WO2019126384A1

WO2019126384A1 - Effective il-12 dosing regimens

Info

Publication number: WO2019126384A1
Application number: PCT/US2018/066596
Authority: WO
Inventors: Lena A. Basile
Original assignee: Neumedicines Inc
Current assignee: Neumedicines Inc
Priority date: 2017-12-19
Filing date: 2018-12-19
Publication date: 2019-06-27
Anticipated expiration: 2020-06-19

Abstract

Disclosed herein are methods of using IL-12 comprising low dosages of IL-12, and methods related to the timing of multiple dosages of IL-12, particularly when IL-12 is used as an adjunct to another treatment.

Description

EFFECTIVE IL-12 DOSING REGIMENS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/607,904, filed December 19, 2017, and U.S. Provisional Application No. 62/703,264, filed July 25, 2018.

FIELD

[0002] The present disclosure is directed to methods of using IL-12 comprising low dosages of IL-12, and methods related to the timing of multiple dosages of IL-12, particularly when IL- 12 is used as an adjunct to another treatment or as a single agent.

BACKGROUND

[0003] The effector mechanisms of IL-12 in innate resistance and adaptive immunity have been explored in various experimental tumor models, as reviewed by Colombo and Trinchieri. Promising data obtained in preclinical models led to clinical trials in oncology patients with the hope that IL-12 could be a powerful anticancer therapeutic agent. Indeed, immunomodulatory effects of IL-12 have been demonstrated in several human cancer. However, the high doses and repeat-dosing regimens used in oncology trials generally resulted in excessive clinical toxicity that was accompanied by only a modest clinical response. Various studies have implicated interferon gamma (IFN-g), the hallmark of immune activation by IL-12, as an important mediator of antitumor activity.

[0004] Prior IL-12 clinical experience: IL-12 has been studied extensively in clinical trials as a cancer immunotherapeutic using a repeat dose regimen with a cumulative high dose. The data described herein demonstrates that prior applications of IL-12 typically used too high of a dosage, e.g., 500 ng/kg or 300 ng/kg in a repeat dosing regimen of 5x or 2x per week, respectively, while drug-related toxicities were generally up to Grade 3 and reversible (mostly flu-like symptoms, elevated LFTs and transient cytopenias).

[0005] There remains a need in the art for improved formulations of IL-12, methods of making such formulations, and methods of using the same. Based on its hematopoietic and immunomodulatory activities, a recombinant human IL-12 (rHuIL-l2) is now under

development to address the unmet need for a medical countermeasure (MCM) against the hematopoietic syndrome of the acute radiation syndrome (HSARS) that occurs in individuals exposed to lethal radiation, and also to serve as adjuvant therapy that could provide dual hematopoietic and immunotherapeutic benefits in patients with cancer receiving chemotherapy.

SUMMARY

[0006] The present disclosure is directed to methods of treating a patient in need with injectable administration of Interleukin- 12 (IL-12). The patient can be a mammal, such as a human. A patient in need can be a cancer patient.

[0007] It was surprisingly found that IL-12 administered at low doses, and at a single dosage, or infrequently using more than one low dose of IL-12, is highly effective. The effects of these administrations of IL-12 can have long-lasting effects. It is theorized that the

effectiveness of a single IL-12 dosage or very infrequent IL-12 dosages is due to the unexpected, very long exposure of IL-12 after exogenous subcutaneous administration. These effects have been observed to be greater than one week, and estimated by pharmacokinetic models to be up to several weeks to one month or more. This long exposure of IL-12 is unexpected because the IL-12 half-life shown in the prior art is reported to be about 5-20 hours. This unexpected long exposure of IL-12 in blood may be due to lymphatic absorption of the drug, which is much slower than capillary absorption. The long-lasting effects of IL-12 in blood may also be due to the endogenous production of IL-12 following administration of a low dose of IL-12.

Alternatively or in addition, the long-lasting effects of IL-12 in blood resulting from

infrequently administering low dosages of IL-12 may avoid tachyplylaxis. This may allow for the activation of feedback loops.

[0008] Thus, in one embodiment, encompassed is a unique dosing schedule for IL-12 and the application of this dosing schedule to adjunctive or single agent therapy. In some cases, the therapy is adjunctive to a cancer treatment. For example, IL-12 can be administered as adjunctive therapy to a radiation or chemotherapy or immunotherapy cancer treatment. In some cases, the therapy comprises a therapy for HSARS.

[0009] The subject who is to receive treatment is generally a mammal, preferably a human.

A therapeutically effective dose of IL-12 is generally less than 1000 ng/kg/day and preferably less than 500 ng/kg/day. However, even lower doses of IL-12 are effective, such as doses of less than 100 ng/kg/day, especially when more than one dose is administered to the subject at varying time intervals. Exemplary dosages of IL-12 are described herein. Thus, embodiments further include repeated administration, i.e., more than one administration of IL-12, at certain time intervals following the initial administration. Subsequent doses of IL-12 may be the same or different from the initial dose.

[0010] In addition, the IL-12 dose can be administered for any therapeutically effective duration of time as described herein, such as but not limited to, 1 day up to 1 year or any time point in-between, including for example 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. An IL-12 dose may also be administered for periods of longer than 1 year, e.g., over a several year period.

[0011] In general, the frequency of IL-12 dosing can be less than once a week, with preferred dosing regimens of about 2 to about up to 4 weeks, or other time periods as described herein. In some cases, the dosing regimen is approximately every two weeks. In some cases, IL- 12 is given as a maintenance dose following a therapeutic dosing regimen. In some of such cases, these maintenance dosing regimens will be at a frequency of once a month, once every two months or once every three to four months, or other time periods as described herein.

[0012] Also described herein are methods of adjusting a dosing regimen based on

characteristics of an individual patient. Example characteristics include the use of biomarkers informative of the efficacy or adverse effects of a particular therapeutic regimen. This disclosure describes several phenomena that can guide the dosage amount and frequency of administration of IL-12. Examples include PK, terminal half-life of IL-12, and the ability of IL-12 to traffic major peripheral blood cells out of the peripheral blood and/or to sites of injury or disease, including injury by T cells. Thus, the disclosures includes dosing regimens for IL-12 to various PD markers, such as cell trafficking and the presence of Th2 cytokines. These markers can be informative of a therapeutic need for, an amount of, and a timing for a subsequent dose of IL-12.

[0013] For example, the frequency can be modified based upon various benchmarks indicating the need for a further dose of IL-12. These benchmarks are characterized in the present application and include the half-life of IL-12 in the target patient population. In some cases, the half-life can be affected by a particular type of cancer or other disease. Other benchmarks include the ability of subsequent doses of IL-12 to traffic major peripheral blood cells into sites of injury, such as a tumor, a wound, or a site of organ damage. Still other benchmarks include the presence of IL-10 or any other T Helper 2 cell (Th2) cytokine, such as IL-2, IL-4, IL-3, IL-5, IL-6, IL-13, IL-25, IL-31, or IL-33. In some cases, the presence of Th2 cytokines is undesirable and indicates that IL-12 should not be dosed and/or that the IL-12 dosage should be decreased. [0014] In some embodiments, the time between a first dose and a second dose of IL-12 can be 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.

In some embodiments, the time between doses can vary. In other embodiments, the IL-12 dosing frequency can be every 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In one embodiment, the dosing frequency is determined by measuring the IL-12 exposure in blood (PK). Alternatively or in addition, the dosing frequency can be determined by IL-l2’s downstream pharmacodynamic effects (PD) in blood. Examples of such effects include exposure to IFN-g or other cytokines or chemokines in the target patient population, such as a patient population having a particular type of cancer. In some of such embodiments, the first and the second administrations of IL-12 can give similar PK and/or PD exposure in blood via PK and PD parameters such as C_max, AUC, etc. Similar PK and/or PD exposure can indicate a time that IL-12 is essentially cleared from the blood. In some cases, a second dose of IL-12 will result in optimal efficacy if administered after IL-12 is essentially cleared from the blod. See e.g., data described in Example 5 below.

[0015] In another embodiment, encompassed is the discovery that the trafficking of major peripheral blood cells can be related to the efficacy of IL-12. For example, data described herein show that these trafficked cells can move into sites of injury following IL-12 administration. Examples of sites of injury include a tumor, a wound, or a site of organ damage. Thus, the ability of IL-12 to traffic peripheral blood cells is another key pharmacodynamic (PD) parameter that can guide effective dosing regimens. Thus, in addition to the PK profile or terminal half- life of IL-12, another parameter of IL-12 which impacts the frequency of an IL-12 dose administration is the ability of subsequent doses of IL-12 to similarly traffic cells out of the peripheral blood in patients receiving the IL-12 adjunctive or single agent therapy.

[0016] IL-12 administration time points for optimal results of peripheral blood cell trafficking can be characterized for different target patient populations. Methods of measuring an increase in PBC trafficking to site of interest (e.g., site of disease or injury) include, for example, measuring an increase as compared to a base line measurement in cytotoxic T lymphocytes (CTL) precursors directed to a tumor in peripheral blood (peripheral CTLp) within a few days of IL-12 administration (e.g.,“circulating antitumor CTLp”). Examples of useful measuring techniques include a high-efficiency limiting dilution analysis technique and by staining peripheral blood lymphocytes (PBLs) with a tumor-specific antigen or antibody recognized by T cells. Another method of measuring an increase in PBC trafficking to a site of interest is by evaluating infiltration of target tissue, such as neoplastic or tumor tissue, by T cells by immunohistochemistry.

[0017] Thus, in some embodiments, a subsequent dose of IL-12 is administered when the initial increase in PBC (T cells, NK cells, monocytes, red blood cells, reticulocytes and/or platelets) trafficking to a target site has (a) begun to decrease or (b) when the amount has reached baseline levels present in the subject or patient population prior to IL-12 administration. An“increase” in PBC (T cells, NK cells, monocytes, red blood cells, and/or platelets) cell trafficking to a site of interest can be, for example, an about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or an about 45% increase (e.g., as measured by an increase in CTLp, or an increase in T cells at the target site as measured by

immunohistochemistry). Moreover, in some cases IL-12 traffics all major peripheral blood cells to sites of injury or disease. Thus, in some cases these markers can be useful in characterizing a therapeutic need for an IL-12 dosage.

[0018] Yet another benchmark that can be used to determine IL-12 dosing frequency for a particular patient population is the presence of various cytokines in a patient’s serum. For example, IL-10 and other Th2 cytokines can serve as a guidepost to effective dosing. If IL-10 or other Th2 cytokines are observed in peripheral blood, then either the IL-12 dose and/or frequency of IL-12 administration may not be optimal for efficacy or the timing may not be optimal for a subsequent dose. Accordingly, in another embodiment, a subsequent dose of IL-12 is not administered to a subject when an increase in serum of IL-10 or any other Th2 cytokine, such as IL-4, is observed as compared to baseline levels in the subject or patient population present prior to IL-12 administration.

[0019] An“increase” in IL-10 or any other Th2 cytokine, such as IL-4, can be, for example, an about 5%, about 10%, about 15%, about 20%, or about 25% increase or more as compared to baseline levels of the same cytokine (e.g., as measured by a typical immunoassay, such as an enzyme-linked immunosorbent assay (ELISA).).

[0020] In some cases, IL-12 can be administered as an adjunctive therapy to a chemotherapy cancer treatment with one cycle of chemotherapy, more than one cycle of chemotherapy, or with each cycle of chemotherapy. The IL-12 dose can be given before, during, or after the chemotherapy cycle, with exemplary time points of IL-12 administration being up to about 96 hours before or after initiation of the chemotherapy cycle.

[0021] In general, if IL-12 is being administered as an adjunctive therapy to a radiation cancer treatment, IL-12 will be given in repeat doses as radiation is generally fractionated into small, frequent dosing. In some embodiments, an IL-12 dose is given with each dose of radiation, either before, during, or after administration of a dose of radiation. The IL-12 dose can be given before, during, or after the radiation, with exemplary time points of IL-12 administration being up to about 96 hours before or after initiation of the radiation.

[0022] In another embodiment, encompassed is a dosing schedule of IL-12 for maintenance following administration of the adjunctive therapy. Maintenance doses of IL-12 can be given for any desirable time period, for example up to years following the adjunctive therapy. In some embodiments, a maintenance dose of IL-12 can be given at intervals of about one to about three months following adjunctive therapy of IL-12 with either radiation or chemotherapy.

[0023] Thus, IL-12 can be administered for any therapeutically effective duration of time, such as but not limited to, 1 day up to 1 year or any time point in between, including for example 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. An IL-12 dose may also be administered for periods of longer than 1 year, e.g., over a several year period.

[0024] The IL-12 dose, either as a single dose or in repeated dose regimens, can be administered via a weight-based dosing or it can be a fixed dosing regimen. A fixed dose is the preferred embodiment. For example, for weight-based dosing, the IL-12 dose amount can be any dosage amount as described herein, e.g., from about 1 ng/kg up to about 2000 ng/kg, or less than about 2000 ng/kg. In other embodiments, the dose of IL-12 is less than about 1000 ng/kg, less than about 500 ng/kg, about 300 ng/kg, less than about 300 ng/kg, about 200 ng/kg, less than about 200 ng/kg, about 100 ng/kg, less than about 100 ng/kg, about 100 ng/kg or less, about 50 ng/kg or less, or about 10 ng/kg or less.

[0025] For a fixed IL-12 dosing regimen, the amount of IL-12 administered can be about 2 pg up to about 20 pg, or any amount in-between these two values, with a preferred dosing range of about 5 pg to about 15 pg, or any amount in-between these two values.

[0026] In another embodiment, IL-12 is administered by an injectable delivery route selected from the group consisting of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intratumorally, or epidural routes.

[0027] In yet another embodiment, IL-12 is administered near a site of a tumor or cancer.

[0028] Provided herein are methods of treating a subject in need with an exogenous IL-12 composition comprising: a) administering a first treatment comprising administering a first dose of IL-12 to the subject; and b) administering a second treatment comprising administering a second dose of IL-12 to the subject, wherein the second treatment is administered after a first non-treatment interval of at least 8 days. In some embodiments, the second treatment elicits a therapeutic response that is not diminished by tachyphylaxis. In some embodiments, the method further comprises administering a third treatment comprising administering a third dose of IL-12 to the subject, wherein the third treatment is administered after a second non-treatment interval of at least 8 days. In some embodiments, the method further comprises administering a fourth treatment comprising administering a fourth dose of IL-12 to the subject, wherein the fourth treatment is administered after a third non-treatment interval of at least 8 days. In some embodiments, the method further comprises administering a fifth treatment comprising administering a fifth dose of IL-12 to the subject, wherein the fifth treatment is administered after a fourth non-treatment interval of at least 8 days. In some embodiments, a non-treatment interval is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, a non-treatment interval is no more than 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some

embodiments, a non-treatment interval is at least 1 2, 3, or 4 weeks. In some embodiments, a non-treatment interval is no more than 1, 2, 3, or 4 weeks. In some embodiments, a non treatment interval is at least 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, a non-treatment interval is not more than 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0029] In some embodiments, a dose is administered before, during, or after a cycle of chemotherapy. In some embodiments, the dose is administered 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days before or after a cycle of chemotherapy.

[0030] In some embodiments, a dose is administered before, during, or after a cycle of radiation therapy. In some embodiments, the dose is administered 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days before or after a cycle of radiation therapy. In some embodiments, the radiation therapy comprises low-dose Total Skin Electron Beam Therapy (LD-TSEBT).

[0031] In some embodiments, the second non-treatment interval is different than the first non-treatment interval. In some embodiments, the third non-treatment interval is different than at least one of the second non -treatment interval and the first non-treatment interval. In some embodiments, the fourth non-treatment interval is different than at least one of the third non treatment interval, the second non-treatment interval, and the first non-treatment interval. [0032] In some embodiments, the third dose elicits a therapeutic response that is not diminished due to tachyphylaxis. In some embodiments, the fourth dose elicits a therapeutic response that is not diminished due to tachyphylaxis. In some embodiments, the fifth dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

[0033] In some embodiments, at least one dose comprises between 2-20 pg of IL-12. In some embodiments, the at least one dose comprises between 5-15 pg of IL-12. In some embodiments, the at least one dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,

9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,

19.5, or 20 pg of IL-l2.

[0034] In some embodiments, each dose comprises between 2-20 pg of IL-12. In some embodiments, each dose comprises between 5-15 pg of IL-12. In some embodiments, each dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,

13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of IL-l2.

[0035] In some embodiments, the at least one dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg). In some embodiments, at least one dose comprises

0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350

375, or 400 ng/kg.

[0036] Also provided herein are methods of treating a subject in need with an exogenous IL- 12 composition comprising administering a first treatment comprising administering a first dose of IL-12 to the subject, wherein the first dose comprises between 2-20 pg of IL-12. In some embodiments, the first dose comprises between 5-15 pg of IL-12. In some embodiments, the first dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,

12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of IL-l2.

[0037] In some embodiments, the first dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg). In some embodiments, the first dose comprises 0.5, 1, 2, 3,

4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ng/kg.

[0038] In some embodiments, the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant. In some embodiments, the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL. In some embodiments, the pharmaceutical composition comprises IL-12 at a concentration of 20 pg/mL. In some embodiments, the buffer comprises sodium phosphate. In some embodiments, the

pharmaceutical composition comprises 10 mM sodium phosphate. In some embodiments, the salt comprises sodium chloride. In some embodiments, the pharmaceutical composition comprises 150 mM sodium chloride. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the non-ionic surfactant comprises poloxamer 188. In some embodiments, the pharmaceutical composition comprises 0.1% (w/v) poloxamer 188. In some embodiments, the pharmaceutical formulation comprises a pH of between 5.0 to 8.0. In some embodiments, the pharmaceutical formulation comprises a pH of 6.0.

[0039] In some embodiments, the method comprises treating hematopoietic syndrome of the acute radiation syndrome (HSARS) in the subject. In some embodiments, the method comprises treating cutaneous T-cell lymphoma (CTCL) in the subject.

[0040] In some embodiments, the method further comprises adjusting a length of a non treatment interval prior to a treatment based on a time point at which the subject is expected to have completed a direct response to the first dose of IL-12. In some embodiments, the method further comprises adjusting a length of a non-treatment interval prior to a treatment based on a time point at which the subject is expected to have completed an indirect response to the first dose of IL-12. In some embodiments, the method further comprises adjusting a length of a non treatment interval prior to a treatment based on a time point at which the a previous treatment is expected to no longer exert a pharmacodynamic effect on the subject.

[0041] In some embodiments, the method further comprises assessing a level of IL-12 in the subject's blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of IL-12 is above a threshold amount. In some embodiments, the threshold amount is a level of IL-12 in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

[0042] In some embodiments, the method further comprises assessing a level of at least one of INF-gamma, IL-2, IL-10, IL-18, or CXCL10 in the subject's blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of the at least one of INF-gamma, IL-2, IL-10, IL-18, or CXCL10 is above a threshold amount. In some embodiments, the threshold amount is a level of the at least one of INF-gamma, IL-2, IL-10, IL- 18, or CXCL10 in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

[0043] In some embodiments, the method further comprises assessing a level at least one of lymphocytes, neutrophils, platelets, and reticulocytes in the subject's blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of the at least one of lymphocytes, neutrophils, platelets, and reticulocytes is below a threshold amount. In some embodiments, the threshold amount is a level of the at least one of lymphocytes, neutrophils, platelets, and reticulocytes in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

[0044] Also provided herein are methods of treating a subject in need with an exogenous IL- 12 composition comprising: a) administering a first dose of IL-12 to the subject; and b) administering a second dose of IL-12 to the subject at least 8 days after administering the first dose.

[0045] Also provided herein are methods of treating a subject in need with an exogenous IL- 12 composition comprising: a) administering a first dose of IL-12 to the subject; and b) administering a second dose of IL-12 to the subject, wherein the second dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

[0046] In some embodiments, the second dose is administered at a time point that reduces a likelihood that the subject will develop tachyphylaxis. In some embodiments, the second dose is administered at least 8 days after administering the first dose. In some embodiments, the second dose is administered at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, or 31 days after the first dose. In some embodiments, the second dose is administered no more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

28, 29, 30, or 31 days after the first dose. In some embodiments, the second dose is

administered at least 1 2, 3, or 4 weeks after the first dose. In some embodiments, the second dose is administered 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the first dose.

[0047] In some embodiments, the method further comprises administering a third dose of IL-12 after administering the second dose, wherein the third dose elicits a therapeutic response that is not diminished due to tachyphylaxis. In some embodiments, the method further comprises administering a fourth dose of IL-12 after administering the third dose, wherein the fourth dose elicits a therapeutic response that is not diminished due to tachyphylaxis. In some embodiments, the method further comprises administering a fifth dose of IL-12 after

administering the fourth dose, wherein the fifth dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

[0048] In some embodiments, at least one dose comprises between 2-20 pg of IL-12. In some embodiments, the at least one dose comprises between 5-15 pg of IL-12. In some embodiments, the at least one dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19

19.5, or 20 pg of IL-l2.

[0049] In some embodiments, the at least one dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg). In some embodiments, at least one dose comprises

375, or 400 ng/kg.

[0050] In some embodiments, the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant. In some embodiments, the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL In some embodiments, the pharmaceutical composition comprises IL-12 at a concentration of 20 pg/mL. In some embodiments, the buffer comprises sodium phosphate. In some embodiments, the

[0051] Also provided herein are methods of treating a subject in need with an exogenous IL- 12 composition comprising: a) administering a first single low dose of IL-12, wherein IL-12 can be detected in a sample of the subject's blood, serum, and/or plasma for at least one week; and b) administering at least one subsequent dose of IL-12 at a time point when the amount of IL-12 in the subject's blood is no longer observable. In some embodiments, the IL-12 dosing schedule results in preventing the occurrence of tachyphylaxis. In some embodiments, a subsequent dose of IL-12 is administered at a time point when peripheral blood cell trafficking to a site of injury or disease is decreasing. In some embodiments, the peripheral blood cells are selected from the group consisting of NK cells, monocytes, red blood cells reticulocytes, platelets, and any combination thereof. In some embodiments, a subsequent does of IL-12 is not administered when one or more Th2 cytokines are detectable in the subject's blood, serum, and/or plasma. In some embodiments, if one or more Th2 cytokines are detectable in the subject's blood, serum, and/or plasma, then the subsequent dosage of IL-12 is decreased as compared to the prior IL-12 dosage. In some embodiments, if one or more Th2 cytokines have increased in the subject's blood, serum, and/or plasma sample, as compared to baseline levels of the same cytokine present in serum of either the subject or the patient population for the subject, then the subsequent dosage of IL-12 is decreased as compared to the prior IL-12 dosage. In some embodiments, the Th2 cytokine is selected from the group consisting of IL-2, IL-4, IL-3, IL-5, IL-6, IL-10, IL-13, IL-25, IL-31, and IL-33.

[0052] In some embodiments, the IL-12 is detectable in the subject's blood, serum, and/or plasma due to lymphatic absorption of IL-12. In some embodiments, the IL-12 is detectable in the subject's blood, serum, and/or plasma at least in part due to endogenous production of IL-12 stimulated by the exogenous IL-12 administration.

[0053] In some embodiments, the subsequent dose of IL-12 is administered at least 2 weeks after the first IL-12 dose. 98. The method of any one of embodiments 87 to 97, wherein the method is used as adjunctive therapy to a radiation cancer treatment. In some embodiments, the method is used as adjunctive therapy to a chemotherapy cancer treatment.

[0054] In some embodiments, the IL-12 dose is a weight-based dosage amount. In some embodiments, the IL-12 dose is a fixed dosage amount.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figures 1A-B. Plasma Concentration Profiles of rHuIL-12 over time. A) Plasma concentration vs. time for rHuåL-l2 at 2, 5, 10, 12, 15 and 20 pg doses in the FIH study. B) Plasma concentration vs. time for rHuåL-l2 (12 pg ) and for IFN-g and CXCL10 (C-X-C motif chemokine 10) in subjects treated with rHuIL-l2 at 12 pg in the phase lb expansion study.

[0056] Figures 2A-H. Transient Hematological Changes with Different rHuIL-12 Doses and Placebo in the FIH Study. The percentage changes from baseline count after treatment with rHuIL-l2 (2, 5, 10, 12, 15, or 20 pg) or placebo are shown as follows: A) lymphocytes/rHuIL-l2; B) lymphocytes/placebo; C) neutrophils/rHuIL-l2; D)

neutrophils/placebo; E) platelets/rHuIL-l2; F) platelets/placebo; G) reticulocytes/rHuIL-l2; H) reti culocyte s/pl aceb o .

[0057] Figure 3A-F. Transient Hematological Changes with 12 pg dose of rHuIL-12 or Placebo in Phase lb Expansion Study. Standard hematologic methods were used to determine cell counts at the indicated time points. The mean percentage of baseline count after treatment with rHuåL-l2 (12 pg) or placebo are shown as follows: A) lymphocytes, neutrophils, platelets, and reticulocytes after treatment with rHuåL-l2; B) lymphocytes, neutrophils, platelets, and reticulocytes after treatment with placebo; C) CD45⁺, CD3⁺, CD4⁺ and CD8⁺ cells after rHuIL-l2; D) CD45⁺, CD3⁺, CD4⁺ and CD8⁺ cells after rHuIL-l2; D) NK and CD34⁺ after placebo; E) NK and CD34⁺ cells after rHuIL-l2; F) NK and CD34⁺ cells after placebo. NK cells were defined as CD45⁺CDl6⁺CD56⁺ triple positive lymphocytes.

[0058] Figures 4A-B. Effect of rHuIL-12 or Placebo on rHuIL-12Rjl2 Positivity and CD56 Mean Fluorescence Intensity in the Phase lb Expansion Study. Flow cytometry was used to determine A) changes in the percentage of rHuIL- 1211b2 (IL-12 receptor b2 subunit) positive NK and CD34⁺ cells in response to rHuåL-l2 (12 pg) or placebo; and B) changes in CD56 mean fluorescence intensity on NK cells in response to dose of rHuåL-l2 (12 pg ) or placebo. NK cells were defined as CD45⁺CDl6⁺CD56⁺ triple positive lymphocytes.

[0059] Figure 5A-B. Effect of rHuIL-12 or Placebo on EPO, IL-18 and CXCL10 levels in the Phase lb Expansion Study. EPO (erythropoietin), IL-18, and CXCL10 were measured at the indicated time points using validated assays. The mean changes from baseline after treatment with rHuåL-l2 (12 pg) or placebo are shown as follows: A) EPO; B) IL-18.

[0060] Figure 6. Survival of rhesus monkeys following exposure to TBI and treatment 24 hours after TBI with either vehicle or rHuIL-l2. Kaplan-Meier plots of survival over the study period are shown for each treatment group. Each dose group comprised 18 animals. Log rank p- values were 0.0305 0.0344, 0.0404, and 0.0265, respectively for the 50 ng/kg, 100 ng/kg, 250 ng/kg and 500 ng/kg dose groups vs. the vehicle-treated control group.

[0061] Figures 7A-E. Blood counts over time in rhesus monkeys exposed to lethal TBI and treated 24 hours after TBI with either vehicle or rHuIL-12 (Average ± SEM). A) platelets; B) mean platelet volume; C) neutrophils; D) lymphocytes; E) reticulocytes. Normal ranges are as follows: platelets, 252 to 612 x l0⁹/L; mean platelet volume, 6.3 to 9.4 x l0⁹/L; neutrophils, 1.21 to 10.29 x l0⁹/L; lymphocytes, 1.85 to 8.71 x l0⁹/L; reticulocytes, 29.9 to 103.9 x l0⁹/L.

[0062] Figures 8A-D. Identification of Bone Marrow Regeneration Islands. (A)

Histopathological identification of regenerating bone marrow. Clusters of cells appearing in otherwise ablated bone marrow were scored as one regenerating island. Left panel, ablated bone marrow; middle panel, regenerating bone marrow; right panel, non-irradiated bone marrow. (Olympus BX41 compound microscope; Infinity Analyze software v5.0, magnification: lOx). (B) Quantification of number of islands of regeneration for individual treatment groups (left panel, p<0.0l for 500 ng/kg group vs. control) and the combined rHUIL-l2-treated groups vs. vehicle-treated control (right panel, p<0.05). (C) Quantification of area of regeneration for individual treatment groups (left panel, p<0.05 for 50 and 500ng/kg groups vs. control) and the combined rHuIL-l2-treated groups vs. vehicle-treated control (right panel, p<0.05). (D) Quantification of megakaryocytes for individual treatment groups (left panel) and the combined rHuIL- 12 -treated groups vs. vehicle-treated control (right panel).

[0063] Figures 9A-D. Blood counts over time in surviving vs. non-surviving rhesus monkeys exposed to lethal TBI and treated 24 hours after TBI with either vehicle or rHuåL-l2 (Average ± SEM). A) lymphocytes; B) neutrophils; C) platelets; D) reticulocytes. Normal ranges are as follows: lymphocytes, 1.85 to 8.71 x l0⁹/L; neutrophils, 1.21 to 10.29 x l0⁹/L; platelets, 252 to 612 x l0⁹/L; reticulocytes, 29.9 to 103.9 x l0⁹/L.

[0064] Figures 10A-B. Shows the results of measuring the amount of IL-12 in the blood stream of a Rhesus monkey #2001 (A) and #2501 (B) administered a single dose of IL-12 using ELISA and using a Multiplex kit from Meso Scale Discovery.

[0065] Figure 11. Decrease in Physical Activity. Average score for decrease in activity, calculated per live animal, by dose groups. Scoring rubric: 1 = slight decrease; 2 = moderate decrease; 3 = severe decrease.

[0066] Figure 12. Decrease in Appetite Score. Average score for the decrease in appetite, calculated per live animal, by dose groups. Scoring rubric: 1 = slight decrease; 2 = moderate decrease; 3 = severe decrease.

[0067] Figure 13. Body Weight over Time. Average percent body weight (± standard error of the mean) relative to baseline body weight, by dose group, over time.

[0068] Figure 14. Depicts a final structural model of IL-12 following SC dosing in humans and monkeys. BSV = Inter-individual variability, CL = Systemic clearance, CLd =

Intercompartmental distribution, CLdt = Distribution to deep tissue, Vc = Central volume of distribution, Vdt = Volume of distribution to deep tissue, Vp = Volume of peripheral

compartment, Kaf = absorption rate to the capillaries, Kas = absorption rate to the lymphatic system, F = absolute bioavailability, and Frel = relative amount of the dose to the lymphatic system.

[0069] Figures 15A-H: Shows the Goodness-of-Fit (GOF), impact of BLQ, of the structural model of IL-12 following SC dosing in humans and monkeys, plotted in Figs. 15A-D using original model parameters, while GOF plots using a refined model, M3 method for BQL, are shown in Figs. 15E-H.

[0070] Figure 16: Shows a PK graph for IL-12 exposure following a first injection (time = zero) and then a second injection 28 days later for two individual non-human primate (NHP) (rhesus) monkeys, one male and one female (Rhesus #2001 and #2501). [0071] Figure 17: Shows a PK graph for IL-12 exposure following a first injection (time = zero) and then a second injection 28 days later for NHP (rhesus) monkeys, one male and one female (average data for exposure from two monkeys).

[0072] Figure 18: Shows pharmacodynamics for Interferon-gamma exposure following a first injection (time = zero) of IL-12 and then a second injection of IL-12 at 28 days later for two individual NHP (rhesus) monkeys, one male and one female (Rhesus #2001 and #2501).

[0073] Figure 19: Shows pharmacodynamics for Interferon-gamma exposure following a first injection (time = zero) of IL-12 and then a second injection of IL-12 at 28 days for NHP (rhesus) monkeys, one male and one female (average data for exposure from two monkeys).

[0074] Figure 20: Shows hematology changes from baseline for lymphocytes in rhesus monkeys following a first injection of IL-12 (time=zero) and then a second injection of IL-12 at day 28. Group 1 comprised 8 monkeys and group 2 comprised two monkeys. Both groups had an equal male to female ratio.

[0075] Figure 21: Shows hematology changes from baseline for platelets in rhesus monkeys following a first injection of IL-12 (time=zero) and then a second injection of IL-12 at day 28. Group 1 comprised 8 monkeys and group 2 comprised two monkeys. Both groups had an equal male to female ratio.

[0076] Figure 22: Shows the amount of human IL-12 (pg/mL) over time following administration of a weight-based IL-12 dose to human patients with Cutaneous T cell 25 Lymphoma (CTCL) in a clinical trial. IL-12 was administered in combination with low-dose Total Skin Electron Beam Therapy (LD-TSEBT), followed by maintenance doses of IL-12 in the absence of LD-TSEBT. IL-12 (150 ng/kg) was administered in week 1, study day 2 and in week 3 on study day 15. The pharmacokinetic (PK) profile of IL-12, with circulating blood levels of IL-12 analyzed, is shown in Figure 22. IL-12 levels were determined prior to dosing on study 30 days 2 and 15, and for up to 72 hrs after dosing. Circulating levels of IL-12 usually reached a peak 5 or 24 hrs after administration on either study day 1 or study day 15. The mean peak level of IL-12 was highest 5 hrs after administration for both study day 1 and study day 15.

[0077] Figures 23A-C: Figure 23 A) and Figure 23 B) show swimmer and waterfall plots, respectively, of clinical responses in human patients with CTCL that were administered IL-12 in combination with low-dose total skin electron beam therapy (LD-TSEBT). Figure 23 C) shows analysis of PD-l expression in patient peripheral blood mononuclear cells after being cultured for 20 hours. [0078] Figure 24A-C: Figure 24 A) shows circulating levels of IL-12 following a 100 mg/kg subcutaneous administration of IL-12 in Rhesus monkeys. Figure 24 B) shows circulating levels of IL-12 following a 316 mg/kg subcutaneous administration of IL-12 in Rhesus monkeys. Figure 24 C) shows circulating levels of IL-12 following a 1,000 mg/kg subcutaneous administration of IL-12 in Rhesus monkeys.

[0079] Figure 25: Figure 25 shows plasma concentration vs. time profiles for IFN-g following subcutaneous administration of IL-12 in Rhesus monkeys.

DETAILED DESCRIPTION

I. OVERVIEW

[0080] Interleukin- 12 (IL-12), a heterodimeric cytokine with p40 and p35 subunits, is well- known for its pleiotropic effects. Several in vitro studies in the early-mid nineties reported that IL-12 is capable of stimulating hematopoiesis synergistically with other cytokines. The hematopoiesis-promoting activity of IL-12 was suggested to be due to a direct action on bone marrow stem cells, thereby promoting the proliferation and/or differentiation of hematopoietic progenitor cells. In addition to these hematopoietic effects of IL-12, the cytokine has demonstrated potent immunomodulatory effects. For example, IL-12 has been shown to play a role in the interaction between the innate and adaptive arms of immunity. IL-12 has also been shown to enhance cytolytic activity of macrophages, T cells, and natural killer (NK) cells, and to stimulate the differentiation of naive T helper (Th) cells into Thl (T-helper cell type 1) cells. These immunomodulatory effects of IL-12 have been widely studied in pre-clinical models of diseases such as cancer, viral and parasitic infections, and allergy. The importance of immune- mediated effects of IL-12 has been documented clinically by the discovery that immunodeficient patients experiencing recurrent infections harbor mutations in IL-12 or IL-12 receptor components. IL-12 therapy also has been shown to restore resistance to infection after injury.

[0081] Generally the production of IL-12 stimulates the production of INF -g, which, in turn, enhances the production of IL-12, thus forming a positive feedback loop. In in vitro systems, it has been reported that IL-12 can synergize with other cytokines (IL-3 and SCF for example) to stimulate the proliferation and differentiation of early hematopoietic progenitors (Jacobsen S E, et ak, J Exp. Med., 2: 413-8 (1993); Ploemacher et ah, Leukemia , 7: 1381-8 (1993); Hirao et ah, Stem Cells , 13: 47-53 (1995)).

[0082] However Gollob and colleagues found that induction of IFN-g was markedly attenuated after repeat dosing of rHuIL-l2 (recombinant human interleukin- 12), indicating a tachyphylactic response. This inability to maintain IFN-g release may have contributed to reduced tumor response rates in previous clinical trials of IL-12, where high doses and frequent administration regimens were used. As these early phase trials did not meet expectations, further clinical development of IL-12 was not pursued.

[0083] Other examples of the use of IL-12 are described in US 2013-0259828 Al for“Uses of IL-12 and the IL-12 receptor positive cell in tissue repair and regeneration”; US 2013- 0129674 Al for“IL-12 formulations for enhancing hematopoiesis”; US 2012-0190909 Al and US 2011-0206635 Al, both for“Uses of IL-12 in hematopoiesis”; US 2012-0189577 Al for “Use of IL-12 to increase survival following acute exposure to ionizing radiation”; US 2010- 0278777 Al for“Method for treating deficiency in hematopoiesis”; US 2010-0278778 Al for “Method for bone marrow preservation or recovery”; and US Patent No. 7,939,058 for“Uses of IL-12 in hematopoiesis.”

[0084] The present disclosure describes a single, low dose of IL-12 which provides dual hematopoietic and immunologic benefits.

[0085] The most surprising discovery detailed herein is that following a single

administration of a low dose of IL-12, the IL-12 exhibits a very long PK terminal half-life (predicted to be up to one month) and the trafficking of all major peripheral blood cells to tissues. Therapeutic effects in humans can be obtained via dosing schedules based on this long half-life and other dosing parameters described herein.

[0086] Specifically, it was surprisingly found that IL-12 administered at low doses is highly effective. This includes treatment regimens including both a single dose and multiple doses. IL-12 treatment regiments can be susceptible to inducing tachyphylaxis. As an exemplary advantage, the treatment regimens described herein can reduce, prevent, or reverse the effects of tachyphylaxis. In some cases, tachyphylaxis can downregulate the immune stimulating effects of IL-12. Thus, tachyphylaxis can reduce the efficacy of IL-12. It is also theorized that the effectiveness of a single dosage, or very infrequent dosages, can be due to the unexpected very long terminal half-life of IL-12 after exogenous subcutaneous administration, in some cases. In some cases, the terminal half-life of IL-12 is estimated to be several weeks to one month or more.

[0087] This long terminal half-life of IL-12 is unexpected because the terminal half-life shown in the prior art for other forms of manufactured recombinant IL-12 is estimated to be measured in hours, such as 5-20 hours. This unexpected long terminal half-life is due to the final 3-demensional structure of our novel IL-12 called rHuIL-l2-high-gly, and other factors, such as lymphatic absorption of the drug, which is much slower than capillary absorption, and also to the endogenous production of IL-12 following administration of a single low dose of IL- 12, or infrequently administering low dosages of IL-12 via feedback loops.

[0088] Accordingly, In some embodiments, IL-12 is administered to a patient population at a dosing frequency corresponding to twice the terminal half-life of IL-12 in that particular patient population. For example, if IL-12 has a terminal half-life of about 8 days in a particular patient population, then a second dose of IL-12 is administered at about 16 days, plus or minus about 2 days, to members of the patient population.

[0089] In some embodiments, PD parameters are assessed for establishment of an effective dosing regimen. Interferon-gamma can be a hallmark signal related to the immune stimulating activity of IL-12. Moreover, tachyphylaxis that downregulates these immune-stimulating activity of IL-12 can often be shown by measurement of IFN-g (we show this is figure 25) following dosing of IL-12 at various intervals. However, it is notable that INF-g release is often quite variable in human populations, so the magnitude may be less important than the repeat release upon a second or more doses of IL-12.

[0090] In some embodiments, encompassed is the discovery that the trafficking of major peripheral blood cells is related to the efficacy of IL-12. Data described herein show that following IL-12 administration, these trafficked cells can move into sites of injury, such as a tumor, a wound, or a site of organ damage. Thus, assessing the ability of subsequent doses of IL-12 to traffic cells out of the peripheral blood in patients receiving the IL-12 adjunctive therapy can be informative of further dosing schedules and dosage amounts.

[0091] Therefore, this disclosure describes three phenomena that guide the dosage amount and frequency of administration of IL-12: PK or terminal half-life of IL-12, PD parameters relying primarily on IFN-g or other releasable factors, and the ability of IL-12 to traffic major peripheral blood cells out of the peripheral blood and to sites of injury. Measurement of peripheral blood cell trafficking can be done using techniques known in the art, and IL-12 administration time points for optimal results of peripheral blood cell trafficking can be characterized for different target patient populations.

[0092] The methods described herein have several advantages. For example, the methods described herein do not suffer from the relatively low tumor responses that were observed in other studies using IL-12 as an immunotherapy because the present compositions and methods have: (1) improved or optimal dosing regimens that do not lead to adaptive tachyphylaxis (decreased IFN-g production and other cytokine and chemokine responses after repeated administration); and (2) low dosing regimens do not lead to counterproductive lymphopenia and other cytopenias, including persistent and transient cytopenias.

[0093] Along the lines of downregulation of IL-12 mechanisms of action (tachyphylaxis) is the fact that repeat dosing generally led to decreases in adverse events following the initial dosing. This is an example of the body regulating the downstream mechanisms of action related to IL-12 administration. Of note in this regard is the fact that IL-12, unlike most cytokines, is not constitutively expressed. Endogenous production of IL-12 from dendritic cells and macrophages, or the like, is found after infection, or other events, such as wounding, which stimulate its production. It is also hypothesized that in the very early stage of cancer, IL-12 is endogenously produced in an effort to mount an attack against the cancer, but subsequently as the cancer grows, the cancer develops methods to disable IL-12 production form dendritic cells and macrophages, or the like.

[0094] The methods and composition disclosed herein have reduced toxicity relative to the use of high doses (e.g., 250-1000 ng/kg) or frequent dosing of IL-12 (e.g., daily for 5 days or 2x weekly). Thus, the present methods exhibit lower levels, rates, or severity of cytopenia (e.g., persistent cytopenias). In some embodiments, the methods described herein include a low dose (e.g., up to about 300 ng/kg or about 20-22 Dg (meg)), along with a single dose per cycle (e.g., once every 2-4 weeks or every 3-4 weeks). This exemplary dosing regimen is often sufficient to obtain the desired therapeutic results given the long IL-12 residence time - e.g., up to a month or more.

[0095] IL-12 therapies described herein can exhibit improved efficacy. The methods described herein can reduce the occurrence of tachyphylaxis (dysregulation), as less frequent dosing does not result in diminished release of IL-l2-induced cytokines and chemokines. For example, the present disclosure exhibits improved results relative to Bajetta et ah, Clin. Can. Res., 4:75-85 (1998), which used IL-12 as a cancer therapy in melanoma patients. Bajetta used IL-12 in a 28 day cycle with once weekly dosing of IL-12 at 500 ng/kg for three weeks in the first cycle (on days 1, 8 and 15), followed by a second three week cycle with once a week dosing. The dosing regimen of Bajetta et al. resulted in nearly no IL-12 or INF-g release in peripheral blood at the end of the second cycle (6th dose) as compared to the level of IL-12 and INF-g release after the first dose. Interestingly, in Bajetta et al., trafficking of peripheral blood cells was also diminished on the 6th dose as compared with the first dose. Moreover, overall toxicity was reported to decrease in the second cycle as compared with the first cycle. [0096] The compositions and methods disclosed herein do not suffer from the same problems. Dosing is often not repeated until after the levels of exogenous IL-12 and the resulting endogenous release of IL-12 is sufficiently diminished. Such exemplary dosing regimens result in improved efficacy. Also in the Bajetta study, IL-10 was shown to be increased in both the first dose and after the sixth dose. In the present disclosure, the production of IL-10 or other Th2 cytokines can serve as a landmark of unproductive IL-12 dosing in both the dose level and the dose frequency.

[0097] Another exemplary advantage of the compositions and methods disclosed herein includes improved efficacy because patients are often not heavily pre-treated and

immunosuppressed. In some aspects, patients can be first line (cancer treatment-naive). This can maximize immune-mediated responses. In some aspects, the compositions and methods described herein address shortcomings of IL-12 therapies that failed to explore åL-l2’s ability to stimulate hematopoiesis. In some embodiments, the methods and compositions described herein mitigate cancer treatment-induced hematological toxicity in addition to direct anti-cancer effect and eliminate counterproductive lymphopenia.

II. METHODS OF TREATMENT

A. Conditions

[0098] In some embodiments, the subject to be treated has a cancer which is solid tumor type of cancer, a non-solid tumor type of cancer, a hematopoietic cancer, or a leukemia.

Preferred non-solid tumor cancers treatable with the methods disclosed herein include but are not limited to leukemias. In addition, examples of types of cancer treatable with the methods disclosed herein include but are not limited to, a solid tumor, carcinomas, sarcomas,

lymphomas, cancers that begin in the skin, and cancers that begin in tissues that line or cover internal organs. In another embodiment, examples of such types of cancer include, but are not limited to, brain cancer, including glioblastoma, neuroblastoma, leukemias, lymphomas, thyroid cancer, head and neck cancer, skin cancer, including melanoma, kidney cancer, gastrointestinal cancers, cancer of the digestive system, esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, stomach cancer, small intestine cancer, large intestine (colon) cancer, rectal cancer, gynecological cancers, cervical cancer, ovarian cancer, uterine cancer, vaginal cancer, vulvar cancer, prostate cancer, bladder cancer, endometrial cancer, breast cancer, and lung cancer. B. Dosing

[0099] The IL-12 dose amount can be weight-based dosing or it can be a fixed dosing regimen. An exemplary IL-12 weight-based dose range according to the present disclosure is about 300 ng/kg or less, and preferably about 150 ng/kg or less. In some embodiments, an IL-12 weight-based dose is administered at a dosage of about 400 ng/kg or less, about 375 ng/kg or less, about 350 ng/kg or less, about 325 ng/kg or less, about 300 ng/kg or less, about 275 ng/kg or less, about 250 ng/kg or less, about 225 ng/kg or less, about 200 ng/kg or less, about 175 ng/kg or less, about 150 ng/kg or less, about 125 ng/kg or less, about 100 ng/kg or less, about 75 ng/kg or less, about 50 ng/kg or less, about 25 ng/kg or less, about 20 ng/kg or less, about 15 ng/kg or less, about 10 ng/kg or less, about 5 ng/kg or less, about 4 ng/kg or less, about 3 ng/kg or less, about 2 ng/kg or less, about 1 ng/kg or less, or about 0.5 ng/kg. In some embodiments, an IL-12 weight-based dose is administered at a dosage of 400 ng/kg, 375 ng/kg, 350 ng/kg, 325 ng/kg, 300 ng/kg, 275 ng/kg, 250 ng/kg, 225 ng/kg, 200 ng/kg, 175 ng/kg, 150 ng/kg, 125 ng/kg, 100 ng/kg, 75 ng/kg, 50 ng/kg, 25 ng/kg, 20 ng/kg, 15 ng/kg, 10 ng/kg, 5 ng/kg, 4 ng/kg, 3 ng/kg, 2 ng/kg, 1 ng/kg, or 0.5 ng/kg.

[0100] Exemplary human IL-12 dosages can also include, but are not limited to, about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 pg/dose.

[0101] For a fixed IL-12 dosing regimen, examples of the amount of IL-12 administered can be about 2 pg up to about 20 pg, or any amount in-between these two values, with a preferred dosing range of about 5 pg to about 15 pg, or any amount in-between these two values. In some embodiments, the fixed dose amount of IL-12 can be about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about

17.5, about 18, about 18.5, about 19, about 19.5, or about 20 pg. In some embodiments, the amount of IL-12 administered in a single dose can be 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,

8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 pg C. Methods of Administration

[0102] IL-12 can be administered to the subject in many ways. These methods of administration include intravenous, subcutaneous, intraperitoneal, intradermal, or the like. Another method of administration of IL-12 is via continuous infusion. The continuous infusion method has the advantage of delivery a low dose of IL-12 over longer time period, which can add to the effectiveness.

D. Treatment Regimens

[0103] The preferred dosing protocols are consistent with the parameters detailed above, e.g., subsequent doses of IL-12 given at about twice the terminal half-life of IL-12 for a particular patient population. A second parameter relates to the ability of a subsequent dose of IL-12 to traffic peripheral blood cells to a target site, such as a site of injury or disease. A third parameter is the ability of IL-12 to increase serum levels of IL-10 and other Th2 cytokines, which is undesirable. These parameters generally result in IL-12 administration at a frequency of no more than every two weeks, preferably every 3-4 weeks, such as when IL-12 is given along with chemotherapy, immunotherapies or radiation therapy. For chemotherapy regimens, the preferred dosing protocol would be a single dose of IL-12 per cycle of chemotherapy, generally within 48 hours after chemotherapy administration. For radiation protocols, preferred protocols combining radiation with IL-12 entail administration of IL-12 every 2-4 weeks during the therapy. Immunotherapeutic protocols generally allow for administration every 2-4 weeks. In addition, for either chemotherapy, immunotherapeputic or radiation protocols, after completing the adjunctive therapeutic regimen, responsive patients can receive a maintenance dose of IL-12 either monthly, or every two months, or every three to 4 months. The maintenance dose of IL-12 will be at the same as the last dose of IL-12 given during the chemotherapy, immunotherapy or radiation regimen, or a lower dose, such as 1/3 or ½ or ¼ of the last dose given during the therapeutic regimen of chemotherapy or radiation.

[0104] In some embodiments, the IL-12 dosing frequency can be every about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or about 31 days. In some embodiments, the dosing frequency may be determined by measuring the terminal half-life of IL-12 in the target patient population, such as a patient population having a particular type of cancer, where the dosing frequency is preferably about twice the IL- 12 terminal half-life, plus or minus about 2 days. The time between treatments or doses of IL- 12 can be a non-treatment interval. In some embodiments, the non-treatment interval between a first dose of IL- 12 and a second dose of IL-l2 can be or can be about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the non-treatment interval is or is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the non-treatment interval is no more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the non-treatment interval is or is at least 1, 2, 3, or 4 weeks. In some embodiments, the non-treatment interval is no more than 1 2, 3, or 4 weeks. In some embodiments, the non-treatment interval is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the non-treatment interval is not more than 1, 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, or 12 months.

[0105] In some embodiments, the non-treatment interval may be determined by measuring the terminal half-life of IL-12 in the target patient population, such as a patient population having a particular type of cancer, where the non-treatment interval is preferably about twice the IL-12 terminal half-life, plus or minus about 2 days.

[0106] In general, if IL-12 is being administered as an adjunctive therapy to a chemotherapy cancer treatment, then an IL-12 dose will be given with each cycle of chemotherapy. Most chemotherapeutic regimens are administered about every 3 weeks or about 21 days. The IL-12 dose can be given before, during, or after the chemotherapy cycle, with exemplary time points of IL-12 administration being up to about 96 hours before or after initiation of the chemotherapy cycle. In some embodiments, the IL-12 dose can be given about 90, about 94, about 72, about 68, about 62, about 56, about 48, about 42, about 36, about 35, about 34, about 33, about 32, about 31, about 30, about 29, about 28, about 27, about 26, about 25, about 24, about 23, about 22, about 21, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 hours, about 1 hour, or less than 1 hour before or after the initiation of the chemotherapy cycle.

[0107] In general, if IL-12 is being administered as an adjunctive therapy to a radiation cancer treatment, IL-12 will be given in repeat doses as radiation is generally fractionated into small, frequent dosing. In some embodiments, an IL-12 dose is given with each dose of radiation, either before, during, or after administration of a dose of radiation. The IL-12 dose can be given before, during, or after the radiation, with exemplary time points for the initiation of IL-12 administration being up to about 96 hours before or after initiation of the radiation. In some embodiments, the IL-12 dose can be given about 90, about 94, about 72, about 68, about 62, about 56, about 48, about 42, about 36, about 35, about 34, about 33, about 32, about 31, about 30, about 29, about 28, about 27, about 26, about 25, about 24, about 23, about 22, about 21, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 hours, about 1 hour, or less than 1 hour before or after the initiation of the radiation. IL-12

administration is expected to be continued after the completion of the radiation course, which is usually a finite period of time of about one to three weeks or longer, as a maintenance therapy given about every 2-4 weeks generally or monthly or every other month.

[0108] In another embodiment, encompassed is a dosing schedule of IL-12 for maintenance following administration of the adjunctive therapy involving any cancer therapy modality, for example, radiation, chemotherapy and immunotherapy. Maintenance doses of IL-12 can be given for any desirable time period, for example up to years following the adjunctive therapy.

In some embodiments, a maintenance dose of IL-12 can be given at intervals of about one to about three months following adjunctive therapy of IL-12 with either radiation, chemotherpay or immunotherapy, e.g., at about 1, about 2, about 3, or about 4 months following either radiation or chemotherapy, or at the following intervals following adjunctive therapy of IL-12 with either radiation or chemotherapy: about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, or about 15 weeks.

[0109] The compositions and methods described herein comprising IL-12 can be used in combination with or adjunct to a variety of chemotherapeutic agents. Classes of

chemotherapeutic agents include the alkylating agents, antitumor antibiotics, plant alkaloids, antimetabolites, hormonal agonists and antagonists, and a variety of miscellaneous agents.

[0110] The classic alkylating agents are generally highly reactive compounds that have the ability to substitute alkyl groups for the hydrogen atoms of certain organic compounds.

Alkylation of nucleic acids, primarily DNA, is the critical cytotoxic action for most of these compounds. The damage they cause can interfere with DNA replication and RNA transcription. The classic alkylating agents include mechlorethamine, chlorambucil, melphalan,

cyclophosphamide, ifosfamide, thiotepa and busulfan.

[0111] A number of nonclassic alkylating agents also damage DNA and proteins through other mechanisms, such as methylation or chloroethylation, that differ from the classic alkylators. The nonclassic alkylating agents include dacarbazine, carmustine, lomustine, cisplatin, carboplatin, procarbazine and altretamine. The clinically useful antitumor drugs can be natural products of various strains of the soil fungus Streptomyces. They produce their tumoricidal effects by one or more mechanisms. The antibiotics are often capable of binding DNA, usually by intercalation, with subsequent unwinding of the helix. This distortion impairs the ability of the DNA to serve as a template for DNA synthesis, RNA synthesis, or both. These drugs may also damage DNA by the formation of free radicals and the chelation of important metal ions.

[0112] Some drugs may also act as inhibitors of topoisomerase II, an enzyme critical to cell division. Drugs of this class include doxorubicin (Adriamycin), daunorubicin, idarabicin, mitoxantrone, bleomycin, dactinomycin, mitomycin C, plicamycin and streptozocin.

[0113] Plants have provided some of the most useful antineoplastic agents. Three groups of agents from this class are the Vinca alkaloids (vincristine and vinblastine), the

epipodophyllotoxins (etoposide and teniposide) and paclitaxel (Taxol). The Vinca alkaloids can bind to microtubular proteins found in dividing cells and the nervous system. This binding alters the dynamics of tubulin addition and loss at the ends of mitotic spindles, resulting ultimately in mitotic arrest. Similar proteins make up an important part of nervous tissue; therefore, these agents are sometimes neurotoxic. The epipodophyllotoxins can inhibit topoisomerase II and therefore sometimes have profound effects on cell function. Paclitaxel has complex effects on microtubules.

[0114] The antimetabolites include structural analogs of normal metabolites that are required for cell function and replication. They typically work by interacting with cellular enzymes.

Among the many antimetabolites that have been developed and clinically tested are

methotrexate, 5-fluorouracil (5-FU), floxuridine (FUDR), cytarabine, 6-mercaptopurine (6- MP), 6-thioguanine, deoxycoformycin, fludarabine, 2-chlorodeoxyadenosine, and hydroxyurea.

[0115] Endocrine manipulation is an effective therapy for several forms of neoplastic disease. A wide variety of hormones and hormone antagonists have been developed for potential use in oncology. Examples of available hormonal agents are diethylstilbestrol, tamoxifen, megestrol acetate, dexamethasone, prednisone, aminoglutethimide, leuprolide, goserelin, flutamide, and octreotide acetate.

[0116] The compositions and methods described herein comprising IL-12 can be used in combination with or adjunct to a variety of radiotherapeutic devices, compositions, and methods. In some embodiments, the radiation is electromagnetic or particulate in nature. Electromagnetic radiation includes, but is not limited to, x-rays and gamma rays. Particulate radiation includes, but is not limited to, electron beams, proton beans, neutron beams, alpha particles, and negative pimesons. Examples of radiation therapies include external beam radiation therapy, such as three-dimensional conformal radiation therapy (3-D CRT), intensity modulated radiation therapy (IMRT), image guided radiation therapy (IGRT), stereotactic radiation therapy, intraoperative radiation therapy, proton beam therapy, and neutron beam therapy. Examples of stereotactic radiation therapy include stereotactic radiosurgery, stereotactic body radiation therapy, and stereotactic ablative radiotherapy, including the stereotactic radiation therapies that involve Axesse, CyberKnife, Gamma Knife, Edge, Novalis, Primatom, Synergy, X-Knife,

TomoTherapy, Trilogy, Truebeam, Versa HD or View Ray machines.

[0117] Radiotherapy can be administered by a conventional radiological treatment apparatus and methods, or by intraoperative and sterotactic methods. Radiation may also be delivered by other methods that include, but are not limited to, targeted delivery, systemic delivery of targeted radioactive conjugates and brachytherapy. Examples of brachytherapy include intracavitary treatment and interstitial treatment.

[0118] Thus, IL-12 can be administered for any therapeutically effective duration of time, such as but not limited to, 1 day up to 1 year or any time point in between, including for example 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. An IL-12 dose may also be administered for periods of longer than 1 year, e.g., over a several year period.

[0119] In some embodiments, following a single dose of IL-12, the PK terminal half-life of the administered IL-12 can be about 1 week up to about 31 days, or any time point in-between these two values. For example, following a single dose of IL-12, the PK terminal half-life of the administered IL-12 can be about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or about 31 days.

[0120] Moreover, the low IL-12 dosage, combined with the unique dosing profile, exhibits a significant safety and efficacy profile. This is unexpected and surprising given past toxicities and adverse events associated with IL-12 use. Moreover, based on the data described herein, previous IL-12 clinical studies dosed too much IL-12, and too frequently. This is significant as too high a dose of IL-12, combined with dosing too frequently, increases toxicity and reduces efficacy by downregulation of therapeutic processes, such as release of INF-g and other cytokines & chemokines (IL-15, IL-18, EPO, CCL2 (MCP-l), CCL4 (Mip-lb), CXCL9 (Mig), CXCL10 (IP- 10).

[0121] For treatment of cancer, combining IL-12 with chemotherapy, radiation or immunotherapy can result in enhanced antigen presentation by damaged tumor cells which synergizes with IL-l2-induced adaptive immune system activation. Moreover, a single dose of IL-12 per chemotherapy cycle, which generally are given about every 3-4 weeks, eliminates tachyphylaxis; allows time for system to be responsive at the next chemotherapy cycle. For IL- 12 combined with radiation as currently practiced, the repeat dosing on IL-12 will be once every two to four weeks, depending on the timing of the radiation fractionation. See e.g., US

2012/0190909, for“Uses of IL-12 in Hematopoiesis,” which describes the use of IL-12 as adjunctive therapy for radiation or chemotherapy. For use of IL-12 with currently available immunotherapies, the dosing frequency generally will be about every 2-4 weeks following by maintenance dosing as described herein.

[0122] The Examples below report the results of two clinical studies conducted in parallel to animal efficacy studies to evaluate the safety and tolerability of single, low doses of rHuIL-l2 in normal healthy subjects. The first-in-human (FIH) phase 1, dose-escalation study identified the maximum tolerated dose (MTD) of rHuåL-l2 based on stringent toxicity criteria and the subsequent phase lb expansion study demonstrated the safety of the rHuIL-l2 at the MTD in a larger sample of healthy adults. An additional important objective of these safety studies was to assess the safety of initiating development of rHuIL-l2 as a multilineage hematopoietic and immunotherapeutic agent in patients with cancer who are receiving aggressive chemotherapy, where single doses of IL-12 are planned to be used once per chemotherapy cycle.

E. Companion Diagnostics

[0123] Also disclosed herein are dosing schedules for IL-12 and the application of these dosing schedules to adjunctive therapy, such as adjunctive therapy to a cancer treatment. For example, IL-12 can be administered as adjunctive therapy to a radiation or chemotherapy cancer treatment. In general, the frequency of IL-12 dosing is expected to be about 2 to about 4 weeks. However, the frequency can be modified based upon various benchmarks indicating the need for a further dose of IL-12 or based on patient convenience where dosing of IL-12 occurs near the time of a standard of care therapy. These benchmarks are characterized in the present application and include: (1) the terminal half-life of IL-12 in the target patient population, such as a patient population having a particular type of cancer, (2) PD parameters, such as the release of IFN-g or other factors, particularly Th2 factors, such as the presence of IL-10 or any other T Helper 2 cell (Th2) cytokine, such as IL-2, IL-4, IL-3, IL-5, IL-6, IL-13, IL-25, IL-31, IL-33, (3) the ability of subsequent doses of IL-12 to traffic major peripheral blood cells (T cells, NK cells, monocytes, neutrophils, red blood cells, and/or platelets, and progenitor and/or stem cells such as CD34+ or mesenchymal cells released from the bone marrow) into sites of injury, such as a tumor, a wound, or a site of organ damage.

[0124] In other embodiments of the invention, the IL-12 dosing frequency can be every 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In one embodiment of the invention, the dosing frequency can be determined by measuring the terminal half-life of IL-12 in the target patient population, such as a patient population having a particular type of cancer, but if this is not practical, dosing of IL-12 in a cancer population will be every 2-4 weeks, preferably every three weeks, aligning with the standard of care cancer therapy regimen. In the oncology setting, it is important to consider the nature of the standard of care cancer therapy in terms it ability to release tumor associated antigens, or neo-antigens, as the effect of these released antigens will be maximized in the presence of IL-12.

[0125] In another embodiment, encompassed is the discovery that the trafficking of major peripheral blood cells (T cells, NK cells, monocytes, red blood cells, reticulocytes and/or platelets) is related to the efficacy of IL-12, as the data described herein describe that following IL-12 administration, these trafficked cells are moving into sites of injury, such as a tumor, a wound, or a site of organ damage. Thus, in addition to the PK profile or terminal half-life of IL- 12, the PD profile, as discussed above, another parameter of IL-12 which impacts the frequency of an IL-12 dose administration is the ability of subsequent doses of IL-12 to traffic cells out of the peripheral blood in patients receiving the IL-12 adjunctive therapy.

[0126] Measurement of peripheral blood cell trafficking can be done using techniques known in the art ( see e.g. , Mortarini et ah, Cancer Research, 60, 3559-3568 (July 1, 2000)), and IL-12 administration time points for optimal results of peripheral blood cell trafficking can be characterized for different target patient populations. Methods of measuring an increase in PBC (T cells, NK cells, monocytes, red blood cells, reticulocytes and/or platelets) trafficking to site of interest (e.g., site of disease or injury) include, for example, measuring an increase as compared to a base line measurement in cytotoxic T lymphocytes (CTL) precursors directed to a tumor in peripheral blood (peripheral CTLp) within a few days of IL-12 administration (e.g., “circulating antitumor CTLp”). Examples of useful measuring techniques include a high- efficiency limiting dilution analysis technique and by staining peripheral blood lymphocytes (PBLs) with a tumor-specific antigen or antibody recognized by T cells. Another method of measuring an increase in PBC trafficking to a site of interest is by evaluating infiltration of target tissue, such as neoplastic or tumor tissue, by T cells by immunohistochemistry.

[0127] Thus, in one embodiment of the invention, a subsequent dose of IL-12 is

administered when the initial increase in PBC (T cells) trafficking to a target site has (a) begun to decrease or (b) when the amount has reached baseline levels present in the subject or patient population prior to IL-12 administration. An“increase” in PBC (T cells, NK cells, neutrophils, monocytes, red blood cells, and/or platelets or progenitor or stem cells characterized by CD34+) cell trafficking to a site of interest can be, for example, an about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or an about 45% increase (e.g., as measured by an increase in any of the peripheral blood cells (T cells, NK cells, neutrophils, monocytes, red blood cells, and/or platelets or progenitor or stem cells characterized by CD34+), or an increase in lymphocytes, particularly CD8+ cells at the target site as measured by

immunohi stochemi stry) .

[0128] Yet another benchmark that can be used to determine IL-12 dosing frequency for a particular patient population is the presence of IL-10, or any other Th2 cytokine, such as IL-4, in serum, which is undesirable. Prior studies have shown that IL-12 administration can increase levels of IL-10 in serum. Mortarini et ah, Cancer Research, 60, 3559-3568 (July 1, 2000).

Thus, IL-10 and other Th2 cytokines can serve as a guidepost to effective dosing. If IL-10 or other Th2 cytokines are observed in peripheral blood, then either the IL-12 dose and/or frequency of IL-12 administration is too high. Accordingly, in another embodiment of the invention, a subsequent dose of IL-12 is not administered to a subject when an increase in serum of IL-10 or any other Th2 cytokine, such as IL-4, is observed in peripheral blood as compared to baseline levels in the subject or patient population prior to IL-12 administration. An“increase” in IL-10 or any other Th2 cytokine, such as IL-4, can be, for example, an about 5%, about 10%, about 15%, about 20%, or about 25% increase, or about 50% or about 100% or more as compared to baseline levels of the same cytokine (e.g., as measured by an enzyme-linked immunosorbent assay (ELISA)).

III. COMPOSITIONS

A. Recombinant Human IL-12

[0129] For general descriptions relating to 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 et al., J. Exp. Med., 170: 827-45 (1989)). Generally IL-12 stimulates the production of interferon-g (INF-g) from natural killer (NK) cells and T cells (Lertmemongkolchai et al., J. of Immunology, 166: 1097-105 (2001); Cui et al., Science, 278: 1623-6 (1997); Ohteki et al., J. Exp. Med., 759: 1981-6 (1999); Airoldi et al., J. of Immunology, 165: 6880-8 (2000)), favors the differentiation of T helper 1 (TH1) cells (Hsieh et al., Science, 260: 547-9 (1993); Manetti et al., J. Exp. Med., 177: 1199-1204 (1993)), and forms a link between innate resistance and adaptive immunity. IL-12 has also been shown to inhibit cancer growth via its immuno-modulatory and anti -angiogenesis effects (Brunda et al., J. Exp. Med., 178: 1223-1230 (1993)); Noguchi et al., Proc. Natl. Acad. Sci. U.S.A., 93: 11798-11801 (1996); Giordano et al., J. Exp. Med., 194: 1195-1206 (2001); Colombo et al, Cytokine Growth factor, Rev., 13: 155-168 (2002); Yao et al., Blood, 96: 1900-1905 (2000)). IL-12 is produced mainly by dendritic cells (DC) and phagocytes (macrophages and neutrophils) once they are activated by encountering pathogenic bacteria, fungi or intracellular parasites (Reis et al., J. Exp. Med., 186: 1819-1829 (1997); Gazzinelli et al., J. Immunol., 153: 2533-2543 (1994); Dalod et al., J. Exp. Med., 195: 517-528 (2002)). The IL-12 receptor (IL-12 R) is expressed mainly by activated T cells and NK cells (Presky et al., Proc. Natl. Acad. Sci. U.S.A., 93: 14002-14007 (1996); Wu et al., Eur. J. Immunol., 26: 345-50 (1996)). Each reference in this paragraph is incorporated by reference herein.

B. Pharmaceutical Compositions

[0130] In some embodiments, the active agents described herein, such as IL-12, including recombinant human IL-12, are formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active agents into preparations that are used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack

Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds.,

Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated by reference for such disclosure.

[0131] In some embodiments, the active agents described herein are administered either alone or in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition. Administration of the active agents and compositions described herein can be affected by any method that enables delivery of the active agents to the site of action. These methods include, though are not limited to delivery via enteral routes (including oral, gastric or duodenal feeding tube, rectal suppository and rectal enema), parenteral routes (injection or infusion, including intraarterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous), inhalational, transdermal, transmucosal, sublingual, buccal and topical (including epicutaneous, dermal, enema, eye drops, ear drops, intranasal, vaginal, and intrauterine) administration, although the most suitable route may depend upon for example the condition and disorder of the recipient. By way of example only, the active agents described herein can be administered locally to the area in need of treatment, by for example, local infusion during surgery, topical application such as creams or ointments, injection, catheter, implant, or inserted device. The administration can also be by direct injection at the site of a diseased tissue or organ.

[0132] In some embodiments, pharmaceutical compositions are formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen -free water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0133] Pharmaceutical compositions for parenteral administration include aqueous and non- aqueous (oily) sterile injection solutions of the active inhibitors which may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the inhibitors to allow for the preparation of highly concentrated solutions.

[0134] Pharmaceutical compositions can also include surfactants, dispersing agents, and/or viscosity modulating agents. These agents include materials that can control the diffusion and homogeneity of a drug through liquid media or a granulation method or blend method. In some embodiments, these agents also facilitate the effectiveness of a coating or eroding matrix.

Exemplary diffusion facilitators/dispersing agents include, e.g., hydrophilic polymers, electrolytes, Tween ® 60 or 80, PEG, Tyloxapol, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and the carbohydrate-based dispersing agents such as, for example, hydroxypropyl celluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K 15M, and HPMC K100M), carboxymethylcellulose sodium, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,

hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate

(HPMCAS), noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA ), vinyl pyrrolidone/vinyl acetate copolymer (S630), 4-(l,l,3,3-tetramethylbutyl)- phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68®, F88®, and F108®, which are block copolymers of ethylene oxide and propylene oxide; and poloxamer 188); and poloxamines (e.g., Tetronic 908®, also known as Poloxamine 908®, which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany,

N. J.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate copolymer (S-630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 4000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates, chitosans and combinations thereof. Plasticizcers such as cellulose or triethyl cellulose can also be used as dispersing agents. In some cases, the pharmaceutical composition comprises a surfactant at between 0.01% and 0.5% (w/v). In some instances, the pharmaceutical composition comprises a surfactant at 0.01%, 0.05%,

O.1%, 0.2%, 0.4%, or 0.5% (w/v). [0135] The pharmaceutical composition can also include salts. In some cases, the salt can be sodium chloride. In some embodiments, the salt can be at a concentration of between 10 mM and 250 mM. In some embodiments, the salt is at a concentration of 10, 25, 50, 100, 125, 150, 175, 200, 225, or 250 mM.

[0136] Salts can be dissolved in buffered solutions, including, but not limited to, a phosphate buffered saline solution or sodium phosphate, are utilized as diluents in the art, and can also provide pH control or maintenance. In some instances, the formulation has a pH of between 5.0 and 8.0. In some cases, the formulation has a pH of 5.0, 5.5, 6.O., 6.5, 7.0, 7.5, or 8.0.

C. Dosage Forms

[0137] In some embodiments, the pharmaceutical composition is packaged for single use. In some embodiments, the pharmaceutical composition is packaged for multiple uses in a 2 mL clear glass vial. NM-IL-12 should be stored at 2° to 8°C. It should not be frozen.

[0138] IL-12 can be administered to the subject in many ways. These methods of administration include intravenous, subcutaneous, intraperitoneal, intradermal, or the like.

Another method of administration of IL-12 is via continuous infusion. The continuous infusion method has the advantage of delivery a low dose of IL-12 over longer time period, which can add to the effectiveness of the compositions and methods described herein.

[0139] The subject who is to receive treatment is a generally a mammal, preferably a human. A therapeutically effective dose of IL-12 is generally less than 1000 ng/kg/day and preferably less than 500 ng/kg/day. However, even lower doses of IL-12 are effective, such as doses of less than 100 ng/kg/day, especially when more than one dose is administered to the subject at varying time intervals. Exemplary dosages of IL-12 are described herein. Thus, some embodiments further include repeated administration, i.e., more than one administration of IL- 12, at certain time intervals following the initial administration. Subsequent doses of IL-12 may be the same or different from the initial dose.

[0140] The IL-12 dose amount can be, for example, weight-based dosing or a fixed dosing regimen in some embodiments. The IL-12 weight-based dose can be any dosage amount as described herein, e.g., from about 1 ng/kg up to about 2000 ng/kg, or less than about 2000 ng/kg. In some embodiments, the dose of IL-12 is less than about 1000 ng/kg, less than about 500 ng/kg, about 300 ng/kg, less than about 300 ng/kg, about 200 ng/kg, less than about 200 ng/kg, about 100 ng/kg, less than about 100 ng/kg, about 100 ng/kg or less, about 50 ng/kg or less, or about 10 ng/kg or less. In addition, the IL-12 fixed dosing regimen encompasses any fixed IL- 12 dose amount described herein, e.g., about 2 pg up to about 20 pg, or any amount in-between these two values, with a preferred dosing range of about 5 pg to about 15 pg, or any amount in- between these two values.

[0141] In addition, the IL-12 dose can be administered for any therapeutically effective duration of time as described herein, such as but not limited to, 1 day up to 1 year or any time point in-between, including for example 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. An IL-12 dose may also be administered for periods of longer than 1 year, e.g., over a several year period.

IV. DEFINITIONS

[0142] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0143] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0144] As used in the specification and claims, the singular forms“a”,“an” and“the” include plural references unless the context clearly dictates otherwise. For example, the term“a sample” includes a plurality of samples, including mixtures thereof.

[0145] The terms“determining”,“measuring”,“evaluating”,“assessing,”“assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement, and include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing is alternatively relative or absolute.“Detecting the presence of’ includes determining the amount of something present, as well as determining whether it is present or absent.

[0146] The terms“subject,”“individual,” or“patient” are often used interchangeably herein. A“subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. The disease can be endometriosis. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0147] The term“ in vivo” is used to describe an event that takes place in a subject’s body.

[0148] The term“ex vivo” is used to describe an event that takes place outside of a subject’s body. An“ex vivo” assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an“ex vivo” assay performed on a sample is an“in vitro” assay.

[0149] The term“in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the living biological source organism from which the material is obtained. In vitro assays can encompass cell-based assays in which cells alive or dead are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0150] As used herein, the terms“treatment” or“treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject,

notwithstanding that the subject may still be afflicted with the underlying disorder. A

prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0151] As used herein, the term“about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used,“about” will mean up to plus or minus 10% of the particular term.

[0152] “Disease state” refers to a condition present in a mammal whereby the health and well-being of the mammal is compromised.

[0153] “Chemotherapy” refers to agents useful in the treatment of cancer, including cytotoxic agents. The term as used herein includes natural or synthetic agents now known or to be developed in the medical arts. Examples of chemotherapy include the numerous cancer drugs that are currently available. In some embodiments, chemotherapy may include the administration of several state of the art drugs intended to treat the disease state.

[0154] “Hematopoietic disorders (cancers)” generally refers to the presence of cancers of the hematopoietic system such, as leukemias, lymphomas etc.

[0155] “Hematopoietic stem cells” are generally the blood stem cells; there are two types: “long-term repopulating” and“short-term repopulating” hematopoietic stem cells. Short-term repopulating hematopoietic stem cells can generally produce“progenitor cells” for a short period (weeks, months or even sometimes years depending on the mammal).

[0156] “Hematopoietic progenitor cells” are generally the first cells to differentiate from (i.e., mature from) blood stem cells; they then differentiate (mature) into the various blood cell types and lineages.

[0157] “ Interleukin- 12 (IL-12)” refers to any IL-12 molecule that yields at least one of the properties disclosed herein, including native IL-12 molecules, variant 11-12 molecules and covalently modified IL-12 molecules, now known or to be developed in the future, produced in any manner known in the art now or to be developed in the future. Generally, the amino acid sequences of the IL-12 molecule used in the compositions and methods described herein can be derived from the specific mammal to be treated. Thus, for the sake of illustration, for humans, generally human IL-12, or recombinant human IL-12, would be administered to a human, and similarly, for felines, for example, the feline IL-12, or recombinant feline IL-12, would be administered to a feline. Also disclosed herein are embodiments where the IL-12 molecule does not derive its amino acid sequence from the mammal that is the subject of the therapeutic methods disclosed herein. For the sake of illustration, human IL-12 or recombinant human IL- 12 may be utilized in a feline mammal.

[0158] Some embodiments include IL-12 molecules where the native amino acid sequence of IL-12 is altered from the native sequence, but the IL-12 molecule functions to yield the properties of IL-12 that are disclosed herein. Alterations from the native, species-specific amino acid sequence of IL-12 include changes in the primary sequence of IL-12 and encompass deletions and additions to the primary amino acid sequence to yield variant IL-12 molecules.

An example of a highly derivatized IL-12 molecule is the redesigned IL-12 molecule produced by Maxygen, Inc. (Leong et al., Proc Natl. Acad. Sci. U. S. A., 100(3): 1163-8 (Feb. 4, 2003)), where the variant IL-12 molecule is produced by a DNA shuffling method. Modified IL-12 molecules are also included in disclosure, such as covalent modifications to the IL-12 molecule that increase its shelf life, half-life, potency, solubility, delivery, etc., additions of polyethylene glycol groups, polypropylene glycol, etc., in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Each of these references is incorporated herein. 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 that is 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 may be covalently modified. Also as referred to herein, the IL-12 molecule can be produced by various methods known in the art, including recombinant methods. Since it is often difficult to predict in advance the characteristics of a variant IL-12 polypeptide, it will be appreciated that some screening of the recovered variant will be needed to select the optimal variant. A preferred method of assessing a change in the properties of variant IL-12 molecules is via 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 the tendency to aggregate with carriers or into multimers are assayed by methods well known in the art.

[0159] Exemplary IL-12 formulations are described, for example, in US Patent No.

7,939,058, US 2011-0206635, and US 2012-0190909, all for“Uses of IL-12 in Hematopoiesis;” US 2010-0278777 for“Method for Treating Deficiency in Hematopoiesis;” US 2010-0278778 for“Method for Bone Marrow Preservation or Recover;” US 2012-0189577 for“Use of IL-12 to Increase Survival Following Acute Exposure to Ionizing Radiation;” US 2013-0129674 and WO 2011/146574, both for“IL-12 Formulations For Enhancing Hematopoiesis;” US 2013-0259828 and WO 2012/050829, both for“Uses Of IL-12 And The IL-12 Receptor Positive Cell In Tissue Repair And Regeneration;” WO 2012/174056 for“Mitigation Of Cutaneous Injury With IL-12;” and WO 2013/016634 for“Use Of 11-12 To Generate Endogenous Erythropoietin.” Each of these references is incorporated herein.

[0160] “Radiation or radiation therapy or radiation treatment” refers to any therapy where any form of radiation is used to treat the disease state. The instruments that produce the radiation for the radiation therapy are either those instruments currently available or to be available in the future.

[0161] “ Solid tumors” generally is manifested in various cancers of body tissues, such as those solid tumors manifested in lung, breast, prostate, ovary, etc., and are cancers other than cancers of blood tissue, bone marrow or the lymphatic system.

[0162] Plasma terminal half-life (also terminal plasma half-life) is the time required to divide the plasma concentration by two after reaching pseudo-equilibrium, and not the time required to eliminate half the administered dose.

[0163] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

V. DISCUSSION

A. Discussion regarding Example 1

[0164] The first-in-human (FIH) and Phase lb expansion studies reported here were designed to study the safety of rHuIL-l2 when administered as a single SC dose of up to 20 pg in healthy subjects and to characterize the PK profile and hematological and immune-mediated effects of rHuIL-l2.

[0165] The FIH study used a conservative range of single 2 to 20 pg doses, which are equivalent to 0.03 to 0.3 pg/kg in a 70 kg person. The SC route was chosen as it represents the route of choice for a front line medical radiation countermeasure and is also appropriate for cancer patients based on historical data showing that SC administration of IL-12 limited the toxic effects observed with intravenous treatment.

[0166] Per protocol, dose reductions were required in the event that one or more subject experienced a single Grade 3 change in clinical laboratory values, based on a stringent toxicity grading scale for healthy subjects. At the 20 pg dose, the sentinel subject experienced Grade 3 thrombocytopenia and lymphopenia; thus, no additional subjects were dosed in this cohort. An additional cohort was enrolled at the reduced dose level of 15 pg, and one of 4 rHuIL-l2-treated subjects experienced Grade 3 neutropenia. An additional cohort was therefore enrolled for treatment at an intermediary dose of 12 pg. As no Dose-limiting toxicities (DLTs) were observed in this final cohort, the MTD was determined to be 12 pg. None of the drug-related, severe cytopenias observed in the FIH study resulted in any clinically significant manifestations and the absolute platelet and neutrophil counts were well above the levels which are considered predisposing to hemorrhagic or infectious episodes. No non-hematologic DLTs associated with rHuIL-l2 occurred at any doses in the FIH study. The tolerability of the MTD identified in the FIH study (12 pg) was confirmed in the expansion study, as no clinically significant changes in vital signs, ECGs and physical examinations were observed in either study.

[0167] The most common adverse events (AEs) related to rHuIL-l2 treatment in both studies were headache, dizziness, and chills. These AEs were expected and have been documented in previous studies as flu-like symptoms. Incidence of fever was 39% in an earlier study of healthy volunteer that used similar doses of IL-12, while it was 2% in our study. Thus, rHuIL-l2 administered as single, subcutaneous doses up to 12 pg was found to be safe and well tolerated in healthy subjects in these studies.

[0168] Data from these studies showed that rHuIL-l2 induced transient decreases in multiple peripheral blood cell counts, namely lymphocytes, neutrophils, platelets and

reticulocytes as well as subpopulations of lymphocytes, i.e., CD3+, CD4+, and CD8+ cells, NK cells, and CD34+ hematopoietic progenitor cells. All of these cell populations subsequently recovered at different rates and resolved to their pre-dose baseline levels, or slightly above. The early onset and rapid reversibility of these changes indicate that they do not result from suppression of proliferation of hematopoietic precursors and progenitors in bone marrow, which otherwise would be expected to start later and last longer. These transient hematological changes can be explained on the basis of trafficking and redistribution of cells from the central blood compartment, e.g., neutrophil margination and exit into tissues, platelet sequestration in the spleen, and lymphocyte redistribution to lymphoid organs. An additional explanation could be the delayed release of mature cells from the bone marrow post-mitotic compartments. The transient changes in neutrophil and lymphocyte counts observed in our studies have been reported by others. However the rHuIL-l2 induced transient changes in platelets and reticulocytes are novel findings in our study. We have not yet studied the underlying mechanism for these changes. However similar findings with respect to platelets have been reported with rHuIL-lO where mild reversible decline in platelet counts was seen by Sosman et al. In this healthy volunteer study, a relatively large decline was seen in splenic sequestration of platelets. These changes probably led to an increase in peripheral platelet counts.

[0169] In addition to the parameters described above, safety also was assessed by testing for the development of anti-rHuIL-l2 antibodies using a validated assay. Results showed no evidence of immunogenicity associated with rHuåL-l2 in the FIH study (measured after 28 days) or the expansion study (measured after 45 days).

[0170] This is the first report of a detailed PK analysis for IL-12 in human subjects. The validated ELISA used to measure systemic concentrations of IL-12 was sensitive enough to detect IL-12 levels at all dose levels examined. There were no gender differences in IL-12 exposure. Elimination of IL-12 was biphasic with a short initial half-life of 8 to 24 hours measurable in all subjects and a prolonged terminal half-life > 50 hours measurable only in 47% subjects, possibly due to limitation of the IL-12 assay. This terminal half- life possibly represents a slow return of IL-12 to the central compartment from the tissues, and/or slow absorption from the SC space via the lymphatics and/or capillaries. Another explanation could be the secondary expression of endogenous IL-12 produced through a positive feedback loop. In the FIH study, the IL-12 exposure generally increased with increase in the dose, as shown in the Table 4, below (Example 1).

[0171] IL-12 is known to stimulate adhesion molecules. In vitro studies have demonstrated that IL-12 is chemotactic to NK cells and stimulates adhesion of NK cells to endothelial cells and platelets via activation and increased expression of cell-cell adhesion molecules CD56 and CD1 la. The margination effect of lymphocytes and increased expression of adhesion molecules on NK cells was also demonstrated in cancer patients treated with IL-12. CD34 is also an important adhesion molecule required for T cells to enter lymph nodes. In the expansion study, expression of CD56 levels as measured by mean fluorescence intensity increased after rHuIL-l2 administration, while no such increase was seen in response to placebo. There also was an increase in CD34+ cells in response to rHuIL-l2, but not with placebo. These observations provide a mechanistic explanation for the transient hematological changes. In addition, analysis of NK and CD34+ progenitor cells for IL- 1211b2 positivity confirmed that these cells can be directly targeted by rHuIL-l2. During the course of the study, relative percentages of IL- 12Kb2+ cells after rHuIL-l2 treatment were similar to those observed after placebo treatment, suggesting that rHuIL-l2 did not down regulate IL 1211b2 within these two cell populations.

[0172] Overall the hematological changes observed in both studies suggest that rHuIL-l2 induces trafficking of peripheral blood cells from the vasculature to the tissues as a part of normal immune surveillance. Interestingly, the effect of rHuåL-l2 on peripheral blood cells was multilineage, affecting all major blood cell types. To the best of our knowledge, this is the first example of a single cytokine that simultaneously induces a decline followed by recovery of lymphocytes, neutrophils, platelets, reticulocytes as well as subpopulations of lymphocytes. We speculate that these changes are resulting from the activation and distribution of the cells into tissues. Definitive studies are needed to understand this phenomenon. Gately et al have reported animal data that is relevant to our speculation. Studies in IL-12 treated mice revealed focal mononuclear cell infiltrates and livers containing increased numbers of NK cells, CD8+ T cells, and monocytes. Also the liver and splenic lymphoid cells from these IL-l2-treated mice spontaneously secreted IFN-g In vitro, suggesting that they had been induced by IL-12 to produce IFN-g In vivo. This was indeed confirmed as IFN-g levels were detected in the serum of IL-l2-treated mice. Additionally, Mortarini et al have reported a clinical study in metastatic melanoma patients where subcutaneous administration of human recombinant IL-12 promoted the infiltration of neoplastic lesions by CD81 T Cells with a memory phenotype.

[0173] The hallmark effect of IL-12 in immunity is its ability to stimulate the production of IFN-g from NK cells, macrophages and T cells. CXCL10 is a chemokine associated with T-cell responses and leukocyte migration, while IL-18 plays important roles in the development, homeostasis, and functions of CD4+ T cells, CD8+ T cells, and NK cells. Additionally EPO was also analyzed as a systemic measure of rHuåL-l2 activity based on its biological functions, other than erythropoiesis, that have been unraveled after finding EPO receptors on cells other than erythroid progenitors, such as polymorphonuclear leukocytes, megakaryocytes, and endothelial, myocardial, and neuronal cells. EPO production was stimulated by rHuIL-l2 in non-irradiated and irradiated NHP and mice demonstrating a potential central role in mediating the radiomitigation activity. Thus induction and interrelationship between rHuIL-l2 and a battery of these biological response parameters was studied in healthy human subjects.

[0174] The Pharmacodynamic (PD) parameters in these studies were selected based on their relevance to immune-mediated effects of IL-12 that have been well documented in vitro, as well as in animal models and clinical studies. In our studies, IFN-g, IL-18 and CXCL10 were found to be useful as downstream positive biological response parameters in support of the

immunomodulatory activities of rHuIL-l2. The IFN-g response was consistently detected in both studies. The relatively delayed Tmax together with the lack of endogenous IFN-g indicate that this is a true biological IFN-g response, which is known to be mediated via differentiation of TH1 cells. CXCL10 is another sensitive marker of induction of THl-like innate immune activation manifested by IFN-g, as well as directly stimulated by IL-12. Significantly higher levels of CXCL10 were detected in response to rHuIL-l2, although results were confounded by variable endogenous levels. Based on the PD effects observed in these studies, we hypothesize that a single, low dose of exogenous rHuIL-l2 is a trigger for multilineage hematopoietic stimulation and innate immune responses, which is unique and not achievable with currently used hematopoietic growth factors.

[0175] Currently rHuIL-l2 is being developed as a radiation countermeasure under the provisions of FDA’s Animal Rule. Nonclinical studies in rhesus monkeys have shown a similar pattern of transient hematological changes and immune responses with rHuIL-l2 treatment in normal monkeys. Further, in the studies where monkeys were subjected to lethal total body irradiation, single, low doses of rHuIL-l2 in the range of 50 to 500 ng/kg demonstrated an increased survival benefit in the absence of any supportive care (i.e., no antibiotics, fluids or blood products). The safe and well-tolerated human dose of 12 pg (171 ng/kg based on 70 kg human body weight) is within this efficacious range based on exposure parameters (Gluzman- Potlorak et al., manuscript submitted). Based on these results, rHuåL-l2 is being developed under the FDA Animal Rule approval pathway as a frontline therapy in humans to mitigate radiation-induced damage and increase the potential for survival in the event of a nuclear disaster or accident. We also have reported that low-dose, adjuvant IL-12 promotes multilineage hematopoietic recovery from cancer therapy-induced cytopenias, along with concomitant anti tumor responses, in tumor-bearing mice. Thus, results from these animal and human studies of rHuIL-l2 also can be applied towards clinical trials in oncology, with the aim of reducing both toxicity and tachyphylactic effects that result from repeated high-dose regimens of IL-12, thereby augmenting antitumor effects.

[0176] Conclusions: The observations from these studies indicate that a single low dose of rHuIl-l2 administered subcutaneously can elicit hematological and immune-mediated effects without undue toxicity. The potent hematological and immune effects observed in the examples described herein suggest that IL-12, when dosed in a prudent manner, can be safely used to support the hematopoietic system and provide immunotherapeutic benefits in individuals, such as exposed to lethal radiation, as well as in patients with myelosuppression resulting from either chemotherapy or radiation therapy.

B. Discussion regarding Example 2:

[0177] The data from Example 2, which was a randomized, blinded, placebo-controlled study, demonstrate a positive and significant effect of a single, subcutaneous injection of IL-12, over a lO-fold dose range, on survival following lethal total body irradiation (TBI) (700cGy; LD90_/60) in the rhesus monkey model of HSARS. The animal model used in this study has been validated at CiToxLAB North America as an established model of human HSARS, based on the occurrence of similar hematologic effects, infection and hemorrhage following TBI as reported for humans.

[0178] The mechanism by which IL-12 rescues animals following TBI involves the multiple effects of IL-12 on hematopoieses and immune function. Radiation-induced bone marrow suppression was mitigated by IL-12: animals treated with IL-12 showed statistically significant reductions in the occurrence of severe neutropenia and severe thrombocytopenia, as well as attenuated nadirs for lymphocytes, neutrophils, platelets, and reticulocytes. Further, the increase, relative to controls, in mean platelet volume among animals treated with IL-12 suggests that IL-12 promoted release of newly formed platelets from the bone marrow - e.g, peripheral blood cell trafficking to a target site. Quantitative analysis of the number and size of bone marrow regenerative pockets supports the conclusion that IL-12 alone stimulates hematopoiesis, allowing for recovery of all major blood cell components.

[0179] The in vivo observation that IL-12 induced recovery of multiple hematopoietic lineages is consistent with previous reports in which IL-12 stimulated growth of hematopoietic stem cells and progenitors in vitro, and prevented radiation-induced death of hematopoietic stem cells in vivo in a murine model of HSARS. This multilineage hematopoietic effect is also consistent with previous findings in studies of tumor-bearing mice and with the observation that IL-12 receptors are present on hematopoietic stem cells. The observation that blood counts were higher in the surviving IL-12 treated animals than in decedents further supports the conclusion that IL-12 improved survival by regeneration of multilineage bone marrow hematopoiesis.

[0180] Decrease of lymphocyte counts below 0.25 xl0⁹/L has been established as a marker of irreparable lethal bone marrow damage in a large database of human victims of acute radiation. As such, it is important to note that in the study described below, the average lymphocyte nadir was 0.09 xl0⁹/L among decedents in the control group, 0.14 xl0⁹/L among decedents in the rHu-ILl2-treated groups, and 0.27 xl0⁹/L among survivors in all groups. These findings further support the validity of the Rhesus monkey animal model as an accurate representation of human HSARS and its ability to predict effectiveness in humans exposed to lethal radiation.

[0181] Consistent with the reduction in severe neutropenia, the incidence of blood culture positivity for infection was significantly lower in IL-l2-treated groups 4 and 5 (47% and 44%, respectively) compared with than in the control group (86%). These data demonstrate that IL-12, administered after total body irradiation, such as about 24 hours after TBI, in the absence of antibiotics, decreased infectivity of broad-spectrum bacteria. These effects are consistent with and are likely due to the well-known multiple stimulatory effects of IL-12 on innate and adaptive immunity. Previous studies in mice have shown that during the early stages following exposure to lethal radiation, type 1 T-helper cell (Thl) function is reduced due to the suppression of endogenous IL-12 secretion from antigen presenting cells. IL-12 administered after irradiation can promote the proliferation and activation of the surviving natural killer (NK) cells, macrophages and dendritic cells. In the data described herein, it is demonstrated that in irradiated monkeys IL-12 increases plasma levels of IFN-g, which is the hallmark of NK cell activation, as well as IL-18, and IP- 10, similarly observed previosely in non-irradiated monkeys.¹⁵ The tri-directi onal cross-talk between NK, macrophages and dendritic cells further promotes their maturation, leading to the restoration of Thl function and the establishment of early immune competence following TBI.

[0182] Further, continuous production of endogenous IL-12 from pathogen-activated dendritic cells serves as a positive feedback loop and plays a key role in sustaining the initial response to exogenous IL-12. Taken together, these IL-12 generated immune-mediated effects can account in large part for the positive survival benefit observed in Example 2.

[0183] Consistent with the reduction in severe thrombocytopenia, IL-12 treatment in Example 2 was associated with lower severity of hemorrhage for animals that died or were euthanized prior to the scheduled termination on Day 60. In support of the finding that treatment with IL-12 was associated with reduced severe thrombocytopenia and hemorrhage, it was recently reported that hematopoietic stem cells, egakaryocytes and osteoblasts in the bone marrow express the IL-12 receptor b2 subunit (IL- 1211b2), which is primary subunit for IL-12 signaling. The presence of IL-l2R)32 receptors on these key bone marrow cells suggest that through its receptors, rHuIL-l2 may promote proliferation and differentiation of the surviving stem cells and megakaryocytes following exposure to lethal radiation, thereby enhancing platelets regeneration and reducing severe thrombocytopenia. Indeed, quantitative analysis of the bone marrow in the current companion study (described in Example 2) showed that relative to controls, IL-12- treated groups had higher numbers of megakaryocytes. The ability of IL-12 to facilitate regeneration of platelets may be of clinical importance in indications other than HSARS mitigation, such as cancer, as there is currently no available drug that can facilitate platelet recovery following myelosuppressive therapies. [0184] While leucocyte growth factors are recommended for use in victims of radiation, they are not approved by FDA for this indication. One published study of radiation mitigation in NHP demonstrated improved survival following exposure to lethal radiation for animals treated with rHuG-CSF (recombinant human Granulocyte-colony stimulating factor) in combination with intensive, trigger-based medical management (antibiotics, intravenous blood product transfusions, intravenous fluid replacement) compared with that of control animals that received only the medical management.

[0185] Pharmacokinetics and Pharmacodynamics of IL-12: Example 2 also provides an evaluation of IL-12 pharmacokinetics and pharmacodynamics,. In the animal cohort used for pharmacokinetic and pharmacodynamic analyses, blood samples from animals treated with SC rHuIL-l2 were collected at the various time points ranging from pretreatment (approximately 2 weeks prior to irradiation) up to 264 hours after IL-12 dosing (e.g, 11 days after IL-12 dosing). The concentrations of IL-12 and IFN- g in monkey plasma were determined, and then measured using ELISA. In addition, IL-18 and interferon g-induced protein (IP- 10) levels were determined using ELISA methods. Standard non-compartmental analyses were performed using Phoenix™ WinNonlin® Version 6.3 (WinNonlin; Pharsight Corporation, Mountain View, CA).

[0186] A randomized, blinded study comparing a single injection of rHuIL-l2 or vehicle with 18 injections of rHuG-CSF in the NHP model without supportive care was recently conducted. Preliminary analysis confirmed superior survival in the rHuIL-l2-treated group vs. both the control group and a G-CSF-treated group. Notably, G-CSF did not provide any survival benefit compared to control (Gluzman-Poltorak et al, in preparation).

[0187] In parallel to the animal efficacy studies, the safety and tolerability of rHuIL-l2 has been examined in normal healthy subjects per the Animal Rule. A first in human (FIH) study was conducted to determine the safe and well-tolerated doses of rHuIL-l2 via dose escalation (at doses ranging from 2 to 20 pg). The FIH study was followed by a phase lb expansion study at the highest safe and well-tolerated dose from the FIH study of 12 pg

(Gokhale et al, submitted for publication). The 12 pg unit human dose for a 70 kg adult can be converted to 171 ng/kg rhesus monkey dose using a weight based conversion and this dose is within the efficacious dose range as determined in the Rhesus monkeys studies.

C. Discussion regarding Example 4:

[0188] Example 3 describes a PK analysis of IL-12 doses based upon data from four monkey and two human trials to develop a cross-species compartmental population PK IL-12 model. One of the objectives of Example 3 was to refine the previously developed cross-species PK model by investigating the impact of below the limit of quantitation (BLQ) effects on PK parameters of IL-12 and predict the long term effects of IL-12. In particular, to improve the long term prediction of IL-12 concentrations (e.g. beyond 1 or 2 weeks), the population PK model was customized by including a likelihood function that takes into account censoring of below the limit of quatitation (BLQ) values, and the expected distribution of concentrations for samples with a high number of BLQ values (“model refinement”).

[0189] Figure 14 depicts a final structural model of IL-12 following SC dosing in humans and monkeys, with BSV = Inter-individual variability, CL = Systemic clearance, CLd =

Intercompartmental distribution, CLdt = Distribution to deep tissue, Vc = Central volume of distribution, Vdt = Volume of distribution to deep tissue, Vp = Volume of peripheral compartment, Kaf = absorption rate to the capillaries, Kas = absorption rate to the lymphatic system, F = absolute bioavailability and Frel = relative amount of the dose to the lymphatic system. In particular, Figures 15E, F, and G show that for the refined model, there is no effect on C_max (Fig_· 15E), less bias for few low concentrations (Fig. 15F), and a better characterization of the terminal phase (Fig. 15G).

[0190] The impact of including a cumulative distribution function on the BLQ on the systemic parameters of HemaMax (CL, Vc...) was negligible (i.e., less than 1% change).

However, significant changes in mean absorption time (MAT) from capillaries and the lymphatic system in irradiated monkeys and humans were observed: (1) an increase in

MATlymphatic in monkeys was observed; (2) an increase in MATlymphatic in humans was observed; and (3) previously observed gender-related effects are no longer seen. These results are likely to simplify the dosing rationale in humans. Overall, this sensitivity analysis suggests that the refined model resulted in a slight improvement in the quality-of-fit.

D. Discussion regarding Example 5:

[0191] Example 5 describes PK and pharmacological data following in vivo administration of IL-12 dosages to non-human primates at two time points: 0 and 28 days. The PK results for IL-12 (pg/mL vs time) are shown in Figures 16 and 17, the pharmacodynamics for IFN-g (pg/mL vs time) are shown in Figures 18 and 19, and the hematology changes from baseline are shown in Figures 20 (lymphocytes) and 21 (platelets). The data show that IL-12 administration produces a similar PK profile after subcutaneous injection of 250 ng/kg in monkeys. The data also show that IL-12 administration triggers a first modest peak in IFN-g serum levels, but this is then following by a more significant peak several days following IL-12 administration (e.g., 72 - 120 hours following initial IL-12 administration). Similar results are seen with a second IL-12 injection at 28 days. See Figures 18 and 19. Similarly, the data show that IL-12 administration is following by an initial modest increase in lymphocytes, followed by a significant decrease, and then a consistent increase well above the initial lymphocyte increase. This second increase continues for an extended period of time, with similar results shown for a second IL-12 dosage at 28 days. These data support the IL-12 dosing rational described herein where IL-12 dosages can have effects over a prolonged period of time.

VI. EXEMPLARY EMBODIMENTS

[0192] Among the exemplary embodiments are:

[0193] 1. A method of treating a subject in need with an exogenous IL-12 composition comprising: a) administering a first treatment comprising administering a first dose of IL-12 to the subject; and b) administering a second treatment comprising administering a second dose of IL-12 to the subject, wherein the second treatment is administered after a first non -treatment interval of at least 8 days. 2. The method of embodiment 1, wherein the second treatment elicits a therapeutic response that is not diminished by tachyphylaxis. 3. The method of embodiment 1 or 2, further comprising administering a third treatment comprising administering a third dose of IL-12 to the subject, wherein the third treatment is administered after a second non -treatment interval of at least 8 days. 4. The method of any one of embodiments 1 to 3, further comprising administering a fourth treatment comprising administering a fourth dose of IL-12 to the subject, wherein the fourth treatment is administered after a third non-treatment interval of at least 8 days. 5. The method of any one of embodiments 1 to 4, further comprising administering a fifth treatment comprising administering a fifth dose of IL-12 to the subject, wherein the fifth treatment is administered after a fourth non-treatment interval of at least 8 days. 6. The method of any one of embodiments 1 to 5, wherein a non-treatment interval is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. 7. The method of any one of embodiments 1 to 6, wherein a non-treatment interval is no more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. 8. The method of any one of embodiments 1 to 7, wherein a non-treatment interval is at least 1 2, 3, or 4 weeks. 9. The method of any one of embodiments 1 to 8, wherein a non-treatment interval is no more than 1, 2, 3, or 4 weeks. 10. The method of any one of embodiments 1 to 9, wherein a non-treatment interval is at least 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. 11. The method of any one of embodiments 1 to 10, wherein a non-treatment interval is not more than 1 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, or 12 months. [0194] 12. The method of any one of embodiments 1 to 11, wherein a dose is administered before, during, or after a cycle of chemotherapy. 13. The method of embodiment 12, wherein the dose is administered 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days before or after a cycle of chemotherapy.

[0195] 14. The method of any one of embodiments 1 to 13, wherein a dose is administered before, during, or after a cycle of radiation therapy. 15. The method of embodiment 14, wherein the dose is administered 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25

26, 27, 28, 29, 30, or 31 days before or after a cycle of radiation therapy. 16. The method of embodiment 14 or 15, wherein the radiation therapy comprises low-dose Total Skin Electron Beam Therapy (LD-TSEBT).

[0196] 17. The method of any one of embodiments 3 to 16, wherein the second non treatment interval is different than the first non-treatment interval. 18. The method of any one of embodiments 4 to 17, wherein the third non-treatment interval is different than at least one of the second non-treatment interval and the first non -treatment interval. 19. The method of any one of embodiments 5 to 18, wherein the fourth non-treatment interval is different than at least one of the third non-treatment interval, the second non-treatment interval, and the first non-treatment interval.

[0197] 20. The method of any one of embodiments 3 to 19, wherein the third dose elicits a therapeutic response that is not diminished due to tachyphylaxis. 21. The method of any one of embodiments 4 to 20, wherein the fourth dose elicits a therapeutic response that is not diminished due to tachyphylaxis. 22. The method of any one of embodiments 5 to 21, wherein the fifth dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

[0198] 23. The method of any one of embodiments 1 to 22, wherein at least one dose comprises between 2-20 pg of IL-12. 24. The method of embodiment 23, wherein the at least one dose comprises between 5-15 pg of IL-12. 25. The method of embodiment 23, wherein the at least one dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of lL- 12

[0199] 26. The method of embodiment 23, wherein each dose comprises between 2-20 pg of IL-12. 27. The method of embodiment 26, wherein each dose comprises between 5-15 pg of IL- 12. 28. The method of embodiment 26, wherein each dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5

17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of IL-l2.

[0200] 29. The method of any one of embodiments 23 to 28, wherein the at least one dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg). 30. The method of embodiment 29, wherein at least one dose comprises 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ng/kg.

[0201] 31. A method of treating a subject in need with an exogenous IL-12 composition comprising administering a first treatment comprising administering a first dose of IL-12 to the subject, wherein the first dose comprises between 2-20 pg of IL-12. 32. The method of embodiment 31, wherein the first dose comprises between 5-15 pg of IL-12. 33. The method of embodiment 32, wherein the first dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,

8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of IL-l2.

[0202] 34. The method of any one of embodiments 31 to 33, wherein the first dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg). 35. The method of embodiment 34, wherein the first dose comprises 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ng/kg.

[0203] 36. The method of any one of embodiments 1 to 35, wherein the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant. 37. The method of embodiment 36, wherein the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL. 38. The method of embodiment 37, wherein the pharmaceutical composition comprises IL-12 at a concentration of 20 pg/mL. 39. The method of any one of embodiments 36 to 38, wherein the buffer comprises sodium phosphate. 40. The method of any one of embodiments 36 to 39, wherein the pharmaceutical composition comprises 10 mM sodium phosphate. 41. The method of any one of embodiments 36 to 40, wherein the salt comprises sodium chloride. 42. The method of any one of embodiments 36 to 41, wherein the pharmaceutical composition comprises 150 mM sodium chloride. 43. The method of any one of embodiments 36 to 42, wherein the surfactant is a non-ionic surfactant. 44. The method of embodiment 43, wherein the non-ionic surfactant comprises poloxamer 188. 45. The method of any one of embodiments 36 to 44, wherein the pharmaceutical composition comprises 0.1%

(w/v) poloxamer 188. 46. The method of any one of embodiments 36 to 45, wherein the pharmaceutical formulation comprises a pH of between 5.0 to 8.0. 47. The method of embodiment 46, wherein the pharmaceutical formulation comprises a pH of 6.0. [0204] 48. The method of any one of embodiments 1-47, wherein the method comprises treating hematopoietic syndrome of the acute radiation syndrome (HSARS) in the subject. 49. The method of any one of embodiments 1-48, wherein the method comprises treating cutaneous T-cell lymphoma (CTCL) in the subject.

[0205] 50. The method of any one of embodiments 1 to 49, wherein the method further comprises adjusting a length of a non-treatment interval prior to a treatment based on a time point at which the subject is expected to have completed a direct response to the first dose of IL- 12. 51. The method of any one of embodiments 1 to 50, wherein the method further comprises adjusting a length of a non-treatment interval prior to a treatment based on a time point at which the subject is expected to have completed an indirect response to the first dose of IL-12. 52. The method of any one of embodiments 1 to 51, wherein the method further comprises adjusting a length of a non-treatment interval prior to a treatment based on a time point at which the a previous treatment is expected to no longer exert a pharmacodynamic effect on the subject.

[0206] 53. The method of any one of embodiments 1 to 52, wherein the method further comprises assessing a level of IL-12 in the subject's blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of IL-12 is above a threshold amount. 54. The method of embodiment 53, wherein the threshold amount is a level of IL-12 in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

[0207] 55. The method of any one of embodiments 1 to 54, wherein the method further comprises assessing a level of at least one of INF-gamma, IL-2, IL-10, IL-18, or CXCL10 in the subject's blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of the at least one of INF-gamma, IL-2, IL-10, IL-18, or CXCL10 is above a threshold amount. 56. The method of embodiment 55, wherein the threshold amount is a level of the at least one of INF-gamma, IL-2, IL-10, IL-18, or CXCL10 in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

[0208] 57. The method of any one of embodiments 1 to 56, wherein the method further comprises assessing a level at least one of lymphocytes, neutrophils, platelets, and reticulocytes in the subject's blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of the at least one of lymphocytes, neutrophils, platelets, and reticulocytes is below a threshold amount. 58. The method of embodiment 57, wherein the threshold amount is a level of the at least one of lymphocytes, neutrophils, platelets, and reticulocytes in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

[0209] 59. A method of treating a subject in need with an exogenous IL-12 composition comprising: a) administering a first dose of IL-12 to the subject; and b) administering a second dose of IL-12 to the subject at least 8 days after administering the first dose. 60. A method of treating a subject in need with an exogenous IL-12 composition comprising: a) administering a first dose of IL-12 to the subject; and b) administering a second dose of IL-12 to the subject, wherein the second dose elicits a therapeutic response that is not diminished due to

tachyphylaxis.

[0210] 61. The method of embodiment 59 or 60, wherein the second dose is administered at a time point that reduces a likelihood that the subject will develop tachyphylaxis. 62. The method of any one of embodiments 59 to 61, wherein the second dose is administered at least 8 days after administering the first dose. 63. The method of any one of embodiments 59 to 62, wherein the second dose is administered at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days after the first dose. 64. The method of embodiment 63, wherein the second dose is administered no more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days after the first dose. 65. The method of any one of embodiments 59 to 63, wherein the second dose is administered at least 1 2, 3, or 4 weeks after the first dose. 66. The method of any one of embodiments 59 to 65, wherein the second dose is administered 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the first dose.

[0211] 67. The method of any one of embodiments 59 to 66, further comprising

administering a third dose of IL-12 after administering the second dose, wherein the third dose elicits a therapeutic response that is not diminished due to tachyphylaxis. 68. The method of embodiment 67, further comprising administering a fourth dose of IL-12 after administering the third dose, wherein the fourth dose elicits a therapeutic response that is not diminished due to tachyphylaxis. 69. The method of embodiment 68, further comprising administering a fifth dose of IL-12 after administering the fourth dose, wherein the fifth dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

[0212] 70. The method of any one of embodiments 59 to 69, wherein at least one dose comprises between 2-20 pg of IL-12. 71. The method of embodiment 70, wherein the at least one dose comprises between 5-15 pg of IL-12. 72. The method of embodiment 70, wherein the at least one dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of lL- 12

[0213] 73. The method of embodiment 70, wherein the at least one dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg). 74. The method of embodiment 73, wherein at least one dose comprises 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ng/kg.

[0214] 75. The method of any one of embodiments 59 to 74, wherein the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant. 76. The method of embodiment 75, wherein the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL 77. The method of embodiment 76, wherein the pharmaceutical composition comprises IL-12 at a concentration of 20 pg/mL. 78. The method of any one of embodiments 75 to 77, wherein the buffer comprises sodium phosphate. 79. The method of any one of embodiments 75 to 78, wherein the pharmaceutical composition comprises 10 mM sodium phosphate. 80. The method of any one of embodiments 75 to 79, wherein the salt comprises sodium chloride. 81. The method of any one of embodiments 75 to 80, wherein the pharmaceutical composition comprises 150 mM sodium chloride. 82. The method of any one of embodiments 75 to 81, wherein the surfactant is a non-ionic surfactant. 83. The method of embodiment 82, wherein the non-ionic surfactant comprises poloxamer 188. 84. The method of any one of embodiments 75 to 83, wherein the pharmaceutical composition comprises 0.1%

(w/v) poloxamer 188. 85. The method of any one of embodiments 75 to 84, wherein the pharmaceutical formulation comprises a pH of between 5.0 to 8.0. 86. The method of embodiment 85, wherein the pharmaceutical formulation comprises a pH of 6.0.

[0215] 87. A method of treating a subject in need with an exogenous IL-12 composition comprising: a) administering a first single low dose of IL-12, wherein IL-12 can be detected in a sample of the subject's blood, serum, and/or plasma for at least one week; and b) administering at least one subsequent dose of IL-12 at a time point when the amount of IL-12 in the subject's blood is no longer observable. 88. The method of embodiment 87, wherein the IL-12 dosing schedule results in preventing the occurrence of tachyphylaxis. 89. The method of embodiment 87 or 88, wherein a subsequent dose of IL-12 is administered at a time point when peripheral blood cell trafficking to a site of injury or disease is decreasing. 90. The method of embodiment 89, wherein the peripheral blood cells are selected from the group consisting of NK cells, monocytes, red blood cells reticulocytes, platelets, and any combination thereof. 91. The method of any one of embodiments 87 to 90, wherein a subsequent does of IL-12 is not administered when one or more Th2 cytokines are detectable in the subject's blood, serum, and/or plasma. 92. The method of any one of embodiments 87 to 91, wherein if one or more Th2 cytokines are detectable in the subject's blood, serum, and/or plasma, then the subsequent dosage of IL-12 is decreased as compared to the prior IL-12 dosage. 93. The method of any one of embodiments 87 to 92, wherein if one or more Th2 cytokines have increased in the subject's blood, serum, and/or plasma sample, as compared to baseline levels of the same cytokine present in serum of either the subject or the patient population for the subject, then the subsequent dosage of IL-12 is decreased as compared to the prior IL-12 dosage. 94. The method of any one of embodiments 92 to 93, wherein the Th2 cytokine is selected from the group consisting of IL- 2, IL-4, IL-3, IL-5, IL-6, IL-10, IL-13, IL-25, IL-31, and IL-33.

[0216] 95. The method of any one of embodiments 87 to 94, wherein the IL-12 is detectable in the subject's blood, serum, and/or plasma due to lymphatic absorption of IL-12. 96. The method of any one of embodiments 87 to 95, wherein the IL-12 is detectable in the subject's blood, serum, and/or plasma at least in part due to endogenous production of IL-12 stimulated by the exogenous IL-12 administration.

[0217] 97. The method of any one of embodiments 87 to 96, wherein the subsequent dose of IL-12 is administered at least 2 weeks after the first IL-12 dose. 98. The method of any one of embodiments 87 to 97, wherein the method is used as adjunctive therapy to a radiation cancer treatment. 99. The method of any one of embodiments 87 to 97, wherein the method is used as adjunctive therapy to a chemotherapy cancer treatment.

[0218] 100. The method of any one of embodiments 87 to 99, wherein the IL-12 dose is a weight-based dosage amount. 101. The method of any one of embodiments 87 to 99, wherein the IL-12 dose is a fixed dosage amount.

VII. EXAMPLES

[0219] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: First in human study

[0220] The purpose of this example was to demonstrate in healthy subjects the safety of rHuIL-l2 at single, low doses that are appropriate for use as a medical countermeasure for humans exposed to lethal radiation and as an immunomodulatory anti-cancer agent.

[0221] Summary of the Results. Thirty-two subjects were enrolled in the FIH study: 4 active and 2 placebo at rHuIL-l2 doses of 2, 5, 10, 12, and 15 pg; 1 active and 1 placebo at 20 pg. Sixty subjects were enrolled in the expansion study: 48 active and 12 placebo at 12 pg dose of rHuIL-l2. In both studies, the most common adverse events (AEs) related to rHuIL-l2 were headache, dizziness, and chills. No immunogenicity was observed. Elimination of rHuIL-l2 was biphasic, suggesting significant distribution into extravascular spaces. rHuIL-l2 triggered transient changes in neutrophils, platelets, reticulocytes, lymphocytes, natural killer cells, and CD34⁺ hematopoietic progenitor cells, and induced increases in interferon-g and C-X-C motif chemokine 10.

[0222] Conclusion. A single low dose of rHuIl-l2 administered subcutaneously can elicit hematological and immune-mediated effects without undue toxicity. The safety and the potent multilineage hematopoietic/immunologic effects triggered by low-dose rHuåL-l2 support the development of rHuIL-l2 both as a radiation medical countermeasure and as adjuvant immunotherapy for cancer.

[0223] Methods. Two placebo-controlled, double-blinded studies assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of rHuåL-l2. The first-in-human (FIH) dose-escalation study randomized subjects to single subcutaneous injections of placebo or rHuIL-l2 at 2, 5, 10, and 20 pg doses. Due to toxicity, the dose was reduced to 15 pg and then to 12 pg. The phase lb expansion study randomized subjects to the highest safe and well tolerated dose of 12 pg.

[0224] Subjects: A total of 32 male and female healthy subjects, between 18 and 45 years of age, with a body mass index > 19 and < 30 kg/m², were enrolled in the first-in-human (FIH) study and 60 subjects were enrolled in the expansion study.

A. Study Design

[0225] The primary endpoint for both studies was safety and tolerability of rHuIL-l2.

Secondary endpoints were pharmacokinetic and pharmacodynamic profiles.

[0226] In the double-blind FIH study, eligible subjects were enrolled in consecutive cohorts to receive either a single subcutaneous dose of rHuIL-l2 (2, 5, 10, 20 pg) or placebo. Due to the occurrence of DLTs in the first subject treated at the 20 pg dose level, an additional cohort of 6 subjects was randomized to rHuIL-l2 at 15 pg or placebo, and due to the occurrence of a DLT in the final subject in that cohort, an additional cohort of 6 subjects was enrolled for treatment at 12 pg of rHuIL-l2 or placebo. Thus, a total of 32 subjects were enrolled and randomized.

[0227] Study drug was administered subcutaneously on day 1 following baseline

assessments. Blood samples for clinical chemistry and hematology assessments were collected at screening, baseline (day 1), and on days 2 to 7, 11, 14 and 28. PK samples were collected on day 1 (within 1 hour predose, and at 1, 2, 5, 8, 12, and 24 hours postdose) and daily on days 3 to 7, 11, and 14. PD samples were collected at screening, on day 1 (within 1 hour predose; at 5,

12, and 24 hours post dose), and daily on days 3 to 7, 11 and 14. Samples for immunogenicity testing were collected on day 1 (within 1 hour predose) and on day 28.

[0228] In the double-blind Phase lb expansion study, eligible subjects were randomized at a 4: 1 ratio to receive either a single subcutaneous 12 pg dose of rHuåL-l2 or placebo and were dosed in three consecutive groups. Study drug was administered on day 1 following baseline assessments. Blood samples were collected for clinical laboratory investigations and immunogenicity tests from all 60 subjects at screening, daily on days 1 (within 1 hour pre-dose) to 16 and 28. Samples for PK and PD analyses were collected only from the subset of 40 subjects. PK samples were collected on day 1 (within 1 hour pre-dose and at 1, 2, 5, 8, 12 and 24 hr post dose); then daily on days 3 to 7. PD samples were collected on day -1, day 1 (within 1 hour predose and at 5, 12 and 24 hours post dose), then daily on days 3 to 7, 11, and 14.

Peripheral blood samples for the flow cytometry analysis were collected on day-l, day 1 (within 1 hour pre-dose); then daily on days 2 to 7, 9, 11, and 14. Samples for immunogenicity testing were collected on day 1 (within 1 hour pre-dose) and on day 28 (all subjects) and day 45 (only for 34 subjects who consented to additional sampling).

[0229] Analysis of Safety: The safety and tolerability of rHuåL-l2 were assessed by physical examination, vital sign measurements, laboratory tests, ECGs, and monitoring and reporting of adverse events (AEs). As only one dose of rHuIL-l2 was given, subjects were not withdrawn from the study as a result of a DLT. In the expansion study, subjects from all 3 groups were combined to form the placebo and rHuåL-l2 groups for comparison of endpoints.

In both studies, the safety analysis population included all subjects who were randomized.

[0230] Hematological Assessment: A complete blood count (CBC) was done and flow cytometric detection of immunophenotypes within the lymphocyte population, i.e. CD3⁺, CD4⁺, and CD8⁺ lymphocytes, was done. Additionally, NK cells and circulating hematopoietic progenitors were measured by flow cytometry. NK cells were defined to be the

CD45⁺CDl6⁺CD56⁺ lymphocyte population, and circulating hematopoietic progenitors were defined to be CD45⁺CD34⁺ leukocytes. IL- 12RIb2 positivity was further determined on the NK and CD34⁺ cells. Fluorescently labeled antibodies against 7-AAD, CD45, CD56, CD16, and CD34, were purchased from BD Biosciences (San Jose, CA) and the anti-IL-l2 Rj32 antibody^ was purchased from R&D Systems (Minneapolis, MN). [0231] Bioanalytical Assays: All enzyme linked immunosorbent assay (ELISA) methods were validated in human K2 EDTA plasma using commercially available kits and antibodies. These assays were performed at Intertek Pharmaceutical Services, San Diego, CA. Assay systems and LLOQ were as follows: rHuåL-l2 - Quantikine® HS Human IL-12 High

Sensitivity ELISA Kit, (R&D Systems; Minneapolis, MN; Cat. #HSl20, SS120, or PHSl20) and LLOQ = 3.75 pg/mL; IFN-g. Human Interferon-g (IFN-g) ELISA MAX™ Deluxe Sets, (BioLegend Inc.; San Diego, CA; Cat. #430106) and LLOQ = 30.00 pg/mL; EPO - Quantikine® IVD® Erythropoietin ELISA kit, (R&D Systems; Cat. # DEP00) and LLOQ = 2.00 mlU/mL; IL-18 - Human IL-18 ELISA Kit, MBL (Japan), distributed by R&D Systems (Cat #7620) and LLOQ = 25.60 pg/mL; CXCL10 - Quantikine® Human CXCL10 Immunoassay ELISA Kit, (R&D Systems; Cat. #DIPl00) and LLOQ = 12.00 pg/mL. Immunogenicity testing (anti rHuIL-l2 antibodies) used a tiered cut-point titration method validated at Intertek

Pharmaceutical Services, utilizing anti-human IL-12 (p70) antibody (BioLegend Inc.; Cat.

#511002); LLOQ was not applicable.

[0232] Pharmacokinetic and Pharmacodynamic Analyses: Plasma PK analyses for rHuIL-l2, and PD analyses for IFN-g, EPO, IL-18 and CXCL10 were performed using non- compartmental methods within Phoenix™ WinNonlin^® Version 6.3 or higher (Pharsight Corporation; Mountain View, California). Nominal dose and actual weight and sampling times were used. Summary statistics were prepared with WinNonlin and Microsoft^® Excel 2007 (Seattle, WA), while mean and individual concentration graphs were prepared with Prism™ Version 5.0 (GraphPad Software, Inc.; La Jolla, CA). Descriptive statistics were provided for PK and PD by treatment and by gender. Group and gender comparisons of means for C_max, AUCo-_t and AUCo-_¥ values were conducted via a t-test with log-transformed data using

MedCalc^® version 12.2.1.0 (Mariakerke, Belgium).

B. RESULTS

1. Subject Disposition

[0233] In the FIH dose-escalation study, 32 subjects were randomized: 21 subjects were randomized to rHuIL-l2 (4 each at doses of 2, 5, 10, 12, and 15 pg and 1 the 20 pg dose) and 11 were randomized to placebo (2 each for doses of 2, 5, 10, 12, and 15 pg and 1 the 20 pg dose). All 32 subjects completed the study. Nineteen subjects (59%) were males and 13 (41%) were females; mean age was 28 years (range: 18 to 44 years). Twenty-six subjects (81%) were white, 2 (6%) were African American, 2 (6%) were Asian, and 2 (6%) were of other races. [0234] In the subsequent Phase lb expansion study, 60 subjects were randomized 4: 1 to rHuIL-l2 at the MTD identified in the FIH study (12 pg) or placebo; thus 48 subjects were treated with rHuIL-l2 and 12 subjects were treated with placebo. Forty-nine subjects (82%) were males and eleven (18%) were females; mean age was 28 years (range: 18 to 45 years). Forty subjects (67%) were white, 13 (22%) were African American and 7 (11%) were of other races. Fifty-eight subjects completed the study. Two subjects withdrew (one on Day 16 and other on Day 22) for personal reasons unrelated to the study.

2. Safety and Tolerability

[0235] All 32 subjects in the FIH study and all 60 subjects in the phase lb expansion study were analyzed for the primary endpoint.

[0236] FIH Study: No serious AEs or AEs leading to discontinuation and no clinically significant changes in clinical chemistry laboratory evaluations, vital sign measurements, physical examinations, or l2-lead ECGs were observed in either study. All AEs were resolved or recovered by the end of the study. A summary of drug-related AEs in the FIH study is presented in Table 2, below.

Table 2. Number and Percentage of Subjects Experiencing Study Drug

Related Adverse Events in the FIH study _

System Organ Placebo rHuIL- rHuIL- rHuIL-12 rHuIL-12 rHuIL- rHuIL-

Class/ 12 12 10 m 12 m 12 12

Preferred Term (N=ll) 2 m 5 m (N=4) (N=4) 15 m 20 m

_ (N=4) (N=4) _ (N=4) (N=l) n (%) n (%) n (%) n (%) n (%) n (%) n (%)

Blood and

Lymphatic

System Disorders

Leukopenia 1

(100%)

Lymphopenia 1

(100%)

Neutropenia 1 (25%)

Thrombocytope 1 nia (100%)

Gastrointestinal

Disorders

Abdominal 1 (25%)

Distension

Dyspepsia 1 (25%)

Nausea 1

(100%)

Paraesthesia 1 (25%)

Oral

General Disorders

and

Administration

Site Conditions

Chills 1 (25%) 1 (25%) 1 Table 2. Number and Percentage of Subjects Experiencing Study Drug Related Adverse Events in the FIH study

System Organ Placebo rHuIL- rHuIL- rHuIL-12 rHuIL-12 rHuIL- rHuIL- Class/ 12 12 10 pg 12 pg 12 12

Preferred Term (N=ll) 2 pg 5 pg (N=4) (N=4) 15 pg 20 pg

Fatigue 1 (25%)

Feeling - 1

Abnormal (25%)

Feeling Hot 1

(100%)

Injection Site 2 1 (25%) 1

Erythema (18%) (100%)

Injection Site 1 (9%)

Hemorrhage

Injection Site 1 (9%) 1 (25%) 1 (25%)

Pain

Injection Site 1 (9%) 1 (25%)

Reaction

Local Swelling 1 (25%)

Malaise 1

(100%)

Pain 1 (25%)

Infections and

Infestations

Viral Infection 1 (25%) 1 (25%)

Tonsillitis - 1

(25%)

Musculoskeletal

and Connective

Tissue Disorders

Back Pain 1 (25%)

Myalgia 1 (25%) 1 (25%)

Musculoskeleta 1 (9%)

1 Pain

Nervous System

Disorder

[0237] The most frequent AEs related to rHuIL- 12 treatment were headache, dizziness, chills and injection site pain. All the AEs related to rHuIL-l2, were mild (grade 1) to moderate (grade 2), except for three severe (grade 3) hematological AEs reported at the two highest doses. These included lymphopenia (absolute count of 400 lymphocytes/mm³) and thrombocytopenia (absolute counts of 109,000 to 118,000 platelets/mm³ over 4 days) in the single subject treated at the 20 pg dose level, and neutropenia (absolute counts of 700 to 1000 neutrophils/mm³ over 4 days) in 1 subject treated at the 15 pg dose level. The severe AEs started 3 or 4 days after dosing and continued for 3 to 4 days. The 2 events (grade 3 lymphopenia and grade 3 thrombocytopenia) occurring at the 20 pg dose level constituted dose-limiting toxi cities (DLTs), and led to dose reduction of rHuIL-l2 to 15 pg. Another DLT (grade 3 neutropenia) at 15 pg resulted in dose reduction of rHuIL-l2 to 12 pg. None of the severe drug-related AEs required concomitant medication or other action. The maximum tolerated dose of 12 pg was further examined in the subsequent phase lb expansion study.

[0238] Phase lb Expansion Study: A summary of drug-related AEs that occurred in > 5% subjects in the expansion study is presented in Table 3.

Table 3. Study Drug Related Adverse Events Occurring in > 5% of Subjects in

the Phase lb Expansion Study

System Organ Class Placebo rHuIL-12 12 pg

(Preferred Term) _ (N=12) (N=48)

Blood and Lymphatic System Disorders

Lymphadenitis 0 6 (13%)

Thrombocytopenia 0 3 (6%)

Eye Disorders

Photophobia 1 (8%) 0

Gastrointestinal Disorders

Nausea 3 (25%) 7 (15%)

Vomiting 1 (8%) 2 (4%)

General Disorders and Administration

Site Conditions

Chills 1 (8%) 11 (23%)

Fatigue 1 (8%) 7 (15%)

Feeling Hot 3 (25%) 5 (10%)

Injection Site Erythema 0 5 (10%)

Injection Site Pain 1 (8%) 5 (10%)

Injection Site Reaction 0 4 (8%)

Pain 0 3 (6%)

Investigations

Alanine Aminotransferase Increased 0 3 (6%)

Musculoskeletal and Connective Tissue

Disorders

Myalgia 1 (8%) 11 (23%)

Back Pain 0 10 (21%)

Arthralgia 0 3 (6%)

Musculoskeletal Stiffness 1 (8%) 2 (4%)

Neck Pain 1 (8%) 1 (2%)

Nervous System Disorders

Headache 4 (33%) 26 (54%)

Dizziness 0 _ 5 (10%) _

Abbreviations: N = total number of subjects in the dose group; n (%) number (%) of subjects who experienced each type of adverse event

[0239] No subject treated with the single, 12 pg dose of rHuåL-l2 used in this study experienced drug-related severe cytopenias, confirming the safety and tolerability observed in the FIH study. The most frequently reported rHuIL-l2-related AEs were headache, dizziness, chills, fatigue, myalgia and back pain. All the AEs related to rHuåL-l2 were mild or moderate except for episodes of severe chills and dizziness that occurred in the same subject beginning 11 hours after dosing; both events resolved over the next 5 hours, and neither required treatment or other action. Thus, data from the expansion study confirmed the overall safety and tolerability of rHuIL-l2 observed in the FIH study, and expanded the size of the population treated safely at the 12 pg dose level.

3. Pharmacokinetics

[0240] All subjects who received rHuIL-l2 and had evaluable data were included in the pharmacokinetic (PK) analysis. Due to insufficient data in the terminal phase, exposure parameters requiring extrapolation, such as AUCo-_¥, elimination half-life, volume of distribution and clearance, could not be reliably estimated in either study. The initial half-life was calculated within the time frame of 8 to 24 hours after dosing while the terminal half-life was evaluated at later time points; the calculation for each subject required at least 3 descending data points beyond 24 hours after dosing. Plasma concentration vs. time profiles for rHuIL-l2 are presented in Figure 1 A (FIH study) and 1B (expansion study). Figure 1B also shows plasma concentration vs. time profiles for IFN-g and C-X-C motif chemokine 10 (CXCL10; also known as IFN-g- induced protein 10) in relation to rHuIL-l2. These pharmacodynamic (PD) results are discussed with other PD results below.

[0241] FIH Study: Mean PK parameters for rHuIL-l2 after single SC injections in the FIH study are shown in Table 4.

Table 4. PK Parameters for Single Dose rHuIL-12 in the FIH and Phase lb

_ Expansion Studies _

PK Parameter

Dose Statistic Initial Terminal T_max Cmax AUC o-t

(pg) T_½ T_½ (h) (pg/mL) (h*pg/mL)

Study _ (h) (h)

FIH

2 N 4 4 4 4 4

Mean ± NR NR 8.00 5.55 ± 45.28 ± 11.30

SD (8.00 - 1.34

8.00)^a

5 N 4 4 4 4 4

Mean ± NR NR 5.00 16.52 ± 154.22 ±

SD (2.00 - 2.87 56.41

8.00)^a

10 N 3 4 4 4 4

Mean ± 11.8 ± 89.56 ± 8.00 26.39 ± 1169.86 ±

SD 1.79 57.61 (5.00 - 3.90 513.05

8.00)^a Table 4. PK Parameters for Single Dose rHuIL-12 in the FIH and Phase lb

Expansion Studies

PK Parameter

Dose Statistic Initial Terminal T_max C_max AUC _0-t

Mean ± 9.23 ± 48.92 ± NR 8.00 45.41 ± 790.74 ±

SD 5.78 (8.00 - 43.46 305.24

l2.00)^a

15 N 4 4 4 4 4

Mean ± 8.71 ± 90.19 ± 5.00 96.81 ± 1436.67 ±

SD 6.54 68.49 (5.00 - 57.12 602.18

8.00)^a

20 N 1 1 1 1 1

Mean ± 7.22 83.05 l2.00^b 39.50 970.58

SD

Ex

pansion

12 N 27 10 32 32 32

Mean ± 8.74 ± 56.92 ± 8.00 57.67 ± 1016.07 ±

SD 4.73 42.20 (2.00 - 49.84 612.97

24 00)^a

Abbreviations: N = number of subjects with sufficient data; NR = not reported due to insufficient data; N/A = not applicable; SD = standard deviation

a All data are means (± SD), except for T_ma . which is presented as median (range)

b Only 1 subject, range N/A

[0242] Sixteen of the 17 subjects who received rHuåL-l2 (2 to 20 pg doses) had

measureable systemic drug concentrations. The initial and terminal half-lives following an SC dose of rHuIL-l2 could not be determined at the 2 and 5 pg doses due to inadequate data. There were no obvious trends in T_max with increasing doses of rHUIL-l2. The C_max values generally increased with increasing dose, with the exception of the 20 pg cohort, where only 1 subject was dosed. Area-under-the-curve (AUC_0-t) increased consistently as the dose increased from 2 to 15 pg. At 20 pg. there was a decrease in AUC_0.t, based on the available single-subject data.

Overall, increases in rHuIL-l2 dose resulted in greater exposure. There were no gender differences.

[0243] Phase lb Expansion Study: In the expansion study, a subset of 40 of the 60 enrolled subjects participated in the PK study. Of these, 32 were randomized to rHuIL-l2 and 8 were randomized to placebo. Plasma rHuIl-l2 was not detected in any of the 8 subjects who received placebo. All 32 subjects who received rHuIL-l2 had measureable systemic

concentrations after dosing, and-endogenous rHuåL-l2 was detected in 1 of the 32 subjects before treatment with rHuIL-l2. Mean PK parameters are shown in Table 4. Systemic exposure of rHuIL-l2 was variable, i.e., the coefficients of variance for C_max, and AUC_0-t were 86%, 60% respectively. As shown in Figure lb, the rHuIL-l2 concentration versus time profile appeared to be biphasic, with an initial drop in plasma concentrations occurring around 8 hours after dosing and a terminal prolonged phase occurring at approximately 56 hours after dosing, suggesting significant distribution into the extravascular spaces. The slower terminal phase could be assessed only for 10 of the 32 subjects treated with rHuIL-l2. There were no gender differences in rHuIL-l2 exposure. Mean PK parameters from this study were comparable to those observed at the 12 pg SC dose level of rHuIL-l2 in the FIH study.

4. Immunogenicity

[0244] Plasma samples from all subjects in both studies were assayed for the presence of anti-drug antibodies to rHuIL-l2. In both studies, samples from all subjects were tested at baseline (before dosing) and 28 days after dosing. In the Phase lb expansion study,

immunogenicity testing also was performed at day 45 in a subset of 34 subjects who consented to additional sampling. No confirmed anti-drug antibodies to rHuåL-l2 were observed at the specified time points in any subjects in either treatment group in either study. Thus, there was no evidence that any of the PD or PK parameters for rHuåL-l2 were confounded by the presence of anti-rHuIl-l2 antibodies.

5. Pharmacodynamics:

[0245] Hematological Effects: As a part of the biological response to rHuIL-l2, the hematological effects on-various blood cell types were characterized over the course of the study. Neutrophils, platelets, lymphocytes, reticulocytes were examined in both studies, and specific lineages of lymphocytes and progenitor cells were examined in the Phase lb expansion study only. a. Neutrophils, Platelets, Lymphocytes and Reticulocytes

[0246] FIH study: All 32 subjects were monitored for hematological changes during the first 7 days and at return visits on days 11, 14 and 28. Time-dependent, transitory changes in peripheral blood cell counts were seen in subjects treated with rHuIL-l2 over the 28-day period, while counts remained more or less stable following placebo treatment. Figure 2 indicates the percentage change in lymphocyte, neutrophil, platelet, and reticulocyte counts relative to baseline in subjects treated with placebo or rHuåL-l2 at the different dose levels. In rHuåL-l2- treated subjects, lymphocytes and platelets decreased after day 1 and reached their respective nadirs between days 2 and 6; both types of cells then gradually increased, exceeding the baseline level at day 11 and returning to baseline by day 28. Neutrophils initially increased on day 2 and then decreased, reaching the nadir on day 5, and then followed the pattern observed for lymphocytes and platelets. Reticulocytes decreased after day 1, reached the nadir on day 6, and then followed the same pattern observed for lymphocytes, platelets, and neutrophils.

[0247] At the 20 pg dose of rHuIL-l2, the nadirs for lymphocytes, neutrophils, and platelets were 70%, 69%, and 29% below baseline, respectively (based on data from one subject), which constituted DLTs per protocol criteria and led to a dose reduction from 20 pg to 15 pg, as described above. At the 15 pg dose of rHuIL-l2, nadirs for lymphocytes, neutrophils, and platelets ranged from 53% to 69% below baseline, 33% to 64% below baseline, and 17% to 33% below baseline, respectively. A 64% decrease in neutrophils in one subject met the criteria for a DLT, as described above, and resulted in dose reduction to 12 pg. All DLTs were transitory.

[0248] Phase lb Expansion Study: All subjects were monitored for hematological changes during the in-house stay for 16 days and at return visit on day 28. There was no difference in the absolute baseline counts for lymphocytes, neutrophils, and platelets between the treatment groups. These changes were similar to the transient decreases, recovery and increases seen at the 12 pg dose of rHuIL-l2 in the FIH study. Figure 3 panel A describes mean percentage changes from baseline in lymphocytes, neutrophils, platelets, and reticulocytes after rHuåL-l2 treatment. After dosing with rHuIL-l2, treatment-related, transient decreases in lymphocytes and platelets were observed between days 2 and 8; both cell types returned to baseline by days 8 to 9, increased above baseline through day 16, and again returned to baseline by day 28.

Similarly, neutrophils increased on day 2 and then decreased from day 3 to 9, returned to baseline by day 10 and stabilized by day 28. Reticulocytes increased on day 2, decreased from day 4 to 8, returned to baseline on Day 10, increased above baseline between days 10 and 16, and returned to baseline by day 28. None of these changes from baseline in any cell type were deemed to be clinically significant. Similar transient hematological changes were not seen after placebo treatment (Figure 3 A panel B).

[0249] Lymphocyte Subpopulations and Progenitor Cells (Expansion Study Only): In the expansion study, the transient hematological changes described in Figure 3, Panels A and B, were further characterized using flow cytometry to detect specific lineages of blood cells such as CD3⁺, CD4⁺, CD8⁺ and CD45⁺ lymphocytes, NK cells, and CD34⁺ hematopoietic progenitor cells, as well as rHuIL-l2R 2 positivity on NK and CD34⁺ progenitor cells, and mean fluorescence intensity of CD56 on NK cells. [0250] As shown in Figure 3, Panel C, treatment-related decreases for CD3⁺, CD4⁺, CD8⁺, and CD45⁺ lymphocytes were observed from day 2 to 6 after dosing with 12 pg of rHuIL-l2. Values for each lymphocyte subtype returned to baseline by day 7, which paralleled the changes in total lymphocytes (see Figure 3 A), then transiently increased, with a peak around day 11, and again returned to baseline on day 28. The decrease from baseline at the nadir was between 40% and 55% and the increase from baseline at the peak was between 16% and 30%, depending on the cell type. Such changes were not seen with placebo treatment.

[0251] NK cells represented nearly 10% of the total peripheral blood cells, while the CD34⁺ progenitor cells accounted for only 0.1% (data not shown). Figure 3, Panel E, shows rHuIL-l2 treatment-induced transient decreases in both NK and CD34+ cells. NK cells reached a nadir at 20% of the baseline level on day 2, and CD34+ cells reached a nadir at 62% of baseline on day 4. Levels returned to baseline by day 5 for NK cells and by day 7 for CD34⁺ progenitor cells. NK cells increased to baseline by day 5, exceeded baseline starting on day 6, peaked on day 11, and stabilized at a level 150% above baseline on day 14. CD34⁺ cells increased to baseline at day 7, exceeded baseline on day 9, and returned to the baseline level on day 14. Thus, NK cells recovered more rapidly than did CD34+ cells. With placebo treatment, both cell types fluctuated above and below the baseline, but changes were not comparable to those seen with rHuIL-l2 treatment.

[0252] Cell surface expression of IL-l2R]32, a unique subunit of the heterodimeric receptor for rHuåL-l2, was studied to evaluate the effect of rHuåL-l2 on the receptor positivity of NK and CD34⁺ progenitor cells. Over the course of the study, about 50% of the NK and CD34⁺ progenitor cells were IL-l2R]32⁺, which represented approximately 5% and 0.05% of total peripheral blood cells, respectively. As shown in Figure 4A, rHuåL-l2 treatment did not affect the number of IL-l2R]32⁺ cells relative to that observed with placebo, although more fluctuations were seen in the number of receptor-positive CD34⁺ progenitor cells than in the number of receptor-positive NK cells.

[0253] Mean fluorescence intensity of CD56 on NK cells was increased with rHuåL-l2 administration, peaking on day 3 and returning to baseline by day 11 (Figure 4B). No such changes were seen with placebo treatment. Overall, flow cytometric analysis showed that a single administration of rHuIL-l2 to healthy subjects resulted in a rapid decreases in various types of peripheral blood cells followed by recovery at differing rates and extents. 6. Pharmacodynamics: Immunological Effects:

[0254] PD responses to rHuIL-l2 were measured by quantifying levels of IFN-g (both studies) as well as erythropoietin (EPO), interleukin- 18 (IL-18) and CXCL10 (expansion study only).

[0255] FIH Study: IFN-g levels were not quantifiable (i.e., they were below the lower limit of quantification [LLOQ]) in response to 2 and 10 pg doses of rHuIL-l2. At other doses of rHuIL-l2, the T_max of IFN-g (15 to 72 hours after rHuåL-l2) was delayed relative to the T_max of rHuIL-l2 itself (5 to 12 hours after dosing), which is indicative of a PD response (data not shown). However the exposure values for IFN-g were quite variable and the composite data did not support a clear dose-PD or PK-PD relationship. Mean C_max values were 272 ± 176 pg/mL, 215 ± 148 pg/mL, 287 ± 181 pg/mL and 410 pg/mL respectively in response to 5, 12, 15 and 20 pg doses of rHuIL-l2 and the mean AUC_0-t values were 13,962 ± 17,034 h*pg/mL, 13,984 ± 4473 h*pg/mL, 10,822 ± 6,994 h*pg/mL and 27,437 h*pg/mL respectively after 5, 12, 15 and 20 pg doses of rHuIL-l2. The detection of systemic IFN-g after rHuIL-l2 dosing at 5 pg, but not at 10 pg was unexpected.

[0256] Expansion study: Endogenous IFN-g was detected in 1 of the 32 subjects before treatment with rHuIL-l2. A measureable IFN-g response was observed 28 of the 32 subjects treated with rHuIL-l2 (Figure 1B), but in none of the 8 subjects who received placebo (data not shown), indicating a positive PD response to rHuåL-l2 treatment. The median IFN-g T_max value (24 hours) was delayed in time relative to the median rHuIL-l2 T_max value (8 hours; Figure 1B). Mean AETCo-_t value was 27,088±22,380 h*pg/mL. There were no gender differences in IFN-g response to rHuIL-l2 (data not shown).

[0257] Endogenous EPO was detected prior to treatment with study drug in all 32 subjects. Over the 14 day period following SC injection of study drug, EPO levels fluctuated in both treatment groups, without any apparent response to rHuåL-l2 treatment (Figure 5A).

[0258] Endogenous IL-18 also was detected prior to treatment with study drug, and, unlike EPO, showed a definite response to rHuIL-l2 between 36 hours and 14 days after treatment with rHuIL-l2 (Figure 5B). By comparison, IL-18 levels remained relatively close to baseline in the placebo-treated group (Figure 5B). Median T_max (107 hours) for IL-18 in the rHuåL-l2 group was delayed relative to the median T_max for rHuåL-l2 (8 hours; data not shown). Male subjects had a significantly higher (p < 0.0002) IL-18 response than did females (data not shown).

[0259] Like EPO and IL-18, endogenous CXCL10 was detected prior to treatment with study drug. A PD response to rHuIL-l2 was observed between 36 hours and 3 days after rHu_IL-l2 treatment, relative to placebo treatment (Figure 5C). The median CXCL10 T_max (24 hours) in subjects treated with rHuIL-l2 was delayed relative to the median T_max for rHuIL-l2 itself (8 hours; Figure 1B). In the placebo-treated group, only 2 of the 8 subjects showed a measurable CXCL10 response (data not shown), and the mean C_max was significantly lower than that of the rHuIL-l2-treated group (1480.53 pg/mL with placebo vs. 2055.53 pg/mL with rHuIL-l2; p < 0.03 by T-test on log transformed data). This difference may be confounded, however, by the extent and variability of endogenous levels observed in placebo-treated subjects (data not shown). There were no gender differences in the CXCL10 response to rHuIL-l2 (data not shown).

Example 2:

[0260] The purpose of this example was to describe the results of a randomized, blinded, efficacy study of rHuIL-l2 as an radiation medical countermeasure (R-MCM) in a larger group of male and female rhesus monkeys.

1. Methods

[0261] In this blinded study, rhesus monkeys (9 animal s/sex/dose group) were randomized to receive a single subcutaneous injection of placebo (group 1) or rHuIL-l2 at doses of 50, 100, 250, or 500 ng/kg (groups 2-5, respectively), without antibiotics, fluids or blood transfusions, 24 -25 hours after TBI (700 cGy).

[0262] Animals: Rhesus monkeys (Macaca mulatta) (3 to 5 years old, and 3.0 to 5.7 kg at the start of treatment) were housed individually and acclimated for > 5 weeks prior to irradiation. Harlan Teklad Certified Hi-Fiber Primate Diet #7195C (Harlan Laboratories, Indianapolis, Indiana) was provided twice daily.

[0263] Experiment design: The dose of 700 cGy (60 cGy/minute from a Theratron 1000 Co60 source [Best Theratronics; Ottawa, Ontario, Canada]) was based on available historical data from CiToxLAB North America. TBI was conducted with animals in a vertical position, as described previously. For homogenous dose distribution, the first half-dose was delivered anteroposterior and the second half-dose was delivered posteroanterior. Dosimetry was verified to be within 10% of prescribed dose using nanodot chips (Landauer, Inc., Glenwood, Illinois, USA) positioned on the front and back of each animal.

[0264] In the main study, male and female animals (45 each; 9 animals per sex per dose group) were randomized, stratified by body weight to the following doses of clinical grade rHuIL-l2 administered by SC injection between the scapulas approximately 24-25 hours following TBI: vehicle control (Group 1), 50 ng/kg (Group 2), 100 ng/kg (Group 3), 250 ng/kg (Group 4), or 500 ng/kg (Group 5). Clinical signs were recorded twice daily. Animals were euthanized if fulfilling predefined criteria. On day 60 all surviving animals were euthanized.

[0265] Results: Survival rates at Day 60 were 11%, 33%, 39%, 39%, and 50% for groups 1- 5, respectively (log rank p<0.05 for each dose vs. control). rHuIL-l2 also significantly reduced the incidences of severe neutropenia, severe thrombocytopenia, and sepsis (positive

hemoculture). Additionally, bone marrow regeneration following TBI was significantly greater in monkeys treated with rHuIL-l2 than in controls.

[0266] Conclusions: Data from this phase 2-equivalent study demonstrate that a single injection of rHuåL-l2 delivered one day after TBI can significantly increase survival and reduce radiation-induced hematopoietic toxicity and infections. These data significantly advance development of rHuIL-l2 toward approval under the Animal Rule as an effective stand-alone medical countermeasure against the lethal effects of radiation exposure.

2. RESULTS

[0267] Survival: Survival data are present in Figure 6. The administered radiation dose corresponded to an approximate LD90/60 (2/18 animals in the control groups) under the conditions of this experiment (no antibiotics, fluids or blood transfusions). Of the 59 deaths that occurred among rHuIL-l2-treated animals, 58 occurred between Day 9 and Day 24, which is consistent with previously observed rates and timing of death due to HSARS in rhesus monkeys (CiToxLAB historical data), and one death occurred at Day 33. The highest proportion of deaths occurred between Day 11 and Day 21, with Day 14 being the peak day of death for both control and rHuIL-l2-treated groups. The death rate was similar for males and females. All deaths, regardless of cause, were included in the statistical analysis of survival, the primary efficacy endpoint. As noted above, in the control group, 2 of 18 animals survived (11%, both males), while 33% (3 males and 3 females), 39% (4 males and 3 females), 39% (4 males and 3 females), and 50% (5 males and 4 females) of animals survived in rHuåL-l2 treated groups 2-5, respectively. Each rHuIL-l2 treated group showed a statistically significant increase in survival compared with the control group (log rank test p < 0.05). Pair-wise comparison between rHuIL- l2-treated groups showed no significant differences. Possible causes of unscheduled death prior to day 60 were predominantly infection and hemorrhage.

[0268] Clinical signs (vomiting, diarrhea, and body weight) resulting from the TBI generally were similar among all treatment groups and between the sexes. Decreases in activity and appetite, which occurred during the period of blood cell count nadirs and highest rates of infection, hemorrhage and death, were greatest in the control group and smaller in groups treated with rHuåL-l2. Changes in body weight were similar between the groups.

a. Hematology:

[0269] Blood counts for platelets, mean platelet volume, neutrophils, lymphocytes, and reticulocytes are presented in Figure 7 A-E, respectively. Hematology blood samples obtained at a pre-radiation time points were considered to be baseline levels. Blood samples obtained at post-radiation time points of 5, 10, 12, 14, 16 and 18 days corresponded to the period of severe cytopenias seen in the HSARS model. Additional samples obtained at days 30, 45 and 60 were used to evaluate whether levels returned to baseline in surviving animals.

[0270] Platelets: Platelet nadirs occurred at day 12 or 14, depending on the dosing group. Significant thrombocytopenia (<50 xl09 /L) was present over days 10-15. The average platelet nadir in the control group (10.1 xl09 platelets/L) was lower than that for each of the treated groups (12.1, 15.5, 12.7, and 18.6 xl09 platelets/L for groups 2-5, respectively). By day 18, initial recovery was observed among survivors in all groups, with full recovery observed by day 30. The proportions of blood samples with severe thrombocytopenia (platelets <10 xl09 /L) between Day 10 and 18 were 33% in the control group and 34%, 20%, 22% and 12% in rHuIL- 12 treated groups 2-5, respectively. The pair-wise comparison of the proportion of blood samples with severe thrombocytopenia for rHuåL-l2-treated animals and controls was significant for the 500 ng/kg group (Fisher exact test p = 0.0073). Mean platelet volume was increased between day 14 and 18, likely due to the release of young platelets from recovering bone marrow. Average peak values were 8.64 fL in the control group compared with 9.21, 9.13, 9.56 and 9.17 fL in rHuIL-l2 treated groups 2-5, respectively.

[0271] Neutrophils: Neutrophil nadirs occurred between days 10 and 14, depending on dosing group. Severe neutropenia (<50xl06/L) occurred in 100% of animals in the control group and in 88.9%, 77.8%, 83.3%, and 72.2% of animals in rHuIL-l2-treated groups 2-5,

respectively. The average neutrophil nadir of the control group (26 xl06 /L) was lower than that for each of the treated groups (34, 54, 39, and 78 xl06/L in groups 2-5, respectively. Neutrophil recovery began by day 18 and baseline levels were reached by day 30. The percentage of blood samples presenting with severe neutropenia on days 10 to 18 was 67% in the control group compared to 46%, 35%, 46% and 31% in groups 2-5, respectively (Fisher’s exact test p= 0.0196, 0.0005, 0.0278, and <.0001, for comparison of control and groups 2-5, respectively).

[0272] Lymphocytes: Lymphocyte nadirs occurred between days 10 and 16, depending on dosing group. All groups showed severe lymphopenia down to 7 - 10% of pre-radiation levels. The average lymphocytes nadir in the control group (0.143 x 109 /L) was lower than that for each of the treated groups (0.163, 0.213, 0.220, 0.239 xl09/L for groups 2-5, respectively). On Day 18, initial recovery from the nadir was observed in all groups. By day 30, group average levels ranged from 30% to near 60% of the pre-radiation levels, and by days 45 and 60, counts were in the normal range but remained slightly lower than baseline levels. The difference in the lymphocyte nadir between controls and animals treated with rHuIL-l2 was statistically significant in females treated at the 50, 250, and 500 ng/kg dose levels (Sidak adjusted t-test p = 0.0443, 0.0103, and 0.0211, respectively).

[0273] Red blood cells and reticulocytes: The red blood cells nadir occurred on Days 16- 18, and represented a 37% reduction from baseline. Red blood cells nadirs were comparable in all groups (data not shown). Average reticulocyte nadirs were 7.1 x 109/L in the control group and 8.7 x 109/L, 12.1 x 109/L, 9.1 x 109/L, and 9.8 x 109/L in rHuIL-l2-treated groups 2-5, respectively, suggesting a stimulatory effect of rHuåL-l2 on erythropoiesis. However, the differences did not reach statistical significance.

[0274] Relationships Between Hematopoietic Recovery and Survival: Mean

lymphocyte, platelet, neutrophil, and reticulocyte counts were higher in survivors than in decedents among the rHuIL-l2 treated animals (Figure 9). Average platelet, neutrophil, and reticulocyte counts were higher in the survivors treated with rHuåL-l2- than in survivors treated with vehicle control (Figure 9).

[0275] Febrile Neutropenia: A total of 15 animals (8 males; 7 females) had febrile neutropenia, and most (10/15) had been treated at the two highest dose levels (group 4, 250 ng/kg and group 5, 500 ng/kg) of rHuåL-l2. Ten of the 15 animals with febrile neutropenia had a positive hemoculture; the most common bacteria identified were Escherichia coli and

Staphylococcus aureus. Duration of febrile neutropenia was 1 day in all 15 affected animals and resulted in death on the same or next day in 12 of the 15 animals. Three of the 15 animals survived to Day 60 (1 in group 4 and 2 in group 5). Notably, 2 of the 3 surviving animals had negative blood cultures.

b. Microbiology and Pathology

[0276] Infection: In the control group hemoculture positivity was 86%, compared to 65%, 65%, 47% and 44% in rHuIL-l2-treated groups 2-5, respectively. The difference was statistically significant for the two highest doses (Fisher’s exact test p=0.0072 for each group). The decrease in the prevalence of infection was observed for both gram-negative and gram positive bacteria. Bacteriological analysis of heart, kidney, liver, both lungs, brain and spleen performed at necropsy for all animals revealed a lower mean bacterial growth score in rHuIL- l2-treated animals compared to that of controls (Table 5).

Table 5. Macroscopic Organ Hemorrhage Score and Organ Infection Score

Per Animal (Average ± SEM)

GI tract

Total hemorrhage Total infection

hemorrhage

score^a score'

Dose (ng/kg) score^b _

0 8.4 ± 1.6 5.6 ± 1.1.4 12.8 ± 2.2

50 5.1 ± 0.63 3.2 ± 0.75 12.3 ± 3.4

100 6.6 ± 0.73 4.5 ± 0.90 10.6 ± 3.7

250 5.0 ± 1.0 3.1 ± 0.78 8.8 ± 3.1

a Hemorrhage was assessed on necropsy of found dead or preterminally euthanized animals by a pathologist blinded to the therapy group assignment. The following tissues were included in

calculating the mean hemorrhage scores presented in this table: stomach, ileum, jejunum,

duodenum, colon, cecum, rectum, heart, brain, kidneys, liver, lungs, urinary bladder). Hemorrhage score was defined as follows: 0 = absence of hemorrhage; 1 = minimal hemorrhage(s); 2 = slight hemorrhage(s); 3 = moderate hemorrhage(s); 4 = marked hemorrhage(s); and 5 = severe

hemorrhage(s). In each animal scores for all organs were summed, then mean score for each

treatment group was calculated.

bThe following tissues were included in calculating the GI tract hemorrhage score: stomach, ileum, jejunum, duodenum, colon, cecum, and rectum.

cFor infection score determination, organ samples were collected on necropsy and cultured (animals found dead excluded from the analysis), including brain, heart, kidney, liver, both lungs, and spleen.

Bacterial growth was scored for each organ (from 0 to 4). In each animal scores for all organs were summed, then mean score for each treatment group was calculated.

[0277] Escherichia coli and Staphylococcus aureus were the most frequent isolates from organs and hemoculture. Among animals that underwent unscheduled euthanasia, 12 of 16 (75%) control animals had organ cultures that were positive for Escherichia coli compared with 66.7%, 63.6%, 72.7% and 55.6% of animals in rHuåL-l2 treated groups 2-5, respectively.

Similarly, 10 of the 16 (62.5%) control animals that underwent unscheduled euthanasia had organ cultures that were positive for Staphylococcus aureus compared with 50.0%, 54.5%,

54.5% and 44.4% of animals in rHuIL-l2 treated groups 2-5, respectively.

[0278] Hemorrhage: Overall group mean hemorrhage scores for all organs, as well as a separate score for the gastrointestinal system, are shown in Table 5. Although the mean scores were higher in the control group than in all rHuIL-l2-treated groups, the differences did not reach statistical significance, likely due to substantial organ to organ and animal to animal variation. Notably, the proportions of animals that had hemorrhage scores > 4 in at least one organ were higher in the control group than in the groups treated with rHuåL-l2, and brain hemorrhage was found only in 2 animals in the control group. [0279] Pharmacokinetics, Pharmacodynamics and Bone Marrow Assessment: In a companion study, a separate cohort of animals (2 per gender per group) was exposed to the same radiation level as in the survival study and treated with the same dose levels of rHuåL-l2. Blood samples were collected at sequential time points from baseline through day 11 and analyzed for rHuIL-l2, IFN-g, IL-18, and PM0. A dose dependent increase in rHuIL-l2 exposure as well as increased plasma levels of IFN-g, IL-18, and PM0 were observed (Table 6).

o BLQ BLQ 150.20 24868.80 1920.75 228818.77 284.36 16519.02

(NR) (NR) (1207.67) (121013.28) (188.72) (7951.53)

50 14.65 190.61 257.73 6239.20 1831.76 236796.45 1100.67 43932.87

(4.75) (90.51) (120.42) (5395.40) (1275.11) (209796.17) (316.73) (29769.36)

100 39.03 532.72 709.60 22904.40 2362.16 309524.07 1422.17 61099.10

(15.85) (238.16) (422.55) (8449.72) (1028.21) (123378.16) (120.53) (6735.34)

250 69.49 1397.30 1949.15 76621.50 3037.33 391945.00 2313.90 78442.31

(11.47) (203.75) (1200.24) (41016.18) (1339.84) (150071.03)) (1289.69) (17024.71)

500 137.18 2992.78 3094.00 123995.40 4738.03 621328.60 3236.52 118595.07

(38.07) (1307.55) (1195.79) (42927.08) (1511.00) (171280.79) (3060.21) (57862.00)

All data are means (standard deviations).

Abbreviations: BLQ-Below the limit of quantitation; AUC, area under the concentration vs. time curve; 0-t, time 0 to time of assessment; hr, hour; NR, not reported due to < 3 animals contributing data

[0280] All animals were sacrificed at Day 12, which represented the day of estimated maximal bone marrow suppression based on the timing of nadir of blood cell counts in the previous study. Histological analysis of bone marrow showed severe hypocellularity with pockets of regeneration (Figure 8A). Quantitation of the number of regeneration islands, the total area of the regenerating islands, and the number of megakaryocytes in each animal was performed in a blinded analysis. Both the number and the area of the regenerative islands were higher in the rHuIL-l2-treated groups compared with control, and the increases were similar across the rHuIL- 12 -treated groups (Figures 8 B-C). The difference for both parameters reached statistical significance for animal in group 5 (t-test p <0.01 and p <0.05 for number and area of islands of regeneration, respectively), as well as in a pooled comparison (all treated groups versus control group; p=0.0272 and p=0.0311 for number and area of islands of regeneration, respectively). Importantly, the number of megakaryocytes was also higher for all rHuIL-l2- treated groups relative to control, but the differences were not statistically significant (Figure 8D). [0281] Pharmacokinetics and Pharmacodynamics of rHuIL-12 in Irradiated Rhesus Monkeys: An evaluation of pharmacokinetics and pharmacodynamics, and the bone marrow in irradiated animals was conducted using separate animals randomized to the same doses of rHuIL-l2 as in the survival cohort (2 per sex per group). In the animal cohort used for pharmacokinetic and pharmacodynamic analyses, blood samples from animals treated with SC rHuIL-l2 were collected at the following time points: pretreatment (approximately 2 weeks prior to irradiation), 24 hours after irradiation immediately before rHuIL-l2 dosing, and at 1, 3,

5, 8, 12, 24, 48, 72, 96, 120, 144, 240, and 264 hours after rHuIL-l2 dosing. The concentrations of rHuIL-l2 and IFN- g in monkey plasma were determined by validated GLP ELISA methods. rHuIL-l2 was measured using the Human IL-12 HS ELISA kit. The lower limit of quantitation was 3.5 pg/mL in 100% monkey plasma. IFN-g was measured using the Monkey IFN-g ELISA kit. The lower limit of quantitation was 7.5 pg/mL in 100% monkey plasma. Interleukin- 18 (IL-18) and interferon g-induced protein (PM0) levels were determined using non-GLP qualified ELISA methods. IL-18 was assayed using the MBL International Corporation Human IL-18 ELISA. The lower limit of quantitation was 120 pg/mL in 100% monkey plasma. IP- 10 concentrations were determined in plasma using a Quantikine Human CXCL10/IP-10 ELISA. The lower limit of quantitation was 15 pg/mL in 100% monkey plasma. Standard non- compartmental analyses were performed using Phoenix™ WinNonlin® Version 6.3

(WinNonlin; Pharsight Corporation, Mountain View, CA).

[0282] Prodromal and Clinical (Manifest Illness) Observations: Vomiting and diarrhea occurred one or more times over the course of the study in more than half of all animals, with a higher incidence in the first few days following radiation. Incidence of vomiting and diarrhea was similar between the groups (Table 7).

Table 7. Causes of Death at Unscheduled Euthanasia

rHuIL-12 dose Group

(ng/kg) All

Cause of Death 0 50 100 250 500

(N= 16) (N= 12) (N= 11) (N= 11) (N= 9) (N= 59) n (%) n (%) n (%) n (%) n (%)

Infection 13 (81.25) 12 (100) 10 (90.9) 10 8 (88.9) 53

(90.9) (89.8)

Infection + 3 (18.75) 0 1 (9.1) 1 (9.1) 1 (11.1) 6 (10.2) hemorrhage

[0283] Decreased activity was observed primarily between Days 4 and 24, with the largest decrease in the control group and smallest decrease in the group treated with highest rHuIL-l2 dose (500 ng/kg; Figure 11). Intermediate rHuIL-l2 doses resulted in intermediate degrees of decreased activity, suggesting a possible inverse trend with rHuIL-l2 dose. Decreased appetite was observed in two waves (Figure 12): the first, on Days 1 through 4, occurred as an immediate reaction to irradiation; the second, on Days 10 through 34, paralleled the period of highest rates of infection, hemorrhage and death, as described below. During the first wave, the degree of decrease in appetite was similar between the control and rHuåL-l2-treated groups, while during the second wave, a trend toward positive effect of rHuIL-l2 was observed.

Average body weight decreased by 5 to 10% from baseline beginning 3 to 4 days after irradiation and continuing up to Day 30, followed by recovery and subsequent increases above baseline (Figure 13). The decrease in body weight was similar in the control and rHuåL-l2- treated groups.

[0284] Histopathology: A wide range of microscopic findings were observed in numerous organs/tissues from monkeys who died or were euthanized before day 60. Microscopically, TBI-related hemorrhage was noted in numerous organs. Other microscopic findings related to TBI were noted in the bone marrow, lymphoid tissue, gastrointestinal tract, and kidney.

Microscopic changes in many organs including small and large intestines, heart, liver, lungs, mesenteric lymph node, and spleen were considered to be predominantly secondary to episodes of bacteremia/septicemia. In animals surviving to day 60, microscopic findings related to irradiation were noted in lymphoid tissue, gastrointestinal tract, kidney, and bone marrow.

There were no substantial differences between the characteristics or the incidence and/or severity of the microscopic findings in the control group compared to the rHuIL-l2-treated groups, among both pre-terminally euthanized animals, animals that died, and surviving animals euthanized on day 60. Possible causes of death in animals that were euthanized before Day 60 are shown in Table 8.

Table 8. Percentage of Animals Presenting with Selected Early Clinical Signs on One or More Days Following TBI _

rHuIL-12 Dose Group

(ng/kg) _

0 50 100 250 500

Clⁱnⁱcal Sⁱ n 8)

Vomiting 13 (72.2) 12 (66.7) 9 (50) 11 (61.1) 13 (72.2)

Diarrhea 12 (66.7) _ 10 (55 6) 10 (55 6) 12 (66 7) 14 (77 8)

Example 3: Endogenous Expression of IL-12

[0285] The purpose of this example was to evaluate the endogenous expression of IL-12 following a single injectable administration of IL-12. [0286] Two Rhesus monkeys (#2001 and #2501) were administered IM a single dose of IL- 12 250 ng/kg. The quantity of IL-12 in the blood stream of each animal was measured via ELISA at different time points as detailed in Table 9. Additionally, the quantity of IL-12 in each animal was evaluated at different time points using a Multiplex kit from Meso Scale Discovery ( see also Leng et al.,“ELISA and multiplex technologies for cytokine measurement in inflammation and aging research,” J. Gerontol. A. Biol. Sci. Med. Sci., 63(8): 879-884 (Aug. 2008)). Enzyme-Linked Immuno-Sorbant Assay (ELISA), the most widely used and best validated method, is limited by its ability to measure only a single protein in each sample.

Recent developments in serum cytokine quantification technology include multiplex arrays which offer the potential of better evaluating the complexity and dynamic nature of

inflammatory responses. The results are also shown in Figures 10A (Animal #2001) and 10B (Animal #2501).

Table 9

[0287] The results provide evidence in monkeys for the feedback loops generated after a single administration of IL-12, leading to endogenous expression of the IL-12 protein long after the exogenous protein apparently clears. It is projected from these data that the half-life of total IL-12 (exogenous plus endogenous) is at least two weeks to one month and likely longer.

Example 4:

[0288] The purpose of this example was to evaluate human doses of IL-12 for

radioprotection based on pharmacokinetics of IL-12 in a Rhesus monkey animal model.

[0289] The effective human IL-12 dose cannot be assessed in controlled clinical efficacy studies as the intentional exposure of human volunteers to radiation is unethical. Thus, the effective human IL-12 dose must be estimated from animal and clinical data into a translational dose scaling framework to satisfy one of the criteria recommended to address efficacy under the Animal Rule, namely:“The data or information on the pharmacokinetics and

pharmacodynamics of the product or other relevant data or information, in animals and humans, allows selection of an effective dose in humans (21 CFR 3 l4.6l0(a)(l)-(4); 21 CFR

60l.9l(a)(l)-(4))”

[0290] The existing data from four monkey and two human trials have been previously used to examine the feasibility of developing a cross-species compartmental population PK model. The model was tested, validated, and the relationship between covariates and PK parameters was explored.

[0291] A significant number of IL-12 concentrations were below the limit of quatitation (BLQ) at 48 hours post-dose. An increase in the variability of the predicted apparent terminal phase and prediction concentration at 48 hours was observed since BLQ values were set to missing during model built-up. Although this was unlikely to be related to model

misspecification, the uncertainty regarding predicted concentrations post 48 hours gradually increased with elapsed time.

[0292] The objective of this project was to refine the previously developed cross-species PK model by investigating the impact of BLQ effects on PK parameters of IL-12 and predict the long term effects of IL-12. To improve the long term prediction of IL-12 concentrations (e.g. beyond 1 or 2 weeks), the population PK model was customized by including a likelihood function that takes into account censoring of BLQ values, and the expected distribution of concentrations for samples with a high number of BLQ values (“model refinement”). Figure 14 depicts a final structural model of IL-12 following SC dosing in humans and monkeys, with BSV = Inter-individual variability, CL = Systemic clearance, CLd = Intercompartmental distribution, CLdt = Distribution to deep tissue, Vc = Central volume of distribution, Vdt = Volume of distribution to deep tissue, Vp = Volume of peripheral compartment, Kaf = absorption rate to the capillaries, Kas = absorption rate to the lymphatic system, F = absolute bioavailability and Frel = relative amount of the dose to the lymphatic system.

[0293] The original model parameters are shown below in Table 10. Based upon this data, Goodness-of-Fit (GOF), impact of BLQ, of the structural model of IL-12 following SC dosing in humans and monkeys, was plotted as shown in Figs. 15A-D, while GOF plots using a refined model, M3 method for BQL, are shown in Figs. 15E-H. In particular, Figures 15E, F, and G show that for the refined model, there is no effect on C_max (Fig. 15E), less bias for few low concentrations (Fig. 15F), and a better characterization of the terminal phase (Fig. 15G). [0294] Table 11 shows the impact of BLQ on the model parameters: new parameter values, while Table 12 provides a comparison of model parameters, with the change from previous model (%). Finally, Tables 13 and 14 highlight the Absorption Model, PK parameters, in comparing the prior model (Table 13) with the refined model (Table 14). The comparison demonstrates that with the refined model, the previously observed gender-related effects on IL- 12 are no longer seen, along with a small increase in MATlymphatic in monkeys and a small decrease in MATlymphatic in humans.

[0295] Conclusions: The population PK model was customized by including a likelihood function that takes into account censoring of BLQ values, and the expected distribution of concentrations for samples with a high number of BLQ values. This customization resulted in a slight improvement in the quality-of-fit.

[0296] Population PK Model Refinement: Effect on Typical PK Parameters: The impact of including a cumulative distribution function on the BLQ on the systemic parameters of

HemaMax (CL, Vc...) was negligible (i.e., less than 1% change). However, significant changes in mean absorption time (MAT) from capillaries and the lymphatic system in irradiated monkeys and humans were observed: (1) an increase in MATlymphatic in monkeys was observed; (2) an increase in MATlymphatic in humans was observed; and (3) previously observed gender-related effects are no longer seen. These results are likely to simplify the dosing rationale in humans. Overall, this sensitivity analysis suggests that the refined model resulted in a slight improvement in the quality-of-fit.

Table 10: original model parameters for IL-12 SC dosing

Model PK Monkeys Htunans %BSV

Parameter Ili a dialed Non-Irradiated lira dialed Nen-Irradiated

Males Females Males Females Males Females Males Females

Systemic Vc (L) Q. l 85 0.186 0.235 0.235

4.69 4.69 NA

Model Vp (L) 0.194 0.194 0.194 0.194 3.88 .88 3.88 3.88 NA

Vdt (L) 0.158 0.158 0.158 0.158 3.17 .17 3.17 3.17 NA

CL (L.li) 0.115 0.115 0.0881 0.0881 1.09 3.09 0.833 0.833 NA

CLd (L.li) 0.373 0.373 0.373 0.373 3.52 3.52 3.52 3.52 NA

CLdt 0.00610 0.00610 0.00610 0.00610 0.0577 0.057" 0.0577 0.0577 NA

(I II)

Absoiption MAT, 37.6 3 .5 3.94 3,94 50.6 50.6 5.32 5.32 35.2*

Model capillaries

(10

Frel. 23.0 23.0 23.0 23.0 23.0 23.0 23.0 23.0 55.6* capillaries

(%)

MAT, 14.4 11,6 140 114 77.1 62.3 753 609 58.5 lymphatic

0

F 20.4 20.4 23.4 23.4 20.4 20.4 23.4 23.4 36.2

(%)

Male and female monkey BW assumed 3.5 kg, male and female humans assumed 70 kg.

* Correlation coefficient = 0.556. NA = Not Applicable

Table 11: Impact of BLQ on the Model Parameters: New Parameter Values

Model PK Monkevs Hmnans %BSV

Parameter Irradiated Non lrradi ted Irradiated Non lrradiated

Males Females Males Females Males Females Males Females

Systemic Vc (L) 0.185 0.185 0.233 0.233 3.70 3.70 4.67 4.67 NA

Model Vp (L) 0.194 0.194 0.194 0.194 3.89 3.89 3.89 3 89 NA

Vdt (L) 0.158 0.158 0.158 0.158 3.17 3.17 3.17 3.17 NA

CL (I . lit 0.1 14 0.114 0.0879 0.0879 3.08 1.08 0.831 0.831 NA CLd (L h) 0.373 0.373 0.373 0.373 3.53 3.53 3.53 3.53 NA CLdt 0.00610 0.00610 0.00610 0.00610 0.0577 0.0577 0.0577 0.0577 NA (L li)

Absoiption MAT, 7.59 7.59

10.7 10.7 5.34 5.34 43.2

Model capillaries

(h)

Frel, 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 73.8 capillaries

(%)

MAT, 26.5 25.2 129 123 139 132 678 647 lymphatic

(h)

F 22.3 22.3 24.1 24.1 22.3

24.1 24.1 42.6

(%) Table 12: Comparison of Model Parameters: Change from previous model (%)

Model PK Monkevs Humans

Parameter bra dinted Non-brndiated bra dinted Non-bra dint d

Males Females Males Females Males Females Males Females

Systemic Vc (L) -0.5 -0.5 -0.9 -0.9 -0.5 -0.5 -0.4 -0.4

Model Vp (L) 0.0 0.0 0.0 0.0 +0.3 +0.3 +0.3 +0.3

Vdt (L) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

CL (Lit) -0.9 -0.9 0.2 0.2 -0.9 -0.9 0.2 0.2 CLd (L h) 0.0 0.0 0.0 0.0 +0.3 +0.3 +0.3 +0.3 CLdt 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0 h)

Absorption MAT, -79.8 -79.8 -4.3 -4.3 -78.9 -78.9 +0.4 +0.4

Model capillaiies

00

Fi el, +4.3 +4.3 +4.3 +4.3 +4.3 +4.3 +4.3 +4.3 capillaiies

(%)

MAT, +83.3 +117.2 -7.9 +7.9 +80.3 +111.9 10.0 6.2 lymphatic

F +9.3 +9.3 +3.0 +3.0 +9.3 +9.3 +3.0 +3.0

(%)

Table 13: Prior Model PK IL-12 values

Model PK Monkeys Humans

Parameter bradiated Non-bradiatecl bradinted Non-b radiat d

Males Females Males Females Males Females Males Females

Absolution MAT. 37.6 37.6 3.94 3.94 50.6 50.6

5.32 35 2*

Model capillaries

(b)

MAT, 14.4 1 1.6 140 114 77.1 62.3 53 609 53.5 lymphatic

0»)

20.4 20.4 23.4 23.4 20.4 20.4 3.4 23.4 36.2

Table 14: Refined Model PK IL-12 values

Monkeys Humans

PK

Model bradiate d N on-br a that e d Irradiated Non-bracliated %BSV

Parameter

Males Females Males Females Males Females Males Females

MAT,

capillaiies 7.59 7.59 3.77 3.77 10.7 10.7 5.34 5.34 43.2

0i)

Absorption MAT,

Model lymphatic 26.4 25.2 129 123 139 132 678 647 2.7

01)

F (%) 22.3 22.3 24.1 24.1 22.3 22.3 24.1 24.1 42.6 Example 5:

[0297] A study was conducted to evaluate the pharmacodynamics and PK values of IL-12 in vivo in non-human primates (NHP).

[0298] In a first test, the PK for IL-12 exposure was determined following a first IL-12 injection of 250 ng/kg (time = zero) and then a second IL-12 injection of 250 ng/kg 28 days later for two individual non-human primate (NHP) (rhesus) monkeys, one male and one female (Rhesus #2001 and #2501). The individual results are shown in Figure 16, and average results are shown in Figure 17.

[0299] In a second test, pharmacodynamics for Interferon-gamma exposure were determined following a first IL-12 injection of 250 ng/kg (time = zero) and then a second IL-12 injection of 250 ng/kg at 28 days later for two individual NHP (rhesus) monkeys, one male and one female (Rhesus #2001 and #2501) (Figure 18), with average data for exposure from two monkeys shown in Figure 19. These data show that IL-12 administration triggers a first modest peak in IFN-g serum levels, but this is then following by a more significant peak several days following IL-12 administration (e.g., 72 -120 hours following initial IL-12 administration). Similar results are seen with a second IL-12 injection at 28 days.

[0300] In a third test, hematology changes from baseline for lymphocytes (Figure 20) and platelets (Figure 21) in rhesus monkeys were measured following a first IL-12 injection of 250 ng/kg (time=zero) and then a second IL-12 injection of 250 ng/kg at day 28. Group 1 comprised 8 monkeys and group 2 comprised two monkeys. The results show that IL-12 administration is following by an initial modest increase in lymphocytes, followed by a significant decrease, and then a consistent increase well above the initial lymphocyte increase. This second increase continues for an extended period of time, with similar results shown for a second IL-12 dosage at 28 days.

Example 6:

[0301] IL-12 has been subcutaneously administered to human cancer patients in several dosing regimens involving multiple dosing, both weight-based dosing and fixed dosing. In a clinical trial with Cutaneous T cell Lymphoma (CTCL) patients, IL-12 was administered in combination with low-dose Total Skin Electron Beam Therapy (LD-TSEBT, followed by maintenance doses of IL-12 in the absence of LD-TSEBT. IL-12 (150 ng/kg) was administered in week 1, study day 2 and in week 3 on study day 15. Subsequently IL-12 (100 ng/kg) was administered once every 4 weeks, namely in week 7, week 11, week 15, week 19, week 23, week 27, week 31, week 35, week 39, and week 43.

[0302] The pharmacokinetic (PK) profile of IL-12, with circulating blood levels of IL-12 analyzed, is shown in Figure 22. IL-12 levels were determined prior to dosing on study days 2 and 15, and for up to 72 hrs after dosing. Circulating levels of IL-12 usually reached a peak 5 or 24 hrs after administration on either study day 1 or study day 15. The mean peak level of IL-12 was highest 5 hrs after administration for both study day 1 and study day 15.

[0303] A second cohort of CTCL patients was administered a fixed dose of IL-12. In week 1, study day 2 and in week 3, study day 15, in combination with LD-TSEBT, patients were administered 12pg IL-12. 12pg is equivalent to a l70ng/kg dose for a 70kg body weight. A maintenance dose of IL-12 (lOpg) was then administered once every 4 weeks, namely in week 7, week 11 , week 15 , week 19, week 23 , week 27, week 31 , week 35 , week 39, week 43 , week 47, week 51, week 55, week 59, week 63, week 67, week 71, week 75, week 79, week 83, week 87, week 91, week 95, week 99 and week 103.

[0304] Clinical responses are summarized in Figure 23 A and Figure 23 B. The response rate (complete response + partial response) was 100% for 7 patients with clinical stage IB disease. Of these seven patients, four patients with stage IB disease achieved a complete response with durations of 19 to greater than 50 weeks. The overall response rate was 80% for all patients enrolled. Table 15 summarizes LD-TSEBT Toxicity Alone (data from Stanford’s historical database) vs. IL-12 combined with LD-TSEBT.

Table 15

[0305] Patient peripheral blood mononuclear cells were analyzed for PD-l expression, after being cultured for 20hr. Patients with complete response or partial response tend to have low PD-l expression following IL-12 + LD-TSEBT, which remains low in follow up. Non responders (except for one patient who a short-lived PR), all late stage patients, are seen to have an increase in PD-l expression by week 15 as shown in Figure 23 C.

Example 7

[0306] The effect of repeated IL-12 administration on circulating levels of IL-12 was evaluated in Rhesus monkeys. IL-12 was administered subcutaneously on Days 1, 3, 5, 7, 9, 11, and 13. Figure 24A shows circulating levels of IL-12 following a 100 mg/kg subcutaneous administration of IL-12. Figure 24B shows circulating levels of IL-12 following a 316 mg/kg subcutaneous administration of IL-12. Figure 24C shows circulating levels of IL-12 following a 1,000 mg/kg subcutaneous administration of IL-12. Figures 24A-C demonstrate that IL-12 exposure is reduced with repeat dosing. The IL-12 half-life ranged from 8.5 hours to 41.7 hours. Figure 25 demonstrates that IFN-g is reduced with repeat IL-12 administrations, and that high and frequent dosing with IL-12 results in tachyphylaxis.

[0307] These data in the examples above support the IL-12 dosing rational described herein where IL-12 dosages can have effects over a prolonged period of time.

[0308] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method of treating a subject in need with an exogenous IL-12 composition comprising a) administering a first treatment comprising administering a first dose of IL-12 to the subject; and

b) administering a second treatment comprising administering a second dose of IL-12 to the subject, wherein the second treatment is administered after a first non-treatment interval of at least 8 days.

2. The method of claim 1, wherein the second treatment elicits a therapeutic response that is not diminished by tachyphylaxis.

3. The method of claim 1 or 2, further comprising administering a third treatment comprising administering a third dose of IL-12 to the subject, wherein the third treatment is administered after a second non-treatment interval of at least 8 days.

4. The method of any one of claims 1 to 3, further comprising administering a fourth treatment comprising administering a fourth dose of IL-12 to the subject, wherein the fourth treatment is administered after a third non-treatment interval of at least 8 days.

5. The method of any one of claims 1 to 4, further comprising administering a fifth treatment comprising administering a fifth dose of IL-12 to the subject, wherein the fifth treatment is administered after a fourth non-treatment interval of at least 8 days.

6. The method of any one of claims 1 to 5, wherein a non-treatment interval is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.

7. The method of any one of claims 1 to 6, wherein a non-treatment interval is no more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.

8. The method of any one of claims 1 to 7, wherein a non-treatment interval is at least 1 2, 3, or 4 weeks.

9. The method of any one of claims 1 to 8, wherein a non-treatment interval is no more than 1, 2, 3, or 4 weeks.

10. The method of any one of claims 1 to 9, wherein a non-treatment interval is at least 1 2,

3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

11. The method of any one of claims 1 to 10, wherein a non-treatment interval is not more than 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

12. The method of any one of claims 1 to 11, wherein a dose is administered before, during, or after a cycle of chemotherapy.

13. The method of claim 12, wherein the dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

14. The method of any one of claims 1 to 13, wherein a dose is administered before, during, or after a cycle of radiation therapy.

15. The method of claim 14, wherein the dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days before or after a cycle of radiation therapy.

16. The method of claim 14 or 15, wherein the radiation therapy comprises low-dose Total Skin Electron Beam Therapy (LD-TSEBT).

17. The method of any one of claims 3 to 16, wherein the second non-treatment interval is different than the first non-treatment interval.

18. The method of any one of claims 4 to 17, wherein the third non-treatment interval is different than at least one of the second non-treatment interval and the first non-treatment interval.

19. The method of any one of claims 5 to 18, wherein the fourth non-treatment interval is different than at least one of the third non-treatment interval, the second non-treatment interval, and the first non-treatment interval.

20. The method of any one of claims 3 to 19, wherein the third dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

21. The method of any one of claims 4 to 20, wherein the fourth dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

22. The method of any one of claims 5 to 21, wherein the fifth dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

23. The method of any one of claims 1 to 22, wherein at least one dose comprises between 2- 20 pg of IL-12.

24. The method of claim 23, wherein the at least one dose comprises between 5-15 pg of IL- 12

25. The method of claim 23, wherein the at least one dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of IL-l2.

26. The method of claim 23, wherein each dose comprises between 2-20 pg of IL-12.

27. The method of claim 26, wherein each dose comprises between 5-15 pg of IL-12.

28. The method of claim 26, wherein each dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of IL-12.

29. The method of any one of claims 23 to 28, wherein the at least one dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg).

30. The method of claim 29, wherein at least one dose comprises 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ng/kg.

31. A method of treating a subject in need with an exogenous IL-12 composition comprising administering a first treatment comprising administering a first dose of IL-12 to the subject, wherein the first dose comprises between 2-20 pg of IL-12.

32. The method of claim 31, wherein the first dose comprises between 5-15 pg of IL-12.

33. The method of claim 32, wherein the first dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,

6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,

17.5, 18, 18.5, 19, 19.5, or 20 pg of IL-l2.

34. The method of any one of claims 31 to 33, wherein the first dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg).

35. The method of claim 34, wherein the first dose comprises 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ng/kg.

36. The method of any one of claims 1 to 35, wherein the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant.

37. The method of claim 36, wherein the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL.

38. The method of claim 37, wherein the pharmaceutical composition comprises IL-12 at a concentration of 20 pg/mL.

39. The method of any one of claims 36 to 38, wherein the buffer comprises sodium phosphate.

40. The method of any one of claims 36 to 39, wherein the pharmaceutical composition comprises 10 mM sodium phosphate.

41. The method of any one of claims 36 to 40, wherein the salt comprises sodium chloride.

42. The method of any one of claims 36 to 41, wherein the pharmaceutical composition comprises 150 mM sodium chloride.

43. The method of any one of claims 36 to 42, wherein the surfactant is a non-ionic surfactant.

44. The method of claim 43, wherein the non-ionic surfactant comprises poloxamer 188.

45. The method of any one of claims 36 to 44, wherein the pharmaceutical composition comprises 0.1% (w/v) poloxamer 188.

46. The method of any one of claims 36 to 45, wherein the pharmaceutical formulation comprises a pH of between 5.0 to 8.0.

47. The method of claim 46, wherein the pharmaceutical formulation comprises a pH of 6.0.

48. The method of any one of claims 1-47, wherein the method comprises treating hematopoietic syndrome of the acute radiation syndrome (HSARS) in the subject.

49. The method of any one of claims 1-48, wherein the method comprises treating cutaneous T-cell lymphoma (CTCL) in the subject.

50. The method of any one of claims 1 to 49, wherein the method further comprises adjusting a length of a non-treatment interval prior to a treatment based on a time point at which the subject is expected to have completed a direct response to the first dose of IL-12.

51. The method of any one of claims 1 to 50, wherein the method further comprises adjusting a length of a non-treatment interval prior to a treatment based on a time point at which the subject is expected to have completed an indirect response to the first dose of IL-12.

52. The method of any one of claims 1 to 51, wherein the method further comprises adjusting a length of a non-treatment interval prior to a treatment based on a time point at which the a previous treatment is expected to no longer exert a pharmacodynamic effect on the subject.

53. The method of any one of claims 1 to 52, wherein the method further comprises assessing a level of IL-12 in the subject’s blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of IL-12 is above a threshold amount.

54. The method of claim 53, wherein the threshold amount is a level of IL-12 in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

55. The method of any one of claims 1 to 54, wherein the method further comprises assessing a level of at least one of INF-gamma, IL-2, IL-10, IL-18, or CXCL10 in the subject’s blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of the at least one of INF-gamma, IL-2, IL-10, IL-18, or CXCL10 is above a threshold amount.

56. The method of claim 55, wherein the threshold amount is a level of the at least one of INF-gamma, IL-2, IL-10, IL-18, or CXCL10 in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

57. The method of any one of claims 1 to 56, wherein the method further comprises assessing a level at least one of lymphocytes, neutrophils, platelets, and reticulocytes in the subject’s blood before a treatment and increasing a length of a non-treatment interval prior to the treatment if the level of the at least one of lymphocytes, neutrophils, platelets, and reticulocytes is below a threshold amount.

58. The method of claim 57, wherein the threshold amount is a level of the at least one of lymphocytes, neutrophils, platelets, and reticulocytes in the blood of a healthy individual or in the blood of the subject prior to administrating the dose of IL-12.

59. A method of treating a subject in need with an exogenous IL-12 composition comprising: a) administering a first dose of IL-12 to the subject; and b) administering a second dose of IL-12 to the subject at least 8 days after administering the first dose.

60. A method of treating a subject in need with an exogenous IL-12 composition comprising: a) administering a first dose of IL-12 to the subject; and

b) administering a second dose of IL-12 to the subject, wherein the second dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

61. The method of claim 59 or 60, wherein the second dose is administered at a time point that reduces a likelihood that the subject will develop tachyphylaxis.

62. The method of any one of claims 59 to 61, wherein the second dose is administered at least 8 days after administering the first dose.

63. The method of any one of claims 59 to 62, wherein the second dose is administered at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days after the first dose.

64. The method of claim 63, wherein the second dose is administered no more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days after the first dose.

65. The method of any one of claims 59 to 63, wherein the second dose is administered at least 1 2, 3, or 4 weeks after the first dose.

66. The method of any one of claims 59 to 65, wherein the second dose is administered 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the first dose.

67. The method of any one of claims 59 to 66, further comprising administering a third dose of IL-12 after administering the second dose, wherein the third dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

68. The method of claim 67, further comprising administering a fourth dose of IL-12 after administering the third dose, wherein the fourth dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

69. The method of claim 68, further comprising administering a fifth dose of IL-12 after administering the fourth dose, wherein the fifth dose elicits a therapeutic response that is not diminished due to tachyphylaxis.

70. The method of any one of claims 59 to 69, wherein at least one dose comprises between 2-20 pg of IL-12.

71. The method of claim 70, wherein the at least one dose comprises between 5-15 pg of IL- 12

72. The method of claim 70, wherein the at least one dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5,

5.5, 6 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5,

17, 17.5, 18, 18.5, 19, 19.5, or 20 pg of IL-l2.

73. The method of claim 70, wherein the at least one dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg).

74. The method of claim 73, wherein at least one dose comprises 0.5, 1, 2, 3, 4, 5, 10, 15,

20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ng/kg.

75. The method of any one of claims 59 to 74, wherein the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant.

76. The method of claim 75, wherein the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL.

77. The method of claim 76, wherein the pharmaceutical composition comprises IL-12 at a concentration of 20 pg/mL.

78. The method of any one of claims 75 to 77, wherein the buffer comprises sodium phosphate.

79. The method of any one of claims 75 to 78, wherein the pharmaceutical composition comprises 10 mM sodium phosphate.

80. The method of any one of claims 75 to 79, wherein the salt comprises sodium chloride.

81. The method of any one of claims 75 to 80, wherein the pharmaceutical composition comprises 150 mM sodium chloride.

82. The method of any one of claims 75 to 81, wherein the surfactant is a non-ionic surfactant.

83. The method of claim 82, wherein the non-ionic surfactant comprises poloxamer 188.

84. The method of any one of claims 75 to 83, wherein the pharmaceutical composition comprises 0.1% (w/v) poloxamer 188.

85. The method of any one of claims 75 to 84, wherein the pharmaceutical formulation comprises a pH of between 5.0 to 8.0.

86. The method of claim 85, wherein the pharmaceutical formulation comprises a pH of 6.0.

87. A method of treating a subject in need with an exogenous IL-12 composition comprising: a) administering a first single low dose of IL-12, wherein IL-12 can be detected in a sample of the subject’s blood, serum, and/or plasma for at least one week; and

b) administering at least one subsequent dose of IL-12 at a time point when the amount of IL-12 in the subject’s blood is no longer observable.

88. The method of claim 87, wherein the IL-12 dosing schedule results in preventing the occurrence of tachyphylaxis.

89. The method of claim 87 or 88, wherein a subsequent dose of IL-12 is administered at a time point when peripheral blood cell trafficking to a site of injury or disease is decreasing.

90. The method of claim 89, wherein the peripheral blood cells are selected from the group consisting of NK cells, monocytes, red blood cells reticulocytes, platelets, and any combination thereof.

91. The method of any one of claims 87 to 90, wherein a subsequent does of IL-12 is not administered when one or more Th2 cytokines are detectable in the subject’s blood, serum, and/or plasma.

92. The method of any one of claims 87 to 91, wherein if one or more Th2 cytokines are detectable in the subject’s blood, serum, and/or plasma, then the subsequent dosage of IL-12 is decreased as compared to the prior IL-12 dosage.

93. The method of any one of claims 87 to 92, wherein if one or more Th2 cytokines have increased in the subject’s blood, serum, and/or plasma sample, as compared to baseline levels of the same cytokine present in serum of either the subject or the patient population for the subject, then the subsequent dosage of IL-12 is decreased as compared to the prior IL-12 dosage.

94. The method of any one of claims 92 to 93, wherein the Th2 cytokine is selected from the group consisting of IL-2, IL-4, IL-3, IL-5, IL-6, IL-10, IL-13, IL-25, IL-31, and IL-33.

95. The method of any one of claims 87 to 94, wherein the IL-12 is detectable in the subject’s blood, serum, and/or plasma due to lymphatic absorption of IL-12.

96. The method of any one of claims 87 to 95, wherein the IL-12 is detectable in the subject’s blood, serum, and/or plasma at least in part due to endogenous production of IL-12 stimulated by the exogenous IL-12 administration.

97. The method of any one of claims 87 to 96, wherein the subsequent dose of IL-12 is administered at least 2 weeks after the first IL-12 dose.

98. The method of any one of claims 87 to 97, wherein the method is used as adjunctive therapy to a radiation cancer treatment.

99. The method of any one of claims 87 to 97, wherein the method is used as adjunctive therapy to a chemotherapy cancer treatment.

100. The method of any one of claims 87 to 99, wherein the IL-12 dose is a weight-based dosage amount.

101. The method of any one of claims 87 to 99, wherein the IL-12 dose is a fixed dosage amount.