual patient is performed in accordance with iRANO.

European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and EORTC Brain Cancer Module (EORTC QLQ-BN20): EORTC QLQ-C30

- 56 045900 is a 30-parameter questionnaire that assesses health-related quality of life (HRQoL) in patients with malignant tumors using 15 scales (single- or multi-parameter), each with possible scores ranging from 0 to 100. Of the 30 dimensions, 24 are collected into 9 multidimensional scales representing different aspects of HRQoL: 5 functional scales (physical, role, emotional, cognitive and social), 3 symptom scales (fatigue, pain and nausea) and 1 global health status measure. The remaining 6 single-parameter scales assess symptoms: dyspnea, loss of appetite, sleep disturbance, constipation and diarrhea, and the financial burden of treating the disease. Higher scores indicate better HRQoL for the global health measure and functional scales and poorer HRQoL for the symptom scales.

The EORTC QLQ-BN20 is a 20-item assessment of QoL specific to brain neoplasms and is intended to complement the EORTC QLQ-C30 in assessing health-related quality of life. The EORTC QLQ-BN20 assesses disease symptoms, side effects of treatment and some specific psychosocial issues important for patients with malignant brain tumors using 4 scales (assessment of future uncertainty, visual impairment, motor impairment and communication deficits) and 7 separate parameters (assessment of other symptoms of the disease [eg, headaches and paroxysms] and toxic effects of treatment [eg, hair loss]). Possible scores range from 0 to 100; higher scores indicate poorer HRQoL.

Mini-Mental State Examination: The Mini-Mental State Examination (MMSE©) is a concise quantitative measure of cognitive status in adults. It can be used to screen for cognitive impairment to assess the severity of cognitive impairment at a given point in time and to monitor the progress of cognitive changes in an individual over time. In this study, the MMSE assessment is part of the neurological examination performed in the context of disease assessment.

MMSE is performed on day 1/baseline, at the end of each treatment cycle, and every 8 weeks during the observation period. MMSE assessments coincide with the disease assessment plan, but should be performed before disclosing the results of the radiological assessment to the patient. The MMSE can be performed at the start of the next scheduled treatment administration. During the survival follow-up period, MMSE should continue to be performed at every other survival visit (every 8 weeks) if the patient has not yet progressed.

The overall MMSE score has a possible range of 0 (worst) to 30 (best).

Security procedures.

On day 1 of cycle 1 and on all subsequent days of treatment, vital signs, including temperature, blood pressure, pulse, and resting respirations, along with weight, should be collected prior to the infusion and approximately 15 minutes after completion of the infusion. A complete physical examination and 12-lead ECG are performed at the beginning of each cycle.

Procedures for research biological tumor markers.

Biological markers of interest that are analyzed by immunohistochemistry (IHC) include, but are not limited to, EGFRvIII and biological markers of cell proliferation (eg, Ki67). Expression levels (mRNA and/or protein) of PD-L1, GITR and LAG-3, as well as tumor-infiltrating lymphocyte lineage markers (CD4, CD8, CD25, FoxP3) were analyzed in tumor biopsy samples to investigate the potential effect of REGN2810.

Tumor tissue samples can be used to extract tumor DNA and RNA and subsequently analyze putative genetic biological markers relevant to the study treatment and glioblastoma. A blood sample is collected for germline DNA isolation on day 1/baseline (pre-dose) or at any study visit if collection on day 1/baseline is possible. Tumor DNA analysis includes (but is not limited to) MGMT promoter methylation status, IDH1 mutational status, microsatellite instability (MSI), and tumor mutational burden (both may predict response to REGN2810 and other immunotherapies). Analyze genetic variants in tumor (somatic) and germline DNA that may influence disease progression, drug response, and possible toxicity. Germline DNA is also used for comparison with tumor DNA to investigate potential new genetic variants underlying malignancy processes.

Results.

REGN2810 in combination with hfRT is safe and well tolerated in patients with nGBM. Administration of REGN2810 in combination with hfRT inhibits tumor growth and/or promotes tumor regression in patients with nGBM compared to standard of care therapy. Patients with nGBM treated with REGN2810 and hfRT demonstrate longer OS compared with standard of care therapy.

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Example 11: Clinical Study of REGN2810 in Patients with Advanced Cutaneous Squamous Cell Carcinoma.

This example describes a phase 2 study that was conducted to confirm the positive results observed in patients with advanced CSCC in the phase 1 study (see Examples 7, 8 and 9 herein).

Objectives of the study.

The primary objective of this study is to evaluate the clinical benefit of REGN2810 monotherapy for patients with metastatic (nodal or distant) cutaneous squamous cell carcinoma (CSCC) (group 1) or unresectable locally advanced CSCC (group 2), as measured by total response rates (ORR).

The secondary objectives of the study are: (i) assessment of ORR; (ii) assessment of duration of response, progression-free survival (PFS) and overall survival (OS); (iii) assessment of complete response rate (CR); (iv) evaluation of the safety and tolerability of REGN2 810; (v) evaluation of the pharmacokinetics (PK) of REGN2810; (vi) assessing the immunogenicity of REGN2810; and (vii) assessing the impact of REGN2810 on quality of life using the EORTC QLQ-C30.

Research plan.

This represents a phase 2, non-randomized, multicenter, 2-arm study of REGN2810 at a dose of 3 mg/kg administered intravenously (IV) every 2 weeks in patients with advanced CSCC. The study has 2 groups. Group 1 for patients with metastatic CSCC. Group 2 for patients with unresectable locally advanced CSCC. All patients undergo screening procedures to determine eligibility within 28 days prior to initial administration of REGN2810.

After a screening period of up to 28 days, patients receive up to twelve 56-day (8-week) treatment cycles for up to 96 weeks of treatment. Each patient receives 3 mg/kg REGN2810 IV on days 1, 15±3, 29±3, and 43±3 during each treatment cycle. Tumor assessments are performed at the end of each treatment cycle. Extended safety assessments should be performed on Day 1 of each cycle, and standard safety assessments should be performed at each REGN2810 dosing visit.

The patient receives treatment until completion of the 96-week treatment period or until disease progression, unacceptable toxicity, withdrawal of consent, or confirmed CR. Patients with confirmed CR after a minimum of 48 weeks of treatment may elect to interrupt treatment and continue with all relevant study evaluations (eg, effect evaluations).

Duration of the study.

Screening (up to 4 weeks), up to 96 weeks of treatment and up to 6 months of follow-up.

Study population.

Patients with metastatic CSCC or unresectable locally advanced CSCC.

Study treatment.

REGN2810 3 mg/kg administered IV over 30 minutes every 14 days for 96 weeks.

Research Variables.

The primary efficacy measure for this study is ORR during 12 treatment cycles. Overall response rates are assessed separately for patients with metastatic CSCC or unresectable locally advanced CSCC: for patients in arm 1, Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 is used to determine ORR. For patients in group 2, composite response criteria are used to determine the ORR. In patients achieving CR, tumor biopsies are used in the final determination of complete versus partial response (PR).

Secondary outcome measures of effect are: duration of response; duration of disease control; PFS; OS; CR frequency; change in patient-reported outcome measures in EORTC QLQC30; adverse events (AE); serum REGN2810 concentrations; and antibodies against REGN2810.

Procedures and assessments.

Tumor imaging (computed tomography [CT] or magnetic resonance imaging [MRI]) and digital medical photography (for lesions that are externally visible) are performed to measure tumor burden and characterize the response profile of the study treatment using response criteria.

Physical examination, laboratory tests, vital signs, electrocardiogram (ECG), pregnancy test for women of childbearing potential, and recording of AEs and concomitant drug therapy are performed to ensure patient safety and to characterize the safety profiles for the study treatment.

Other assessments include: peripheral blood samples for RK; peripheral blood samples to evaluate antibodies against REGN2810; tumor biopsy; and quality of life assessments.

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Results.

Study enrollment has been completed and results to date are at the level of Phase I results (described herein in Examples 7, 8 and 9), where patients demonstrate inhibition of tumor growth when administered REGN2810. Patients with metastatic CSCC who have been treated with prior therapy and who have not undergone surgery demonstrate complete response, partial response, or stable disease when treated with the anti-PD-1 antibody REGN2810.

Example 12: Clinical Study of REGN2810 in Patients with Advanced Basal Cell Carcinoma.

A phase 2 study was conducted to confirm the positive results observed in patients with advanced SCD in the phase 1 study (see Examples 7 and 8 herein).

Objectives of the study.

The primary objective of the study is to evaluate the overall response rate (ORR) for metastatic basal cell carcinoma (SBC) (arm I) or unresectable locally advanced SCC (arm II) when treated with REGN2810 monotherapy in patients who have progressed on a pathway inhibitor Hedgehog (HHI) or intolerant to previous HHI therapy.

The secondary objectives for both Cohort I and Cohort II are: (i) assessment of ORR according to the research review; (ii) assessment of duration of response, progression-free survival (PFS) and overall survival (OS); (iii) assessment of complete response rate (CR); (iv) evaluation of the safety and tolerability of REGN2810; (v) evaluation of the pharmacokinetics (PK) of REGN2810; (vi) assessing the immunogenicity of REGN2810; and (vii) assessing the impact of REGN2810 on quality of life using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) and Skindex-16.

Research plan.

This represents a phase 2, non-randomized, multicenter, 2-arm trial for REGN2810 at a dose of 350 mg administered intravenously (IV) every 3 weeks (Q3W) in patients with advanced SCD who have experienced disease progression on HHI therapy or are intolerant to HHI therapy. previous HHI therapy. The study has 2 groups. Group 1 for patients with metastatic SCD. Group 2 for patients with unresectable locally advanced SCD. All patients undergo screening procedures to determine eligibility within 28 days prior to initial administration of REGN2810. There is no randomization or placebo control.

After a screening period of up to 28 days, patients receive up to 93 weeks of treatment. Each patient receives a dose of REGN2810 350 mg IV every 3 weeks. The infusion time for REGN2810 is approximately 30 minutes (±10 minutes). Tumor assessments are performed at the end of each treatment cycle, 5 treatment cycles at 9 weeks, followed by 4 treatment cycles at 12 weeks). Extended safety assessments are performed on Day 1 of each cycle, and standard safety assessments are performed at each REGN2810 dosing visit.

The patient receives treatment until completion of the 93-week treatment period or until disease progression (PD), unacceptable toxicity, withdrawal of consent, or confirmation of CR. Patients with confirmed CR after a minimum of 48 weeks of treatment may elect to interrupt treatment and continue with all relevant study evaluations (eg, effect evaluations). Patients who discontinue study treatment due to PD return to the clinic 30 days (range: 28 days to 42 days) after the last study treatment to complete end-of-study (EOS) assessments. After the EOS visit, patients are monitored for survival status until death, loss to follow-up, or study end.

Duration of the study.

After a screening period of up to 28 days, patients receive up to 93 weeks of treatment. After the visit at the end of the study, there is an observation period consisting of periods of 28 days. Patients are monitored for survival status until death, loss to follow-up, or study end.

Study population.

Patients with metastatic (group 1) or unresectable locally advanced (group 2) SCD who suffered disease progression on HHI therapy or were intolerant to prior HHI therapy.

Study treatment

The study treatment contained 350 mg of REGN2810 administered IV over 30 minutes (±10 minutes) every 3 weeks (Q3W) for up to 93 weeks.

Endpoints.

The primary performance measure for this study is ORR. ORR is estimated from

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Claims (25)

useful for patients with metastatic SCD (group 1) or unresectable locally advanced SCD (group 2). For patients in group 1 (metastatic SCD), Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 is used to determine the ORR. Clinical response criteria can be used for patients with externally visible target lesions if all metastatic lesions are not measurable by RECIST (as may be the case in patients with bone-only metastases). For patients in group 2 (unresectable locally advanced SCD), clinical criteria are used to determine the ORR. Composite response criteria are used for patients with injuries that are measurable by both the clinical response criteria and RECIST 1.1. Secondary endpoints are: (i) duration of response; (ii) PFS; (iii)OS; (iv) CR frequency; (v) change in patient-reported outcome scores in the EORTC QLQ-C30 and Skindex-16; (vi) adverse events (AE); (vii) serum REGN2810 concentrations; and (viii) anti-REGN2810 antibodies. Procedures and assessments. Tumor imaging (computed tomography [CT] or magnetic resonance imaging [MRI]) and digital medical photography (for externally visible lesions) are performed to measure tumor burden and characterize the response profile of the study treatment using response criteria. Physical examination, laboratory tests, vital signs, electrocardiogram (ECG), pregnancy test for women of childbearing potential, and recording of AEs and concomitant drug therapy are performed to ensure patient safety and to characterize the safety profiles for the study treatment. Other assessments include blood samples for PK, blood samples to evaluate antibodies against REGN2810, tumor biopsies, biological markers and quality of life assessments. Results. Based on the Phase 1 results (see Examples 7 and 8 herein), administration of REGN2810 is expected to result in tumor regression in patients with advanced basal cell carcinoma who have demonstrated disease progression on treatment with a Hedgehog pathway inhibitor (HHI) or are intolerant to previous HHI therapy. Patients demonstrate complete response, partial response or stable disease when treated with REGN2810. The scope of the present invention is not to be limited to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described in the present description will be apparent to those skilled in the art from the foregoing description and accompanying drawings. It is intended that such modifications fall within the scope of the appended claims. CLAIM 1. A method of treating or inhibiting the growth of a primary and secondary tumor in an individual, comprising: (a) selecting an individual with a cancer; and (b) administering to the individual combined fractionated radiation therapy with one or more doses and one or more doses of a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to programmed cell death protein 1 (PD-1) wherein the anti-PD-1 antibody or antigen binding fragment thereof comprises the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable region (HCVR) and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the light chain variable region ( LCVR), HCDR1 contains the amino acid sequence SEQ ID NO:3; HCDR2 contains the amino acid sequence SEQ ID NO:4; HCDR3 contains the amino acid sequence SEQ ID NO:5; LCDR1 contains the amino acid sequence SEQ ID NO:6; LCDR2 contains the amino acid sequence SEQ ID NO:7; LCDR3 contains the amino acid sequence of SEQ ID NO:8, and each dose of anti-PD-1 antibody or antigen binding fragment thereof contains an amount in the range of 5 to 20 mg/kg body weight of the individual and fractionated radiation therapy is administered to the primary tumor rather than the secondary tumors, and combined use results in tumor regression in both the primary tumor and the secondary tumor, the growth of each primary tumor and secondary tumor in the patient is inhibited at least 60% more than in the patient who received the antibody alone or who received radiation alone therapy. - 60 045900 2. The method according to claim 1, where each dose of an antibody against PD-1 or an antigen-binding fragment thereof contains 5 or 10 mg/kg of the individual's body weight. 3. The method according to claim 1, where each dose contains 20-600 mg of an antibody against PD-1 or an antigen-binding fragment thereof. 4. Method according to any one of claims 1-3, where each dose of radiation contains 2-80 Gray (Gy). 5. The method of claim 4, wherein each dose of anti-PD-1 antibody contains 10 mg/kg of the individual's body weight and each dose of radiation therapy contains 20-50 Gy. 6. The method according to claim 5, where fractionated radiation therapy contains 2-10 fractions. 7. The method according to claim 6, where the fractionated radiation therapy contains 30 Gy in 5 fractions. 8. The method according to claim 6, where the fractionated radiation therapy contains 27 Gy in 3 fractions. 9. The method according to any one of claims 1 to 8, wherein each dose of the anti-PD-1 antibody or antigen binding fragment thereof is administered 0.5 to 4 weeks after the immediately preceding dose. 10. The method of claim 9, wherein each dose of the anti-PD-1 antibody or antigen-binding fragment thereof is administered 2 weeks after the immediately preceding dose. 11. The method according to any one of claims 1 to 10, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered simultaneously with radiation therapy. 12. The method of claim 10, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered before or after radiation therapy. 13. The method according to any one of claims 1 to 12, where the administration of the combination leads to an enhanced therapeutic effect compared to the administration of only the antibody or antigen-binding fragment thereof or the administration of radiation therapy alone. 14. The method of claim 13, wherein the enhanced therapeutic effect includes an effect selected from the group consisting of tumor regression, abscopal effect, inhibition of tumor metastasis, reduction of metastatic damage over time, reduced use of chemotherapeutic or cytotoxic agents, reduced tumor burden, increased progression-free survival, improved overall survival, complete response, partial response, and stable disease. 15. The method of claim 14, wherein the growth of each primary tumor and secondary tumor is inhibited by at least 50% compared to the individual dosed prior to the anti-PD-1 antibody. 16. The method according to any one of claims 1 to 15, where the individual is resistant to previous therapy or insufficiently responds to it or has had a relapse after it. 17. The method according to any one of claims 1 to 16, wherein the malignant tumor is a recurrent or metastatic malignant tumor. 18. The method according to any one of claims 1 to 17, which further includes administering to the individual an additional therapeutic agent or therapy, wherein the additional therapeutic agent or therapy is selected from the group consisting of surgery, a chemotherapeutic agent, a cancer vaccine, a ligand inhibitor programmed death 1 (PD-L1), lymphocyte activation gene 3 (LAG3) inhibitor, cytotoxic T lymphocyte associated protein 4 (CTLA-4) inhibitor, glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody, T-containing inhibitor -cell immunoglobulin and mucin-3 (TIM3), B- and T-lymphocyte attenuator inhibitor (BTLA), T-cell immunoreceptor inhibitor with Ig and ITIM domains (TIGIT), CD47 inhibitor, indoleamine 2,3-dioxygenase inhibitor (IDO ), anti-CD3/anti-CD20 bispecific antibody, vascular endothelial growth factor (VEGF) antagonist, angiopoietin-2 (Ang2) inhibitor, transforming growth factor β (TGFe) inhibitor, CD38 inhibitor, epidermal growth factor receptor (EGFR) inhibitor, granulocyte- macrophage colony-stimulating factor (GM-CSF), cyclophosphamide, tumor-specific antigen antibody, bacillus Calmette-Guérin vaccine, cytotoxin, interleukin-6 receptor (IL-6R) inhibitor, interleukin-4 receptor (IL-4R) inhibitor, IL-6 inhibitor 10, IL-2, IL-7, IL-21, IL-15, antibody-drug conjugate, anti-inflammatory drug and dietary supplement. 19. The method of claim 18, wherein the additional therapeutic agent is an anti-GITR antibody. 20. The method of claim 18, wherein the additional therapeutic agent is cyclophosphamide. 21. The method of claim 18, wherein the additional therapeutic agent is GM-CSF. 22. The method of claim 18, wherein the additional therapeutic agent is selected from the group consisting of docetaxel, carboplatin, paclitaxel, cisplatin, gemcitabine and pemetrexed. 23. The method according to any one of claims 1 to 22, wherein the anti-PD-1 antibody is administered intravenously, subcutaneously or intraperitoneally. 24. The method according to any one of claims 1 to 23, wherein the malignant tumor includes a solid tumor. 25. The method of claim 24, wherein the solid tumor is selected from the group consisting of colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, cancer -