Il-12 for radiation protection and radiation-induced toxicity mitigation

Abstract

To provide therapeutic agents that can increase protection or mitigate of the effects of exposure to ionizing radiation useful for increasing the survival of normal cellular, tissue, organ and system and restoration of these functions after accidental exposure to ionizing radiation or in a radiation therapy setting.SOLUTION: Provided is a pharmaceutical composition for protecting a subject after exposure to radiation, the subject being undergoing radiation therapy for head and neck cancer, and the pharmaceutical composition comprising a therapeutically effective amount of substantially isolated rIL-12, thereby reducing radiation-induced damage to one or more of the subject's immune system, gastrointestinal system, and mucosal tissue.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This application is a continuation of U.S. Provisional Patent Application No. 61 / 588,098, filed January 18, 2012, and U.S. Provisional Patent Application No. 61 / 588,098, filed December 6, 2012. This application claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 734,364, filed on 2004 / 02 / 11.

[0002] The present disclosure relates generally to novel methods and compositions for radioprotection and / or radiation-induced toxicity mitigation. In particular, the present disclosure provides methods and compositions for radioprotection and / or radiation toxicity mitigation for the treatment of radiation-induced toxicity and acute radiation syndrome associated with the treatment of cutaneous T-cell lymphoma. [Background technology]

[0003] The following includes information that may be useful in understanding various aspects and embodiments of the present disclosure. No admission is made that any of the information set forth herein is prior art or relevant to any presently described or claimed invention, or that any publication or document specifically or implicitly cited by reference is prior art.

[0004] Humans and animals are highly susceptible to radiation-induced damage, resulting in cellular, tissue, organ, and whole-body damage. During accidental radiation exposure, such as in a nuclear explosion or disaster scenario, many victims suffer from varying degrees of acute radiation syndrome (ARS). The immediate goal in a radiation disaster is quite different from cancer radiotherapy. In such a disaster scenario, initial efforts require reaching as many victims as possible with potentially life-prolonging treatments so that victims can be successfully prioritized and subsequently receive intensive medical care determined by their individual condition and suffering. Another aspect of accidental or intentional radiation disasters is that life-saving medications or treatments must remain effective long after the radiation disaster. This requirement arises because it takes time to mobilize medical staff, medications / treatments, and equipment to the disaster site so that life-saving medications or treatments can be administered to victims in need.

[0005] Furthermore, radiation-induced damage to cells, tissues, organs, and systems can be the result of radiation exposure during the treatment of diseases such as cancer, or accidental radiation exposure due to disasters involving the release of radiation, such as nuclear explosions. More than 40% of cancer patients require radiation therapy in the management of their disease. Although radiation therapy improves survival for a significant number of cancer patients, both acute radiation toxicity (which occurs during or shortly after the course of clinical radiation therapy) and late toxicity (which occurs months to years after the completion of radiation therapy) jeopardize the overall outcome of successfully treated cancer patients.

[0006] For example, cutaneous T-cell lymphoma (CTCL) accounts for approximately 4% of all cases of non-Hodgkin's lymphoma and is generally characterized in part by the malignant proliferation of skin-homing T helper cells within the epidermis and outer dermis. The most common subgroup of CTCL is mycosis fungoides (MF). The exact etiology of CTCL is unknown, but genetic, infectious, and environmental factors have been suggested. The incidence of CTCL increases with age, with the average age of onset being between 50 and 60 years. CTCL affects twice as many men as women. The disease is less common in children, but can affect people of all ages. The early course of CTCL patients typically progresses from limited erythema to more generalized erythema, plaques, tumors, and eventually lymph node or visceral metastases. Patients with CTCL are classified according to a clinical staging system based on the extent of skin involvement (T stage) and the presence of lymph node and visceral metastases (TNM system). The two most common subtypes of CTCL are mycosis fungoides, which is often indolent (slow-growing) in the early stages, and a more aggressive form called "Sézary syndrome." Other less common CTCL subtypes include cutaneous CD30-expressing anaplastic large cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, CD8-expressing aggressive epidermotropic T-cell lymphoma, and gamma-delta T-cell lymphoma. Traditional therapy for patients with CTCL can include topical and systemic therapy. The most common therapies include psoralen-exposed ultraviolet A (PUVA), total skin electron beam therapy (TSEBT), and topical and systemic chemotherapy.

[0007] TSEBT has been used to treat CTCL since the 1950s. Total skin electron beam therapy (TSEBT) or partial skin electron beam therapy (PSEBT) is an effective treatment for cutaneous T-cell lymphoma (CTCL) and mycosis fungoides (MF). Conventional total skin electron irradiation (TSEI) for mycosis fungoides (MF) causes radiation toxicity, which leads to poor patient compliance, prolongs treatment duration, and requires treatment interruption. While extending the total treatment time may spare tumor cells and reduce the chance of cure, delivering the total radiation dose over a shorter period of time provides greater radiobiological benefit and results in more adequate tumor control. TSEI is typically administered daily (5 days a week), which inevitably results in severe radiation-related toxicity, requiring treatment interruption and an extension of the total treatment duration. This can reduce radiobiological efficacy and affect the final treatment outcome and disease-free status.

[0008] Currently, there are drugs that can protect cells and tissues from radiation therapy used in cancer, but none have proven very effective. With regard to accidental or intentional radiation exposure, to date there are no known drugs that can significantly extend survival when administered long after exposure. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is an unmet need for methods, agents, and / or compositions that can prevent or reduce radiation-induced damage to cells, tissues, organs, and systems, thereby increasing the likelihood of recovery of health after acute or chronic radiation exposure. The present disclosure provides methods and therapeutic agents that can enhance prevention or reduction of the effects of exposure to ionizing radiation, which are useful for increasing survival and recovery of function of normal cells, tissues, organs, and systems after accidental exposure to ionizing radiation or in the setting of radiation therapy. [Means for solving the problem]

[0010] In one aspect, a method is provided for protecting a subject from system, organ, tissue or cellular damage following exposure of the subject to ionizing radiation, comprising administering to the subject a dose of a therapeutically effective amount of a pharmaceutical composition comprising substantially isolated IL-12, thereby reducing the system, organ or tissue and / or cellular damage resulting from the radiation.

[0011] In one aspect, the radiation is administered as an acute lethal or near-lethal dose sufficient to produce the characteristics associated with acute radiation injury. In another aspect, the radiation injury in the subject is a chronic or systemic injury.

[0012] In one aspect, the radiation exposure results in total body irradiation, hi one embodiment, the radiation dose is from about 0.7 Gy to about 50 Gy, depending on the quality factor of the ionizing radiation source, as described herein.

[0013] In one embodiment, the system, organ or tissue to be protected is selected from the group consisting of bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lungs, kidneys, skin and brain.

[0014] In one embodiment, the effective amount of IL-12 is less than 300 ng / kg. In one embodiment, the effective amount of IL-12 is administered in two or more doses, each dose being less than 50 ng / kg. In one embodiment, one or more effective doses of IL-12 are less than 200 ng / kg. In one embodiment, one or more effective doses of IL-12 are less than 100 ng / kg. In one embodiment, the effective amount of IL-12 is administered in two or more doses, each dose being less than 30 ng / kg.

[0015] In one embodiment, one or more effective doses of IL-12 are administered before radiation exposure. In one embodiment, one or more effective doses of IL-12 are administered before and after radiation exposure. In one embodiment, one or more effective doses of IL-12 are administered after radiation exposure. In one embodiment, one or more effective doses of IL-12 are administered more than 24 hours after radiation exposure. In one embodiment, one or more effective doses of IL-12 are administered more than 48 hours after radiation exposure. In one embodiment, one or more effective doses of IL-12 are administered more than 72 hours after radiation exposure. In one embodiment, one or more effective doses of IL-12 are administered more than 96 hours after radiation exposure. In one embodiment, one or more effective doses of IL-12 are administered more than 120 hours after radiation exposure.

[0016] In one embodiment, administered IL-12 protects skin tissue from radiation damage. In one embodiment, administered IL-12 induces the production of erythropoietin. In one embodiment, the production of erythropoietin enhances the defense against system, organ, tissue, or cell damage.

[0017] In one embodiment, the systems, organs or tissues to be protected include the kidneys and lungs, hi one embodiment, the systems, organs or tissues to be protected include the brain and cardiovascular system.

[0018] In one aspect, an effective amount of IL-12 protects more than one system, organ, and / or tissue from radiation damage. In one embodiment, the system, organ, or tissue protected is selected from the group consisting of bone marrow, gastrointestinal system, lymphatic system, immune system and / or tissue, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lungs, kidneys, skin, nails, sweat glands, and brain.

[0019] In another aspect, the radiation is received during treatment for a disease and / or disorder associated with CTCL while the subject is undergoing radiation therapy. In one embodiment, the disease and / or disorder associated with CTCL is mycosis fungoides. In another embodiment, the disease and / or disorder associated with CTCL is Sézary syndrome. In one embodiment, the radiation exposure is associated with treatment of CTCL using electron beam therapy.

[0020] In one embodiment, one or more effective doses of IL-12 are administered subcutaneously. In one embodiment, one or more effective doses of IL-12 are administered intravenously. In one embodiment, one or more effective doses of IL-12 are administered topically. In one embodiment, IL-12 is administered near the site of injury to a sensitive organ. In one embodiment, the subject is undergoing radiation therapy for CTCL, and IL-12 is administered at or near the site of irradiation.

[0021] In one embodiment, the radiation is administered as a fractionated dose in two or more fractions. In another embodiment, the radiation is administered as a fractionated dose in a hyperfractionated regimen. In another embodiment, the radiation is administered as a fractionated dose in an accelerated fractionated regimen.

[0022] In one embodiment, an effective amount of IL-12 is administered in one or more doses, each dose being less than 30 ng / kg. In another embodiment, an effective amount of IL-12 is administered in one or more doses, each dose being less than 50 ng / kg. In another embodiment, one or more effective doses of IL-12 are less than 100 ng / kg. In another embodiment, one or more effective doses of IL-12 are less than 200 ng / kg. In one embodiment, the effective amount of IL-12 is less than 300 ng / kg.

[0023] In one embodiment, one or more effective doses of IL-12 are administered before radiation exposure. In another embodiment, one or more effective doses of IL-12 are administered before and after radiation exposure. In another embodiment, one or more effective doses of IL-12 are administered after radiation exposure.

[0024] In certain embodiments, the one or more effective doses of IL-12 are administered more than about 24 hours, about 48 hours, about 72 hours, about 96 hours, or about 120 hours after radiation exposure.

[0025] In one aspect, one or more effective doses of IL-12 are administered topically, subcutaneously, intradermally, intravenously, intraperitoneally, intramuscularly, epidurally, parenterally, intranasally, and / or intracranially. In one embodiment, IL-12 is administered intradermally. In another embodiment, IL-12 is administered intratumorally.

[0026] In one embodiment, IL-12 is administered near, adjacent to or at the site of injury to a susceptible organ.

[0027] In one embodiment, the subject is undergoing radiation therapy for head and neck cancer and IL-12 is administered at or near the radiation site.

[0028] In one embodiment, the administered IL-12 protects mucosal tissue from radiation damage.

[0029] In one aspect, the radiation damage is caused by a nuclear explosion, hi another embodiment, the radiation damage is caused by the emission of radiation from an ionizing radiation source.

[0030] In one aspect, the radiation damage is caused by a radiation therapy treatment modality. In another embodiment, the treatment modality comprises external beam radiation therapy. In one aspect, the external beam radiation therapy comprises three-dimensional conformal radiation therapy (3-D CRT). In another aspect, the external beam radiation therapy is selected from the group consisting of intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), tomotherapy, stereotactic body radiation therapy, stereotactic body radiation therapy, photon beam, electron beam, and proton beam therapy.

[0031] In other embodiments, the radiation therapy comprises internal beam radiation therapy or brachytherapy. In other embodiments, the radiation therapy comprises systemic radiation therapy. In other embodiments, the radiation therapy comprises radioimmunotherapy (RIT).

[0032] In one embodiment, a pharmaceutical composition is provided comprising IL-12 in a suitable formulation for delivery to a subject in need of prevention of radiation-induced damage.

[0033] In one aspect, the administered IL-12 induces production of at least one of erythropoietin, chemokines, cytokines, IFN-g, MCP-1, IL-15, IL-18, IP-10, MG, Mip1 beta or I-TAC, eotaxin, eotaxin-3, TARC, and IL-8. In some embodiments, production of erythropoietin enhances protection from system, organ, tissue, and / or cell damage.

[0034] In one embodiment, the systems, organs and / or tissues protected include the bone marrow and gastrointestinal system. In another embodiment, the systems, organs and / or tissues protected include the kidneys and lungs. In another embodiment, the systems, organs or tissues protected include the brain and cardiovascular system.

[0035] In one embodiment, a typical pharmaceutical composition can protect or prevent cells, tissues, and / or organs from damage following exposure to radiation. For example, in some embodiments, a typical pharmaceutical composition can protect or prevent damage in hematopoietic tissues, blood, lymph, bone marrow parenchymal cells, circulating myeloblasts, circulating small lymphocytes, platelets, white blood cells, red blood cells, skin and oral mucosa, basal layer of skin, basal cells, epidermis, stem cells, digestive organs and system, stomach, intestine, intestinal epithelium, colon, rectum, male and female reproductive systems, germ cells, testes, ovaries, oocytes, liver, thyroid, vascular endothelium, blood vessels, eyes, lens, cardiovascular system, endothelium, heart, lungs, bone and cartilage, connective tissue, liver, kidneys, CNS, sensory organs, glial cells, and adrenal medulla.

[0036] In one embodiment, the subject requires radiation therapy for the cancer. In another embodiment, the subject also requires chemotherapy.

[0037] In one embodiment, the cancer is a solid tumor. In another embodiment, the solid tumor comprises a sarcoma, carcinoma, or lymphoma. In another embodiment, the cancer is selected from the group consisting of lung, breast, prostate, pancreatic, ovarian, bladder, head and neck, thyroid, brain, liver, gallbladder, skin, colon, and kidney cancer. In one embodiment, the solid tumor is a poorly reoxygenating tumor.

[0038] In one embodiment, each dose of IL-12 is from about 1 ng / kg to less than about 2000 ng / kg, and said dose is administered by a delivery route selected from the group consisting of intradermal, intramuscular, intraperitoneal, intramuscular, intravenous, parenteral, intranasal, intracranial, topical, subcutaneous, and epidural routes.

[0039] The invention described and claimed herein has many features and embodiments, including but not limited to those shown or described or referenced in this Summary. It is not intended to be all-inclusive, and the invention described and claimed herein is not limited to or by the features and embodiments specified in this Summary. Further embodiments will be disclosed in the Detailed Description below. [Brief explanation of the drawings]

[0040] [Figure 1A] Figure 1A shows that exemplary recombinant murine IL-2 (e.g., m HemaMax) administered at least 24 hours after TBI extended survival time in irradiated mice. (a) Animals were administered vehicle or recombinant murine IL-12 at a nominal dose of 100 ng / mouse at 24 and 72 hours after 8 Gy (LD86 / 30) TBI. Vehicle and recombinant murine IL-12 were injected subcutaneously. The vehicle was PBS. The delivered dose of recombinant murine IL-12 was estimated to be 10 ng / mouse because subsequent testing showed that the actual delivered dose was approximately 10% of the intended dose, likely due to adhesion of recombinant murine IL-12 to vial and syringe surfaces. [Figure 1B] Figure 1B shows that exemplary recombinant murine IL-12 (e.g., m HemaMax) administered at least 24 hours after TBI extended survival time in irradiated mice. (b) Animals were administered vehicle or a single nominal dose of 300 ng / mouse recombinant murine IL-12 at 24, 48, or 72 hours after 9 Gy (LD100 / 30) TBI. Vehicle and recombinant murine IL-12 were injected subcutaneously. The vehicle was PBS. The delivered recombinant murine IL-12 dose was estimated to be 30 ng / mouse because subsequent testing showed that the actual recombinant murine IL-12 dose delivered was approximately 10% of the intended dose, likely due to adhesion of the recombinant murine IL-12 to vial and syringe surfaces. [Figure 1C] Figure 1C shows that exemplary recombinant murine IL-12 (e.g., m HemaMax) administered at least 24 hours after TBI extended survival time in irradiated mice. (c) Animals were administered vehicle or a single low dose of recombinant murine IL-12 (2 ng / mouse or 18 ng / mouse) 24 hours after 7.9 Gy (LD85 / 30) TBI. Vehicle and recombinant murine IL-12 were injected subcutaneously. The vehicle was P5.6TT. [Figure 2]

[0023] Figure 1 shows that the efficacy of recombinant murine IL-12 in extending survival time is independent of radiation dose in mice. Animals were subjected to total body irradiation (TBI) with increasing radiation doses of 8.6 Gy (LD70 / 30), 8.8 Gy (LD90 / 30), and 9.0 Gy (LD100 / 30), followed by administration of recombinant murine IL-12 at a dose of 20 ng / mouse 24 hours post-irradiation. Mice were monitored for survival until day 30. Vehicle was P5.6TT. [Figure 3A]Figure 3A shows that administration of recombinant murine IL-12 increased plasma recombinant murine IL-12 and IFN-γ levels in irradiated and non-irradiated mice. Animals were administered recombinant murine IL-12 subcutaneously at a dose of 10 ng / mouse in the absence of irradiation or 24 hours after LD90 / 30 TBI. Plasma concentrations of recombinant murine IL-12 and IFN-γ were measured by ELISA in blood samples collected at the indicated time points. n=3 for each time point. [Figure 3B] Figure 3B shows that administration of recombinant murine IL-12 increased plasma recombinant murine IL-12 and IFN-γ levels in irradiated and non-irradiated mice. Animals were administered recombinant murine IL-12 subcutaneously at a dose of 20 ng / mouse in the absence of irradiation or 24 hours after LD90 / 30 TBI. Plasma concentrations of recombinant murine IL-12 and IFN-γ were measured by ELISA in blood samples collected at the indicated time points. n=3 for each time point. [Figure 3C] Figure 3C shows that administration of recombinant murine IL-12 increased plasma recombinant murine IL-12 and IFN-γ levels in irradiated and non-irradiated mice. Animals were administered recombinant murine IL-12 subcutaneously at a dose of 40 ng / mouse in the absence of irradiation or 24 hours after LD90 / 30 TBI. Plasma concentrations of recombinant murine IL-12 and IFN-γ were measured by ELISA in blood samples collected at the indicated time points. n=3 for each time point. [Figure 3D] Figure 3D shows that administration of recombinant murine IL-12 increased plasma recombinant murine IL-12 and IFN-γ levels in irradiated and non-irradiated mice. Animals were administered recombinant murine IL-12 subcutaneously at a dose of 200 ng / mouse in the absence of irradiation or 24 hours after LD90 / 30 TBI. Plasma concentrations of recombinant murine IL-12 and IFN-γ were measured by ELISA in blood samples collected at the indicated time points. The y-axis scale in (d) is 8-fold greater than that in (a) and (b) and 5-fold greater than that in (c). n=3 for each time point. [Figure 4]Figure 1 shows that an optimal recombinant murine IL-12 dose of 20 ng / mouse increased plasma EPO concentrations in irradiated mice. Animals were administered recombinant murine IL-12 subcutaneously at doses of (a) 10 ng / mouse, (b) 20 ng / mouse, (c) 40 ng / mouse, or (d) 200 ng / mouse in the absence of irradiation or 24 hours after an LD90 / 30 TBI. Plasma concentrations of EPO were measured by ELISA in blood samples taken 12 hours after administration of recombinant murine IL-12. [Figure 5-1] Photographs showing that recombinant murine IL-12 promotes hematopoietic recovery in irradiated mice. Representative sections of femoral bone marrow from unirradiated, untreated mice stained for IL-12Rβ2 (orange) are shown in (a). Animals were subjected to TBI (8.0 Gy) and then subcutaneously administered vehicle (P5.6TT) or recombinant murine IL-12 (20 ng / mouse) at the indicated time points after irradiation (b–f). An additional group of mice received recombinant human IL-12 24 hours after TBI (g). Femoral bone marrow was immunohistochemically stained for IL-12Rβ2 (orange) 12 days after irradiation. The bone marrow of vehicle-treated mice lacked IL-12Rβ2-expressing cells and showed no signs of hematopoietic regeneration (b), whereas mice treated with recombinant murine IL-12 showed hematopoietic reconstitution and the presence of IL-12Rβ2-expressing megakaryocytes, myeloid progenitor cells, and osteoblasts (c-f). Mice administered recombinant human IL-12 showed IL-12Rβ2-expressing osteoblasts but lacked megakaryocytes (g). Magnification = 100x. [Figure 5-2] Continued from Figure 5-1. [Figure 6]Mouse bone marrow hematopoietic stem cells, osteoblasts, and megakaryocytes express IL-12Rβ2. Tissue sections obtained 30 days (a and c) and 12 days (b) after TBI (according to the protocol described in Figure 5) were immunohistochemically stained for IL-12Rβ2 (a and b, upper panels), the hematopoietic stem cell marker Sca-1 (a, lower panel), and the osteoblast marker osteocalcin (b, lower panel), or both IL-12Rβ2 and Sca-1 (c). Both immature and mature megakaryocytes also showed strong immunohistochemical staining, indicating the presence of IL-12Rβ2 (c). The red arrow in (a) indicates hematopoietic stem cells expressing IL-12Rβ2, while the black arrow indicates those that do not express IL-12Rβ2. In IL-12Rβ2 and Sca-1 double staining (c), IL-12Rβ2 stains pink, while Sca-1 stains brown. Subpopulations of stem cells co-expressing IL-12Rβ2 and Sca-1, as well as subpopulations expressing only IL-12Rβ2 or Sca-1, are shown (c). Magnification = 100x. [Figure 7] Figure 1 shows that low doses of recombinant murine IL-12 suppress radiation-induced intestinal injury in mice. (a) IL-12Rβ2 expression in jejunal crypts and (b) suppression of jejunal expression of LGR5, a marker of GI stem cell injury. Mice were subcutaneously administered vehicle (P5.6TT) or the indicated doses of recombinant murine IL-12 in the absence of irradiation or 24 hours after TBI (8.6 Gy). Three days after irradiation, jejunal tissues were removed and immunohistochemically stained for IL-12Rβ2 (a) or LGR5 (b). Representative images show brown LGR5 as indicated by the arrow. Magnification = 400. [Figure 8] Figure 1 shows similar exposure to species-specific equivalent doses of recombinant murine IL-12 and recombinant human IL-12 in mice and rhesus monkeys. A plot of plasma AUClast of recombinant murine IL-12 versus mouse dose in the absence of irradiation was linear at doses from 10 ng / mouse to 40 ng / mouse. Plasma AUClast of recombinant human IL-12 at monkey-equivalent doses of 20 ng / kg and 80 ng / kg was consistent with the prolonged dose-dependent increase in recombinant murine IL-12 exposure in mice. [Figure 9A] Figure 9A shows that administration of an exemplary recombinant human IL-12 (e.g., HemaMax) increased plasma IFN-γ concentrations in non-irradiated rhesus monkeys. (a) Temporal kinetics of IFN-γ compared to recombinant human IL-12. Animals were administered recombinant human IL-12 subcutaneously at doses of 250 ng / Kg or 1000 ng / Kg in the absence of irradiation. Plasma concentrations of recombinant human IL-12 and IFN-γ were measured by ELISA in blood samples collected at the indicated time points. n=3 per group for each time point. [Figure 9B] Figure 9B shows that administration of an exemplary recombinant human IL-12 (e.g., HemaMax) increased plasma IL-18 and EPO concentrations in non-irradiated rhesus monkeys. (b) Temporal kinetics of IL-18 and EPO. Animals were subcutaneously administered recombinant human IL-12 at doses of 250 ng / Kg or 1000 ng / Kg in the absence of irradiation. Plasma concentrations of IL-18 and EPO were measured by ELISA in blood samples collected at the indicated time points. n=3 per group for each time point. [Figure 9C] Figure 9C shows that administration of an exemplary recombinant human IL-12 (e.g., HemaMax) increased plasma IL-15 and neopterin concentrations in non-irradiated rhesus monkeys. (c) Temporal kinetics of IL-15 and neopterin. Animals were subcutaneously administered recombinant human IL-12 at doses of 250 ng / Kg or 1000 ng / Kg in the absence of irradiation. Plasma concentrations of IL-15 and neopterin were measured by ELISA in blood samples collected at the indicated time points. n = 3 per group per time point. n = 1 for neopterin. [Figure 10]Photographs showing that NHP and human bone marrow and small intestine express IL-12Rβ2. NHP and human femoral bone marrow (a) and jejunum / ileum (b) tissues were immunohistochemically stained for IL-12Rβ2. (a) Progenitor cells and megakaryocytes expressing IL-12Rβ2 are shown. Adipocytes did not express IL-12Rβ2. (b) Intestinal crypts expressing IL-12Rβ2 are shown. Lymphoid cells in the lamina propria and submucosal regions also expressed IL-12Rβ2. C = crypt; LP = lamina propria. Magnification was 40x for (a) and 100x for (b). [Figure 11] Figure 1 shows that recombinant human IL-12, initiated at least 24 hours after irradiation, increased survival in unsupported monkeys. Individual treatment groups (a) and pooled recombinant human IL-12 treatment groups (b) are shown. Animals were subjected to an LD50 / 30 TBI on day 0 and then administered vehicle (P5.6TT) or recombinant human IL-12 subcutaneously at the indicated dosing schedule. Supportive care was prohibited throughout the study. Animals were monitored for survival up to 30 days. a One animal was removed from the study due to a broken tooth. [Figure 12] Figure 1 shows that administration of recombinant human IL-12 attenuated nadir leukopenia (A) and thrombocytopenia (B) in irradiated non-supportive rhesus monkeys. Animals were subjected to an LD50 / 30 TBI on day 0. Animals were administered subcutaneously with vehicle (P5.6TT) or recombinant human IL-12 at doses of 100 ng / Kg or 250 ng / Kg 24 hours after TBI. Blood samples were collected at the indicated times, and leukocytes and platelets were counted using an automated hematology analyzer. [Figure 13A] Figure 13A shows that irradiated rhesus monkeys receiving recombinant human IL-12 lost less weight than animals receiving vehicle. Weights in kg are shown for the 100 ng / kg dose group. Monkeys were subjected to an LD50 / 30 TBI on day 0 and then administered vehicle (P56TT) or recombinant human IL-12 subcutaneously at the indicated dosing schedule. Supportive care was prohibited throughout the study. Body weights were recorded every other day until day 30. [Figure 13B]Figure 13B shows that irradiated rhesus monkeys receiving recombinant human IL-12 lost less weight than animals receiving vehicle. Weights in kg for the 250 ng / kg dose group are shown. Monkeys were subjected to an LD50 / 30 TBI on day 0 and then administered vehicle (P5.6TT) or recombinant human IL-12 subcutaneously at the indicated dosing schedule. Supportive care was prohibited throughout the study. Body weights were recorded every other day until day 30. [Figure 13C] Figure 13C shows that irradiated rhesus monkeys receiving recombinant human IL-12 lost less weight than animals receiving vehicle. Body weights are shown as a percentage of the 100 ng / Kg dose group. Monkeys were subjected to an LD50 / 30 TBI on day 0 and then administered vehicle (P5.6TT) or recombinant human IL-12 subcutaneously at the indicated dosing schedule. Supportive care was prohibited throughout the study. Body weights were recorded every other day until day 30. [Figure 13D] Figure 13D shows that irradiated rhesus monkeys receiving recombinant human IL-12 lost less weight than animals receiving vehicle. Body weights are shown as a percentage of the 250 ng / Kg dose group. Monkeys were subjected to an LD50 / 30 TBI on day 0 and then administered vehicle (P5.6TT) or recombinant human IL-12 subcutaneously at the indicated dosing schedule. Supportive care was prohibited throughout the study. Body weights were recorded every other day until day 30. [Figure 14]This figure shows a multilevel model of the mechanism of action of recombinant human IL-12 in extending survival after radiation exposure. Current evidence suggests that recombinant human IL-12 induces responses at at least four levels in the body. In the Level 1 response, recombinant human IL-12 promotes the proliferation and activation of existing radiation-sensitive immune cells, namely, NK cells, macrophages, and dendritic cells. Recombinant human IL-12-induced increases in plasma IL-15 and IL-18 also promote NK cell maturation, leading to the release of IFN-γ, which in turn positively influences the production of endogenous IL-12 from macrophages and dendritic cells, and possibly NK cells. These events enhance innate immune capabilities early after administration of recombinant human IL-12. In the Level 2 response, recombinant human IL-12 promotes the proliferation and differentiation of viable hematopoietic stem cells, osteoblasts, and megakaryocytes, into specific cellular components that ensure optimal hematopoiesis. Recombinant human IL-12-induced secretion of EPO from CD34+, IL-12Rβ2-positive bone marrow cells may also suppress local overproduction of IFN-γ in the bone marrow, thereby creating an environment that promotes hematopoietic cell proliferation. Hematopoietic regeneration in the bone marrow enhances innate and adaptive immune capabilities. In a level 3 response, recombinant human IL-12 maintains GI stem cells, leading to reduced pathogen leakage, increased food consumption, and decreased diarrhea. In a level 4 response, recombinant human IL-12 may directly increase the renal release of EPO, a cytoprotective factor that promotes cell survival in a diverse set of organs / tissues. The sustained production of endogenous IL-12, primarily derived from dendritic cells activated by pathogens and / or EPO, serves as a positive feedback loop and likely plays an important role in sustaining the initial response to exogenous recombinant human IL-12 for several weeks after radiation exposure. ↑ = increase; ↓ = decrease; HSC = hematopoietic stem cell; NK cells = natural killer cells. [Figure 15]

[0023] Figure 1 shows an exemplary demonstration of IL-12 efficacy in achieving a 3.5-fold increase in survivors after exposure to radiation (LD90). Results from a dose-finding study demonstrated a survival benefit at LD90 in rhesus monkeys in the absence of supportive care. All protocols were performed in accordance with GLP and data were generated based on a blinded study design. [Figure 16] FIG. 1 shows a demonstration that exemplary IL-12 (HemaMax) treatment is associated with reduced bleeding scores in irradiated NHPs (LD90 / 60). [Figure 17] FIG. 1 shows the efficacy of exemplary IL-12 (HemaMax) in stimulating BM regeneration after lethal radiation exposure. [Figure 18] An example of a pocket of regeneration is shown in NHP bone marrow 12 days after lethal irradiation (700 cGy). The pocket is defined by the presence of H&E staining. H&E stained areas are fewer and smaller in vehicle-treated animals (A-C). An increased frequency of stained areas and a larger stained area are seen in HemaMax-treated NHPs (D-G). Magnification 4X. [Figure 19] FIG. 10 shows another example of the effectiveness of rIL-12 HemaMax in stimulating BM regeneration after lethal radiation exposure. [Figure 20] FIG. 1 shows a demonstration of the efficacy of rIL-12 HemaMax treatment with reduced incidence of sepsis in irradiated NHPs. [Figure 21A] Figure 21A shows the demonstration of efficacy of rIL-12 based on secondary endpoints: Hematology: Lymphocytes. [Figure 21B] Figure 21B shows demonstration of efficacy of rIL-12 based on secondary endpoints: Hematology: Neutrophils. [Figure 21C] Figure 21C shows demonstration of efficacy of rIL-12 based on secondary endpoints: Hematology: Platelets. [Figure 22] Demonstration of the effectiveness of HemaMax against radiation complex injury (RCI) is presented. [Figure 23]FIG. 1 shows a demonstration of the efficacy of rMuIL-12 in accelerating wound closure (reduction in wound size) and attenuating combined injury in irradiated mice (2-4 hours post-exposure). [Figure 24] Photographs demonstrating the efficacy of rMuIL-12 in accelerating wound closure (reduction in wound size) and attenuating combined injury in irradiated mice (2-4 hours post-exposure). [Figure 25] Photographs demonstrating the efficacy of rMuIL-12 in accelerating wound closure (reduction in wound size) and attenuating combined injury in irradiated mice (2-4 hours post-exposure). [Figure 26] FIG. 1 shows a demonstration of the efficacy of rMuIL-12 in accelerating wound closure and reducing combined injury in irradiated mice (24 hours post-exposure). [Figure 27] Photographs showing a demonstration of the efficacy of rMuIL-12 in accelerating wound closure and reducing combined injury in irradiated mice (24 hours post-exposure). [Figure 28] FIG. 1 shows the plasma concentration-time profile of HemaMax after SC administration in non-irradiated and irradiated monkeys. [Figure 29] FIG. 1 shows plasma concentration-time profiles (log scale) of HemaMax after IV administration (250 ng / kg) in non-irradiated and irradiated monkeys. [Figure 30] FIG. 1 shows plasma concentration-time profiles (linear scale) of HemaMax after IV administration (250 ng / kg) in non-irradiated and irradiated monkeys. [Figure 31] Figure 1 shows the pharmacodynamics of IFN-γ. IFN-γ response after administration of HemaMax. [Figure 32] FIG. 1 shows the pharmacodynamics of IFN-γ following IV administration of HemaMax in non-irradiated and irradiated monkeys. [Figure 33] FIG. 1 shows EPO-pharmacodynamics following SC administration of HemaMax in non-irradiated and irradiated monkeys. [Figure 34] FIG. 1 shows the pharmacodynamics of EPO- following IV administration of HemaMax in non-irradiated and irradiated monkeys. [Figure 35] FIG. 1 shows the pharmacodynamics of IL-18 after SC administration of HemaMax in non-irradiated and irradiated monkeys. [Figure 36] FIG. 1 shows the pharmacodynamics of IL-18 after IV administration of HemaMax in non-irradiated and irradiated monkeys. [Figure 37] FIG. 1 shows the pharmacodynamics of IL-15 after SC administration of HemaMax in non-irradiated and irradiated monkeys. [Figure 38] FIG. 1 shows the pharmacodynamics of IL-15 after IV administration of HemaMax in non-irradiated and irradiated monkeys. DETAILED DESCRIPTION OF THE INVENTION

[0041] Accordingly, the present disclosure relates generally to novel methods and compositions for radioprotection and / or mitigation of radiation-induced toxicity associated with accidental radiation exposure (such as in a nuclear detonation or disaster scenario) and / or radiation therapy, such as the treatment of diseases and / or disorders associated with cutaneous T-cell lymphoma using electron beam therapy.

[0042] For example, the use of ionizing radiation or nuclear bombs as weapons of terrorism is now recognized as a serious public health threat. Nuclear detonations in populated areas, terrorist radioactive (e.g., "dirty") bombs, or attacks on nuclear power plants would result in mass casualties requiring immediate medical treatment. Exposures of approximately 4 Gy result in 50% of deaths within 60 days without medical intervention. The majority of deaths resulting from exposures of at least 2-10 Gy or more are attributable to the combined effects of immune, hematopoietic, and gastrointestinal (GI) failure, as these are the most radiosensitive tissues. No FDA-approved therapeutic agents exist that can increase survival by simultaneously promoting or accelerating recovery of the immune, hematopoietic, and GI compartments after radiation injury.

[0043] In the case of a radiological disaster or terrorist act affecting a large number of civilians, the goal would be to provide robust frontline medical care that would increase the chances of survival for exposed or potentially exposed individuals. One of the challenges in such cases is that medical care and treatment may not be available immediately after radiation exposure. It may take more than 24 hours to mobilize medical teams and the necessary life-saving medications and equipment to the scene of a radiological disaster.

[0044] Because medical care is not immediately available, medical interventions that can increase the chances of survival as first-line treatments must be effective when administered long after radiation exposure. This presents a practical challenge in that total body irradiation (TBI) causes significant apoptosis of rapidly dividing cells in radiation-sensitive organs, such as peripheral blood, bone marrow, and the GI tract, beginning immediately after radiation exposure. Furthermore, the likelihood of successfully administering life-saving treatment to an exposed individual decreases exponentially after radiation injury. Thus, the effectiveness of administering countermeasures that can mitigate radiation-induced damage rapidly decreases over time.

[0045] Accordingly, certain aspects of the present disclosure generally relate to novel methods and compositions for radioprotection and / or mitigation of radiation-induced toxicity resulting from acute radiation exposure.

[0046] In other aspects, the present disclosure also provides methods and compositions for radioprotection or radiation toxicity mitigation for the treatment of diseases and / or disorders associated with cutaneous T-cell lymphoma using electron beam therapy.

[0047] Aspects and embodiments of the present disclosure address the unmet need for drugs that can spare cancerous tissue from the killing effects of radiation while simultaneously protecting and / or regenerating normal tissue. To date, there are no approved drugs with these properties. Amifostine, a chemo- and radioprotectant, is the only approved radiomitigating drug.

[0048] Amifostine has been used therapeutically to (1) reduce the incidence of neutropenia-associated fever and infections caused by DNA-binding chemotherapy agents, including alkylating agents (e.g., cyclophosphamide) and platinum-containing drugs (e.g., cisplatin); (2) reduce the cumulative nephrotoxicity associated with platinum-containing drugs; and (3) reduce the incidence of xerostomia in patients undergoing radiation therapy for head and neck cancer. However, amifostine has the ability to promote tumor cell proliferation and protect normal tissues. Therefore, this drug should be used with caution in cancer patients. Serious side effects of amifostine include hypotension (seen in 62% of patients), erythema multiforme, Stevens-Johnson syndrome and toxic epidermal nephrolysis, immune hypersensitivity syndrome, erythroderma, anaphylaxis, and unconsciousness (rare).

[0049] Small molecule kinase inhibitors are being developed as early chemoprotective agents, but it is unclear whether these drugs also protect cancer cells. In particular, there are no known radiomitigation drugs that simultaneously have antitumor activity. Recombinant human and / or murine IL-12 is the only radiomitigation drug in development that has been shown to have dual effects in animal models.

[0050] Recombinant human and / or murine IL-12 is non-cancerous after radiation exposure, but can protect and regenerate damaged tissue. Concomitant with its protective and regenerative properties after radiation, recombinant human and / or murine IL-12 can suppress the growth of cancer cells. No other known drug has these dual effects.

[0051] CTCL As used herein, cutaneous T-cell lymphoma (CTCL) represents a group of lymphoid malignancies affecting the skin. Primary cutaneous T-cell lymphoma (CTCL) accounts for approximately 60% to 70% of cutaneous lymphomas. Among the variants of CTCL, mycosis fungoides (MF) is the most common. Staging is based on the tumor, node, metastasis (TNM) system. Multiple treatment options exist for skin-confined MF, including phototherapy (psoralen-long-wave ultraviolet A therapy - PUVA), topical nitrogen mustard, carmustine BCNU, radiation therapy such as total skin electron beam therapy (TSEBT), topical steroids, interferon alpha, retinoids such as bexarotene, receptor-targeted cytotoxic fusion proteins (e.g., denileukin diftitox), and extracorporeal photophoresis. Because MF is indolent but recurrent, patients with MF often require multiple therapies.

[0052] Cutaneous T-cell lymphoma is a group of lymphoproliferative disorders characterized by the localization of neoplastic T-lymphocytes to the skin. Cutaneous T-cell lymphoma (CTCL) is a class of non-Hodgkin's lymphoma, a type of cancer of the immune system. Unlike most non-Hodgkin's lymphomas (which generally involve B cells), CTCL is caused by mutations in T cells. Malignant T cells in the body first migrate to the skin, where various lesions appear. These lesions change shape as the disease progresses, typically beginning as what appears to be a very itchy skin rash and eventually forming plaques and tumors before metastasizing to other parts of the body.

[0053] CTCL is a malignant proliferation derived from a clone of skin-infiltrating CD41 T lymphocytes. Clinical presentation of CTCL can include a wide range of findings, ranging from limited skin erythema and plaques without evident peripheral blood or lymph node involvement to widespread skin metastases of tumors or erythroderma with blood, lymph node, or visceral disease.

[0054] As used herein, cutaneous T-cell lymphoma includes, but is not limited to, the following types or categories: mycosis fungoides, Pagetoid reticulosis, Sézary syndrome, granulomatous lax cutis, lymphomatoid papulosis, pityriasis lichenoides chronica, pityriasis lichenoides acute, CD30-positive cutaneous T-cell lymphoma, secondary cutaneous CD30-positive large cell lymphoma, non-mycosis fungoides CD30-negative cutaneous large cell lymphoma, pleomorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma, adult T-cell lymphoma / leukemia (human T-cell lymphotropic virus [HTLV] positive), extranodal natural killer (NK) / T-cell lymphoma, nasal type, primary cutaneous peripheral T-cell lymphoma, unspecified (PTCL-U).

[0055] As used herein, a person affected by CTCL can include the following clinical and / or subclinical manifestations characteristic of a CTCL-associated condition: WHO-EORTC classification Insidious clinical behavior mycosis fungoides Mycosis fungoides Variants and Subtypes Folliculotropic mycosis fungoides Pagetoid reticulosis Granulomatous loose skin Primary CD30-positive lymphoproliferative disorders Primary cutaneous anaplastic large cell lymphoma Lymphomatoid papulosis Subcutaneous panniculitis-like T-cell lymphoma (provisional) Primary cutaneous CD4-positive small to medium pleomorphic T-cell lymphoma (tentative) Aggressive clinical behavior Sézary syndrome Adult T-cell leukemia / lymphoma.

[0056] Additionally, CTCL may also include or be characterized by the following features and / or classifications:

[0057] Primary cutaneous CD30-positive lymphoproliferative disorders The term CD30-positive lymphoproliferative disorders includes disease entities such as anaplastic large cell lymphoma (primary cutaneous and systemic types) and lymphomatoid papulosis. Although sometimes pathologically equivocal, these disease entities are clinically distinct. Therefore, clinicopathologic correlation in the management of these diseases is desirable.

[0058] Anaplastic large cell lymphoma (ALCL) is a primary cutaneous form that manifests as a solitary nodule or ulcerative tumor (>2 cm) in patients without concurrent mycosis fungoides or lymphomatoid papulosis or a history of these conditions and without evidence of extradermal disease. Extradermal spread, primarily to regional lymph nodes, occurs 10% of the time. The disease is multifocal in the skin approximately 30% of the time. CD30-positive (>75%) membrane staining is observed in large lymphocytes or large clusters of CD30-positive atypical lymphocytes with pleomorphic or multinuclei and nucleoli. Numerous mitotic figures can be observed. Unlike systemic anaplastic large cell lymphoma, anaplastic lymphoma kinase (ALK) staining is usually negative. A useful tool for distinguishing cutaneous anaplastic large cell lymphoma from systemic anaplastic large cell lymphoma is testing for the presence of the t(2;5) translocation. This translocation is often, but not always, present in systemic anaplastic large cell lymphoma, but is not usually present in primary cutaneous cases. Distinction from lymphomatoid papulosis is not always possible based on histological criteria. Immunologically, atypical lymphocytes are CD4 positive with variable loss of CD2, CD3, or CD5. Staging is required, as with other non-Hodgkin's lymphomas (e.g., using computed tomography [CT] scans, bone marrow examination, and blood tests). Patients may experience spontaneous remission followed by relapse. If spontaneous remission does not occur, radiation therapy, surgical resection, or both are preferred. Chemotherapy is reserved for patients with systemic disease.

[0059] Lymphomatoid papulosis manifests as recurrent crops of self-healing, reddish-brown, centrally hemorrhagic or necrotic papules and nodules on the trunk or extremities. These may progress to papulovesicular or pustular lesions. These lesions are much smaller (<2 cm) than anaplastic large cell lymphoma. Lesions spontaneously resolve over 4 to 6 weeks, leaving behind hyperpigmented or atrophic scars. Outbreaks of variable frequency and / or intensity may occur in different patients. Lymphomatoid papulosis is clinically benign, although clonal T-cell gene rearrangements may be present in 60 to 70% of cases. Hodgkin's disease, mycosis fungoides, or cutaneous anaplastic large cell lymphoma are present in 20% of cases.

[0060] Subcutaneous panniculitis-like T-cell lymphoma In subcutaneous panniculitis-like T-cell lymphoma, erythematous subcutaneous nodules appearing as crops are localized to the extremities or trunk. These lesions may be confused with benign panniculitis and are often accompanied by fever, chills, weight loss, and fatigue. They may also be associated with hemophagocytic syndrome, which may have a rapidly progressive downward course. Spread to extradermal sites is rare. Histologically, early lesions may show focal atypical lobular lymphocytic infiltration of the subcutaneous fat, which may be confused with benign subcutaneous panniculitis. Later, infiltration of the fat by pleomorphic lymphoid cells, with rimming of individual adipocytes by tumor cells, is accompanied by frequent mitoses, karyorrhexis, and fat necrosis. Histiocytic panniculitis (histiocytophagous red and white blood cells) may also complicate the histological picture. Immunologically, atypical lymphocytes stained positive for CD3 and CD8, demonstrating clonal rearrangement of T-cell receptor genes. At least two groups of subcutaneous panniculitis-like T-cell lymphoma can be distinguished, with distinct histologies, phenotypes, and prognoses. Cases with an alpha / beta-positive T-cell phenotype are usually CD8+, characterized by recurrent disease limited to the subcutaneous tissue (without dermal or epidermal involvement), and tend to have an indolent clinical course. The WHO-EORTC term subcutaneous panniculitis-like T-cell lymphoma refers exclusively to the alpha / beta phenotype. Affected patients have previously been treated with chemotherapy or radiation therapy, but patients receiving systemic steroid therapy appear to be clinically well controlled. Similar-appearing lymphomas with a gamma / delta phenotype are CD8- and CD56+. Histologically, infiltration may not be limited to the subcutaneous tissue, and the course is aggressive. In the WHO-EORTC classification, this lymphoma is considered a distinct disease entity and is included in the group of cutaneous gamma / delta-positive lymphomas in the provisional category. Clinically, this lymphoma is more aggressive, with spread to mucosal and other extranodal sites.

[0061] Primary cutaneous CD4-positive small to medium pleomorphic T-cell lymphoma This condition presents as solitary or localized plaques or tumors on the face, neck, and / or upper trunk. The disease generally has an indolent course, and solitary lesions can be treated by surgical excision or radiation therapy. Histologically, there is infiltration of the dermis and subcutaneous tissue by CD3+, CD4+ malignant cells, and localized epidermotropism may be present.

[0062] Provisional categories such as primary aggressive epidermotropic CD8-positive cytotoxic T-cell lymphoma and primary cutaneous CD4-positive small-medium pleomorphic T-cell lymphoma are also included. Cutaneous gamma / delta-positive T-cell lymphoma also belongs to this category. Sézary syndrome is included, as is mycosis fungoides.

[0063] Adult T-cell lymphoma / leukemia Most patients with adult T-cell lymphoma / leukemia have antibodies to HTLV-1, a virus endemic to southwestern Japan, South America, Central Africa, and the Caribbean. Adult T-cell lymphoma / leukemia develops in 1-5% of seropositive individuals, often 20 years after exposure. In acute cases, skin lesions, hepatosplenomegaly, lytic bone lesions, and infections are present, along with elevated white blood cell (WBC) counts and hypercalcemia. In chronic and smoldering cases, the rash is characterized by papules, nodules, plaques, or pruritic erythroderma, which may resemble mycosis fungoides histologically and clinically. Cells with hyperlobate nuclei (cloverleaf pattern) infiltrate the dermis and subcutaneous tissue. Epidermotropism with Autrie microabscesses can be identified in one-third of cases. Immunologically, malignant cells are positive for CD2, CD3, and CD5 but negative for CD7. CD4 and CD25 are positive. T cell gene rearrangements are clonal, and the HTLV-1 genome is integrated into the genome of tumor cells. Standard chemotherapy treatment does not appear to affect survival. The use of zidovudine and infiltration is supported. The prognosis for patients with adult T-cell lymphoma / leukemia is poor, with a median survival of 6 months for acute disease and 24 months for chronic disease.

[0064] Nasal extranodal NK / T cell lymphoma In nasal extranodal NK / T-cell lymphoma, a disease characterized by small, medium, and large cells, the nasal cavity / nasopharynx and the skin of the trunk and extremities are affected by multiple plaques and tumors. These lesions are frequently accompanied by systemic symptoms such as fever and weight loss, and an associated hemophagocytic syndrome may be present. Skin lesions can be primary or secondary. Because both primary and secondary lesions are clinically aggressive and require the same type of treatment, distinguishing between the two types of skin lesions appears unnecessary. This condition is more common in men and is more prevalent geographically in Asia, Central America, and South America. Cutaneous and subcutaneous infiltration, with lymphoid cells invading vascular walls and obliterating vascular spaces, leads to tissue necrosis and ulceration. Malignant cells are usually CD2- and CD56-positive (NK phenotype) and CD3-positive in the cytoplasm, but not on the surface. The cells contain cytotoxic proteins (T-cell intracellular antigen 1 [TIA-1], granzyme B, and perforin). Epstein-Barr virus (EBV) testing is generally positive. Rarely, the cells may have a true cytotoxic T-cell phenotype. Nasal-type extranodal NK / T-cell lymphoma is an aggressive disease requiring systemic therapy, although experience with systemic chemotherapy has generally been poor.

[0065] Primary cutaneous peripheral T-cell lymphoma, unspecified PTCL-U is a heterogeneous disease entity presenting with localized or generalized patches, nodules, and / or tumors. By definition, this group excludes all three provisional categories of PTCL outlined in the WHO-EORTC classification. The absence of previous or concurrent macules or plaques consistent with mycosis fungoides distinguishes these lesions from classic mycosis fungoides due to the diffuse large cell transformation. A pleomorphic infiltrate of small and large lymphocytes is found diffusely infiltrating the dermis. Large neoplastic T cells are present in greater than 30%. The immunophenotype is generally CD4+. Immunologically, most neoplastic lymphocytes display an aberrant CD4-positive phenotype with clonal rearrangement of T-cell receptor genes. CD30 staining results are negative. Patients with PTCL-U generally have a poor prognosis and should be treated with systemic chemotherapy. The 4-year survival rate approaches 22%. A small percentage of patients may experience spontaneous remission, but more aggressive behavior is more likely. Systemic lymphoma staging and multiagent chemotherapy are recommended. If patients have solitary or localized disease, radiation therapy could be considered as initial therapy.

[0066] Primary cutaneous progressive epidermotropic CD8-positive cytotoxic T-cell lymphoma Primary cutaneous progressive epidermotropic CD8-positive cytotoxic T-cell lymphoma is a clinically aggressive and (occasionally) disseminated disease presenting with tumors with eruptive papules, nodules, and central ulceration. This entity may also present with superficial macules and / or plaques. Affected patients are typically treated with anthracycline-based systemic chemotherapy. Histologically, epidermotropism with infiltration and disruption of adnexal skin structures and angiocentricity with vascular invasion can be observed. Malignant cells are CD3- and CD8-positive and contain cytotoxic proteins. Clonal T-cell gene rearrangements are observed. EBV testing is generally negative in primary progressive epidermotropic CD8-positive cytotoxic T-cell lymphoma.

[0067] Mycosis fungoides is the most common type of cutaneous T-cell lymphoma (44%), and as such, some authors use the term synonymously with cutaneous T-cell lymphoma. Cutaneous T-cell lymphoma is a relatively common clonal proliferation of T helper cells, and more rarely, T suppressor / killer cells or NK cells, which usually present as a widespread, chronic skin rash. Mycosis fungoides itself is often an epidermotropic disease characterized by the development of patches and tumors composed of small to medium-sized skin-homing T cells, some (or rarely all) of which have convoluted cerebriform nuclei. The term mycosis fungoides was first used by French dermatologist Alibert in 1806 to describe a severe form of the disease in which large, necrotic tumors resembling mushrooms were present on the patient's skin. Approximately 1,000 new cases of mycosis fungoides occur per year (i.e., 0.36 cases per 100,000 population). The condition is more common in black patients than in white patients (incidence ratio = 1:6) and occurs more frequently in men than in women (male to female ratio = 2:1). Although the most common age of onset is 50 years, mycosis fungoides can also be diagnosed in children and adolescents, with apparently similar outcomes. Variants of mycosis fungoides recognized by the WHO / EORTC include Sézary syndrome, folliculotropic mycosis fungoides, granulomatous lax skin, and Pagetoid reticulosis (Boehringer-Kropf disease).

[0068] Sézary syndrome Sézary syndrome accounts for approximately 5% of all cases of mycosis fungoides. Patients with Sézary syndrome have generalized exfoliative erythroderma and lymph node disease, as well as other evidence of significant malignant T-cell clones in the blood, such as circulating atypical T lymphocytes with cerebriform nuclei (>1000 / mm3) or the same clonal T-cell gene rearrangements found in the skin. (See image below.) T-cell gene rearrangements are demonstrated by molecular or cytogenetic techniques and / or proliferation of cells with a malignant T-cell immunophenotype (an increase in CD4+ cells with a CD4 / CD8 ratio >10 and / or proliferation of T cells with loss of one or more normal T-cell antigens [e.g., CD2, CD3, CD5]). Circulating malignant cells tend to be CD7- and CD26-negative. While Sézary syndrome may be part of a continuum from erythrodermic mycosis fungoides, the WHO / EORTC classification of cutaneous lymphomas considers its behavior to be "aggressive."

[0069] Folliculotropic mycosis fungoides Folliculotropic mycosis fungoides presents with follicular papules, patchy alopecia, and comedones, particularly on the head and neck. Atypical lymphocytic infiltration is present in the epithelium of the hair follicles, and mucinous degeneration of the hair follicles (follicular mucin deposition) may be present. Due to the depth of invasion, topical therapy may be ineffective.

[0070] Pagetoid reticulosis Pagetoid reticulosis or Bohringer-Kropf disease presents as solitary, asymptomatic, well-circumscribed, red, scaling spots or macules on the extremities that may gradually enlarge. A massive, fully epidermal infiltrate of atypical lymphocytes is present. The prognosis is excellent, with radiation therapy or surgical excision being the treatment of choice. The term Pagetoid reticulosis should be restricted to the localized type and not used to describe the disseminated type (Ketron-Goodman type).

[0071] Granulomatous loose skin Granulomatous loose skin is a condition characterized by the gradual development of ptotic loose skin, most commonly in the axillary and groin areas. Histologically, granulomatous infiltrates are present, with multinucleated giant cells with elastophagocytosis and a near-complete loss of elastin in the dermis (as evidenced by elastin staining). Disease recurrence is common after surgical intervention. Radiation may be useful, but its experience in this disease is limited. One-third of patients have been reported to have concomitant Hodgkin's lymphoma or mycosis fungoides.

[0072] Granulomatous cutaneous T-cell lymphomas are rare, and therefore, limited data are available regarding their clinicopathologic and prognostic features. Patients with granulomatous mycosis fungoides or granulomatous lax cutis exhibit overlapping histologic features. The development of large skin wrinkles in granulomatous lax cutis clinically distinguishes this condition from granulomatous mycosis fungoides.

[0073] Of all primary cutaneous lymphomas, 65% are T-cell. The most common immunophenotype is CD4+. Because the term cutaneous T-cell lymphoma encompasses a wide variety of diseases, there is no common pathophysiology for these diseases. Mycosis fungoides is a malignant lymphoma characterized by the clonal expansion of CD4+ (or helper) memory T cells (CD45RO+), which normally patrol the skin and home in to their target sites. The malignant clone often lacks normal T-cell antigens such as CD2, CD5, or CD7. Normal and malignant cutaneous T cells reach their target sites on the skin via interactions with cutaneous capillary endothelial cells. Cutaneous T cells express cutaneous lymphocyte antigen (CLA), an adhesion molecule that mediates T lymphocyte ligation to endothelial cells in cutaneous postcapillary venules through its interaction with E-selectin. Further promoting the propensity of cutaneous T cells to reach their intended site on the skin is the release by keratinocytes of cytokines that extravasate into the dermis, coat the luminal surface of dermal endothelial cells, and upregulate adhesion molecules in the cutaneous capillary endothelial lumen that react with CC chemokine receptor 4 (CCR4) found on cutaneous T cells.

[0074] The cells exude into the dermis, exhibiting an affinity for the epidermis and clustering around Langerhans cells (seen microscopically as Pautlier microabscesses). However, malignant cells that adhere to the skin retain the ability to exit the skin via afferent lymphatics. They migrate to lymph nodes and then return to the blood via efferent lymphatics, joining the circulating population of CLA-positive T cells. Thus, mycosis fungoides is essentially a systemic disease, even when the disease appears to be in its early stages and is clinically limited to the skin.

[0075] Treatment of CTCL Treatment for patients with CTCL includes local and systemic therapies. The most common treatments include, but are not limited to, psoralen-ultraviolet 2 (PUVA), electron beam therapy, including topical and total skin electron beam therapy (TSEBT), and local and systemic chemotherapy, or combinations thereof in multimodal therapy.

[0076] JPEG2025148358000002.jpg50150

[0077] In one embodiment, the treatment for CTCL is electron beam therapy. In one embodiment, the treatment for CTCL is local electron beam therapy. In one embodiment, the treatment for CTCL is total skin electron beam therapy. In one embodiment, the treatment for CTCL is electron beam therapy in combination with at least one other treatment modality and / or therapeutic agent.

[0078] As used herein, treatment modalities and / or agents suitable for use in combined modality therapy in conjunction with electron beam therapy may include, for example, moisturizing creams, PUVA, bexarotene, topical steroids, extracorporeal photophoresis, UVB phototherapy, interferon, nitrogen mustard, methotrexate cream, BCNU cream, nitrogen mustard, topical radiation therapy, systemic chemotherapy, etanercept, Ontak, and antifungal cream. These prior treatments are a diverse mix of topical and systemic therapies.

[0079] As used herein, other agents suitable for topical or combination modality therapy with TSEBT for the treatment of CTCL include, for example, denileukin diftitox (Ontak); (2000) bexarotene (Targretin) retinoid; (2006) vorinostat (Zolinza) hydroxymate histone deacetylase (HDAC) inhibitor; (2009) romidepsin (Istodax) cyclic peptide histone deacetylase (HDAC) inhibitor; off-label therapeutic agents such as, for example, topical and oral corticosteroids; bexarotene (Targretin) gel and capsules; carmustine These may include benzodiazepines (BCNU, nitrosoureas); mechlorethamine (nitrogen mustard); phototherapy (broad and narrow band UVB or PUVA); conventional radiation therapy; photophoresis; interferon; alemtuzumab (Campath-1H); methotrexate; pentostatin and other purine analogs (fludarabine, 2-deoxychloroadenosine); liposomal doxorubicin (Doxil); gemcitabine (Gemzar); cyclophosphamide; bone marrow / stem cells; allogeneic transplantation; forodesine (inhibits purine nucleoside phosphorylase); and / or panobinostat.

[0080] General Aspects of Electron Beam Therapy EBT is one of the most effective therapies for CTCL. Unfortunately, most patients develop dose-limiting toxicities and cannot undergo repeated courses or larger doses of EBT. Total skin EBT (TSEBT) can be considered as initial therapy for patients with widespread, thick plaques. This is because the effective depth of treatment with TSEBT is greater than that of topical nitrogen mustard or phototherapy, but this is usually reserved for later stages of disease due to the potential for cumulative radiation toxicity. EBT may also be appropriate for patients with rapidly progressive disease and those who have failed other therapies, such as topical nitrogen mustard, bexarotene gel, and / or phototherapy. Many of these patients are likely to benefit from TSEBT and topical EBT for adequate disease control. The most dramatic responses have been observed in patients with neoplastic disease, i.e., disease with thick plaques, and nearly all such patients are considered appropriate candidates for total skin irradiation. However, the majority of patients treated with total skin irradiation eventually develop recurrent disease, although long-term remissions have been reported. Additionally, acute side effects such as epithelial lysis, hypohidrosis, blisters / skin ulcers, mucositis, and alopecia can occur in most patients treated with EBT.

[0081] Maintenance therapy for EBT Because the majority of patients treated with total skin irradiation eventually develop recurrent disease, various adjuvant or maintenance therapies are used after completion of electron beam therapy. These include topical nitrogen mustard, PUVA, oral etretinate, extracorporeal photophoresis, and systemic chemotherapy. Topical nitrogen mustard in Aquaphor provides the dual benefit of treating residual disease and softening the skin, which is often chronically dry after completion of TSEB therapy. These maintenance therapies delay the time to recurrence, but there is little evidence of improved long-term disease-free survival.

[0082] Biological response modifiers Biological response modifiers may provide useful treatments for patients with CTCL. These include interferons, cytokines, various retinoids, and combinations thereof. Alpha interferon is an effective single agent (response rate: 50%), typically administered at 3-5 million units per week. Its effectiveness is limited by the development of antibodies and its generalized, flu-like symptoms, and remission duration is usually short, with a median of 6 months. Combination therapy of interferon with PUVA or retinoids has been highly effective, even in patients with stage IV disease or tumors. There are two classes of retinoid receptors: RAR and RXR. Upon entering cells, retinoids bind to the receptor, form RAR and RXR heterodimers, translocate into the nucleus, and interact with transcription factors. Thus, retinoids interact with gene promoters and regulate transcription. Well-known retinoids, such as acitretin, etretinate, and 13-cis retinoic acid, interact with RAR receptors, whereas targretin is a novel RXR-selective retinoid. All of these retinoids have been used in CTCL. Small studies have shown similar efficacy (50-60% response rate) for etretinate and 13-cis retinoic acid.

[0083] Localized disease can be treated with low-energy X-rays or electrons. Electrons have an inherent advantage over X-rays because electron penetration depth can be controlled by appropriate selection of electron energy. The relative dose contribution to subcutaneous and deeper tissues is greater even with low-energy photons compared to electrons. For indurated plaques, electron energies as low as 6 MeV are generally sufficient. Low-energy electrons have a relative "skin-sparing" effect, which may indicate the use of a bolus. Lower-energy electrons have a relative "skin-sparing" effect, meaning that the maximum dose is actually deep relative to the skin surface. Because MF lesions are very superficial, it is desirable to have the maximum dose at the skin surface. This can be achieved by using a 0.5-1.0 cm thick tissue-equivalent bolus of material. To treat individual lesions, an electron energy should be selected that provides sufficient penetration depth throughout the entire depth of the plaque, macular, or tumor lesion, exceeding at least 0.5 cm of penetration. For common or thin plaques, treatment with 6-9 MeV electrons using a 1.0 cm bolus is usually sufficient. Exophytic tumors may require 9-12 MeV electrons. A margin of up to 2 cm is recommended, but may depend on location and proximity to sensitive tissue.

[0084] TSEBT The ability to irradiate the entire skin depends on advances in electron beam therapy, where the depth-dose characteristics of electron beams allow for treatment of large surfaces of the skin in a single field, concentrating the radiation dose in the epidermis and upper dermis while limiting the dose to the deep dermis and subcutaneous tissue.

[0085] A linear accelerator accelerates electrons, which are then produced to strike a target to produce high-energy photons (X-rays). The basic approach of the "Stanford technique" is to replace the target at the end of the linear accelerator with an electron scattering foil, thereby producing a divergent electron beam. The patient could stand about 10 feet / 3 meters from the end of the accelerator and have her or his entire surface treated with a broad electron beam. By using a multi-field technique, it was possible to irradiate the entire skin surface. At Stanford, a four-field technique was first used, and later six-field treatments were introduced.

[0086] In general, dosimetry for total-skin electron beam irradiation improves as the number of treatment fields increases. With four-field treatments, there is significant overlap of adjacent fields, creating "hot spots" that can result in long-term telangiectasia, subcutaneous fibrosis, and even necrosis. These complications can be compounded with fractionation programs using larger doses per fraction or fewer fractions per week. In a typical configuration, patients are treated in an upright position at a distance of 3.5 m from the isocenter (electron source). To attenuate and further scatter the electrons, a 3 / 8-inch / 1 cm Lucite plate is placed as close as possible to the patient's body surface. During treatment, the device is angled upward or downward at an 18 Å angle. The combination of these two fields for treating each body surface results in a very uniform dose distribution at the patient's surface and minimizes photon contamination, which is greatest at the central axis of the electron beam. Patients are currently treated with a six-field technique, including anterior, posterior, and four opposing oblique fields. A full "cycle" of treatment is administered over two days. On day 1, the anterior and two posterior oblique fields are treated at each of the two accelerator angles. On day 2, the posterior and two anterior oblique fields are treated at each of the two accelerator angles. The dose administered in each cycle is approximately 1.5–2 Gy. Most patients tolerate approximately 2 Gy per cycle, but lower doses are used for patients with erythroderma, atopic skin, or a previous course of electron beam therapy. The prescribed total dose is approximately 30–36 Gy administered over approximately 9–10 weeks. A 1-week split was introduced after a dose of approximately 18–20 Gy was delivered to provide some relief of the generalized skin erythema that typically accompanies treatment.

[0087] This typical approach allows certain areas of the body surface to be "shadowed" and receive a relatively lower total radiation dose. These areas include the top of the scalp, perineum, and soles of the feet. Other areas may be problematic for individual patients due to their constitution, such as the lower chest in some women and the subcutaneous tissue layers in obese individuals. To compensate for this effect, we typically treat the perineum and soles with approximately 6 MeV (using a 1 cm tissue-equivalent bolus) for a total dose of 20 Gy, with daily fractions of approximately 1.0 Gy. Only in the presence of scalp lesions is supplemental therapy administered to the scalp vertex, which may cause permanent alopecia. If necessary, supplemental therapy is also administered to the chest and subcutaneous tissue layers in individual patients. Additionally, some patients with multiple isolated tumor lesions receive a boost therapy for these tumors at the start of electron therapy to reduce their thickness and allow for better penetration by the electrons. Typically, a dose of approximately 15 Gy in fractions of approximately 1.5–3.0 Gy using approximately 6–9 MeV electrons is sufficient for this purpose. In the standard course of treatment, only the eyes are shielded. If disease is present on the face or scalp, an internal lead eye shield with an inner coating of paraffin or dental acrylic is used. The shield is placed under the eyelid after local anesthesia of the eye. If disease is not present in these areas, an external lead eye shield taped over the closed eyes is used. Additionally, if no scalp or facial lesions are present, a scalp shield is used after the 25 Gy dose to promote adequate hair regrowth. Complete scalp shielding is contraindicated and may result in spread of disease to this area. Individual shielding is used as needed in the clinical situation. For example, some patients have minimal penetration into the dermis and deeper tissues, thus resulting in fewer side effects. TSEBT should be considered as initial treatment for patients with thickened plaques because TSEBT is more effective at reaching deeper plaques than topical therapies such as nitrogen mustard and phototherapy. In patients with rapidly progressing disease and those experiencing failure of topical therapy, TSEBT can be an effective therapy for achieving disease control. The total dose delivered to the skin is usually about 30-36 Gy over about 8-10 weeks.The overall clinical response rate after TSEBT is nearly 100%, with complete response rates ranging from 98% in the limited plaque stage to 40% in the tumor stage. However, the majority of patients treated with TSEBT experience recurrent disease. Maintenance and adjuvant therapy is often used after TSEBT to delay the time to recurrence.

[0088] In one embodiment, the patient was treated with high-dose (about 30 Gy) local and / or total skin electron beam therapy. In another embodiment, the patient was treated with low-dose (about 4 Gy) local and / or total skin electron beam therapy.

[0089] Accordingly, aspects and embodiments of the present disclosure provide therapeutic compositions comprising IL-12 (recombinant human interleukin-12), including IL-12 formulations, for treating, reducing, or preventing radiation-induced damaging effects, including acute radiation syndrome and / or radiation-induced cytotoxicity, in humans associated with local or total-body skin electron beam irradiation.

[0090] IL-12 As used herein, an exemplary recombinant murine IL-12 (e.g., a suitable recombinant murine IL-12 preparation, including, e.g., a glycosylated form of recombinant murine IL-12 produced in CHO cells; hereinafter "recombinant murine IL-12") was obtained from Peprotech (Rocky Hill, NJ, USA) or provided to Neumedicines by SBH Sciences (Natick, MA, USA). An exemplary recombinant human IL-12, i.e., rHuIL-12 (e.g., a suitable recombinant human IL-12 preparation, including, e.g., a glycosylated form of recombinant human IL-12 produced in CHO cells; hereinafter "recombinant human IL-12"), was provided to Neumedicines by SBH Sciences (Natick, MA, USA).

[0091] IL-12 is a heterodimeric cytokine containing p40 and p35 subunits whose role in immunity is well known. Numerous reports over the past 20 years have demonstrated that IL-12 plays an essential role in the interplay between the innate and adaptive arms of immunity by regulating inflammatory responses, innate resistance to infection, and adaptive immunity. Endogenous IL-12 is required for resistance to many pathogens and to transplantable and chemically induced tumors. A notable effect of IL-12 on immunity is its ability to stimulate interferon-γ (IFN-γ) production from natural killer (NK) cells, macrophages, and T cells. Furthermore, several in vitro studies in the early to mid-1990s reported that IL-12 stimulates hematopoiesis synergistically with other cytokines. Because these studies used highly purified progenitor cells or single cells, the hematopoietic-promoting activity of IL-12 is likely due to its direct effect on bone marrow stem cells. The role of IFN-γ in the hematopoietic activity of IL-12 is unclear, as several studies have linked both stimulation and suppression of hematopoiesis to IFN-γ.

[0092] As used herein, exemplary recombinant murine and human IL-12 compositions and formulations may be based on the following sequences, including, for example, fragments, structural homologs, sequence homologs, functional homologs and / or derivatives thereof, in a pharmaceutically acceptable vehicle or carrier.

[0093] rHUIL-12: IL12A(p35) (SEQ ID NO: 1) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRET SFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSS LEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS IL12B(p40) (SEQ ID NO: 2) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLL LLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRG DNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTW STPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

[0094] Mouse IL-12: Mouse IL-12A (p35) (SEQ ID NO: 3) RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSS TTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVG EADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAM Mouse IL-12B (p40) (SEQ ID NO: 4) WELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSSHH LLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQ RDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWST PHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS

[0095] Interleukin 12 (IL-12) has been shown to have radioprotective properties when administered before or immediately after exposure to whole-body radiation (Nata et al. (1994) IL-12 protects bone marrow from and sensitizes intestinal tract to ionizing radiation; J Immunol 153:4230-4237; Chen et al. (2007) IL-12 facilitates both the recovery of endogenous hematopoiesis and the engraftment of stem cells after ionizing radiation; Exp Hematol 35:203-213; U.S. Patent Application Publication No. 20110206635 and U.S. Patent No. 7,939,058). In studies, mice were rescued from the adverse effects of lethal whole-body radiation. The radioprotective effect was reported to reside within an unknown cell population in the bone marrow, likely long-term repopulating hematopoietic stem cells. Another study showed that IL-12 resulted in early recovery of peripheral blood counts in tumor-bearing mice after sublethal radiation therapy (Basile et al. (2008) Multilineage hematopoietic recovery with concomitant antitumor effects using low-dose Interleukin-12 in myelosuppressed tumor-bearing mice. J Transl Med 6:26). In this latter study, IL-12 was shown to be synergistic with radiation in reducing tumor volume. Notably, IL-12 did not increase tumor volume when administered before or after radiation exposure.

[0096] Therefore, IL-12 has the potential for radioprotection of bone marrow after total-body irradiation. However, early studies reported that IL-12 had a radioprotective effect on bone marrow, but sensitized the gastrointestinal (GI) system to radiation damage (Neta et al.). A later report found that the GI sensitizing effect of IL-12 was dependent on the dose of IL-12 administered (Chen et al.). There have been no reports of the radioprotective effect of IL-12 on other tissues or organs other than bone marrow.

[0097] The present invention is based on the surprising and unexpected discovery that certain murine recombinant IL-12 (e.g., m-HemaMax) and human recombinant IL-12 (e.g., HemaMax) have the ability to enhance survival in mice, non-human primates (NHPs), and humans, respectively, including when administered at extended time points following total body irradiation (TBI). Furthermore, aspects of the present invention are based on the surprising discovery that recombinant human IL-12 has the ability to treat, prevent, and / or reduce radiation-induced cytotoxicity or damage associated with radiation therapy, including electron beam therapy. In models of radiation mitigation in which a single, low dose of recombinant murine IL-12 in mice or recombinant human IL-12 in NHPs is administered subcutaneously 24 hours or longer following irradiation, the inventors have discovered that recombinant human IL-12 can provide potent mitigation of radiation damage in multiple tissues, including the immune, bone marrow, and GI compartments, leading to a significant increase in survival in mouse and NHP radiation mitigation models in the complete absence of supportive care. To our knowledge, this is the first report showing the potent radiomitigating effects of a therapeutic agent in mice and NHPs at extended post-irradiation time points, such as 24 hours or longer, following acute ionizing radiation exposure.

[0098] The present invention provides embodiments of the multi-tissue or multi-organ radioprotective effects of IL-12 following radiation exposure, based on the surprising and unexpected discovery that, in addition to protection of the bone marrow compartment, the IL-12-mediated radioprotective effects include protection of a variety of tissues, organs, and systems, including bone marrow, lymphatic system, immune system, mucosal tissues, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lungs, kidneys, skin, and brain, when administered according to aspects and embodiments of the present disclosure.

[0099] For purposes of this disclosure, the following definitions shall be used in their entirety to define terms of art and define the scope of compositions of matter for which protection is sought in the claims.

[0100] As used herein, "subject" means an animal that is the object of treatment, observation, or experiment. "Animal" includes both cold- and warm-blooded vertebrates and invertebrates, such as fish, crustaceans, reptiles, and especially mammals. "Mammals" include, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, as well as prenatal, pediatric, and adult humans.

[0101] As used herein, "prevent" or "protect" means to prevent, in whole or in part, or to ameliorate or control.

[0102] As used herein, the term "treating" refers to both therapeutic treatment and prophylactic or preventative measures, or the administration of an agent suspected of having therapeutic potential.

[0103] The term "pharmaceutically effective amount," as used herein, means the amount of an active compound or pharmaceutical agent that elicits the biological or pharmacological response in a tissue, system, animal or human that is desired by a researcher, veterinarian, physician or other clinician, including relief or amelioration of the disease being treated.

[0104] As used herein, an "effective amount" with respect to a pharmaceutical composition of the present disclosure means an amount sufficient to have utility and to bring about a desired therapeutic endpoint.

[0105] As used herein, radiation-induced damage following total body irradiation (TBI) can affect organs, tissues, and systems associated with the following: bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lungs, kidneys, skin, and brain.

[0106] In certain embodiments, radiation-induced damage or toxicity associated with treatment may include, for example, erythema, hyperpigmentation, itching, alopecia, mucositis, scaling, blisters, and edema of the extremities (hands and feet) associated with total body skin electron beam irradiation. Other side effects may include altered thermoregulation due to radiation-induced normal tissue damage to sweat glands.

[0107] As used herein, radiation exposure can be associated with radiation-induced acute, chronic, and systemic damaging effects. In one aspect, the present disclosure provides therapeutic compositions and methods of use for treating radiation-induced acute damaging effects. In one aspect, the present disclosure provides compositions and methods of use for treating radiation-induced cytotoxicity associated with local and / or total-body skin electron beam irradiation associated with CTCL. Typical damaging effects are not necessarily limited to normal tissues underlying the irradiating rays. Typical damaging effects can extend beyond the treatment site and can include, for example, esophagitis (difficulty swallowing); pneumonia in the lungs (cough, fever, fluid accumulation in the lungs); radiation-induced inflammation of the intestines (diarrhea, cramps, abdominal pain); nausea and vomiting; fatigue, tiredness, diarrhea, headache, tissue swelling, skin erythema, cough, and difficulty breathing. Typical damaging effects can affect skin sites, e.g., erythema, desquamation; oral mucosa, e.g., mucositis; nasopharynx; oropharynx; vocal cords; tonsils; and skin (scaling or cancerous). In certain embodiments, typical effects may include telangiectasia, fibrosis, myelitis, and cartilage fibrosis.

[0108] In certain embodiments, typical radiation-induced damaging effects may also include hematopoietic organ (bone marrow) syndrome, characterized by damage to the most rapidly dividing cells (such as bone marrow, spleen, and lymphatic tissue). Typical symptoms include internal bleeding, fatigue, bacterial infections, and fever.

[0109] In certain embodiments, typical radiation-induced damaging effects may also include gastrointestinal syndrome, characterized by damage to cells that do not divide very rapidly (such as the lining of the stomach and intestines). Typical symptoms include nausea, vomiting, diarrhea, dehydration, electrolyte imbalance, loss of digestive capacity, bleeding ulcers, and hematopoietic organ symptoms.

[0110] In certain embodiments, typical radiation-induced damaging effects may also include mucositis. In one embodiment, the radiation-induced mucositis is oral mucositis.

[0111] In certain embodiments, typical radiation-induced effects may also include central nervous system syndromes, characterized by damage to cells that do not regenerate, such as nerve cells. Typical symptoms include loss of coordination, confusion, fainting, convulsions, shock, and symptoms of the hematopoietic and gastrointestinal syndromes.

[0112] In certain embodiments, typical radiation-induced damaging effects may also include effects on the fetus resulting from prenatal radiation exposure, as the embryo / fetus is particularly sensitive to radiation, especially during the first 20 weeks of pregnancy (embryonic / fetal cells are rapidly dividing).

[0113] In certain embodiments, typical radiation-induced effects can also include damage resulting from ionizing radiation-induced production of radical oxygen species (ROS), including superoxide, hydroxyl radical, nitrate, and peroxynitrite, due to the interaction of ionizing radiation with oxygen and water.

[0114] In one aspect, the present disclosure provides therapeutic compositions and methods of use for treating radiation-induced chronic damaging effects. Chronic radiation effects are clinically significant in all patients, but particularly in patients undergoing total body irradiation (TBI). Total body irradiation is used in some cancer therapies, particularly for patients requiring bone marrow transplantation.

[0115] Typical radiation-induced damaging effects can include features common to premature aging, such as graying of hair, thinning and dry skin, cataract development, premature myocardial fibrosis, myocardial infarction, neurodegeneration, osteopenia / osteomalacia, and neurocognitive deficits.

[0116] In certain embodiments, typical radiation-induced effects may also include fibrosis (replacement of normal tissue with scar tissue, resulting in limited movement in the affected area); intestinal damage resulting in diarrhea and bleeding; memory loss; infertility and / or carcinogenesis / leukemogenesis.

[0117] In one aspect, the present disclosure provides therapeutic compositions and methods of use for treating radiation-induced systemic damage effects. Typical systemic damage effects can include, for example, the acute and chronic effects described above, but have several unique characteristics. In particular, systemic effects include general tiredness and fatigue, and symptoms in non-irradiated areas associated with the persistent circulation of inflammatory cytokines.

[0118] In certain embodiments, systemic damaging effects may include central nervous system syndrome, nausea and vomiting, headache, sweating, tachycardia, gastrointestinal syndrome, breakdown of intestinal crypts and intestinal endothelial cells, dehydration, severe abdominal pain, infection, blood loss; hematopoietic syndrome; decreased peripheral white blood cell count, platelet count, red blood cell count, immunosuppressive syndrome; decreased peripheral blood lymphocyte count; radiation-induced skin syndrome, skin burns (beta burns), erythema / redness of the skin, skin ulceration, heat loss, fluid extravasation, lymphedema, bleeding, and secondary infection.

[0119] In certain embodiments, radiation-induced damaging effects may include mucositis; loss of taste; dry mouth; erythema; damage to the microvasculature, stem cell depletion, fibrosis, lymphedema, delayed wound healing, telangiectasia, sore throat, and ulceration.

[0120] In certain embodiments, the methods and compositions of the present disclosure are useful for treating radiation damage resulting from therapeutic radiation therapy, and typical delivery methods / regimens may include, for example, conventional fractionated radiation therapy, hyperfractionated radiation, hypofractionated radiation, and accelerated fractionated radiation.

[0121] In one embodiment, the treatment modality / regimen is local or total skin electron beam radiation (TSEBT) associated with CTCL treatment. In one embodiment, the electron beam radiation is administered as high dose rate therapy (HDR). In another embodiment, the electron beam therapy is administered as low dose rate electron beam therapy.

[0122] In one embodiment, the treatment modality / plan is hyperfractionated radiation therapy. In hyperfractionated radiation therapy, the goal is to deliver a higher tumor dose while maintaining a clinically acceptable level of long-term tissue damage. The daily dose remains unchanged or increases slightly, while the dose per fraction decreases and the total treatment time remains constant.

[0123] In one embodiment, the treatment modality / regimen is accelerated fractionation, in which the dose per fraction remains constant while the daily dose is increased and the total time of treatment is shortened.

[0124] In one embodiment, the treatment modality / plan is sequential hyperfractionated accelerated radiation therapy (CHART) therapy. CHART therapy is an intensive schedule of treatment in which multiple daily fractionated doses are administered. The rules will be implemented in a short period of time.

[0125] In one embodiment, the treatment modality / plan is IMRT.

[0126] Combination with chemotherapy (combination) Some chemotherapeutic agents can enhance the effects of radiation therapy. In one aspect, aspects and embodiments of the present invention can be used as a combination therapy with existing chemotherapy modalities. The combination (sequential or simultaneous) therapy can be administered simultaneously or in the same formulation.

[0127] "Interleukin-12 (IL-12)" refers to IL-12 molecules that confer at least one hematopoietic property as disclosed herein, including native IL-12 molecules, mutant IL-12 molecules, and covalently modified IL-12 molecules, produced by any method now known or later developed in the art.

[0128] The IL-12 molecule can be present in a substantially isolated form. It will be understood that the product can be mixed with carriers or diluents that do not interfere with the intended purpose of the product and still be considered substantially isolated. The products of the invention can also be in a substantially purified form, which generally comprises about 80%, 85%, or 90%, such as at least about 95%, at least about 98%, or at least about 99%, of the peptide or dry mass of the preparation.

[0129] Generally, the amino acid sequence of the IL-12 molecule used in embodiments of the invention will be derived from the particular mammal being treated by the methods of the invention. Thus, by way of example, for humans, typically human IL-12, or recombinant human IL-12, would be administered to the human in the methods of the invention; similarly, for felines, for example, feline IL-12, or recombinant feline IL-12, would be administered to the feline in the methods of the invention.

[0130] However, certain embodiments in which the IL-12 molecule is not derived from an amino acid sequence from the mammal that is the subject of the therapeutic methods of the present invention are also encompassed by the present invention. By way of example, human IL-12 or recombinant human IL-12 can be used in feline mammals. Yet other embodiments of the present invention include IL-12 molecules in which the native amino acid sequence of IL-12 is altered from the native sequence, but which IL-12 molecule functions to confer the hematopoietic properties of IL-12 disclosed herein. Alterations from the native, species-specific amino acid sequence of IL-12 include alterations to the primary sequence of IL-12, including deletions and additions of primary amino acids, resulting in mutant IL-12 molecules. One example of a highly derivatized IL-12 molecule is the redesigned IL-12 molecule produced by Maxygen, Inc. (Leong SR, et al., Proc Natl Acad Sci USA. 2003 Feb. 4; 100 (3): 1163-8), where the mutant IL-12 molecule is produced by DNA shuffling. Also included in the methods of the present invention are modified IL-12 molecules, such as covalent modifications to the IL-12 molecule that increase shelf life, half-life, potency, solubility, delivery, etc., such as the addition of polyethylene glycol groups, polypropylene glycol, etc., as described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. One type of covalent modification of the IL-12 molecule is introduced into the molecule by reacting targeted amino acid residues of the IL-12 polypeptide with an organic derivatizing agent capable of reacting with selected side chains or the N- or C-terminal residues of the IL-12 polypeptide. Both native sequence IL-12 and amino acid sequence variants of IL-12 can be covalently modified. Also, as referred to herein, IL-12 molecules can be produced by various methods known in the art, including recombinant methods. Other IL-12 variants encompassed by the present invention are those in which the standard sequence has been post-translationally modified, e.g., glycosylated. In some embodiments, IL-12 is expressed in a mammalian expression system or cell line.In one embodiment, IL-12 is produced by expression in Chinese hamster ovary (CHO) cells.

[0131] Because it is often difficult to predict the characteristics of mutant IL-12 polypeptides in advance, several rounds of screening of recovered mutants are necessary to select the optimal mutant. A preferred method for assessing hematological changes that stimulate or enhance the hematopoietic properties of mutant IL-12 molecules is by the lethal irradiation rescue protocol disclosed below. Other potential modifications of protein or polypeptide properties, such as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation, or tendency to aggregate with carriers or into multimers, are assayed by methods well known in the art.

[0132] For a general description of IL-12, see U.S. Patent Nos. 5,573,764, 5,648,072, 5,648,467, 5,744,132, 5,756,085, 5,853,714, and 6,683,046. Interleukin-12 (IL-12) is a heterodimeric cytokine generally described as a proinflammatory cytokine that regulates the activity of cells involved in the immune response (Fitz KM, et al., 1989, J. Exp. Med. 170:827-45). In general, IL-12 stimulates the production of interferon-γ (IFN-γ) from natural killer (NK) cells and T cells (Lertmemongkolchai G, Cai et al., 2001, Journal of Immunology. 166:1097-105; Cui J, Shin T, et al., 1997, Science. 278:1623-6; Ohteki T, Fukao T, et al., 1999, J. Exp. Med. 189:1981-6; Airoldi I, Gri G, et al., 2000, Journal of Immunology. 165:6880-8), and helps the differentiation of T helper 1 (TH1) cells (Hsieh CS, et al., 1993, Science. 260:547-9; Manetti R, et al., 1993, J. Exp. Med. 177:1199-1204) and form the link between innate resistance and adaptive immunity.IL-12 has also been shown to inhibit cancer growth through its immunomodulatory and antiangiogenic effects (Brunda MJ, et al., 1993, J. Exp. Med. 178:1223-1230; Noguchi Y, et al., 1996, Proc. Natl. Acad. Sci. USA 93:11798-11801; Giordano PN, et al., 2001, J. Exp. Med. 194:1195-1206; Colombo MP, et al., 2002, Cytokine Growth Factor Rev. 13:155-168; Yao L, et al., 2000, Blood 96:1900-1905). IL-12 is primarily produced by dendritic cells (DCs) and phagocytes (macrophages and neutrophils) once activated by encountering pathogenic bacteria, fungi, or intracellular parasites (Reis C, et al., 1997, J. Exp. Med. 186:1819-1829; Gazzinelli RT, et al., 1994, J. Immunol. 153:2533-2543; Dalod M, et al., 2002, J. Exp. Med. 195:517-528). The IL-12 receptor (IL-12R) is expressed primarily by activated T cells and NK cells (Presky DH, et al., 1996, Proc. Natl. Acad. Sci. USA 93:14002-14007; Wu CY, et al., 1996, Eur J. Immunol. 26:345-50).

[0133] Generally, IL-12 production stimulates IFN-γ production, which in turn enhances IL-12 production, thus forming a positive feedback loop. In vitro, IL-12 has been reported to synergize with other cytokines (e.g., IL-3 and SCF) to stimulate the proliferation and differentiation of early hematopoietic progenitor cells (Jacobsen SE, et al., 1993, J. Exp Med 2: 413-8; Ploemacher RE, et al., 1993, Leukemia 7: 1381-8; Hirao A, et al., 1995, Stem Cells 13: 47-53).

[0134] In vivo administration of IL-12 has been observed to reduce peripheral blood cell counts and bone marrow hematopoiesis (Robertson MJ, et al., 1999, Clinical Cancer Research 5: 9-16; Lenzi R, et al., 2002, Clinical Cancer Research 8:3686-95; Ryffel B. 1997, Clin Immunol Immunopathol. 83:18-20; Car BD, et al., 1999, The Toxicol Pathol. 27:58-63). Using IFN-γ receptor knockout mice, Eng et al. and Car et al. demonstrated that high doses of IL-12 did not induce the commonly observed toxic effects, i.e., no suppression of hematopoiesis (Eng VM, et al., 1995, J. Exp Med. 181:1893-8; Car BD, et al., 1995, American Journal of Pathology 147:1693-707). This observation suggests that the previously reported general phenomenon of IL-12-promoted enhancement of differentiated hematopoietic cells may be balanced in vivo by the production of IFN-γ, which acts in a predominantly myelosuppressive manner.

[0135] Current evidence suggests that recombinant human IL-12 (e.g., recombinant human IL-12), a typical IL-12 preparation, induces at least four levels of responses in the body (see Figure 14). In Level 1 responses, recombinant human IL-12 promotes the proliferation and activation of existing radiosensitive immune cells, namely, NK cells, macrophages, and dendritic cells. The recombinant human IL-12-induced increase in plasma IL-15 and IL-18 also promotes the maturation of NK cells, which leads to the release of IFN-γ, which in turn promotes the proliferation and activation of macrophages and dendritic cells, and possibly NK cells. It positively influences the production of endogenous IL-12. These events enhance innate immune function early after recombinant human IL-12 administration. In a level 2 response, recombinant human IL-12 promotes the proliferation and differentiation of surviving hematopoietic stem cells, osteoblasts, and megakaryocytes into specific cellular components that ensure optimal hematopoiesis. The secretion of EPO from CD34+, IL-12Rβ2-positive bone marrow cells induced by recombinant human IL-12 also suppresses local overproduction of IFN-γ in the bone marrow, thus creating an environment that promotes hematopoietic cell expansion. Hematopoietic regeneration in the bone marrow enhances both innate and adaptive immune function. In a level 3 response, recombinant human IL-12 preserves GI stem cells, thereby reducing pathogen leakage, increasing food intake, and reducing diarrhea. In a level 4 response, recombinant human IL-12 likely directly increases renal release of EPO (a cytoprotective factor), which enhances cell viability in a wide range of organs and tissues. The continued production of endogenous IL-12, arising primarily from pathogen- and / or EPO-activated dendritic cells, functions as a positive feedback loop and likely plays an important role in maintaining the initial response to exogenous recombinant human IL-12 for several weeks after irradiation.

[0136] IL-12 administration method The present invention provides a method of treatment by administering one or more effective doses of IL-12 to a subject over a period of time to achieve the desired therapeutic effect. The subject is preferably a mammal, such as, but not limited to, a cow, pig, horse, chicken, cat, dog, etc., and most preferably a human.

[0137] A variety of delivery systems are known and can be used for administering IL-12 according to the methods of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing IL-12, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), and nucleic acid constructs containing the IL-12 gene as part of a retroviral or other vector. Methods of introduction include, but are not limited to, topical, subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. For the treatment of CTCL, topical, subcutaneous, intradermal, and systemic delivery are particularly effective.

[0138] IL-12 can be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through the epithelial or mucocutaneous system (e.g., oral, rectal, and intestinal mucosa), and may be administered in conjunction with other biologically active agents. Administration may be systemic or local. Furthermore, it may be desirable to introduce a pharmaceutical composition containing IL-12 into the central nervous system by any suitable route, for example, intraventricular and intrathecal injection. Intraventricular injection may be facilitated, for example, by an intraventricular catheter attached to a container such as an Ommaya container. Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer and a formulation containing an aerosolizing agent. It may be desirable to administer a pharmaceutical composition containing IL-12 locally to the area requiring treatment. This may be achieved, for example, but not limited to, by topical application, by injection, by catheter, by suppository, or by implantation of porous, non-porous, or gelatinous material, for example, a membrane or fiber such as a Silastic™ membrane.

[0139] Other methods of administering IL-12 include delivery in vesicles, particularly liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., "Liposomes in the Therapy of Infectious Disease and Cancer," Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0140] Yet another method of administering IL-12 involves delivery in a controlled release system, in certain embodiments, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). Additionally, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NY (1984); Langer and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983; see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)) or controlled release systems can be placed near the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, "Medical Applications of Controlled Release," supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science, 249: 1527-1533 (1990)).

[0141] Forms and Doses of IL-12 Liquid and lyophilized formulations suitable for injection are particularly effective for use herein in treating CTCL. Suitable dosage forms of IL-12 for use in embodiments of the present invention include physiologically / pharmaceutically acceptable carriers that are essentially nontoxic and nontherapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphoric acid, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, P6N (Neumedicines, Pasadena, Calif.), and PEG. Carriers for topical or gel-based formulations of IL-12 polypeptides include polysaccharides, such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, and wood wax alcohol. For all administrations, conventional depot formulations are suitable. Examples of such formulations include microcapsules, nanocapsules, liposomes, plasters, inhalation forms, nasal sprays, sublingual tablets, and sustained-release preparations.

[0142] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, such as poly(2-hydroxyethyl-methacrylate) described in Langer et al., supra, and Langer, supra, or poly(vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman et al., supra), non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable lactic acid-glycolic acid copolymers, such as Lupron Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. If encapsulated IL-12 polypeptides remain in the body for extended periods, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and potential changes in immunogenicity. Depending on the mechanism involved, rational strategies for stabilization can be devised. For example, if the aggregation mechanism is found to be intermolecular S–S bond formation via thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solution, controlling humidity content, using appropriate additives, and developing specific polymer matrix compositions. Sustained-release IL-12-containing compositions also include polypeptides entrapped in liposomes. Liposomes containing IL-12 polypeptides are prepared by methods known in the art, such as those described in Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Typically, the liposomes are small (approximately 200-800 angstroms), unilamellar, and their lipid content is greater than about 30 mol% cholesterol, with the selected ratio adjusted for optimal Wnt polypeptide therapy. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0143] For the treatment of a disease, a suitable dose of IL-12 polypeptide is a therapeutic dose, as defined above. The type of disease to be treated, the severity and course of the disease, previous therapy, the patient's medical history, and this specification The effectiveness of the present invention will depend on the patient's response to the IL-12 treatment methods disclosed herein, as well as the discretion of the attending physician. In this context, IL-12 is suitably administered to the patient at one time or over a series of treatments.

[0144] Depending on the type and severity of the disease, initial candidate doses for administration to patients range from about 10 ng / kg to 2000 ng / kg, whether by one or more separate administrations or by continuous infusion. Humans can safely tolerate repeated doses of about 500 ng / kg, while single doses of up to about 200 ng / kg are unlikely to produce toxic side effects. For example, doses may be similar to those for other cytokines, such as G-CSF, GM-CSF, and EPO. For repeated administration over several days or longer, treatment is continued, depending on the condition, until a desired suppression of disease symptoms occurs. However, other dosing regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0145] IL-12 can be administered with other cytokines by direct coadministration or sequential administration. When one or more cytokines are coadministered with IL-12, lower doses of IL-12 can be used. A suitable dose of the other cytokine, i.e., a cytokine other than IL-12, is about 1 μg / kg to about 15 mg / kg of cytokine. For example, the dose may be the same as for other cytokines such as G-CSF, GM-CSF, and EPO. The other cytokine(s) may be administered before, simultaneously with, or after administration of IL-12. The cytokine(s) and IL-12 can be combined to form a pharmaceutical composition for simultaneous administration to a mammal. In certain embodiments, the amounts of IL-12 and cytokines are such that a synergistic repopulation of blood cells (or a synergistic increase in proliferation and / or differentiation of hematopoietic cells) occurs in the mammal upon administration of IL-12 and the other cytokines. In other words, the synergistic effect of two or more agents (ie, IL-12 and one or more cytokines) on blood cell repopulation (or hematopoietic cell proliferation / differentiation) is greater than the sum of the individual effects of these molecules.

[0146] Therapeutic formulations of IL-12 are prepared for storage in the form of a lyophilized cake or aqueous solution by mixing IL-12 having the desired purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. (ed.), (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween®, Pluronics™, or polyethylene glycol (PEG).

[0147] As used herein, the term "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical preparation. Suitable buffers are well known in the art and can be found in the literature. Pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, phosphate buffer, arginine buffer, or mixtures thereof. The above buffers are generally used in amounts of about 1 mM to about 100 mM, about 5 mM to about 50 mM, and about 10 to 20 mM. The pH of the buffer solution may be at least 4.0, at least 4.5, at least 5.0, at least 5.5, or at least 6.0. The pH of the buffer solution may be less than 7.5, less than 7.0, or less than 6.5. The pH of the buffer solution may be adjusted to about 4.0 to about 7.5, about 5.5 to about 7.5, about 5.0 to about 6.5, or about 5.5 to about 6.5 using acids or bases known to those skilled in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, sodium hydroxide, and potassium hydroxide. As used herein, when describing pH, "about" means ±0.2 pH units.

[0148] As used herein, the term "surfactant" can include pharmaceutically acceptable excipients used to protect protein formulations against mechanical stresses such as agitation and shear. Examples of pharmaceutically acceptable surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Suitable surfactants include polyoxyethylene sorbitan fatty acid esters such as polysorbate 20 (sold under the trademark Tween 20®) and polysorbate 80 (sold under the trademark Tween 80®). Suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188®. Suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij®. Suitable alkylphenol polyoxyethylene esters are sold under the trade name Triton-X. When polysorbate 20 (Tween 20®) and polysorbate 80 (Tween 80®) are used, they are generally used at concentrations ranging from about 0.001 to about 1%, about 0.005 to about 0.2%, and about 0.01% to about 0.1% w / v (weight / volume).

[0149] The term "stabilizer" as used herein can include pharmaceutically acceptable excipients that protect active pharmaceutical ingredients and / or formulations from chemical and / or physical degradation during manufacturing, storage, and application. The chemical and physical degradation pathways of protein drugs are reviewed by Cleland et al., Crit. Rev. Ther. Drug Carrier Syst., 70(4):307-77 (1993); Wang, Int. J. Pharm., 7S5(2):129-88 (1999); Wang, Int. J. Pharm., 203(1-2):1-60 (2000); Chi et al., Pharm. Res., 20(9):1325-36 (2003). Stabilizers include, but are not limited to, sugars, amino acids, polyols, cyclodextrins (e.g., hydroxypropyl-β-cyclodextrin, sulfobutylethyl-β-cyclodextrin, β-cyclodextrin), polyethylene glycols (e.g., PEG 3000, PEG 3350, PEG 4000, PEG 6000), albumin, human serum albumin (HSA), bovine serum albumin (BSA), salts (e.g., sodium chloride, magnesium chloride, calcium chloride), and chelating agents (e.g., EDTA). As described above, the stabilizer may be present in the formulation in an amount of about 10 to about 500 mM, about 10 to about 300 mM, or about 100 mM to about 300 mM. In some embodiments, exemplary IL-12 can be dissolved in a suitable pharmaceutical formulation in which it is stable.

[0150] IL-12 can also be entrapped in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0151] IL-12 to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. IL-12 is typically stored in lyophilized form or in solution. Therapeutic IL-12 compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0152] For topical application, IL-12 is preferably combined with other ingredients, such as carriers and / or adjuvants. There are no limitations on the nature of such other ingredients, except that they must be physiologically acceptable and effective for the intended use and cannot degrade the activity of the active ingredients of the composition. Examples of suitable vehicles include ointments, creams, gels, or suspensions, with or without purified collagen. The composition can also be impregnated into transdermal patches, plasters, and bandages, preferably in liquid or semi-liquid form.

[0153] To obtain a gel formulation, IL-12 formulated in a liquid composition can be mixed with an effective amount of a water-soluble polysaccharide or synthetic polymer, such as PEG, to form a gel of suitable viscosity for topical application. Polysaccharides that can be used include, for example, cellulose derivatives, such as etherified cellulose derivatives, such as alkylcellulose, hydroxyalkylcellulose, and alkylhydroxyalkylcellulose, such as methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, and hydroxypropylcellulose; starch and fractionated starch; agar; alginic acid and alginates; gum arabic; pullulan; agarose; carrageenan; dextran; dextrin; fructan; inulin; mannan; xylan; arabinan; chitosan; glycogen; glucan; and synthetic biopolymers; as well as gums such as xanthan gum; guar gum; locust bean gum; gum arabic; tragacanth gum; and karaya gum; and derivatives and mixtures thereof. Preferred gelling agents described herein are inert to biological systems, non-toxic, easy to prepare, not too thin or too thick in viscosity, and do not destabilize the IL-12 molecules held therein.

[0154] Preferably, the polysaccharide is an etherified cellulose derivative, more preferably one that is well-defined, purified, and USP listed, such as methylcellulose and hydroxyalkyl cellulose derivatives, such as hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethyl cellulose. Most preferred herein is methylcellulose.

[0155] Polyethylene glycols useful for gelation are typically mixtures of low and high molecular weight PEGs to obtain the appropriate viscosity. For example, a mixture of 400-600 molecular weight PEG and 1500 molecular weight PEG is effective for this purpose when mixed in the appropriate ratio to obtain a paste.

[0156] The term "water-soluble" as applied to polysaccharides and PEG is meant to include colloidal solutions and dispersions. Generally, the solubility of a cellulose derivative is determined by the degree of substitution of ether groups, and the stabilized derivatives useful herein should have a sufficient number of such ether groups per anhydroglucose unit in the cellulose chain to make the derivative water-soluble. An ether substitution degree of at least 0.35 ether groups per anhydroglucose unit is generally sufficient. Furthermore, the cellulose derivative may be in the form of an alkali metal salt, such as a Li, Na, K, or Cs salt.

[0157] When methylcellulose is used in the gel, it preferably comprises about 2-5%, more preferably about 3%, of the gel, and the IL-12 is present in an amount of about 300-1000 mg per ml of gel.

[0158] The effective amount of IL-12 used therapeutically depends, for example, on the therapeutic objectives, route of administration, and patient condition. Therefore, it is necessary for the treating professional to determine the dosage strength and modify the route of administration required to obtain the optimal therapeutic effect. Typically, a physician will administer IL-12 until a dosage that achieves the desired effect is reached. Typical dosages for systemic treatment can range from about 10 ng / kg up to 2000 ng / kg or more, depending on the factors described above. In some embodiments, the dosage range can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, to about 20; to about 30; to about 50; to about 100, to about 200, to about 300, or to about 500 ng / kg. In one embodiment, the dosage is less than 500 ng / kg. In another embodiment, the dosage is less than 300 ng / kg. In another embodiment, the dose is less than about 200 ng / kg. In another embodiment, the dose is less than about 100 ng / kg. In another embodiment, the dose is less than about 50 ng / kg. In other embodiments, the dose can be in the range of about 10-300 ng / kg, 20-40 ng / kg, 25-35 ng / kg, or 50-100 ng / kg. In some embodiments, the appropriate dose can be determined based on the amount of IL-12 administered per surface area of ​​the affected area.

[0159] In one aspect, exemplary therapeutic compositions described herein can be administered in combination with fractionated (irradiation) therapy. In one embodiment, a therapeutically effective dose is administered before each fraction. In one embodiment, a therapeutically effective dose is administered approximately simultaneously with the administration of each fraction. In one embodiment, a therapeutically effective dose is administered 5, 10, 15, 20, 25, 30, 35, 40, 50, or 60 minutes before each fraction; or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after each fraction; or 1, 2, 3, 4, 5, 6, or 7 days before each fraction. In one embodiment, the therapeutically effective dose is administered after each fraction within the range of 5, 10, 15, 20, 25, 30, 35, 40, 50, or 60 minutes after each fraction; or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after each fraction; or 1, 2, 3, 4, 5, 6, or 7 days after each fraction. Alternatively, the therapeutically effective dose is administered 1, 2, 3, 4, 5, 6, or 7 times weekly, biweekly, or bimonthly during or after radiation therapy. In another embodiment, one or more typical doses of IL-12 (1-100 ng / kg) are administered both about 5, 10, 15, 20, 30, 40, 50, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days before and after each radiation dose in a fractionated radiation regimen of 1-10 doses per day for up to 30 days administered as TBI or locally with each radiation source.

[0160] An alternative general proposal is to formulate the IL-12 receptor and develop an effective, but not excessively toxic, drug. IL-12 levels greater than about 0.1 ng / cc can be established in tissues up to the maximum dose not The amount of the drug delivered to the target site or tissue may be determined by continuous infusion, sustained release, topical application, or other empirically determined method. This tissue concentration should be maintained, if possible, by a dosing regimen, such as injections at a prescribed frequency. The progress of this therapy is easily monitored by conventional assays.

[0161] "Near the time of administration of the treatment" refers to administration of IL-12 any reasonable period before and / or after administration of the treatment, e.g., about 1 month, about 3 weeks, about 2 weeks, about 1 week, several days, about 120 hours, about 96 hours, about 72 hours, about 48 hours, about 24 hours, about 20 hours, several hours, about 1 hour, or minutes. Near the time of administration of the treatment also refers to simultaneous or near simultaneous administration of the treatment and IL-12, i.e., administration within minutes to a day.

[0162] "Chemotherapy" refers to any therapy involving natural or synthetic drugs currently known or developed in the medical community. Examples of chemotherapy include some currently available anti-cancer drugs. However, chemotherapy also includes any drug, natural or synthetic, intended to treat a disease state. In certain embodiments of the present invention, chemotherapy may include the administration of several known art drugs intended to treat a disease state. Examples include combination chemotherapy with docetaxel, cisplatin, and 5-fluorouracil for patients with locally advanced squamous cell carcinoma of the head (Tsukuda, M. et al., Int J Clin Oncol. 2004 June; 9 (3): 161-6), and fludarabine and bendamustine in refractory and recurrent indolent lymphoma (Konigsmann M, et al., Leuk Lymphoma. 2004; 45 (9): 1821-1827).

[0163] Typical sources of therapeutic or accidental ionizing radiation as used herein include, for example, alpha, beta, gamma, X-ray, and neutron sources.

[0164] "Radiation therapy" refers to any therapy that uses any form of radiation to treat a disease state. The radiation-producing devices for radiation therapy are those currently available or that become available in the future.

[0165] "High-dose therapeutic modality" refers to a high sublethal or near-lethal dose of therapy. High-dose therapeutic modalities are intended to have a high ability to achieve a therapeutic endpoint, but generally have high associated toxicity. Furthermore, high-dose therapeutic modalities generally exhibit higher hematopoietic damage compared to conventional therapeutic modalities. Protocols for high-dose therapeutic modalities are those currently being used or will be used in the future.

[0166] As used herein, the term "radiotherapy treatment modality" includes both ionizing and non-ionizing radiation sources. Typical ionizing radiation treatment modalities include external beam radiation therapy, intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), X-ray irradiation (e.g., photon therapy), electron beam (e.g., beta-ray irradiation), local and whole-body skin electron beam therapy, megavoltage photon therapy (about 4-10 MeV), proton beam irradiation, high linear energy transfer (LET) particles, stereotactic radiotherapy, gamma knife, linear accelerator-mediated frameless stereotactic radiotherapy, robotic arm-controlled x-ray irradiation delivery systems, radioisotope radiotherapy for organ-specific or cancer cell-specific uptake, radioisotopes bound to monoclonal antibodies for tumor-targeted radiotherapy (or radioimmunotherapy, RIT), brachytherapy (interstitial or intracavitary) high-dose-rate radiation source implants, and permanent radioactive seed implants for organ-specific dose delivery.

[0167] A "dose-intensive treatment regimen" is generally a treatment regimen in which treatment is continuously repeated in an accelerated manner to achieve desirable therapeutic results compared to conventional treatment regimens. The method of the present invention facilitates the use of dose-intensive treatment regimens by reducing or ameliorating the associated hematopoietic toxicity of the treatment, thereby enabling the use of dose-intensive treatment regimens and increasing the success rate of treating certain disease states (see generally Hudis CA, Schmits N, Semin Oncol. 2004 June; 31 (3 Suppl 8): 19-26; Keith B et al., J Clin Oncol. 2004 Feb. 15; 22 (4): 749; author reply 751-3; Maurel J et al., Cancer. 2004 Apr. 1; 100 (7): 1498-506; Atkins CD, J Clin Oncol. 2004 Feb. 15; 22 (4): 749-50).

[0168] "Chemoprotection or radioprotection" refers to protection from, or the apparent reduction of, the hematopoietic toxicity associated with a therapy intended to target a disease state.

[0169] As used herein, "acute radiation syndrome (ARS)" (also known as radiation toxicity or radiation sickness) is characterized by an acute illness caused by lethal or sublethal irradiation of the whole body (or a large portion of the body) with high doses of penetrating radiation over a very short period of time (e.g., just a few minutes). Examples of people who develop ARS include survivors of the atomic bombings of Hiroshima and Nagasaki, first responder firefighters after the Chernobyl nuclear power plant accident in 1986, and some other accidental exposures to sterilizing irradiators. In some embodiments, the radiation doses associated with acute radiation syndrome are usually large (i.e., greater than 0.7 Gray (Gy) or 70 rads). In some embodiments, mild symptoms can be observed with doses as low as 0.3 Gy or 30 rads.

[0170] As used herein, "acute damaging effect" and "damaging effect" include radiation-induced damage resulting from acute lethal and near-lethal radiation doses.

[0171] In some embodiments, typical acute radiation syndromes include three groups: 1) Bone marrow syndrome (sometimes referred to as hematopoietic syndrome): Complete syndrome usually occurs with doses between 0.7 and 10 Gy (70 and 1000 rads), although mild symptoms can occur with doses as low as 0.3 Gy or 30 rads. The survival rate of patients with this syndrome decreases with increasing dose. The primary cause of death is destruction of the bone marrow, resulting in infection and bleeding. 2) Gastrointestinal (GI) syndrome: Complete syndrome usually occurs with doses higher than approximately 10 Gy (1000 rads), although some symptoms can occur with doses as low as 6 Gy or 600 rads. Survival with this syndrome is extremely low. Destructive and irreversible changes in the GI tract and bone marrow usually result in infection, dehydration, and electrolyte imbalance. Death usually occurs within two weeks. 3) Cardiovascular (CV) / Central Nervous System (CNS) Syndrome: The complete syndrome usually occurs at doses higher than approximately 50 Gy (5000 rads), although some symptoms can occur at doses as low as 20 Gy or 2000 rads. Death occurs within 3 days. Death may be due to increased pressure within the trapped calvarium as a result of circulatory collapse and increased fluid contents caused by edema, vasculitis, and meningitis.

[0172] In some embodiments, a typical acute radiation syndrome includes four stages: 1) Prodromal Phase (NVD Phase): The standard symptoms of this phase are nausea, vomiting, loss of appetite, and possibly diarrhea (depending on the dose), occurring several days to several days after exposure. These symptoms may last from minutes to several days (in clusters). 2) Latent Phase: The patient generally looks and feels healthy for several hours or even weeks. 3) Symptomatic Phase: Symptoms vary depending on the specific syndrome (see Table A) and may last from several hours to several months. 4) Recovery or Death: Most patients who do not recover die within a few months of exposure. The recovery process can take from about a few weeks to about two years (Center for Disease Control and Prevention Fact Sheet for Physicians, Acute Radiation Syndrome; Radiation Studies Branch (RSB), Division of Environmental Hazards and Health Effects (EHHE), National Center for Environmental Health (NCEH), Coordinating Center for Environmental Health and Injury Prevention (CCEHIP); 2005).

[0173] JPEG2025148358000003.jpg164150

[0174] "Solid tumor" generally refers to a cancerous presence in body tissues other than the blood, bone marrow, or lymphatic system.

[0175] "Hematopoietic disorder (cancer)" generally refers to the presence of cancer cells originating from the hematopoietic system.

[0176] "Ameliorating a deficiency" refers to a reduction in hematopoietic deficiency, i.e., correction of the deficiency, or partial or complete restoration of normal as defined by current medical practice. Thus, ameliorating a hematopoietic deficiency refers to an increase, stimulation, enhancement, or promotion of hematopoiesis, either general or specific. Amelioration of a hematopoietic deficiency can be observed to be general, i.e., an increase in more than one hematopoietic cell type or lineage, or specific, i.e., an increase in one hematopoietic cell type or lineage.

[0177] "Bone marrow cells" generally refer to cells present and / or residing in the bone marrow compartment of a mammal. Included in the term "bone marrow cells" are cells of hematopoietic origin, such as, but not limited to, hematopoietic repopulating cells, hematopoietic stem and / or progenitor cells, as well as any cells that can be derived from bone marrow, such as endothelial cells, mesenchymal cells, bone cells, neural cells, supportive cells (stromal cells), including, but not limited to, committed stem and / or progenitor cells of these and other cell types and lineages.

[0178] "Hematopoietic cell type" generally refers to various kinds of differentiated hematopoietic cells, but may also include hematopoietic progenitor cells that originate from a particular hematopoietic cell type, e.g., various blast cells for all cell types associated with blood cell production, such as stem cells, progenitor cells, and various lineage cells, e.g., myeloid cells, lymphoid cells, etc.

[0179] "Hematopoietic cell lineage" generally refers to a particular lineage of differentiated hematopoietic cells, e.g., myeloid or lymphoid cells, but may also refer to further differentiated lineages, e.g., dendritic cells, erythroid cells, etc.

[0180] "IL-12-promoted (facilitated) proliferation" of cells generally refers to an increase, stimulation, or enhancement of hematopoiesis that is at least in part attributable to proliferation or expansion in cells present or resident in the mammalian bone marrow, e.g., hematopoietic progenitor cells and / or stem cells, but also includes other cells comprising the microenvironment of the bone marrow niche.

[0181] "Stimulation or enhancement of hematopoiesis" generally refers to an increase in one or more hematopoietic cell types or lineages, and particularly relates to stimulation or enhancement of one or more hematopoietic cell types or lineages when a mammal has a deficiency in one or more hematopoietic cell types or lineages.

[0182] "Hematopoietic long-term repopulating cells" generally are the most primitive blood cells in the bone marrow; they are the blood stem cells responsible for providing lifelong production of various blood cell types and lineages.

[0183] "Hematopoietic stem cells" are generally blood stem cells; there are two types: "long-term repopulating" as defined above and "short-term repopulating" which are capable of producing "progenitor cells" over a short period of time (weeks, months, or even sometimes years, depending on the mammal).

[0184] "Hematopoietic progenitor cells (hematopoietic precursor cells)" are generally the first cells to differentiate (ie, mature) from blood stem cells; they then differentiate (mature) into the various blood cell types and lineages.

[0185] "Hematopoietic support cells" are non-blood cells of the bone marrow; these cells provide "support" for blood cell production. These cells are also called bone marrow stromal cells.

[0186] "Bone marrow preservation" refers to the process by which bone marrow damaged by radiation, chemotherapy, disease, or toxins is maintained in its normal or near-normal state; "bone marrow recovery" refers to the process by which bone marrow damaged by radiation, chemotherapy, disease, or toxins is restored to its normal or near-normal state, or any measurable improvement in bone marrow function is obtained; bone marrow function is the process by which appropriate levels of various blood cell types or lineages are produced from hematopoietic (blood) stem cells.

[0187] "Bone marrow injury" is a pathological process in which bone marrow damaged by radiation, chemotherapy, disease, or toxins is unable to repair itself to normal and therefore is unable to produce enough blood cells to maintain adequate hematopoiesis in a mammal. [Example]

[0188] The invention will now be described in connection with the following examples, which are provided for purposes of illustration only, and the present invention is not limited to these examples, but includes all variations that become apparent as a result of the teachings set forth herein.

[0189] Prior to the experiments described herein, there were no published protocols that enabled compositions and methods comprising IL-12, such as therapeutically effective recombinant human interleukin-12 (IL-12) formulations, to treat radiation-induced damaging effects, including acute radiation syndrome and / or radiation-induced cytotoxicity, in subjects associated with topical and / or systemic skin electrotherapy in the treatment of CTCL. It was.

[0190] Aspects and embodiments of the present disclosure stem from the unexpected discovery that certain IL-12 formulations have surprising and unexpected utility and efficacy when administered to subjects following exposure to acute radiation exposure, including exposure associated with topical and total body skin electron beam therapy. The therapeutic compositions provide potent mitigation of radiation damage to multiple tissues, including the immune, bone marrow, and GI compartments, leading to significant prolongation of survival and / or mitigation of radiation-induced cytotoxicity associated with CTCL therapy.

[0191] As an example, a method for preparing a therapeutically effective radioprotective IL-12 formulation was developed.

[0192] Example 1: Exemplary recombinant mouse IL-12 and exemplary recombinant human IL-12 Exemplary recombinant murine IL-12 (e.g., a suitable recombinant murine IL-12 preparation comprising a glycosylated form of recombinant murine IL-12 produced in CHO cells; hereinafter "recombinant murine IL-12") is obtained from Peprotech (Rocky Hill, NJ, USA) or provided exclusively to Neumedicines by SBH Sciences (Natick, MA, USA). Exemplary recombinant human IL-12, i.e., rHuIL-12 (e.g., a suitable recombinant human IL-12 preparation comprising a glycosylated form of recombinant human IL-12 produced in CHO cells; hereinafter "recombinant human IL-12") is obtained from SBH Sciences (Natick, MA, USA). In the initial mouse survival study, typical lyophilized mouse recombinant IL-12 (e.g., recombinant mouse IL-12) was dissolved in phosphate-buffered saline (PBS) pH=7.2. In all other studies, exemplary murine recombinant IL-12 (e.g., recombinant murine IL-12) and exemplary recombinant human IL-12 (e.g., recombinant human IL-12) were dissolved in a trehalose formulation (P5.6TT). In these embodiments, the trehalose formulation formulation was as follows: 200 mL formulation recipe: 186 mL dH2O, 12 g trehalose, 1.6 mL 5% Tween 20, 1.0 g monobasic sodium phosphate anhydrous, 0.24 g dibasic sodium phosphate anhydrous, adjusted to pH 5.6 with 12.1 M HCl. Recombinant murine IL-12 and recombinant human IL-12 were used in studies in mice and rhesus monkeys, respectively. PBS was used as the vehicle for the initial mouse survival study, as indicated. P5.6TT was used as the vehicle for all other studies.

[0193] Example 2: Survival test Mouse survival studies were performed at BATTS Laboratories (Northridge, CA, USA; HHS OLAW A4475-01) or Roy E. Coats Research Laboratories (University of California, Los Angeles, CA, USA; HHS OLAW A3196-01). Mouse bone marrow isolation was performed at BATTS Laboratories. Female C57BL / 6 mice were obtained from The Jackson Laboratory (Sacramento, CA, USA), and male mice were obtained from Harlan Laboratories (Placentia, CA, USA) or bred at Roy E. Coats Research Laboratories (Coats mice). Coats mice are gnobiotic and therefore have lower radiosensitivity than Harlan mice. Differences in radiation doses in experiments using different mice were therefore due to the higher radiation doses used in the Coats mouse experiments. Coats mice were exposed to radiation doses of 8.6, 8.8, and 9.0 Gy in these studies, whereas Harlan mice were exposed to 8 Gy unless otherwise indicated. Mouse pharmacokinetic (PK) and pharmacodynamic (PD) studies and gastrointestinal (GI) tissue isolation were performed at LAB Research, Inc. (Laval, Quebec, Canada; HHS OLAW A5525-01). Male C57BL / 6 mice were obtained from Charles River Canada, Inc. (Saint-Constant, Quebec, Canada). For radiation-related PK / PD studies, Charles River mice were exposed to 8.6 Gy TBI (LD100 / 30). At all study sites, mice were housed in quarantine for at least one week. Mice used for survival and PK / PD studies were 9 to 10 weeks old, weighed approximately 20 g, and showed no signs of disease.

[0194] Assessment of survival rate On day 0, TBI was administered using a Gammacell® 40 (Theratronics, Ontario, Canada) equipped with a 137Cs source in a specially constructed "pie box" designed to hold the mouse in the center of the irradiator for uniform radiation distribution. The dose was 71 cGy / min in the Coats mouse study and 85 cGy / min in the Harlan mouse study. This dose was expected to cause approximately 90% death within 30 days. Mice received subcutaneous injections of vehicle or the indicated doses of recombinant murine IL-12 at 24, 48, and / or 72 hours after irradiation. Mice were monitored for survival until day 30. During this period, mice were deprived of all supportive care, including antibiotics, to enhance the stringency of the survival protocol. Mice had free access to food and acidified water.

[0195] The radiation dose dependence of the effect of recombinant murine IL-12 was evaluated in mice (n=10 per group; Coats mice). Mice were irradiated with lethal doses of approximately 8.6 Gy, 8.8 Gy, and 9.0 Gy, resulting in LD70 / 30, LD90 / 30, and LD100 / 30, respectively. Animals were administered vehicle or recombinant murine IL-12 at a dose of 20 ng / mouse 24 hours after TBI. Mice were monitored for survival until day 30. Supportive care, including antibiotics, was not allowed during this period. Mice had free access to food and acidified water.

[0196] Example 3: Plasma PK and PD of recombinant murine IL-12 in irradiated and non-irradiated subjects As an example, a method was developed to assess the plasma PK and PD of recombinant murine IL-12 in irradiated and non-irradiated subjects.

[0197] Mice (n = 3 per group) were subcutaneously administered recombinant murine IL-12 at doses of 10 ng / mouse, 20 ng / mouse, 40 ng / mouse, or 200 ng / mouse in the absence of irradiation or 24 h after TBI at LD100 / 30 (8.6 Gy; Charles River mice). Two additional control groups of animals (n = 3 per group) that did not receive recombinant murine IL-12 were either not exposed to radiation or irradiated at 8.6 Gy. Concentrations of recombinant murine IL-12 and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA) in plasma from blood samples collected 45 min and 1.5, 3, 6, 12, 24, 48, and 72 h after administration of recombinant murine IL-12. Plasma erythropoietin (EPO) levels were measured only at 12 h due to limited sample availability.

[0198] Example 4: Bone marrow and GI histopathology As an example, methods were developed to assess bone marrow and GI histopathology.

[0199] For histopathological examination of bone marrow, mice (n = 2 per group) were subjected to 8.0 Gy TBI (Harlan mice, approximately LD40 / 30 in this experiment) and then subcutaneously administered vehicle (P5.6TT) or recombinant murine IL-12 (20 ng / mouse) at (a) 24 hours, (b) 24 hours and 2 days, (c) 24 hours and 3 days, (d) 24 hours and 4 days, or (e) 24 hours and 5 days after irradiation. An additional group of mice (n = 2) received recombinant human IL-12 at 24 hours after TBI. Mice were sacrificed 12 days after irradiation, and femoral bone marrow was provided as paraffin-embedded tissue sections by Cyto-Pathology Diagnostic Center, Inc. (Duarte, CA, USA).

[0200] For GI histopathology, mice (n = 3 per group) were subcutaneously administered vehicle (P5.6TT) or recombinant murine IL-12 at doses of 10 ng / mouse to 200 ng / mouse in the absence of irradiation or 24 h after 8.6 Gy TBI (Charles River mice, LD100 / 30). Mice were sacrificed 3 days after irradiation, and the jejunum was provided as paraffin-embedded tissue sections by Cytopathology Diagnostics Center, Inc. (Duarte, CA, USA).

[0201] Tissue sections were deparaffinized with xylene, rehydrated with decreasing concentrations of ethanol, and subjected to heat-induced epitope retrieval (HIER) to retrieve antigens. Endogenous peroxidase was inhibited with 0.3% H2O2, and background staining was blocked with Background Sniper (Biocare Medical, LLC.; Concord, CA).

[0202] For bone marrow histopathology, tissue sections were incubated with rabbit anti-mouse IL-12 receptor beta 2 subunit (IL-12Rβ2) (Sigma; St. Louis, MO), rabbit anti-mouse osteocalcin (Millipore; Billerica, MA), a marker for osteoblasts, or rabbit anti-mouse Sca-1 (Epitomics; Burlingame, CA), a marker for hematopoietic stem cells. For GI histopathology, sections were incubated with rabbit anti-mouse IL-12Rβ2 or rabbit anti-mouse leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a GI stem cell marker expressed during GI injury. After removal of the primary antibody, tissue sections were incubated with peroxidase-conjugated anti-rabbit IgG (ImmPRESS; Vector Laboratories; Burlingame, CA). After incubation with AEC substrate (ImmPACT AEC; Vector Laboratories; Burlingame, CA), peroxidase-labeled cells developed a red color, which was counterstained with CAT hematoxylin (Biocare Medical, Concord, CA). Tissue sections were then immersed in Vectamount (Vector Laboratories; Burlingame, CA), coverslipped, sealed with clear nail polish, and visualized using an Olympus Compound microscope (Olympus America, Inc; Center Valley, PA) at 100x magnification for bone marrow sections and 400x magnification for jejunum.

[0203] Coexpression of Sca-1 and IL-12Rβ2 on hematopoietic stem cells was assessed by first incubating bone marrow tissue sections with rabbit anti-mouse Sca-1 (Epitomics, Burlingame, CA) followed by incubation with rabbit-on-rodent HRP-polymer (Biocare Medical; Concord, CA) and 3,3'-diaminobenzidine substrate (Biocare Medical, Concord, CA). After treatment with denaturing solution (Biocare Medical, Concord, CA), tissue sections were incubated with rabbit anti-mouse IL-12Rβ2 (Sigma; St Louis, MO) followed by incubation with rabbit-on-rodent AP-polymer (Biocare Medical; Concord, CA) and Warp Red substrate (Biocare Medical, Concord, CA). Tissue sections were then counterstained with CAT hematoxylin and visualized as described above. Using this method, cells that expressed Sca-1 and IL-12Rβ2 stained brown and pink, respectively.

[0204] Example 5: Evaluation using non-human primates (NHPs) Male rhesus monkeys (Macaca mulatta) were purchased from Worldwide Primates, Inc. (Miami, FL, USA). Animals weighing 3.5–5.8 kg were 3–4 years old and allowed to acclimate for at least 7 weeks. All rhesus monkeys included in the study were in good health by physical examination, tested negative for herpes B virus, simian immunodeficiency virus, simian T-lymphotropic virus, and simian retrovirus, and were vaccinated against hepatitis A and measles. Animals were individually housed in stainless steel monkey cages equipped with an automated water supply system. The animal room environment was continuously controlled for temperature (21 ± 3°C), humidity (30%–70%), lighting cycle (12 h on, 12 h off), and ventilation (10–15 air changes / h). Standard, certified, commercially available primate chow was provided to each monkey twice daily. An overnight fast was administered prior to irradiation and necropsy. Animals were habituated to the various procedures using positive reinforcement before the start of the study. Animal health was thoroughly assessed to ensure they were in good condition for the study. All animals received prophylactic analgesia (buprenorphine) from day 5 until the end of the study. Specific euthanasia criteria were included in each experimental protocol to minimize suffering. Continuous clinical care (24 / 7) was provided to ensure prompt intervention if necessary. A team of technicians and veterinarians trained in NHP medicine was responsible for clinical monitoring and provided state-of-the-art medical care.

[0205] Example 6: Allometric Dose Conversion from Mouse to Rhesus Monkey Doses of recombinant murine IL-12 found to be effective against lethal TBI in mice were converted to their equivalent doses in rhesus monkeys based on body surface area. The pharmacokinetic equivalence of the doses was assessed with respect to recombinant human IL-12 stimulation of IFN-γ secretion from peripheral blood mononuclear cells (PBMCs) in vitro and the PK and PD properties of recombinant human IL-12 in vivo.

[0206] Example 7: Isolation of CD14-negative PBMCs and quantification of IFN-γ secretion Human PBMCs collected by apheresis were purchased from AllCells (Emeryville, CA, USA). Mouse and rhesus macaque PBMCs were purchased from Bioreclamation (Liverpool, NY, USA). CD14-negative PBMCs were isolated as follows: Erythrocytes were removed from human PBMCs by a one-step gradient using Ficoll-Hypaque Premium (density = 1.077; GE Healthcare Lifesciences; Piscataway, NJ, USA) and from rhesus and mouse PBMCs by lysis using ACK lysis buffer (Invitrogen; Carlsbad, CA, USA). To isolate IL-12-secreting endogenous monocyte populations, human and rhesus macaque PBMCs were labeled with mouse anti-human CD14PE antibody (AbD Serotec; Raleigh, NC, USA), and mouse PBMCs were labeled with mouse anti-mouse CD14PE antibody (AbD Serotec; Raleigh, NC, USA). Excess antibody was removed, and the cells were incubated with anti-PE antibody-conjugated magnetic beads (Miltenyi Biotec; Auburn, CA, USA). After removal of excess antibody, CD14-positive cells were captured by adsorption onto an LD column (Miltenyi Biotec; Auburn, CA, USA) immobilized on a magnetic field (Quadro MACS®; Miltenyi Biotec; Auburn, CA, USA). The flow-through CD14-negative cells were collected and resuspended in cold fetal bovine serum (FBS) containing 20% ​​dimethyl sulfoxide at a density of 14 × 10 cells / ml from human cells, whereas those from rhesus monkeys and mice were resuspended in RPMI medium containing 10% FBS and antibiotics at a density of 2.14 × 10 cells / ml from rhesus monkeys and mice. IFN-γ was quantified by ELISA in supernatants from 2.5 × 105 human, rhesus monkey, or mouse CD14-negative PBMCs incubated with various concentrations (range: 0–1000 pM) of recombinant human IL-12 or recombinant mouse IL-12 for 16 h at 37°C. All experiments were performed in triplicate.The half-maximal effective concentration (EC50) of IL-12 for stimulating IFN-γ secretion was calculated using a four-parameter logistic fit with SoftMax Pro® software version 3.1 (Molecular Devices; Sunnyvale, CA, USA).

[0207] Example 8: Plasma PK and PD of recombinant human IL-12 in NHPs Recombinant human IL-12 was administered subcutaneously to non-irradiated rhesus monkeys at a dose of 250 ng / kg (n=3) or 1000 ng / kg (n=3). Concentrations of recombinant human IL-12, IFN-γ, and other potential biomarkers of recombinant human IL-12 were measured by ELISA in plasma samples collected before administration of recombinant human IL-12 and at 2, 6, 12, 18, 24, 30, 36, 48, 72, 96, 120, 144, and 168 hours after administration of recombinant human IL-12.

[0208] Example 9: IL-12Rbeta2 expression in NHP and human bone marrow and small intestine Paraffin-embedded tissue sections of NHP and human femoral bone marrow and jejunum / ileum were obtained from Biomax, Inc. (Rockville, MD). NHP and human tissue sections were immunohistochemically stained for IL-12Rβ2 using rabbit anti-human IL-12Rβ2 according to the procedure described in the Mouse Histopathology Study section.

[0209] Example 10: Survival studies in NHPs On day 0, rhesus monkeys habituated to the restraint procedure through positive reinforcement were subjected to a LD50 / 30 dose of 6.7 Gy TBI. Irradiation was administered in two half-dose fractions (ventrodorsal and dorsoventral) at a rate of 55 cGy / min using a cobalt-60 unit (Theratron 780; Theratronics; Ontario, Canada). Irradiation dose was monitored using two dosimeters (Thermoluminescent or NanoDot dosimeter; Landauer Inc.; Glenwood, IL, USA) placed at the corresponding levels of the sternal tip and interscapular region of each animal. After TBI, animals were randomly assigned to receive subcutaneous administration of (a) vehicle (n = 8) at 24 hours after TBI, (b) 100 ng / kg recombinant human IL-12 at 24 hours after TBI (n = 8), (c) 100 ng / kg recombinant human IL-12 at 24 hours and 7 days after TBI (n = 8), (d) 250 ng / kg recombinant human IL-12 at 24 hours after TBI (n = 8), or (e) 250 ng / kg recombinant human IL-12 at 24 hours and 7 days after TBI (n = 8). Animals were monitored for survival and clinical and physical characteristics up to day 30. The primary endpoint was percent survival. Peripheral blood counts, body weight, and clinical signs were assessed as secondary endpoints.

[0210] Blood transfusions and antibiotic use were prohibited during the study. Evidence of pain or discomfort was treated with intramuscular buprenorphine (0.01 mg / kg to 0.05 mg / kg at least every 8 hours). Nutritional support (e.g., liquid feed) was provided if animals exhibited decreased appetite. Clinical signs were monitored at least twice daily, and complete blood counts and body weights were monitored every other day during the study. Hematology samples were analyzed using an automated hematology analyzer (Advia 120; Bayer Diagnostics; Tarrytown, NY, USA). During the study, animals were euthanized if they exhibited respiratory distress, anorexia / hypophagia (complete anorexia for 3 days), weight loss (>20% of baseline weight at 72 hours), unresponsiveness to touch, acute gross blood loss, generalized seizures, or abnormal vital signs. Euthanized animals or those found dead were subjected to a complete gross necropsy, including bacteriological examination. All animals were euthanized at the end of the study on day 31.

[0211] Example 11: Quantification of recombinant murine IL-12 and recombinant human IL-12 and their biomarkers in plasma Blood samples from mice and rhesus monkeys were collected into tubes containing ethylenediaminetetraacetic acid and kept on ice (<30 min) until centrifugation. Samples were centrifuged at 1500 x g for 10 min at 4°C. Plasma was aliquoted and stored at -70°C until use. Recombinant mouse IL-12, recombinant human IL-12, and their potential biomarkers in plasma were assayed by ELISA. ELISA kits for mouse IL-12 (p70) and IFN-γ were obtained from BioLegend (San Diego, CA, USA); those for NHP IL-12 were obtained from BioLegend (San Diego, CA, USA), Mab Tech (Mariemont, OH, USA), and R&D Systems (Minneapolis, MN, USA); those for NHP IFN-γ were obtained from Mab Tech (Mariemont, OH, USA); those for human EPO, IL-18, and IL-15 were obtained from R&D Systems (Minneapolis, MN, USA); and those for neopterin were obtained from GenWay (San Diego, CA, USA). All assays were performed in triplicate according to the manufacturer's instructions, except for the NHP IL-12 assay, in which an in-house reference standard was used instead of the standard provided by the manufacturer.

[0212] statistical analysis Data are expressed as mean ± standard error (SE). Intergroup differences in survival were assessed by Kaplan-Meier survival analysis followed by the Mantel-Cox test for survival time and the Pearson chi-square test for percent survival. Intergroup differences in blood counts were analyzed by the Pearson chi-square test. Variance was consistent except for platelet counts that fell below the transfusion level of 20,000 platelets / μl. The differences between groups in clinical symptoms were evaluated by ANOVA. A P value of <0.05 was defined as the level of statistical significance.

[0213] Example 12: A single low dose of recombinant murine IL-12 administered 24 hours after TBI extended survival in irradiated mice In the first study, 87.5% of mice receiving a nominal dose of 100 ng / mouse recombinant murine IL-12 subcutaneously at 24 and 72 hours after TBI survived to day 30 after 8 Gy, whereas only 14% of vehicle mice survived to day 30 after lethal TBI (P < 0.005) (Fig. 1a). The actual dose of recombinant murine IL-12 delivered in these studies was 10 ng / mouse. Subsequent studies evaluated whether a single dose of recombinant murine IL-12 was sufficient to provide a similar radiation mitigation effect. In these studies, a single nominal dose of recombinant murine IL-12 (300 ng / mouse; the actual delivered dose was 20–30 ng / mouse) significantly prolonged survival when administered at 24 h (P = 0.001), 48 h (P = 0.02), or 72 h (P < 0.03) after 9 Gy TBI, yielding an LD100 / 30 (Figure 1b). Mice treated with recombinant murine IL-12 showed higher survival rates when recombinant murine IL-12 was administered at 24 h compared with 48 h after TBI (Figure 1b). The difference in survival between the vehicle group and mice treated with recombinant murine IL-12 at 24 h after TBI was statistically significant (0% vs. 60%, respectively; P < 0.05) (Figure 1b).

[0214] Recombinant murine IL-12 was reconstituted with P5.6TT, which increased dose delivery to nearly 90% of the intended dose. This improvement resulted in a single recombinant murine IL-12 dose of 2 ng / mouse or 18 ng / mouse providing significantly greater radiation mitigation than vehicle for a TBI dose of 7.9 Gy, which resulted in an LD85 / 30 when administered 24 h post-irradiation (Fig. 1c). At the 2 ng / mouse dose, recombinant murine IL-12 significantly increased survival (P < 0.02) and slightly prolonged survival time (P = 0.07) compared with vehicle. At the 18 ng / mouse dose, recombinant murine IL-12 significantly increased both survival (P < 0.005) and survival time (P < 0.03) compared with vehicle. Animals treated with higher doses of recombinant murine IL-12, such as 160 ng / mouse, had slightly longer survival times compared to the vehicle group, but had lower survival rates compared to animals treated with doses of 2 ng / mouse or 18 ng / mouse (data not shown). Thus, these findings indicate that a dose of approximately 20 ng / mouse is the optimal effective amount of recombinant murine IL-12 to increase survival.

[0215] To assess the relationship between radiation dose and percent survival after treatment with recombinant murine IL-12, three increasing doses of radiation (8.6, 8.8, and 9.0 Gy, corresponding to the resulting LD70 / 30, LD90 / 30, and LD100 / 30, respectively) were tested in mice. Recombinant murine IL-12 at a dose of 20 ng / mouse administered 24 hours after TBI significantly reduced survival time at all three levels of radiation intensity. The survival rate in vehicle-treated animals was significantly increased after 8.6 Gy (LD70 / 3 The survival rates were 20% at 8.8 Gy (LD90 / 30), 10% at 8.8 Gy (LD90 / 30), and 0% at 9.0 Gy (LD100 / 30) (Figure 2). Compared with the vehicle group, treatment with recombinant murine IL-12 resulted in significantly higher survival rates of 80% at LD70 / 30, 60% at LD90 / 30, and 70% at LD100 / 30 (all P < 0.05) (Figure 2), demonstrating radiation dose-dependence for administration of recombinant murine IL-12 at 24 hours post-TBI within the selected window of radiation exposure. Notably, the comparable survival rates following administration of a single fixed dose of recombinant murine IL-12 under increasing radiation doses indicate that the efficacy of recombinant murine IL-12 does not decrease with increasing radiation dose. These data suggest that at radiation doses where immune, bone marrow, and GI damage overlap, recombinant murine IL-12 may result in attenuation of damage in all three radiosensitive tissues, thereby resulting in increased survival that is relatively independent of radiation dose within a specific window of exposure.

[0216] Example 13: Plasma PK and PD of recombinant murine IL-12 in irradiated and non-irradiated mice Plasma concentrations of recombinant murine IL-12 and IFN-γ were measured over 72 hours in two groups of mice administered increasing doses of recombinant murine IL-12 (10 ng / mouse to 200 ng / mouse) in the absence of irradiation or 24 hours after approximately the LD90 / 30 TBI (8.6 Gy). Due to the limitations of recombinant murine IL-12 detection, doses of recombinant murine IL-12 lower than 10 ng / mouse were not evaluated. Recombinant murine IL-12 was detected in the plasma of all animals administered recombinant murine IL-12 (Figure 3), but importantly, it was undetectable in plasma samples from mice not administered recombinant murine IL-12, regardless of the presence or absence of irradiation (data not shown).

[0217] Exposure to recombinant murine IL-12 (area under the curve last; AUClast) increased dose-proportionally from 10 ng / mouse to 40 ng / mouse, regardless of the presence or absence of irradiation (Figure 3 and Table 1).

[0218] [Table 1]

[0219] Interestingly, the maximum plasma concentration (Cmax) of recombinant murine IL-12 was consistently higher in irradiated mice compared to non-irradiated mice at all doses (Figure 3). The exposure (AUClast) to recombinant murine IL-12 at a dose of 200 ng / mouse was disproportionately higher than at lower doses (10 ng / mouse to 40 ng / mouse). This suggests that the PK characteristics of recombinant murine IL-12 are nonlinear at higher doses (Table 1). At doses ranging from 10 ng / mouse to 40 ng / mouse, recombinant murine IL-12 reached Cmax between 3 and 6 hours and was excreted with a half-life of approximately 4 hours (Table 1).

[0220] Administration of recombinant murine IL-12 increased plasma IFN-γ concentrations with a consistent lag time at all doses tested (Figure 3). Importantly, IFN-γ production was not eliminated in irradiated mice (Figure 3). In fact, for all doses of recombinant murine IL-12 except the optimal dose of 20 ng / mouse, plasma IFN-γ levels were higher in irradiated mice compared to non-irradiated mice (Figure 3). Exposure to IFN-γ dose increased proportionally as a function of increasing recombinant murine IL-12 doses from 10 ng / mouse to 200 ng / mouse (data not shown). Importantly, IFN-γ was not detected in the plasma of mice that did not receive recombinant murine IL-12, regardless of the presence or absence of irradiation.

[0221] Because preliminary studies showed that combined administration of recombinant murine IL-12 and EPO in a specific dosing regimen resulted in a substantial increase in survival after lethal radiation exposure (data not shown), we sought to evaluate whether recombinant murine IL-12 could affect plasma EPO levels in irradiated and non-irradiated mice. Due to limited sample availability, plasma EPO levels could only be measured at one early time point, 12 hours after administration of recombinant murine IL-12 (Figure 4). In non-irradiated, untreated animals, EPO was detectable in plasma in the low pg / mL range (Figure 4). Irradiation induced a near-linear increase in plasma EPO levels up to 80 hours after TBI, suggesting that EPO is part of the physiological response to radiation injury (data not shown). However, it is noteworthy that at the optimal dose of 20 ng / mouse 12 hours after administration (36 hours after radiation exposure), recombinant mouse IL-12 substantially increased plasma EPO concentrations above radiation-induced levels (Figure 4), indicating that recombinant mouse IL-12 enhances EPO-mediated physiological responses to radiation, but only at or near the optimal dose level. It is noteworthy that at this optimal dose, plasma EPO levels were also increased in non-irradiated mice (Figure 4). Whether the EPO response to recombinant mouse IL-12 administration occurs within a narrow window within the recombinant mouse IL-12 dose range remains to be evaluated, since a highly enhanced EPO response was observed only after administration of a dose of 20 ng / mouse (Figure 4). It is interesting to note that the IFN-γ response appeared to be suppressed at 20 ng / mouse of recombinant mouse IL-12, the dose at which EPO was upregulated by recombinant mouse IL-12, compared with the other doses evaluated. In a mouse model of multiple sclerosis, administration of EPO was reported to downregulate the inflammatory response and, in particular, suppress IFN-γ.Therefore, these findings suggest that increased plasma EPO levels may play a role in the suppression of plasma IFN-γ levels in irradiated mice receiving recombinant murine IL-12 at a dose of 20 ng / mouse (Fig. 3b), leading to a reduced inflammatory response to radiation.

[0222] Other biomarkers of recombinant murine IL-12 administration, namely tumor necrosis factor alpha (TNF-α) and stem cell factor (SCF), were also screened, but plasma levels of these factors were found to be below the limit of quantification.

[0223] Example 14 Administration of Recombinant Murine IL-12 24 Hours After TBI Ameliorates Radiation-Induced Damage in Mouse Bone Marrow and Small Intestine Femoral bone marrow from irradiated mice treated with vehicle or recombinant murine IL-12 at least 24 hours after TBI (LD30 / 30) was stained for IL-12Rβ2 and evaluated for histological signs of recovery from radiation-induced damage on day 12 after TBI. In contrast, bone marrow from unirradiated, untreated mice was characterized by the presence of IL-12Rβ2-expressing hematopoietic stem cells, immature megakaryocytes with segmented nuclei surrounded by a narrow rim of cytoplasm, mature megakaryocytes with segmented nuclei and abundant cytoplasm, and myeloid progenitor cells at the metamyelocytic stage, as identified by co-staining with Sca-1 (a mouse stem cell marker; see below) (FIG. 5a).

[0224] Bone marrow from mice treated with vehicle alone and subjected to LD30 / 30 TBI was characterized by minimal signs of hematopoietic regeneration and a complete lack of IL-12Rβ2-expressing cells 12 days after irradiation (Figure 5b). In contrast, mice treated with various dose regimens of recombinant murine IL-12 showed various stages of hematopoietic reconstitution characterized by the presence of IL-12Rβ2-expressing myeloid progenitors, megakaryocytes, and osteoblasts (Figure 5c-f). Mice treated with recombinant human IL-12, which was demonstrated not to cross-react with the murine IL-12 receptor, showed some signs of regeneration but lacked megakaryocytes (Figure 5g). However, no increased survival was observed for mice treated with recombinant human IL-12 compared to the vehicle control group.

[0225] To further assess whether the morphologically identified cells were indeed hematopoietic stem cells and osteoblasts, bone marrow tissue sections were stained for the corresponding markers, Sca-1 and osteocalcin, respectively. As shown in Figures 6a and 6b, IL-12Rβ2 expression was observed on cells morphologically identified as hematopoietic stem cells and osteoblasts, which expressed Sca-1 and osteocalcin, respectively. Coexpression of IL-12Rβ2 and Sca-1 in bone marrow tissue sections was also assessed by a double-staining approach. As shown in Figure 6c, distinct subsets of hematopoietic stem cells were co-stained for the presence of both IL-12Rβ2 and Sca-1. Immature and mature megakaryocytes expressing IL-12Rβ2 were also observed in bone marrow tissue sections (Figure 6c). These findings suggest a direct role for the IL-12 signaling pathway in hematopoietic reconstitution.

[0226] Similar to hematopoietic stem cells and osteoblasts in femoral bone marrow, mouse jejunal crypts expressed IL-12Rβ2 (Figure 7a). In the absence of irradiation, administration of recombinant mouse IL-12 at doses up to 200 ng / mouse did not induce damage in jejunal crypts (Figure 7b, upper panel). However, exposure to TBI (8.6 Gy) resulted in substantial jejunal crypt damage 3 days after irradiation, as evidenced by widespread expression of LGR5, a GI stem cell marker shown to be expressed during chemotherapy-induced GI injury. Notably, administration of recombinant mouse IL-12 at low doses ranging from 10 ng / mouse to 40 ng / mouse attenuated radiation-induced jejunal injury in a dose-dependent manner, with no LGR5 expression observed at the optimally effective dose of 20 ng / mouse (Figure 7b, lower panel). On the other hand, administration of recombinant mouse IL-12 at a high dose of 200 ng / mouse exacerbated jejunal damage (Figure 7b, lower panel). As observed for the dose range of recombinant murine IL-12 that results in the optimal increase in survival, these data indicate a window of potential for mitigation of radiation injury by recombinant murine IL-12 at very low doses of the drug that are also effective in mitigating bone marrow injury.

[0227] Example 15: Allometric Dose Conversion from Mouse to NHP To achieve similar radiation mitigation effects in rhesus monkeys, a dose pharmacologically equivalent to that administered to mice should be administered to rhesus monkeys. Based on Food and Drug Administration (FDA) guidelines, the optimal 20 ng / mouse dose (1000 ng / kg) and non-optimal 80 ng / mouse dose (4000 ng / kg) in mice are converted to doses of 250 ng / kg and 1000 ng / kg in rhesus monkeys, respectively. However, the induction of a pharmacologically equivalent response at a species-specific equivalent dose depends on several factors, including similar drug exposure and specific reactivity with the primary target site in both species. Therefore, before evaluating the radiomitigation efficacy of recombinant human IL-12 in NHPs, we first examined the pharmacological equivalence of species-specific equivalent doses.

[0228] Example 16: Recombinant human IL-12 and recombinant mouse IL-12 potently stimulated IFN-γ secretion from human, rhesus monkey, and mouse CD4-negative PBMCs in vitro Target responsiveness to recombinant human IL-12 was assessed by comparing the EC50 values ​​of recombinant human IL-12 and recombinant mouse IL-12 for stimulating IFN-γ secretion from CD4-negative PBMCs. As previously reported

[33] , we observed that recombinant human IL-12 did not cross-react with PBMCs isolated from mice and rats (EC50 > 1000 pM). In contrast, recombinant human IL-12 potently stimulated IFN-γ secretion from both human and rhesus macaque PBMCs, with EC50 values ​​of 2.51 ± 0.51 pM and 1.05 ± 0.10 pM, respectively. The EC50 value of recombinant mouse IL-12 for stimulating IFN-γ secretion from mouse PBMCs was 0.35 ± 0.29 pM. These findings suggest that the responsiveness of monkey and mouse PBMCs to recombinant human IL-12 and recombinant mouse IL-12, respectively, is similar in terms of IFN-γ secretion in vitro.

[0229] Plasma PK of recombinant human IL-12 in rhesus monkeys. The plasma PK of recombinant human IL-12 was investigated in rhesus monkeys after single administration of two doses of recombinant human IL-12, 250 ng / kg and 1000 ng / kg, in the absence of irradiation. After administration, exposure to recombinant human IL-12 (AUClast) increased in a dose-proportional manner (Table 2).

[0230] [Table 2]

[0231] The AUClast of recombinant human IL-12 in rhesus monkeys was perfectly linearly superimposable to the AUClast of recombinant murine IL-12 in mice over the dose range of 10 ng / mouse to 80 ng / mouse (Figure 8), suggesting that the species-specific equivalent doses calculated from the mouse studies resulted in similar drug exposure in monkeys. The 200 ng / mouse dose was not used in this analysis because recombinant murine IL-12 appeared to exhibit different PK characteristics at higher doses (Table 1).

[0232] Recombinant human IL-12 at a single dose of 250 ng / kg or 1000 ng / kg was well tolerated and was not associated with any overt signs of toxicity, except for a transient decrease in appetite in the 1000 ng / kg group.

[0233] Example 16: Administration of recombinant human IL-12 increased plasma concentrations of IFN-γ, IL-15, IL-18, neopterin, and EPO in non-irradiated rhesus monkeys. In monkeys, subcutaneously administered recombinant human IL-12 appeared in plasma immediately after administration and was not detectable after 72 hours (Fig. 9a). Furthermore, as observed in mice with recombinant murine IL-12, recombinant human IL-12 was found to increase plasma IFN-γ concentrations in a dose-proportional manner (Fig. 9a). However, the temporal kinetics of the IFN-γ response in rhesus monkeys differed from that in mice in that the IFN-γ response was delayed for a longer period and was much greater in magnitude (Fig. 9a). Neither recombinant human IL-12 nor IFN-γ was detected in the plasma of monkeys that did not receive recombinant human IL-12.

[0234] Among other potential biomarkers, IL-18 and EPO exposure (AUClast) increased 2.4-fold and 5.1-fold, respectively, when the recombinant human IL-12 dose was increased from 250 ng / kg to 1000 ng / kg (Fig. 9b). Recombinant human IL-12 also increased plasma IL-15 and neopterin concentrations, peaking at 72 and 96 hours, respectively, after administration of recombinant human IL-12 (Fig. 9c). In contrast to previous reports in humans, plasma concentrations of TNF-α and IL-10 were unchanged in rhesus macaques.

[0235] Example 17: NHPs and human bone marrow and small intestine express IL-12R2 IL-12Rβ2 expression was assessed by immunohistochemistry in non-irradiated NHP (rhesus macaque) and human femoral bone marrow and jejunum / ileum. As shown in Figure 10A, NHP and human progenitor cells and megakaryocytes expressed IL-12Rβ2. IL-12Rβ2 expression was also observed on osteoblasts / osteoclasts in bone marrow. However, because the tissues provided were smears and did not contain periosteum or other bone tissue, it was not possible to determine whether these cells were osteoblasts and / or osteoclasts. Bone marrow adipocytes did not stain positive for IL-12Rβ2.

[0236] In the small intestine, IL-12Rβ2 was most commonly expressed in crypts (Fig. 10b). It is unknown whether IL-12Rβ2 expression in intestinal crypts is localized to Paneth cells, pluripotent stem cells, or both. IL-12Rβ2 expression was also observed in lymphoid cells present in the lamina propria and submucosal regions (Fig. 10b). Mucin-secreting goblet cells did not express IL-12Rβ2. Both crypt and lamina propria IL-12Rβ2-expressing cells may represent multifunctional mesenchyme-derived myofibroblasts that can function as crypt-shaping cells that also occupy the stem cell niche and serve as nonprofessional antigen-presenting cells to immunoregulatory cells in the lamina propria. Further studies will establish the cellular and functional characteristics of IL-12Rβ2-expressing cells in intestinal crypts and their supportive role in intestinal regeneration after radiation exposure.

[0237] Example 18: Administration of recombinant human IL-12 increased survival in irradiated non-supportive rhesus monkeys In a pilot study of 40 animals, the survival rates of rhesus monkeys exposed to an LD50 / 30 TBI (6.7 Gy) were measured after treatment with 100 ng / kg or 250 ng / kg recombinant human IL-12 administered 24 hours or 24 hours and 7 days after TBI. This study was conducted in the absence of supportive care, including antibiotics. The doses of recombinant human IL-12 were selected based on PK / PD studies in rhesus monkeys and were equivalent to recombinant mouse IL-12 doses of 8 ng / mouse and 20 ng / mouse, respectively. As shown in Figure 11a, both doses of recombinant human IL-12 suppressed radiation-induced mortality to a similar extent after single or double administration. Overall survival rates were 71% in the 100 ng / kg single-dose treatment group (n = 7) and 75% in all other groups receiving recombinant human IL-12 (n = 8), compared with 50% in the vehicle group. The difference in survival between groups was not statistically significant, most likely due to the small number of animals in each group (n = 8), but also possibly because both recombinant human IL-12 doses were within the effective range. However, analysis of survival across recombinant human IL-12 dosing regimens showed that, when pooled together, monkeys treated with recombinant human IL-12 had significantly higher survival rates than monkeys given vehicle (75% vs. 50%, respectively; P = 0.05) (Fig. 11b).

[0238] Example 19: Changes in blood counts in irradiated non-supporting rhesus monkeys after administration of recombinant human IL-12 Three analyses were performed to assess differences in blood counts over the course of the study. Blood counts were analyzed from day 1 to day 30. In the first analysis, animals treated with recombinant human IL-12 had significantly higher white blood cell and platelet counts near the nadir on days 12 and 14 at the 100 ng / kg and 250 ng / kg doses compared to vehicle-treated animals (Figure 12).

[0239] In a second analysis, in which blood counts were analyzed from day 1 to day 14, the day the animals died, recombinant human IL-12-treated animals had higher platelet counts at nadir (days 12-14) compared with vehicle-treated animals (P = 0.079 for the 250 ng / kg group and P = 0.02 for the 100 ng / kg twice-treated group). Furthermore, compared with the vehicle group, recombinant human IL-12-treated animals had significantly higher numbers of white blood cells (P < 0.01 for the 250 ng / kg group and P < 0.04 for the 100 ng / kg twice-treated group) and reticulocytes (P < 0.04 for the 250 ng / kg group and P < 0.001 for the 100 ng / kg group) at nadir (days 12-14). The same trends were evident for neutrophil, basophil and lymphocyte counts, but they did not reach an acceptable level of statistical significance.

[0240] In a third analysis, we assessed the number of animals that achieved clinically low platelet counts during the study. This analysis revealed a notable difference between the vehicle and recombinant human IL-12 groups in platelet counts that fell below the threshold level of 20,000 platelets / μL, a level that typically requires platelet transfusion. In the recombinant human IL-12 250 ng / kg group, 4 of 16 animals (25%) had platelet counts that fell below the transfusion threshold of <20,000 platelets / μL at nadir (days 12–14), whereas 12 of 15 vehicle animals (80%) had platelet counts below the threshold level during the same period (P=0.007).

[0241] Overall, these findings indicate that recombinant human IL-12 increases white blood cells, platelets, and reticulocytes just before the day animals began to die from radiation toxicity (day 13, Figure 11a). Interestingly, vehicle-treated animals that survived to day 30 also showed a rapid recovery in blood counts that was statistically indistinguishable from those in the recombinant human IL-12 group. These findings suggest that death may occur in animals that do not show a robust recovery in blood counts near the nadir. The validity of this hypothesis was assessed by comparing blood counts of animals stratified by mortality status, i.e., animals that survived to day 30 with those that died after day 12. For this analysis, blood counts on the day before death were taken as those of animals that died after day 12. The comparison date for surviving animals in each group was the average day of death for the individual group (days 14–18). This analysis demonstrated that, regardless of the specific treatment group, animals that survived to day 30 had significantly higher numbers of platelets, neutrophils, leukocytes, reticulocytes, and lymphocytes than animals that died after day 12 (P < 0.001 to P < 0.05). When compared by treatment group, animals treated with 100 ng / kg recombinant human IL-12 had significantly higher numbers of neutrophils, leukocytes, and lymphocytes than vehicle-treated animals in both the survivor and nonsurvivor groups (P < 0.001 for all three cell types). Furthermore, animals treated with 100 ng / kg recombinant human IL-12 had numerically higher platelet and reticulocyte counts. These findings suggest that recombinant human IL-12-induced increases in blood cell counts near the nadir may play an important role in promoting survival after radiation exposure.

[0242] Example 20: Clinical and physical characteristics of irradiated non-supportive rhesus monkeys after administration of recombinant human IL-12 Animals administered recombinant human IL-12 at a dose of 100 ng / kg (once or twice) had consistently higher mean body weights from days 14 to 30 than the vehicle group (Fig. 13a). Animals treated with recombinant human IL-12 at a dose of 100 ng / kg (once or twice) or 250 ng / kg (once) had less weight loss from days 14 to 30 than vehicle-treated animals (Fig. 13c and d). Although the between-group differences in body weight and weight loss were not statistically significant, when the analysis of weight loss was limited to day 12 (approximately the day of the nadir of blood counts and the day after animals began to die (Fig. 11a)), pooled recombinant human IL-12-treated animals had significantly less weight loss than vehicle-treated animals (95.3 ± 0.8% vs. 91.6 ± 1.5%, respectively; P = 0.04). Logistic regression showed that weight loss after day 12 was a strong predictor of survival (P<0.001). Appetite and physical activity improved in recombinant human IL-12-treated animals, and the incidence of diarrhea and black or red stools was reduced in the 250 ng / kg twice-dosing regimen group; however, other clinical signs (appetite, physical activity, diarrhea, and stool color) were not significantly different from the vehicle group. However, the above-mentioned clinical signs predicted death after day 12 by logistic regression (P=0.002 for decreased appetite, P<0.001 for decreased physical activity, P=0.04 for the incidence of diarrhea, and P=0.008 for the incidence of black or red stool). Clinical signs of severe ill health and stress, including chronic anorexia, sunken eyes, dehydration, hunched and / or hunched posture, and weakness, began around day 14, with no significant between-group differences in incidence or onset. All adverse clinical signs were consistent with acute radiation syndrome after exposure to radiation.

[0243] Gross pathology and organ and blood culture bacteriology evaluations were performed on all animals that died or were euthanized before the end of the study. There were no gross lesions associated with recombinant human IL-12. The incidence of bleeding was 12.5% ​​(1 / 8 animals) in pooled animals treated with 100 ng / kg or 250 ng / kg recombinant human IL-12 compared to 50% (2 / 4) in vehicle animals. While all of the dead animals (4 / 8 animals) in the vehicle group were observed to have died, only one animal in the recombinant human IL-12 group was observed to have died, and eight animals were humanely euthanized before the end of the study. The diagnosis of sepsis was confirmed by the isolation of the same bacterial strain in at least two organs of all 13 animals.

[0244] In the vehicle group, 75% (3 / 4) of the animals that died showed a combination of bacteria most likely from the intestinal and skin flora, while 25% (1 / 4) showed organ infections caused solely by bacteria most likely from the skin flora. In the various recombinant human IL-12 treatment groups, 8 of 9 animals (89%) showed a combination of bacteria from the intestinal and skin flora, including 2 animals that also showed organ infections caused solely by bacteria most likely from the environment. The other animal (1 / 9) showed organ infections caused solely by bacteria most likely from the skin flora. These results suggest that opportunistic infections were present in all animals that died before the end of the study in this animal model of acute radiation syndrome.

[0245] Aspects and embodiments of the present disclosure are generally based on the principle that radiation damage caused by TBI in a dose-dependent manner consequently affects immune, hematopoietic, and GI tissues, as these tissues are the most radiosensitive targets in the body. Lymphocytes are the cells most sensitive to radiation toxicity and are the first to be depleted from the circulation at irradiation doses above approximately 2 Gy. Lymphocyte loss is followed by a decrease in granulocytes and then a decrease in platelet levels over a period of days. Acute-onset anemia can occur secondary to hemorrhage. At doses >4 Gy, radiation has deleterious effects on the GI epithelium / endothelium, and the resulting clinical symptoms are due to a combination of hematopoietic and GI toxicity and include nausea, vomiting, diarrhea, headache, fatigue, fever, and abdominal pain.

[0246] It is also recognized that deaths from immune and hematopoietic toxicity occur due to infection and / or bleeding resulting from thrombocytopenia resulting from immune impairment, while deaths from GI toxicity are often due to multi-organ failure, severe sepsis, and bleeding complications. In the case of radiological attack, radiation mitigation drugs with multi-tissue effects that can mitigate immune, hematopoietic, and GI toxicity when administered after radiation exposure would be useful.

[0247] The examples described herein clearly demonstrate that recombinant human IL-12, a typical IL-12 preparation, prevented death due to radiation-induced damage / toxicity in both mice and monkeys after administration of a single low dose. In both mice and monkeys, recombinant human IL-12 increased survival when administered at 24 hours or longer after radiation exposure in the absence of supportive care, including oral or topical antibiotics. In irradiated mice and monkeys, recombinant human IL-12 promoted survival at various levels by stimulating the immune system in the peripheral blood and extravascular space, promoting hematopoietic regeneration in the bone marrow, reducing tissue damage in the small intestine, and inducing systemic anti-apoptotic and anti-inflammatory effects throughout the body.

[0248] In one embodiment, the optimal mouse dose that produced these radiation mitigating effects is approximately 20 ng / mouse. Methods for extrapolating the equivalent human dose are well known in the art. This dose is less than previous reports of the efficacy of recombinant human IL-12 in radioprotection and as a hematological adjuvant in cancer therapy through the use of the formulated protein in this study.

[0249] Furthermore, the prolonged administration of recombinant human IL-12, 24 hours after irradiation, appears to act by a somewhat different mechanism when compared to our previous studies in which recombinant human IL-12 was administered either before or immediately after radiation exposure. Evidence for this is provided by a comparison of bone marrow recovery in this mouse radiation mitigation study compared to previous studies in mice. In this study, bone marrow recovery appears to be much slower, which may be due to the timing of administration of recombinant human IL-12 (24 hours before TBI in the previous study vs. 24 hours after TBI in this study). There is a possibility.

[0250] Furthermore, as further demonstration of efficacy, typical IL-12 (recombinant human IL-12, e.g., recombinant human IL-12) significantly reduced radiation-induced expression of LGR5, a stem cell marker that also serves as a marker of GI injury, when administered 24 hours after radiation exposure. Typical IL-12 (recombinant human IL-12, e.g., recombinant human IL-12) administered at approximately 24 hours after TBI at doses of approximately 10 ng / mouse to approximately 40 ng / mouse reduced radiation-induced LGR5 expression. In contrast, approximately 200 ng / mouse (recombinant mouse IL-12, recombinant mouse IL-12) administered at approximately 24 hours after TBI appeared to exacerbate radiation-induced GI injury, as evidenced by increased LGR5 expression.

[0251] This finding is consistent with earlier reports that high doses of IL-12 exacerbated radiation damage to the GI tract. Data obtained in both mice and rhesus monkeys showed a significant increase in body weight in animals treated with recombinant murine IL-12 and recombinant human IL-12, respectively, after irradiation (Figure 13), thereby providing further support for the GI protective effect of recombinant human IL-12 treatment.

[0252] Furthermore, as further evidence of efficacy, recombinant human IL-12 was confirmed in monkeys to reduce radiation toxicity and increase survival in mice. Administration of recombinant human IL-12 to rhesus monkeys 24 hours after irradiation significantly increased survival (P = 0.05, pooled treatment groups vs. vehicle control). Recombinant human IL-12-treated monkeys had significantly higher platelet, white blood cell, and reticulocyte counts at nadir, lower incidence of bleeding, and higher body weight from days 12 to 30.

[0253] Furthermore, thrombocytopenia was less severe in animals treated with recombinant human IL-12 than in animals treated with vehicle. Furthermore, a significant difference was observed between the vehicle- and recombinant human IL-12-treated groups in terms of platelet counts falling below the threshold level of 20,000 platelets / μL, a level typically requiring platelet transfusion. In the 250 ng / kg recombinant human IL-12 group, only 4 of 16 animals (25%) had platelet counts below the transfusion threshold of <20,000 platelets / μL at nadir (days 12–14), whereas 12 of 15 animals (80%) had platelet counts below that threshold during the same period (P=0.007).

[0254] In another example demonstrating efficacy, recombinant human IL-12 was administered after TBI, i.e., at the earliest 24 hours after irradiation (a window considered to be the minimum time required for mobilization of medical personnel and resources to the affected area), which is considered a life-saving intervention in the event of a radiological disaster. These findings provide evidence that recombinant human IL-12, when administered as a single low dose after TBI, mitigates radiation-induced toxicity in at least three major systems affected by radiation: the immune system, the bone marrow compartment, and the GI tract.

[0255] An additional event related to the alleviation of radiation toxicity by recombinant human IL-12 is the stimulation of anti-apoptotic / anti-inflammatory effects through the release of EPO, a known general protector of tissues from cytotoxic damage via anti-apoptotic / anti-inflammatory mechanisms. Several interdependent networks underlie the radiation-mitigating effects of recombinant human IL-12. IL-12 is known to be a central regulator of cellular immune responses and regulates the synthesis and secretion of several immune mediators. In cancer patients, intraperitoneal / intravenous / subcutaneous administration of IL-12 increased peritoneal and serum levels of IFN-γ, TNF-α, IL-10, IL-8, VEGF, IP-10, and neopterin. In the present disclosure described herein, administration of recombinant human IL-12 dose-dependently increased plasma IFN-γ levels in both mice and monkeys. IFN-γ orchestrates many different cellular programs through transcriptional regulation of numerous genes, resulting in advanced immune surveillance and immune system effectiveness against infection.

[0256] In addition to IFN-γ, recombinant human IL-12 increased plasma levels of EPO in mice and IL-15, IL-18, neopterin, and EPO in monkeys. IL-15 and IL-18, alone and / or together, play important roles in the development, homeostasis, and function of CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and NK T cells. In synergy with IL-12, IL-18 stimulates the production of IFN-γ in T helper 1 cells. Neopterin, an autooxidation product of 7,8-dihydroneopterin, reflects the activity of IFN-γ and, consequently, is considered an indicator of systemic immune activation.

[0257] The Examples described herein demonstrate that recombinant human IL-12 stimulated the production of EPO in mice and NHPs, a finding indicating that EPO may play a central role in mediating the radiomitigation activity of recombinant human IL-12.

[0258] EPO may have immunomodulatory, neuroprotective, and cardioprotective effects. EPO promotes cell survival, modulates surface antigen expression, and increases IL-12 secretion in dendritic cells, the most potent antigen-presenting cells. This suggests that the immunomodulatory function of EPO may be mediated in part by dendritic cells, which in turn may induce specific T cell responses. The cytoprotective effects of EPO have been linked, at least in part, to its antioxidant, anti-inflammatory, and anti-apoptotic activities. In various models of cytotoxicity induced by toxins, ischemia, hypoxia, or oxidative stress, EPO reduces apoptosis in neurons, vascular smooth muscle cells, cardiomyocytes, and endothelial cells, while increasing cellular antioxidant capacity and / or reducing oxidative damage in kidney, neurons, and retinal pigment epithelial cells.

[0259] The mechanism underlying radiation mitigation provided by exogenous recombinant human IL-12 demonstrates a multilevel response orchestrated by exogenous delivery of recombinant human IL-12 (Figure 14). Current evidence indicates that recombinant human IL-12 induces at least four levels of responses by directly activating IL-12 receptors on (a) immune cells in peripheral blood and bone marrow (level 1), (b) other important cells in the bone marrow niche, such as hematopoietic stem cells and osteoblasts (level 2), (c) GI stem cells (level 3), and possibly (d) kidney cells (level 4), thereby releasing the cytoprotective factor EPO after radiation exposure (Figure 14).

[0260] The most direct response is the recombinant human IL-12-induced Level 1 response, which is associated with key radioresistant cells of the immune system. Immune cells undergo apoptosis in a sequence dependent on their radiation sensitivity (B cells > T regulatory cells > T helper cells > T cytotoxic cells > T memory cells > NK cells). The immune cells that may remain functional for 24 hours or longer after irradiation are those with the lowest radiation sensitivity, namely, NK cells and differentiated cells such as macrophages and dendritic cells. Therefore, recombinant human IL-12 administered after irradiation can initiate a Level 1 response by promoting the proliferation and activation of surviving NK cells, macrophages, and dendritic cells. Three-way crosstalk between NK cells, macrophages, and dendritic cells further promotes their maturation and expansion through cytokines identified as biomarkers of innate immune recovery, namely, IFN-γ, IL-15, IL-18, and neopterin. This three-way crosstalk further leads to the production of endogenous IL-12 secreted by dendritic cells (Figure 14). As a result, early immune competence is established by innate immune mechanisms after TBI. The sustained production of endogenous IL-12 from pathogen-activated dendritic cells also serves as a positive feedback loop, likely playing an important role in sustaining the initial response to exogenous recombinant human IL-12 for several weeks after radiation exposure (Figure 14). Evidence for the sustained production of endogenous IL-12 after exogenous administration of recombinant human IL-12 is the presence of IL-12Rβ2 on hematopoietic cells 12 days after TBI only in mice treated with recombinant human IL-12.

[0261] Recombinant human IL-12 initiates a level 2 response by interacting with primary bone marrow cells involved in hematopoiesis. In bone marrow, residual hematopoietic stem cells, osteoblasts, and megakaryocytes remain surviving for 24 hours after exposure to lethal doses of radiation and may be functional cell types. The presence of IL-12Rβ2-expressing stem / progenitor cells, megakaryocytes, and / or osteoblasts in bone marrow from mice, NHPs, and humans indicates that these cells are direct targets of recombinant human IL-12. Through its receptor, recombinant human IL-12 initiates a level 2 response by promoting the proliferation and differentiation of surviving stem cells after radiation exposure, leading to hematopoietic regeneration (Figure 14). Osteoblast activation appears to be essential for the survival, expansion, and homing of hematopoietic stem cells and megakaryocytes. We have shown that exposure to lethal doses of radiation leads to a specific expansion of the osteoblast niche, thereby allowing a surviving pool of radioresistant osteoprogenitor cells to proliferate in close proximity to endosteal bone areas. Relatively long-lived surviving megakaryocytes were also found near the endosteal surface of bone trabeculae, rather than in their usual paravenous sinus locations. Megakaryocytes release factors that stimulated the expansion of the osteoblastic niche. Consistent with these findings, immunohistochemical studies in our study revealed a similar cellular organization in mouse bone marrow, showing an osteoblastic niche adjacent to bone, cellular islands consisting of megakaryocytes and hematopoietic stem cells. In CD34+, IL-12Rβ2-positive bone marrow cells, recombinant human IL-12 increased EPO secretion but, in contrast to its traditional action in mature lymphocytes, reduced IFN-γ secretion [unpublished data from our laboratory], providing an environment that promotes the expansion of hematopoietic stem cells and ultimately leads to the regeneration of mature blood cells, including platelets and leukocytes (Figure 14). EPO also contributes to the generation of such an optimal environment by suppressing the overproduction of inflammatory cytokines, such as IFN-γ, IL-6, IL-2, and TNF-α, from T cells. The suppression of IFN-γ production by EPO is consistent with our findings showing that plasma IFN-γ levels were suppressed in irradiated mice at a dose of recombinant human IL-12 (20 ng / mouse) that substantially increased plasma EPO levels.Furthermore, the increase in plasma EPO concentrations was due, at least in part, to the increased monkey plasma levels of pro-inflammatory cytokines such as IL-2, IL-6 and TNF-α following administration of recombinant human IL-12. This explains the lack of growth in

[0262] Recombinant human IL-12 initiates a Level 3 response by regenerating intestinal crypt cells and protecting GI stem cells that ensure intestinal integrity (Figure 14). Recombinant human IL-12 provides integrity of the intestinal cell-cell boundary, which reduces pathogen leakage, increases food absorption, and reduces diarrhea. Reduction of "leaky gut syndrome" provides additional immune-related benefits by reducing pathogen entry into the peripheral circulation (Figure 14). Recombinant human IL-12 results in GI recovery and thus a higher chance of survival after lethal radiation exposure.

[0263] Recombinant human IL-12 initiates the Level 4 response by increasing plasma levels of EPO, likely by enhancing its release from the kidney following direct activation of its renal receptor. Given its antioxidant, anti-inflammatory, and anti-apoptotic activities, EPO serves as a general cytoprotective factor in the body, increasing cell viability in a diverse set of organs and tissues, including the brain, peripheral nerves, heart, kidney, skin, and intestine. EPO may also protect key cells involved in the Level 1 and 2 survival effects of recombinant human IL-12, namely, niche myeloid cells, as well as mature and immature dendritic cells, macrophages, and NK cells, from radiation toxicity. Mature dendritic cells may also release IL-12 in response to EPO and / or IFN-γ, resulting in a positive feedback loop that amplifies events initially initiated by exogenous administration of recombinant human IL-12.

[0264] Finally, sustained production of endogenous IL-12 induced by a single dose of exogenous recombinant human IL-12 in irradiated immunocompromised hosts is another important survival benefit. This sustained endogenous production of IL-12 is primarily the result of a level 1 recombinant human IL-12-induced response. Furthermore, bacterial and pathogenic products that gain access to the circulation after radiation injury can activate dendritic cells, promoting innate and adaptive responses and further leading to the release of endogenous IL-12. Thus, recombinant human IL-12 may promote the proliferation of surviving immune cells, cells of the bone marrow niche, namely osteoblasts and megakaryocytes, and hematopoietic stem cells, and may provide protection from radiation injury to critical intestinal stem cells through various feedback loops. These feedback loops promote the production of endogenous IL-12, IFN-γ, and soluble factors such as EPO, enabling the regeneration of the hematopoietic system and the restoration of immune and GI function (Figure 14).

[0265] These examples were the first to demonstrate that recombinant human IL-12 mitigates radiation-induced damage in NHPs, an animal model closely related to humans. Importantly, for the FDA Animal Rule path to approval, allometric dose conversion from mice to rhesus monkeys allowed for the identification of equivalent doses that resulted in similar recombinant human IL-12 exposure in monkeys. Despite similar PK properties, the IFN-γ response to recombinant human IL-12 appeared to be stronger in monkeys compared to mice. 100 ng / kg Similar survival rates in rhesus monkeys after receiving single or two doses of recombinant human IL-12 at 250 ng / kg or 250 ng / kg suggest that recombinant human IL-12 may be effective even at low doses. Importantly, the doses of recombinant human IL-12 used in the NHP studies correspond to human doses of approximately 30 ng / kg and 80 ng / kg, respectively. In cancer patients, IL-12 has been administered intravenously, intraperitoneally, or subcutaneously at doses ranging from 3 ng / kg to 600 ng / kg as monotherapy or as part of combination therapy for the treatment of various cancers.

[0266] When administered subcutaneously, IL-12 has been shown to be effective in treating rheumatoid arthritis when administered twice weekly at doses ranging from 300 ng / kg to 500 ng / kg for up to 3 years. In our study, recombinant human IL-12 was administered at a single dose of 1000 ng / kg. It was well tolerated in monkeys after a single dose or up to seven doses (data not shown), with no significant toxicity. There were no obvious symptoms.

[0267] Studies in monkeys have shown that the effective dose in mice and monkeys is very low, and the dose of recombinant human IL-12 required for radiation mitigation is substantially lower than the dose of IL-12 previously used in cancer patients, thus suggesting a more favorable safety profile of recombinant human IL-12 in radiation victims. Given the expected safety profile of recombinant human IL-12, it is envisioned that the drug could be distributed to everyone in the vicinity of a radiological event, even in the absence of knowledge of the actual level of radiation exposure.

[0268] As shown in this study, potent radiomitigation effects in mice and NHPs can be achieved using a single, very low nanogram per kilogram dose of recombinant human IL-12. The single, very low dose of recombinant human IL-12 required for its radiomitigation effect highlights its efficacy and anticipated safety in humans.

[0269] These findings indicate that recombinant human IL-12 may serve as a novel intervention for use as frontline therapy to prevent death due to radiation injury. A first-in-human Phase I study to evaluate the safety and pharmacokinetic and pharmacodynamic profiles of recombinant human IL-12, as well as further efficacy studies in animals, are ongoing. The cumulative results of these human and animal studies will enable the determination of a predicted effective dose of recombinant human IL-12 in humans under the Animal Rule, which requires efficacy to be determined in animal models and safety to be determined in humans.

[0270] Example 21: Radioprotection of typical tissues by administration of IL-12 IL-12 is clinically evaluated as a radioprotectant for the prevention of early and late effects and tissue reactions following radiation therapy (RT) for cancer in suitable subjects. Suitable subjects may include, for example, humans, mice, rats, guinea pigs, dogs, or primates, including rhesus monkeys. Radiation therapy (RT) may include single or fractionated doses of heavy charged particles (e.g., X-rays), fission-spectrum neutrons, or gamma rays.

[0271] For example, IL-12 is clinically evaluated as a radioprotectant for the prevention of radiation-induced injury, including alopecia, xerostomia, and mucositis, in subjects undergoing radiotherapy (RT) for head and neck cancer using a rat RT model to examine the protective effects of IL-12 after IV and subcutaneous (SC) administration in the mucositis model. Rats (5 per group) are administered IL-12 at a human equivalent dose of 1–100 ng / kg IV or SC, and their heads and necks are exposed to 15.3 Gy of gamma radiation at 0.5, 2, 4, and 8 hours after IL-12 administration. Doses of 1–50 Gy can also be used. For 1–10 days after treatment, the rats' oral cavity is inspected for signs of mucositis. Mucosal erythema and mucosal edema are scored according to a 0–5 and 0–2 scale, respectively, and the scores are summed to indicate overall mucositis. The mean mucositis score for untreated animals is calculated. Rats are protected from mucositis for up to several hours when IL-12 is administered IV or SC. Rats receiving SC, but not IV, administration of IL-12 are also protected from mucositis for several hours after administration. Similar histopathological and functional assessments are performed to determine protection against alopecia and xerostomia.

[0272] Other radiation-induced damage from radiotherapy can also be measured after single or fractionated doses, including, for example, xerostomia, mucositis, and / or alopecia, to assess the protective effect of IL-12 treatment when administered in conjunction with radiotherapy for head and neck cancer. For example, the incidence of esophagitis and pneumonitis can be used to assess the protective effect of IL-12 treatment in conjunction with radiotherapy for thoracic cancer. For example, the effect on lower gastrointestinal mucositis or dermatitis after radiotherapy for pelvic cancer can be used to assess the protective effect of IL-12 treatment in conjunction with radiotherapy for abdominal or pelvic cancer, including renal, gastric, pancreatic (e.g., pancreatic), gallbladder, bladder, prostate, or gynecological cancer. The effect on lower gastrointestinal mucositis can include rectal and urinary toxicity. Additionally, levels of viability, LRG5 expression (as a measure of GI injury markers), bone marrow damage (measured by platelet, white blood cell, and reticulocyte counts), histopathological evaluation, cell, tissue, or organ-specific proteomic or molecular markers, apoptosis, or tissue or organ edema can also be used to assess radioprotective efficacy. In one embodiment, exemplary IL-12 compositions and treatment methods were effective in preventing and / or mitigating radiation-induced side effects associated with various radiation-based therapeutic modalities in the treatment of pancreatic cancer.

[0273] Biological endpoints in subjects such as rhesus monkeys and mice exposed to about 5-50 Gy TBI, e.g., 6.7 Gy TBI or equivalent fractionated irradiation at a clinically relevant dose (e.g., about 0.1 to about 2 Gy per fraction), are measured following treatment with 100 ng / kg or 250 ng / kg recombinant human IL-12 (or the equivalent doses of 8 ng / mouse and 20 ng / mouse recombinant murine IL-12 doses).

[0274] In one arm of the study, one or more typical doses of IL-12 (1-100 ng / kg) will be administered approximately 5, 10, 15, 20, 30, 40, 50, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and 1, 2, 3, 4, 5, 6, and 7 days before each dose of radiation administered as a TBI or topically in a fractionated dose regimen of 1-10 doses per day for up to 30 days using each individual radiation source.

[0275] In another arm of the study, each individual radiation source will be administered in a 1-10 fractionated dose schedule per day for up to 30 days, with each dose administered as a TBI or locally. One or more typical doses of IL-12 (1-100 ng / kg) will be administered approximately 5, 10, 15, 20, 30, 40, 50, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 hours, and 1, 2, 3, 4, 5, 6, and 7 days after each dose.

[0276] In another arm of the study, one or more typical doses of IL-12 (1-100 ng / kg) will be administered both before and after approximately 5, 10, 15, 20, 30, 40, 50, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and 1, 2, 3, 4, 5, 6, and 7 days before and after each dose of radiation administered as a TBI or locally in a fractionated dose schedule of 1-10 doses per day for up to 30 days using each individual radiation source.

[0277] Example 22: Protection against acute radiation-induced mucositis Oral mucositis is induced in test subjects (e.g., monkeys or mice) by administering a radiation dose of 1 to 50 Gy in a single or multifractionated dose. On day 0, the left cheek pouch is harvested, immobilized, and irradiated under anesthesia while the remainder of the animal is shielded with a lead cover. Radiation is generated using an X-ray, neutron, or gamma ray source at an appropriate focal length.

[0278] The IL-12 composition is administered according to Example 21 by an appropriate route of administration (e.g., daily subcutaneous injection) starting 5 days before radiation (day -5) and continuing until day 15 (day +15). The control group consists of irradiated animals that receive vehicle only from days -5 to +15.

[0279] Assessment of mucositis: The progression of mucositis is monitored daily. Starting on day 6 post-irradiation, animals are anesthetized, and the left cheek pouch is harvested and photographed every other day. At the end of the clinical phase of the study, the film is developed, and the resulting photographs are randomly numbered and scored by two observers in a blinded manner. A 0-5 scoring system is used, with the following numerical scores applied to cheek lesions: 0, normal mucosa 1. Erythema and vasodilation 2, severe erythema and vasodilation, with superficial erosion of the mucosa, leaving patches of bare mucosa. 3. Severe erythema, vasodilation, and ulceration in one or more areas. The cumulative size of the ulcers covers 25% of the buccal mucosa. Pseudomembrane formation is observed. 4. Severe erythema and vasodilation. The cumulative size of the ulcers covers approximately half of the buccal mucosa. Loss of mucosal flexibility. 5. Diffuse and widespread ulceration. Loss of flexibility. Cheek pouches can only be partially removed from the mouth. This can be done.

[0280] In this model, a score of 3 corresponds to a clinically significant National Cancer Institute or WHO score of 3. Oral mucositis (OM) severity is calculated using the scores for each treatment group on each observation day (mean ± SE). The severity scores are used to quantitate the results as the percentile of days with a score of 3 or greater. It is also expressed as a fraction.

[0281] Initial results indicate significant radioprotection at appropriate doses of IL-12 with various administrations in all fractionated or single dose irradiation modalities.

[0282] Example 23: Radioprotection of IL-12 in the esophagus Esophagitis is a significant toxicity of radiotherapy for thoracic cancer. We investigated the radioprotective effect of IL-12 in the mouse esophagus. IL-12 was administered to mice via gavage of a liposomal formulation. Normal mice were treated with IL-12 at the dose described in Example 21 immediately before 28 Gy upper body irradiation. After irradiation, the esophagus was removed and esophageal progenitor (SP) and differentiated (NSP) cells were isolated by cell sorting to evaluate esophagitis. IL-12 may be administered after radiotherapy, in combination with or instead of pre-irradiation administration of IL-12.

[0283] Additional mice are administered 3LL cells intratracheally to induce orthotopic carina lung tumors. IL-12 is administered intraesophagically to mice bearing lung orthotopic tumors before receiving 20 Gy upper body irradiation. IL-12 may be administered after radiation therapy, in combination with or instead of pre-irradiation IL-12 administration. IL-12 uptake is quantified in the liver, peripheral blood, and lung orthotopic tumors 10, 30, and 60 minutes after intraesophageal administration. The esophagus is removed, and esophageal progenitor (SP) and differentiated (NSP) cells are isolated by cell sorting.

[0284] Results also indicate that IL-12 ameliorates radiation-induced esophagitis without compromising the efficacy of radiation therapy. Mice receiving IL-12 prior to 28 Gy upper body irradiation exhibit increased survival compared with mice receiving irradiation alone. Mice bearing lung orthotopic tumors receiving IL-12 immediately prior to 20 Gy upper body irradiation exhibit increased survival compared with mice receiving irradiation alone.

[0285] Example 24: Exemplary tissue radioprotection by administration of IL-12 IL-12 is clinically evaluated as a radioprotectant for the prevention of radiation-induced toxicity following radiation therapy, including electron beam irradiation, for the treatment of CTCL in appropriate subjects. Suitable subjects may include, for example, humans, mice, rats, guinea pigs, dogs, or primates, including rhesus monkeys. Radiation therapy (RT) may include local or total skin electron beam irradiation (including high-dose-rate and low-dose-rate). For example, IL-12 is clinically evaluated as a radioprotectant for the prevention of radiation-induced injury, including erythema, ulceration, hair loss, dry skin, hyperpigmentation, eye irritation, and transient loss of fingernails, in subjects receiving electron beam therapy (local or total skin), using a rat RT model to examine the protective effects of IL-12 after IV and subcutaneous (SC) administration.

[0286] Rats (5 per group) are administered IL-12 IV or SC at human-equivalent doses of 1-100 ng / kg, and their affected areas are exposed to 4-36 Gy of electron beam radiation at 0.5, 2, 4, and 8 hours after IL-12 administration. Doses of 1-50 Gy may also be used. Relevant tissues of the rats are examined for signs of radiation cytotoxicity for 1-10 days after treatment.

[0287] The above radiation-induced cytotoxicity was scored according to a rating scale of 0-5 and 0-2, respectively, and the scores were summed to indicate the overall condition. The mean score of the untreated animals was calculated. The rats were treated with Il Rats receiving IL-12 SC, but not IV, are also protected from side effects for up to several hours after administration. Similar histopathological and functional assessments are performed to assess protection from other cytotoxic endpoints.

[0288] Biological endpoints are measured in subjects such as rhesus monkeys and mice exposed to approximately 1 to 50 Gy of electron beam irradiation after treatment with 100 ng / kg or 250 ng / kg recombinant human IL-12 (or the equivalent doses of 8 ng / mouse and 20 ng / mouse recombinant murine IL-12).

[0289] In one arm of the study, one or more typical doses of IL-12 (1-100 ng / kg) will be administered approximately 5, 10, 15, 20, 30, 40, 50, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and 1, 2, 3, 4, 5, 6, and 7 days before each dose of radiation administered as TSEBT or topically in a 1-10-fraction dose regimen for up to 30 days using each individual radiation source.

[0290] In the other arms of the study, each individual radiation source will be administered in a 1-10 fractionated dose schedule per day for up to 30 days, either as TSEBT or topically. One or more typical doses of IL-12 (1-100 ng / kg) will be administered approximately 5, 10, 15, 20, 30, 40, 50, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2, 22, 23, or 24 hours after each dose. The doses will be 1-10 times daily for up to 30 days.

[0291] In another arm of the study, one or more typical doses of IL-12 (1-100 ng / kg) will be administered both before and after approximately 5, 10, 15, 20, 30, 40, 50, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and 1, 2, 3, 4, 5, 6, and 7 days before and after each dose of radiation administered as TSEBT or topically using each individual radiation source in a 1-10-fractionated dose schedule for up to 30 days.

[0292] Example 25: Typical TSEBT Treatment Regimen In a typical protocol, Hoppe et al., Hematologic Therapy, Stanford University ( TSEBT is administered using a technique similar to that first developed in [The Journal of Radiation Oncology, Vol. 16, pp. 347-354; 2003]. A typical 6 MeV, six-dual-feed technique is used, with the patient standing at six angular positions around the vertical axis to expose the entire body surface to the beam. The positions are divided into two-day cycles. On day 1, the patient is treated with a vertical anterior field and two oblique posterior fields, and on day 2, with a vertical posterior field and two oblique anterior fields. Two gantry angles are used for each of the six positions. The two gantry angles, 90° and 19°, are selected so that the central axis passes above and below the patient, thereby avoiding the X-ray effects of the beam in the anterior direction. The feed size is set to 40 cm / 40 cm, and the dose rate is set to 888 MU / min (at a distance of 1.6 m), corresponding to 1.3 Gy / min at the center of the patient's face. Treatment of one field will take approximately 30 seconds.

[0293] Eye shields consisting of 3 mm of lead are used in the field where the patient faces the accelerator (except in patients who have had cataract surgery). Starting approximately midway through the procedure, the toes and fingers are shielded with 3 mm of lead to prevent overdosage.

[0294] Patients are treated with a source-to-skin distance of 370 cm, standing approximately 20 cm behind a 0.5 cm-thick, 12 m² acrylic panel. The panel acts as an energy absorber, meaning that the deep dose is attenuated closer to the body surface, resulting in a more adequate dose at the most superficial skin layers. The panel also improves dose uniformity, especially in oblique planes. Additional treatment is given to shadowed, superficial areas of the body that receive a relatively lower dose, such as the scalp, perineum, soles of the feet, and lower chest or other skin creases. Typically, 6 MeV electron therapy using a 1 cm bolus is administered to these areas. Patients with thick tumors (up to 2 cm) are treated with a local electron field prior to TSEBT, typically using 15 Gy in five fractions. In this setting, our experience has shown that tumors often shrink during treatment and become very thin at the end of treatment, resulting in complete remission. Patients treated with high-dose TSEBT received a total dose of 30 Gy with 20 Gy of booster therapy to shadowed areas, while patients treated with low-dose TSEBT received a total dose of 4 Gy with 4 Gy of booster therapy to shadowed areas.

[0295] Therefore, the radioprotective efficacy of IL-12 treatment will be determined for patients in each of the following categories: mycosis fungoides; mycosis fungoides variants and subtypes; folliculotropic mycosis fungoides; Sézary syndrome; primary cutaneous CD8 aggressive epidermotropic T-cell lymphoma (tentative); cutaneous g / d T-cell lymphoma (tentative); primary cutaneous CD4 small-medium pleomorphic T-cell lymphoma (tentative).

[0296] Typical P6N preparation At least 100 μL of a 100 μg / mL solution of exemplary murine recombinant IL-12, a heterodimer composed of subunits encoded by the distinct subunits p35 (IL-12A gene) and p40 (IL-12B gene), is formulated in 20 mL of the IL-12 vehicle P6N (Neumedicines, Pasadena, California). Prior to injection, the 100 μg / mL solution of IL-12 is either (1) diluted 1 / 100 with P6N to obtain a 1 μg / mL, 1 ng / μL dosing solution, or (2) diluted 1 / 1000 with P6N to obtain a 0.1 μg / mL, 100 pg / μL dosing solution, or (3) diluted 1 / 10000 with P6N to obtain a 0.01 μg / mL, 10 pg / μL dosing solution. The three solutions obtained after dilution are swirled or gently tapped and then used immediately (eg, injected).

[0297] Example 26: Clinical Trials A clinical trial was conducted to demonstrate the efficacy of human and murine recombinant IL-12 in synergizing with electron beam therapy (EBT) for the treatment of CTCL to produce an antitumor response while simultaneously mitigating radiation-induced normal tissue damage.

[0298] In this study, healthy volunteers received a single subcutaneous injection of 2, 5, 10, 12, 15, or 20 μg of recombinant human and / or murine IL-12 in six cohorts. Each cohort included two sentinel subjects (Group 1) followed by four additional subjects (Group 2) if the sentinel subjects did not exhibit dose-limiting toxicity after a 7-day observation period. Group 1 subjects in each cohort were randomized in a 1:1 ratio to receive either placebo or recombinant human and / or murine IL-12. Group 2 subjects in each cohort were randomized in a 1:3 ratio to receive either placebo or recombinant human and / or murine IL-12, respectively. Toxicity was graded according to the FDA-modified toxicity grading criteria for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials.

[0299] Patients with CTCL were monitored for dose-limiting toxicity prior to dose escalation according to pre-specified dose escalation / discontinuation rules, which were defined in the protocol as a single Grade 3 (severe) adverse event or two or more Grade 2 (moderate) adverse events attributable to recombinant human and / or murine IL-12. Thirty-two subjects enrolled and completed the study, including 19 men (59%) and 13 women (41%), ranging in age from 18 to 44 years. The majority of subjects were Caucasian (81%). It was.

[0300] The safety and tolerability profile of recombinant human and / or murine IL-12 was found to be tolerable in healthy subjects at single subcutaneous (sc) doses of 2, 5, 10, and 12 μg. There were no serious adverse events or study discontinuations due to adverse events.

[0301] Pharmacokinetic parameters of recombinant human and / or murine IL-12 after escalating doses of 2, 5, 10, 12, 15, and 20 μg resulted in greater exposure as indicated by increasing Cmax values. The pharmacodynamic response to recombinant human and / or murine IL-12 was measured by quantifying IFN-γ levels over time after administration of 2, 5, 10, 12, 15, and 20 μg.

[0302] In this study, 60 subjects aged 18 to 45 years were randomized in a 4:1 ratio to receive recombinant human and / or murine IL-12 or placebo. Results of the blinded data to date from this study confirm the safety of the 12 μg subcutaneous dose of recombinant human and / or murine IL-12 (approximately 177 ng / kg in a 70 kg subject), which was found to be optimal in the Phase 1a study. Importantly, the equivalent monkey dose to the 12 μg human dose, approximately 500 ng / kg, was shown to be effective both in increasing survival in lethally irradiated monkeys and in providing radioprotection of specific tissues, such as the gastrointestinal tract and bone marrow.

[0303] Additionally, additional secondary endpoints were investigated in this study: 1) response rate (complete / partial) in CTCL patients treated with recombinant human and / or murine IL-12; 2) frequency of refractory disease in patients treated with recombinant human and / or murine IL-12; and 3) immune and cytokine responses over time in patients treated with this treatment regimen. and 4) the frequency of improved clinical response in patients treated with this regimen. .

[0304] Additionally, biological correlates of response will be assessed, including levels of IFN-γ production, natural killer cell activity, infiltration of skin lesions by CD8-positive cells, lymphocyte IL-12Rβ2 expression, transcript protein levels and signal transducers and activators of IL-12 signaling, and induction of apoptosis in tumor cells in the skin of patients treated with this dosing regimen.

[0305] Example 27: Exemplary rIL-12 efficacy studies using various endpoints Typical efficacy studies of rIL-12 using various endpoints (survival, reduction in bleeding, BM regeneration, reduction in sepsis, secondary endpoints (lymphocyte, neutrophil, and platelet counts) and RCI radiation combined injury) were presented.

[0306] The following dose-ranging studies demonstrated survival benefit at the LD90 in rhesus monkeys in the absence of supportive care, under GLP, blinded conditions: 1. Sample size n=90; 18 cases / group (9 men, 9 women); 2. Received radiation = 7.0 Gy, LD90 3. Exemplary recombinant human IL-12 (HemaMax Product) was prepared as a P6NF formulation according to GMP. 4. Administration: 1X SC (subcutaneous) injection; 50-500 ng / kg rIL-12 preparation 24-25 hours after irradiation 5. No supportive care: fluids, antibiotics, or blood products; and 6. Statistical significance: p<0.04 for each treatment group vs. control.

[0307] As shown in Figure 15, the efficacy of a typical IL-12 formulation / composition was measured after exposure to radiation (LD90). This has been demonstrated in its ability to achieve an approximately 3.5-fold increase in survival.

[0308] As shown in Figure 16, the efficacy of an exemplary IL-12 formulation / composition was assessed based on its ability to reduce bleeding. This was demonstrated in

[0309] As shown in Figures 17-19, the efficacy of exemplary IL-12 formulations / compositions was demonstrated in their ability to induce bone marrow regeneration.

[0310] As shown in Figure 20, the effectiveness of an exemplary IL-12 formulation / composition was demonstrated in its ability to reduce sepsis.

[0311] As shown in Figures 21A-C, the efficacy of exemplary IL-12 formulations / compositions was demonstrated in their ability to promote recovery of lymphocyte, neutrophil and platelet counts.

[0312] As shown in Figure 22, the efficacy of a typical IL-12 formulation / composition was demonstrated in its ability to promote recovery from combined RCI radiation injury. In the RCI study, a single low dose of HemaMax's murine counterpart (rMuIL-12), administered 24 hours after irradiation, was found to increase the rate of wound closure, promote skin remodeling, and increase survival after lethal radiation exposure compared to placebo-treated mice. This is the first demonstration of the versatile, broad-spectrum therapeutic potential of HemaMax as a Rad-MCM. In Figures 22A-C, the previously unidentified role of IL-12 in stimulating wound healing is demonstrated in normal, uninjured (A) and wounded, irradiated skin tissue (B and C). In uninjured skin, the IL-12 receptor is found to be highly expressed on progenitor cells in the basement membrane of the dermis and in the sebaceous glands underlying hair follicles. These progenitor cells are the primary mediators of re-epithelialization after skin injury. The bottom of the figure shows that IL-12 receptor expression is highly upregulated at the wound surface after full-thickness injury equivalent to a third-degree burn. These data demonstrate that injured skin is primed for stimulation with HemaMax (rHuIL-12) after skin injury, which leads to early wound closure (see Data Quadrant in the attached Quad Chart). Figure 1: Skin is "primed" for stimulation with murine IL-12: IL-12RB2, a receptor for HemaMax, is expressed in (A) uninjured skin and (B, C) wound tissue from irradiated mice with full-thickness injury. (A) In unirradiated, uninjured skin, IL-12RB2 is expressed in progenitor cells contained in the basement membrane (BM) of the dermis and in the sebaceous gland (SEB) underlying the hair follicle. BM- and SEB-derived progenitor cells are primary mediators of re-epithelialization after skin injury. (B) IL-12RB2, the receptor for HemaMax, is upregulated in skin with full-thickness injury. Granulation tissue IL-12RB2 is expressed primarily in macrophages (M), with some expression also observed in polymorphonuclear neutrophils (PMN) and fibroblasts (F). (C) Wound edge epithelium showing increased expression of IL-12RB2 in basement membrane cells 48 hours after irradiation / wounding. Data suggest an accelerated entry into the proliferative phase of wound healing. do.

[0313] Example 28: Efficacy test in wound healing As shown in Figures 23, 24 and 25, the efficacy of rMuIL-12 in accelerating wound closure (reduction in wound size) and mitigating complex damage in irradiated mice (24 hours post-exposure) was demonstrated.

[0314] The experimental model / protocol includes the following parameters: C57B1 / 6 mice (3 females, 3 males); Subjects received 500 cGy total body irradiation; A 10mm full-thickness injury was induced on the subject's back and dressed with Tegaderm™; Subjects were treated with the topical administration of: 4% carboxymethylcellulose or 4% carboxymethylcellulose plus rMuIL-12 (100 ng / mL) every 2–3 days; and The subject's wounds were measured, treated, and re-bandaged as necessary.

[0315] Example 29: 24-hour relaxation study: wound healing As shown in Figures 26-27, the efficacy of rMuIL-12 in accelerating wound closure and mitigating complex injury in irradiated mice (24 hours post-exposure) was demonstrated.

[0316] Experimental model: The protocol includes the following:

[0317] C57B1 / 6 mice (3 females, 3 males); Subjects received 500 cGy total body irradiation; A 10mm full-thickness injury was induced on the subject's dorsum; Subjects were given a Tegaderm™ dressing to cover the wound site; Treatments were administered 0–24 h after injury; Treatment groups were treated as follows: 1. Group 1: 4% carboxymethylcellulose (topical) 2. Group 2: 4% carboxymethylcellulose + topical rMuIL-12 (100 ng / mL) on the same day 3. Group 3: 4% carboxymethylcellulose + topical rMuIL-12 (100 ng / mL) x 24 h 4. Group 4: 4% carboxymethylcellulose + topical rMuIL-12 (100 ng / mL) x 24 h + rMuIL -12(20ng)sc 5. Group 5: rMuIL-12 (20 ng) scx 24 hours (single dose) Subjects / wounds were measured, treated, and re-bandaged as needed every 2-3 days.

[0318] Example 30: Pharmacokinetic and Pharmacodynamic Studies The pharmacokinetic (PK) and pharmacodynamic (PD) parameters of recombinant human IL-12 (e.g., HemaMax™) were evaluated in non-irradiated and irradiated monkeys. HemaMax was administered to irradiated and non-irradiated monkeys. The PK of HemaMax was similar in both sets of animals, although there was a slight trend toward a longer half-life in the irradiated animals. In general, pharmacodynamic markers suggested a more pronounced PD response after irradiation. In irradiated animals, plasma concentrations of IFN-γ and IL-18 increased most clearly, reflecting a corresponding increase in HemaMax dose (see Figures 28-38).

[0319] Pharmacokinetics and pharmacodynamics of HemaMax™ in non-irradiated and irradiated monkeys.

[0320] [Table 3]

[0321] [Table 4]

[0322] [Table 5]

[0323]

Table 6

[0324] Pharmacodynamics IFN-γ:

[0325]

Table 7

[0326]

Table 8

[0327]

Table 9

[0328]

Table 10

[0329] Pharmacodynamics EPO:

[0330]

Table 11

[0331]

Table 12

[0332]

Table 13

[0333]

Table 14

[0334] Pharmacodynamics IL-18:

[0335] [Table 15]

[0336] [Table 16]

[0337] [Table 17]

[0338] [Table 18]

[0339] Pharmacodynamics IL-15:

[0340] [Table 19]

[0341] [Table 20]

[0342] [Table 21]

[0343] [Table 22]

[0344] Example 31: Demonstration of efficacy in human subjects The efficacy of exemplary recombinant IL-12 treatment has been demonstrated in human subjects in clinical trials.

[0345] Acute radiation syndrome hematopoietic syndrome (HSARS) is a condition that affects the whole body or a significant part of the body. HemaMax™ (recombinant human interleukin-12 [rHuIL-12]) is an acute disease caused by partial-body irradiation. HemaMax™ (recombinant human interleukin-12 [rHuIL-12]) was developed as a single-dose, first-line point-of-care radiation palliative medical countermeasure (MCM) to stimulate multilineage hematopoiesis and mitigate bone marrow injury after lethal radiation exposure in events such as nuclear weapon detonations, industrial radiological accidents, and radiation therapy errors. HemaMax was granted accelerated approval by the FDA for review and approval under animal regulations due to its safety determined in humans, with parallel efficacy studies in mitigating radiation damage in rhesus monkeys and mice.

[0346] Clinical results showed that HemaMax was found to be safe and well tolerated at doses of 2, 5, 10, and 12 μg in a First in Human (FIH) dose-escalation study. This subsequent Phase 1b, single-dose, randomized, double-blind, placebo-controlled study was conducted to further evaluate and confirm the safety, tolerability, pharmacokinetics, and pharmacodynamics of the 12 μg dose of HemaMax™ (rHuIL-12) in 60 healthy subjects.

[0347] Subjects who met eligibility criteria were randomized to HemaMax or placebo in a 4:1 ratio on Day 1. A single 12 μg dose of HemaMax or placebo was administered subcutaneously on Day 1. Subjects continued as inpatients through Day 16. Subjects returned to the clinic for two outpatient visits on Days 28 and 45. Adverse events were graded based on clinical significance and the FDA toxicity grading criteria for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials. Safety data were monitored throughout the study by a Safety Review Committee (SRC).

[0348] The primary endpoints were safety and tolerability based on the number and percentage of subjects reporting adverse events. Adverse events were graded based on clinical significance and the FDA toxicity grading criteria for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials. Safety data were monitored throughout the study by a Safety Review Committee (SRC).

[0349] Secondary endpoints evaluated the pharmacokinetic (PK) and pharmacodynamic (PD) profile and immunogenicity of the 12 μg dose of HemaMax using validated bioanalytical methods.Exploratory endpoints included evaluation of biological response parameters related to the stimulatory properties of HemaMax on multilineage hematopoiesis.

[0350] The study met its primary and secondary endpoints. No deaths, serious adverse events (SAEs), or study discontinuations due to adverse events (AEs) were reported. There were no clinically significant abnormalities in vital signs, ECGs, or safety laboratory tests. Neutrophils, A transient decrease in platelet and lymphocyte counts was observed.

[0351] PK parameters were reproducible compared to the FIH study. Mean Cmax and AUClast were 57±50 pg / mL and 1034±631 hr*pg / mL, respectively, and mean T1 / 2 was 117±22 hours. The PD profile demonstrated a robust IFN-γ response following administration of 12 μg HemaMax.

[0352] None of the subjects developed anti-HemaMax antibodies.

[0353] Among the biological response parameters, induction of the chemotactic cytokine IP-10 was seen to be promising following administration of 12 μg of HemaMax.

[0354] In conclusion, HemaMax was found to be safe and well tolerated in healthy subjects at a single subcutaneous dose of 12 μg.

[0355] While the present invention has been disclosed with respect to specific embodiments, it will be apparent that other embodiments and variations of the present invention can be devised by those skilled in the art without departing from the true spirit and scope of the present invention, and the appended claims are intended to cover all such embodiments and equivalent variations.

[0356] All patents, publications, scientific articles, websites, and other documents and materials referenced or mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each such reference document and material is incorporated by reference to the same extent as if it were individually incorporated by reference in its entirety or as if set forth herein in its entirety. Applicant reserves the right to physically incorporate into this specification any and all materials and information from such patents, publications, scientific articles, websites, electronically available information, and other reference materials or documents.

[0357] The specific methods and compositions described herein are representative of preferred embodiments and are illustrative rather than limiting the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the appended claims. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein can suitably be practiced in the absence of any element or limitation not specifically disclosed herein as essential. Thus, for example, in each example herein, in an embodiment or example of the invention, any of the terms "comprising," "consisting essentially of," and "consisting of" can be substituted for either of the other two terms herein. Furthermore, terms such as "comprising," "including," and "containing" should be read expansively and without limitation. The methods and processes illustratively described herein can suitably be practiced with steps in a different order and are not necessarily limited to the steps set forth in the specification or claims. Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Under no circumstances should this patent be construed as limited to the particular examples or embodiments or methods specifically disclosed herein. Under no circumstances should this patent be construed as limited by statements made by examiners or other officials or employees of the Patent and Trademark Office, unless such statements are specific and absent any qualifications or reservations expressly adopted in applicant's reply.

[0358] The terms and expressions which have been used are used as terms of description and not as terms of limitation, and it is not intended that such terms and expressions be used to exclude equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as set forth in the claims. Thus, while the invention has been specifically disclosed by preferred embodiments and optional features, it is understood that modifications and variations of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the invention as defined by the appended claims.

[0359] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also constitutes part of the invention. This includes the generic description of the invention with any proviso or negative limitation removing any subject matter from the genus, whether or not the excised material is specifically recited herein.

[0360] Other embodiments are within the scope of the following claims. When describing a Markush group, one skilled in the art will recognize that the invention is also described in terms of individual members or subgroups of members of the Markush group.

[0361] References

[0362] [Table 23]

[0363] [Table 24]

[0364] [Table 25]

[0365] [Table 26]

[0366] Table 27

[0367] Table 28

[0368] Table 29

[0369]

Table 30

[0370] Table 31

[0371] Table 32

[0372]

Table 33

[0373] Table 34

[0374] Table 35

[0375] Table 36

[0376] Table 37

[0377] Table 38

[0378] Table 39

[0379] Table 40

[0380] Table 41

[0381] Table 42

Claims

1. 1. A method for protecting a subject from system, organ, tissue or cellular damage following exposure of the subject to ionizing radiation, comprising: A method comprising administering to a subject a dose of a therapeutically effective amount of a pharmaceutical composition comprising substantially isolated rIL-12, thereby reducing system, organ or tissue and / or cellular damage caused by radiation.

2. 10. The method of claim 1, wherein the radiation is received as an acute lethal or near-lethal dose sufficient to produce properties associated with acute radiation injury.

3. 3. The method of claim 2, wherein the radiation exposure results in total body irradiation.

4. 10. The method of claim 1, wherein the radiation dose is from about 0.7 Gy to about 50 Gy.

5. 10. The method of claim 1, wherein the radiation is administered as a fractionated dose in two or more fractions.

6. 6. The method of claim 5, wherein the radiation is administered as fractionated doses in a hyperfractionated regimen.

7. 6. The method of claim 5, wherein the radiation is received as fractionated doses in an accelerated fractionation regimen.

8. 10. The method of claim 1, wherein an effective amount of IL-12 protects more than one system, organ and / or tissue from radiation damage.

9. 7. The method of claim 6, wherein the system, organ or tissue to be protected is selected from the group consisting of bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lungs, kidneys, skin and brain.

10. 7. The method of claim 6, wherein the two organs protected are the bone marrow and the gastrointestinal system.

11. 7. The method of claim 6, wherein the two organs protected are bone marrow and skin.

12. 2. The method of claim 1, wherein the effective amount of IL-12 is less than 300 ng / kg.

13. 13. The method of claim 12, wherein the effective amount of IL-12 is administered in two or more doses, each dose being less than 50 ng / kg.

14. 10. The method of claim 1, wherein the effective amount of one or more of IL-12 is less than 200 ng / kg.

15. 10. The method of claim 1, wherein the effective amount of one or more of IL-12 is less than 100 ng / kg.

16. 10. The method of claim 1, wherein one or more effective doses of IL-12 are administered prior to radiation exposure.

17. 10. The method of claim 1, wherein one or more effective doses of IL-12 are administered before and after radiation exposure.

18. 10. The method of claim 1, wherein one or more effective doses of IL-12 are administered after radiation exposure.

19. 19. The method of claim 18, wherein the one or more effective doses of IL-12 are administered more than 24 hours after radiation exposure.

20. 19. The method of claim 18, wherein the one or more effective doses of IL-12 are administered more than 48 hours after radiation exposure.

21. 19. The method of claim 18, wherein the one or more effective doses of IL-12 are administered more than 72 hours after radiation exposure.

22. 19. The method of claim 18, wherein the one or more effective doses of IL-12 are administered more than 96 hours after radiation exposure.

23. 19. The method of claim 18, wherein the one or more effective doses of IL-12 are administered more than 120 hours after radiation exposure.

24. 10. The method of claim 1, wherein one or more effective doses of IL-12 are administered subcutaneously.

25. 10. The method of claim 1, wherein one or more effective amounts of IL-12 are administered intravenously.

26. 10. The method of claim 1, wherein one or more effective doses of IL-12 are administered intramuscularly.

27. 10. The method of claim 1, wherein the IL-12 is administered near the site of injury to a susceptible organ.

28. 10. The method of claim 1, wherein the subject is undergoing radiation therapy for head and neck cancer and the IL-12 is administered at or near the radiation site.

29. 29. The method of claim 28, wherein the administered IL-12 protects mucosal tissue from radiation damage.

30. 10. The method of claim 1, wherein the radiation damage is caused by a nuclear explosion.

31. The method of claim 1 , wherein the radiation damage is caused by the treatment modality of radiation therapy.

32. 32. The method of claim 31, wherein the treatment modality comprises external beam radiotherapy.

33. 33. The method of claim 32, wherein the external beam radiotherapy comprises three-dimensional conformal radiation therapy (3-D CRT).

34. 33. The method of claim 32, wherein the external beam radiation therapy is selected from the group consisting of intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), tomotherapy, stereotactic radiotherapy, stereotactic body radiation therapy, photon beam, electron beam, and proton beam therapy.

35. 32. The method of claim 31, wherein the radiation therapy comprises internal beam radiation therapy or brachytherapy.

36. 32. The method of claim 31, wherein the radiation therapy comprises systemic radiation therapy.

37. A pharmaceutical composition comprising IL-12 in a suitable formulation for delivery to a subject in need of prevention of radiation-induced damage.

38. 10. The method of claim 1, wherein one or more effective amounts of IL-12 are administered intradermally.

39. The method of claim 1, wherein the administered IL-12 induces the production of erythropoietin.

40. 40. The method of claim 39, wherein the production of erythropoietin enhances protection from system, organ or tissue damage.

41. 41. The method of claim 40, wherein the systems, organs or tissues protected include the bone marrow and gastrointestinal system.

42. 41. The method of claim 40, wherein the systems, organs or tissues protected include the kidneys and lungs.

43. 41. The method of claim 40, wherein the systems, organs or tissues to be protected include the brain and cardiovascular system.

44. 10. The method of claim 1, wherein the subject in need of treatment is in need of radiation therapy for cancer.

45. 45. The method of claim 44, wherein the subject in need of treatment also requires chemotherapy.

46. 46. ​​The method of claim 45, wherein the cancer is a solid tumor.

47. 47. The method of claim 46, wherein the solid tumor comprises a sarcoma, carcinoma, or lymphoma.

48. 48. The method of claim 47, wherein the cancer is selected from the group consisting of lung, breast, prostate, pancreas, ovary, bladder, head and neck, thyroid, brain, skin and kidney.

49. 10. The method of claim 1, wherein each dose of IL-12 is from about 1 ng / kg to less than about 2000 ng / kg, and said dose is administered by a delivery route selected from the group consisting of intradermal, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, and epidural routes.

50. 13. The method of claim 12, wherein the effective amount of IL-12 is administered in two or more doses, each dose being less than 30 ng / kg.

51. 1. A method for protecting a subject from system, organ, tissue or cellular damage following exposure of the subject to ionizing radiation, comprising:

1. A method comprising administering to a subject a dose of a therapeutically effective amount of a pharmaceutical composition comprising substantially isolated rIL-12 in a pharmaceutically acceptable carrier, thereby reducing system, organ or tissue and / or cellular damage caused by radiation.

52. 52. The method of claim 51, wherein the radiation is administered as electron beam therapy for the treatment of CTCL.

53. 53. The method of claim 52, wherein the electron beam therapy is focal electron beam therapy.

54. 53. The method of claim 52, wherein the electron beam therapy is total skin electron beam therapy.

55. 55. The method of claim 53 or 54, wherein the radiation is received as high-dose fractions.

56. 55. The method of claim 53 or 54, wherein the radiation is received as low-dose fractions.

57. 52. The method of claim 51, wherein the radiation is administered for the treatment of a disease and / or disorder associated with CTCL.

58. 58. The method of claim 57, wherein the CTCL-associated disease and / or disorder is mycosis fungoides and / or Sézary syndrome.

59. 58. The method of claim 57, wherein the system, organ or tissue to be protected is selected from the group consisting of bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lungs, kidneys, skin and brain.

60. 58. The method of claim 57, wherein the organs protected are the skin, nails and sweat glands.

61. 58. The method of claim 57, wherein the organs protected are the immune system and the skin.

62. 52. The method of claim 51, wherein the effective amount of IL-12 is less than 300 ng / kg.

63. 63. The method of claim 62, wherein the effective amount of IL-12 is administered in two or more doses, each dose being less than 50 ng / kg.

64. 52. The method of claim 51, wherein the effective amount of one or more of IL-12 is less than 200 ng / kg.

65. 52. The method of claim 51, wherein the effective amount of one or more of IL-12 is less than 100 ng / kg.

66. 52. The method of claim 51, wherein the one or more effective doses of IL-12 are administered prior to radiation exposure.

67. 52. The method of claim 51, wherein one or more effective doses of IL-12 are administered before and after radiation exposure.

68. 52. The method of claim 51, wherein the one or more effective doses of IL-12 are administered after radiation exposure.

69. 69. The method of claim 68, wherein the one or more effective doses of IL-12 are administered more than 24 hours after radiation exposure.

70. 69. The method of claim 68, wherein the one or more effective doses of IL-12 are administered more than 48 hours after radiation exposure.

71. 69. The method of claim 68, wherein the one or more effective doses of IL-12 are administered more than 72 hours after radiation exposure.

72. 69. The method of claim 68, wherein the one or more effective doses of IL-12 are administered more than 96 hours after radiation exposure.

73. 69. The method of claim 68, wherein the one or more effective doses of IL-12 are administered more than 120 hours after radiation exposure.

74. 52. The method of claim 51, wherein one or more effective doses of IL-12 are administered subcutaneously.

75. 52. The method of claim 51, wherein the one or more effective amounts of IL-12 are administered intravenously.

76. 52. The method of claim 51, wherein one or more effective amounts of IL-12 are administered locally.

77. 52. The method of claim 51, wherein one or more effective amounts of IL-12 are administered intradermally.

78. 52. The method of claim 51, wherein one or more effective doses of IL-12 are administered intratumorally.

79. 52. The method of claim 51, wherein the IL-12 is administered near the site of injury to a susceptible organ.

80. 52. The method of claim 51, wherein the subject is undergoing radiation therapy for CTCL and IL-12 is administered at or near the radiation site.

81. 58. The method of claim 57, wherein the administered IL-12 protects skin tissue from radiation damage.

82. 52. The method of claim 51, wherein the administered IL-12 induces production of at least one of erythropoietin, chemokine, cytokine, MCP-1, IFN-g, IL-15, IL-18, IP-10, MG, Mip1 beta or I-TAC, eotaxin, eotaxin-3, TARC, and IL-8.

83. 81. The method of claim 80, wherein the production of erythropoietin enhances protection from system, organ, tissue or cell damage.

84. 81. The method of claim 80, wherein the systems, organs or tissues protected include the bone marrow and gastrointestinal system.

85. 82. The method of claim 81, wherein the systems, organs or tissues to be protected include the kidneys and lungs.

86. 52. The method of claim 51, wherein the systems, organs or tissues to be protected include the brain and cardiovascular system.

87. 63. The method of claim 62, wherein the effective amount of IL-12 is administered in two or more doses, each dose being less than 30 ng / kg.

88. 63. The method of claim 62, wherein the IL-12 is formulated with P6N.

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