WO2018132539A1 - Safety and pharmacodynamic activity of a toll-like receptor 7/8 agonist - Google Patents

Abstract

Provided are methods of increasing innate and adaptive immune activation in a human subject by administering a composition comprising the TLR7/8 agonist MED 19197, preferably via intratumoral injection into a cutaneous or subcutaneous tumor.

Description

SAFETY AND PHARMACODYNAMIC ACTIVITY

OF A TOLL-LIKE RECEPTOR 7/8 AGONIST

BACKGROUND

[0001] Cancer continues to be a major global health burden. In the United States, it is the second most common cause of death after heart disease, accounting for nearly one in every 4 deaths (American Cancer Society, 2014). The 5-year survival rate for all cancers diagnosed between 1999 and 2006 is 68%, which is 18% higher than the rate reported between 1975 and 1977, likely reflecting progress in diagnosing certain cancers earlier and improvements in treatment (American Cancer Society, 2014). Unfortunately, despite indisputable progress in the treatment of cancer, there continues to be an unmet medical need for more effective and less toxic therapies, especially for those patients with advanced disease that do not respond or have become resistant to existing therapies.

[0002] The most effective treatment for localized solid cancers is to remove the tumor by surgery followed by post-operative chemotherapy or radiation treatment. However, this approach is not suitable for many cancers since many patients are not candidates for surgical procedure due to tumor size, location of the tumor, and/or stage of the cancer. In some cases, even after surgery, the overall survival rates for some patients are not promising. Therefore, therapies including chemotherapy and cancer immunotherapy are additional options for cancer treatment.

[0003] Toll-like receptor (TLR) agonists are small nucleoside analogues that have shown efficacy towards a variety of tumors. TLR agonists are used for cancer immunotherapy to stimulate the immune system locally against cancer cells (Shi et al, 2016). Systemic administration of TLR agonists results in the stimulation of the immune system of the entire body, which can have highly undesirable side effects, such as patient discomfort, while delivering just a small portion of the entire administrated dose to the tumor. Therefore, local delivery of TLR agonists is a preferred approach for administration, such as in dermal applications.

[0004] Thus, TLR agonists are used as a potent modulator for the topical treatment for genital warts (Beutner et al, 1998) and superficial basal cell carcinomas. They are also introduced as a treatment of malignant skin lesions including melanoma (Singh et al, 2014) and basal cell carcinoma. TLR agonists induce pro-inflammatory cytokines and chemokines in-vitro and in-vivo that attract immune cells to the site of administration. Immune cells then attack cancer cells at the site resulting in elimination of cancer cells. Studies have shown that direct injection of TLR agonists into the tumor site as an immune- stimulant generated antitumor CD8 T cell response for the treatment of low-grade lymphoma with low-dose radiotherapy.

[0005] MEDI9197 (also known as 3M-052/S-36862) is a small molecule

imidazoquinoline agonist that activates human TLR7 and TLR8 receptors, which directly activate innate immune cells. This results in innate and adaptive immune modulation such as activation of co- stimulatory molecules, production of antitumor and antiviral cytokines, and stimulation of adaptive immunity. As such, compounds similar to MEDI9197 have shown broad antiviral, antitumor and adjuvant functions both preclinically and clinically (reviewed in Tomai 2006).

[0006] Nonclinical data with numerous TLR 7/8 agonists administered

systemically have demonstrated tumor growth inhibition and eradication in some tumor models (Sidky et al, 1992; Dumitru et al, 2009). Clinically, TLR 7/8 agonists administered systemically have shown some antitumor activity (Dudek et al, 2007; Dummer et al, 2008); however, side effects like lymphopenia, anemia, and flu-like symptoms (fever, chills, fatigue, headache), nausea and vomiting (Dudek et al, 2007; Witt et al, 1993; Savage et al, 1996), which are presumably due to broad spectrum systemic cytokine production, have limited their clinical utility. Therefore, MEDI9197 has been developed specifically for intratumoral (IT) injection and limitation of systemic exposure (Smirnov et al, 2011).

[0007] Various nonclinical mouse tumor studies have demonstrated the antitumor

activity of MEDI9197 (Mullins et al, 2016). However, the safety, tolerability, optimal dosing, and pharmacokinetic (PK)/pharmacodynamic (PD) profiles of MEDI9197 in human patients has not been previously established.

SUMMARY OF THE INVENTION

[0008] Some of the main aspects of the present invention are summarized below.

Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0009] The disclosure provides, for the first time, results of a Phase I, multicenter, open- label, and dose-escalation study of MEDI9197 to evaluate the safety, tolerability,

pharmacokinetics, pharmacodynamics, and preliminary anti-tumor activity in adult human subjects with selected advanced solid tumors. MEDI9197 is generally well tolerated within the dose range of 0.005 to 0.037 mg every four weeks in adult patients with advanced solid tumors. MEDI9197 induces both local and systemic pharmacodynamic (PD) effects, with a trend for dose response.

[0010] In one aspect, the invention provides a method of increasing immune activation in a human subject, the method comprising administering to the subject a composition comprising 0.005-0.037 mg/mL MEDI9197, wherein the composition is administered via injection into a tumor. In certain embodiments, the subject has increased circulating levels of one or more of IFN-γ, CXCL10, or CXCL11 within 24 hours of administration, compared with circulating levels prior to administration; thereby increasing immune activation in the subject.

[0011] In another aspect, the invention provides a method of increasing inflammation in a human subject, the method comprising administering to the subject a composition comprising 0.005-0.037 mg/mL MEDI9197, wherein the composition is administered via injection into a tumor; and wherein the subject has increased infiltration in the tumor of immune cells expressing one or more of CD40, CD8, CD56, or PD-L1 within 3 weeks of administration, compared with expression prior to administration, thereby increasing inflammation in the subject.

[0012] In a further aspect, the invention provides a method of trafficking immune cells

(e.g. T cells and/or NK cells) to a site of inflammation in a human subject, the method comprising administering to the subject a composition comprising 0.005-0.037 mg/mL MEDI9197, wherein the composition is administered via injection into a tumor. In some embodiments, a blood sample from the subject displays one or more of an increase in T_HI signature expression, an increase in Type 1 IFN signature expression, a decrease in CD8a transcript expression, or a decrease in NK cell signature expression within 24 hours of administration, compared with expression prior to administration, thereby trafficking NK and T cells to the site of inflammation.

[0013] The invention also provides the use of an injectable composition comprising

0.005-0.037 mg/mL MEDI9197 to increase immune activation in a human subject, wherein the subject has increased circulating levels of one or more of IFN-γ, CXCL10, or CXCL11 within 24 hours of administration, compared with circulating levels prior to administration.

[0014] The invention further provides the use of an injectable composition comprising

0.005-0.037 mg/mL MEDI9197 to increase inflammation in a human subject, wherein the subject has increased infiltration, at the injection site, of immune cells expressing one or more of CD40, CD8, CD56, or PD-L1 within 3 weeks of administration, compared with expression prior to administration.

[0015] The invention additionally provides the use of an injectable composition

comprising 0.005-0.037 mg/mL MEDI9197 to traffic immune cells (e.g. T cells and/or NK cells) to a site of inflammation in a human subject. In some embodiments, a blood sample from the subject displays one or more of an increase in T_HI signature expression, an increase in Type 1 IFN signature expression, or a decrease in CD8a transcript expression within 24 hours of administration, compared with expression prior to administration.

[0016] In some embodiments, the concentration of MEDI9197 in the composition is

0.037 mg/mL. In other embodiments, the concentration of MEDI9197 in the composition is 0.012 mg/mL. In certain embodiments, the concentration of MEDI9197 in the composition is 0.005 mg/mL.

[0017] In some instances, the injectable composition comprises sesame oil and

dehydrated alcohol. Optionally, the composition can comprise butylated hydroxyanisole (BHA). The composition can comprise about 92.47% (w/w) sesame oil. The composition can comprise about 7.5% (w/w) dehydrated alcohol. The composition can comprise about 0.03% (w/w) BHA.

[0018] The human subject can have a cutaneous or subcutaneous tumor. In certain embodiments, injection of the composition comprising MED 19197 is intratumoral.

[0019] Thus, the invention includes a method of treating a cutaneous or subcutaneous tumor in a human subject, the method comprising administering to the subject via intratumoral injection a composition comprising 0.005-0.037 mg/mL MED 19197, 92.47% (w/w) sesame oil, 7.5% (w/w) dehydrated alcohol, and 0.03% (w/w) BHA. In some embodiments, the concentration of MED 19197 in the composition is 0.005 mg/mL. In other embodiments, the concentration of MED 19197 in the composition is 0.012 mg/mL. In still other embodiments, the concentration of MED 19197 in the composition is 0.037 mg/mL.

[0020] In some embodiments of the invention, the peak plasma concentration of

MEDI9197 is <100 pg/mL in the subject following administration of a composition comprising 0.005-0.037 mg/mL MEDI9197.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows the structure of MEDI9197.

[0022] FIG. 2 shows the design of the dose escalation study.

[0023] FIG. 3 shows a schematic of dose escalation study schedule.

[0024] FIG. 4A-4D show plasma concentrations of MEDI9197 after first IT

administration. Peak plasma levels of MEDI9197 are less than -100 pg/mL (0.1 ng/mL) after IT injection of MEDI9197 in Cohort 1.5 (0.05455 mg) (FIG. 4A), Cohort 1 (0.037 mg) (FIG. 4B), Cohort -1 (0.012 mg) (FIG. 4C), and Cohort -2 (0.005 mg) (FIG. 4D). Minimal effective concentration of MEDI9197 that induced cytokines in vitro in human PBMCs (59 ng/mL) is outside of the graphs axis, and is ~600x higher than peak plasma levels observed after IT administration of MEDI9197.

[0025] FIG. 5A-5C show that MEDI9197 demonstrates evidence of PD effects in a subset of patients. Local PD effects were assessed by immunohistochemistry (IHC) in longitudinal biopsies. FIG. 5A shows representative images of CD8 (T cells), PD-L1 (tumor and immune cells), CD40 (myeloid and B cells), and CD56 (NK cells) IHC staining observed in patient tumors at pre-treatment and at day 22 in Cohort 1 (0.037 mg). FIG. 5B shows the fold change of CD8 (circles), PDL1 (squares), CD40 (triangles), and CD56 (diamonds) via IHC staining in tumors from patients within Cohorts 1 (0.037 mg), -1 (0.012 mg), and -2 (0.005 mg) at day 22, relative to baseline values. Quantifications were performed by Definiens Quantitative analysis. FIG. 5C shows a summary of PD effects, where fold increase is defined as a 2-fold increase over baseline.

[0026] FIG. 6 shows that treatment with MEDI9197 is associated with increased innate and adaptive gene signatures in a subset of patients. Log2 fold-change three weeks after treatment initiation, relative to pre-treatment values of the expression of transcripts or gene signatures, of tumors from patients within Cohorts 1, -1, and -2 are shown and ordered by unsupervised clustering. The x-axis shows the patient identification number, with the cohort in parentheses.

[0027] FIG. 7A-7D show MEDI9197 PD effects via gene expression in blood samples from Cohort 1 patients. Longitudinal gene expression in the blood showing increases in T_HI (FIG. 7A) and Type 1 interferon (FIG. 7B) gene expression signatures and transient decreases in CD8A transcript (FIG. 7C) and NK signature (FIG. 7D) after intratumoral MEDI9197 dosing at Cohort 1 (0.037 mg, n=6), Cohort -1 (0.012 mg, n=8), and Cohort -2 (0.005 mg, n=6) at Day 1 (designated by j).

[0028] FIG. 8A-8I show increased peripheral cytokine levels within 24 hours of

MEDI9197 administration. FIG. 8A-8F show systemic PD effects in Cohorts 1 (n=6), -1 (n=9), and -2 (n=8) following a first IT injection of MED 19197. Longitudinal plasma levels of IFNy (FIG. 8A), CXCL10 (FIG. 8B), CXCL11 (FIG. 8C) demonstrate elevations in cytokines, which peak 18-24 hours after injection. IL-6 (FIG. 8D), IL-10 (FIG. 8E), and TNFa (FIG. 8F) show no significant elevation in cytokine levels. Peak plasma cytokine levels of individual patients from the three cohorts are shown for IFNy (FIG. 8G), CXCL10 (FIG. 8H), and CXCL11 (FIG. 81). Circles show Cohort 1 (A-I); squares show Cohort -1 (A-C, G-I) or Cohort -2 (D-F); triangles show Cohort -1 (D-F) or Cohort -2 (A-C, G-I). Error bars represent standard error of the mean. The horizontal lines in G, H, and I represent the median. *=p<0.05

[0029] FIG. 9A-9I show blunted elevation of peak cytokine levels after second IT

administration of MEDI9197. FIG. 9A-9F show systemic PD effects in Cohort 1 following two ΓΤ injections. Longitudinal plasma levels of IFNy (FIG. 9A), CXCL10 (FIG. 9B), CXCL11 (FIG. 9C), IL-6 (FIG. 9D), IL-10 (FIG. 9E), and TNFa (FIG. 9F) demonstrate blunted elevation in peak plasma levels after a second intratumoral injection (n=5) compared to the 1^st injection (n=6) peak cytokine levels for Cohort 1. Peak plasma cytokine levels of individual patients in Cohort 1 (which occurred between 18-24 hours post- injection, n=4) are shown for IFNy (FIG. 9G), CXCL10 (FIG. 9H), and CXCL11 (FIG. 91). Error bars represent standard error of the mean.

[0030] FIG. 10A-10B show the metabolic response in a patient after intratumoral (IT) administration of 0.012 mg of MEDI9197. FIG. 10A shows PET scans at Baseline and 197 days after repeat MED 19197 injections in injected (top) and non-injected (bottom) rhabdomyosarcoma lesions in Patient #20018560005. Data demonstrate a decrease in the FDG uptake. FIG. 10B shows tumor size of the MED 19197 injected lesion and a non- injected lesion. Injections of MEDI9197 (black arrows), biopsies (red dashes), and palliative radiation (pink box) to the injected lesion are also shown. SUV = standardized uptake value. FDG = [18F]-2-fluoro-2-deoxy-D-glucose. x-axis shows time in monthly intervals.

[0031] FIG.ll A-I show the individual patient peripheral cytokine levels for each cohort.

These figures confirm the results in FIG. 8D-8F further demonstrating that IL-6 (FIG. 11A- 11C), IL-10 (FIG. 11D-11F), and TNFa (FIG. 11G-11I) show no significant elevation in cytokine levels across Cohorts 1 (n=6), -1 (n=9), and -2 (n=8) following a first ΓΤ injection of MEDI9197.

[0032] FIG. 12 shows that treatment with MEDI9197 results in TLR7 and 8 pathway activation a subset of patients. Log2-fold change 3 weeks after treatment initiation relative to pretreatment values of the expression of transcripts of tumors from patients in cohorts 1, -1, and -2 are shown and ordered by unsupervised clustering. The range of log₂ fold change is truncated to a range of -5 to 5. The x-axis shows the patient identification number, the cohort in parentheses, and tumor type. TLR7 and 8 downstream genes were derived from Guiducci et al.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Handbook of Pharmaceutical Excipients (7th ed., Rowe et al. eds., 2012); Martin's Physical Pharmacy and Pharmaceutical Sciences (6th ed., Sinko, 2010); Remington: The Science and Practice of Pharmacy (21st ed., Univ. Sci. Philadelphia ed., 2005); Current Protocols in Molecular Biology (Ausubel et al. eds., 2016); Molecular Cloning: A Laboratory Manual (4th ed., Green and Sambrook eds., 2012); Lewin's Genes XI (11th ed., Krebs et al. eds., 2012); DNA Cloning: A Practical Approach, Volumes I and II (2d ed., Glover and Hames eds., 1995); Protein Engineering: A Practical Approach (1st ed., Rees et al. eds. 1993); Culture Of Animal Cells (6th ed. Freshney, 2010); Antibodies: A Laboratory Manual (2nd ed., Greenfield ed., 2013); Antibody Engineering (2d ed.,

Borrebaeck ed., 1995).

[0034] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. For example, Dictionary of Pharmaceutical Medicine (3rd ed. Nahler and Mollet eds., 2013); The Dictionary of Cell and Molecular Biology (5th ed. J.M. Lackie ed., 2013), Oxford Dictionary of Biochemistry and Molecular Biology (2d ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skill with general definitions of some terms used herein.

[0035] Any headings provided herein are not limitations of the various aspects or

embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0036] All of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.

I. Definitions

[0037] As used in this specification and the appended claims, the singular forms "a,"

"an," and "the" include plural referents, unless the context clearly dictates otherwise. The terms "a" (or "an") as well as the terms "one or more" and "at least one" can be used interchangeably.

[0038] Furthermore, "and/or" is to be taken as specific disclosure of each of the two

specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0039] Wherever embodiments are described with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are included.

[0040] Units, prefixes, and symbols are denoted in their Systeme International de Unites

(SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1- 10, from 1-8, from 3-9, and so forth.

[0041] An "isolated" molecule is one that is in a form not found in nature, including those which have been purified. In some embodiments, an isolated molecule is substantially pure. As used herein, the term "substantially pure" refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

[0042] A "label" is a detectable compound that can be conjugated directly or indirectly to a molecule, so as to generate a "labeled" molecule. The label can be detectable on its own (e.g., radioisotope labels or fluorescent labels) or can catalyze chemical alteration of a substrate compound or composition that is detectable (e.g., an enzymatic label).

[0043] The terms "inhibit," "block," and "suppress" are used interchangeably and refer to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity. For example, "inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence.

[0044] An "active agent" is an ingredient that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human body. The active agent can be in association with one or more other ingredients, and can be, but is not necessarily, in a finished dosage form. The terms "active agent" and "drug substance" are used

interchangeably herein.

[0045] An "effective amount" of an active agent is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose. [0046] The term "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. human albumin), a preservative (e.g. sodium benzoate), an absorption promoter to enhance bioavailability and/or other conventional solubilizing or dispersing agents.

[0047] A "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and laboratory animals including, e.g., humans, non-human primates, canines, felines, porcines, bovines, equines, rodents, including rats and mice, rabbits, etc.

[0048] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to

alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully "treated" for a disease or disorder according to the methods provided herein if the patient shows, e.g., total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.

[0049] "Prevent" or "prevention" refer to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of prevention include those prone to have or susceptible to the disorder. In certain embodiments, a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who has not been subject to the methods of the invention. [0050] An "adverse event" (AE) is any untoward medical occurrence in a subject or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. (ICH Guideline for Good Clinical Practice E6(R1).)

[0051] A "serious adverse event" (SAE) is any AE that results in death, is immediately life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability/incapacity, is a congenital anomaly/birth defect in offspring of the subject, and/or is an important medical event that may jeopardize the subject or may require medical intervention to prevent one of the outcomes listed above.

[0052] "Cytokine release syndrome" (CRS) is a systemic inflammatory response caused by activated T cells and characterized by nausea, headache, tachycardia, hypotension, rash, and shortness of breath.

[0053] A "cycle" is the period of time between dose administrations. For example, if a pharmaceutical composition is administered every two weeks for a period of eight weeks, administration would occur over four cycles of fourteen days each. Typically, the day of administration is day 1 of a cycle.

II. Immune Response Modifiers and Composition

[0054] Compositions used in the present invention comprise an immune response

modifier (IRM). IRMs have been shown to induce the production of certain cytokines, such as interferon alpha (IFN-a), tumor necrosis factor alpha (TNF-a), and certain interleukins, indicating that these compounds can inhibit tumor cell growth and virus production. The ability to modulate the immune response by inducing cytokine biosynthesis also makes IRMs useful as a vaccine adjuvant. In the present invention, the IRM is a Toll-like receptor 7/8 (TLR7/8) agonist, MEDI9197, also known as S-36862 and 3M-052 (FIG. 1).

[0055] Any pharmaceutically acceptable form of the IRM and its salts can be used,

including isomers (e.g., diastereomers and enantiomers), solvates, polymorphs, and the like. In particular, the invention specifically includes each of the compound's enantiomers as well as racemic mixtures of the enantiomers. [0056] MEDI9197 may be synthesized by synthetic routes that include processes analogous to those well known in the chemical arts. The starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, WI) or are readily prepared using methods well known to those skilled in the art (e.g., prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, N.Y., (1967-1999 ed.); Alan R. Katritsky, Otto Meth-Cohn, Charles W. Rees, Comprehensive Organic Functional Group Transformations, v 1-6, Pergamon Press, Oxford, England, (1995); Barry M. Trost and Ian Fleming, Comprehensive Organic Synthesis, v. 1-8, Pergamon Press, Oxford, England, (1991); or Beilsteins Handbuch der organischen Chemie, 4, Aufl. Ed. Springer-Verlag, Berlin, Germany, including supplements (also available via the Beilstein online database)); see also US Patent

Publication No. 2016/0271059.

[0057] The IRM can be purified using standard methods in the art. Purification methods include, for example, chromatography, such as high pressure liquid chromatography (HPLC), solvent extraction, and precipitation.

[0058] In some embodiments, the composition for use in the invention comprises about

0.0012 to about 0.055 mg/mL IRM (MEDI9197). For example, the composition can comprise about 0.0012, 0.0025, 0.005, 0.012, 0.037, or 0.055 mg/mL IRM. The amount of IRM may vary according to the subject treated and the intended indication.

[0059] In some embodiments, the composition is administered to the patient in an

injection volume of about 1 mL injection. In other embodiments, the composition is administered in an injection volume of less than about 1 mL. For example, the composition can be administered in an injection volume of about 100 μί, about 150 μί, about 200 μί, about 250 μί, about 300 μί, about 350 μί, about 400 μί, about 450 μί, about 500 μί, about 550 μί, about 600 μί, about 650 μί, about 700 μί, about 750 μί, about 800 μί, about 850 μί, about 900 μί, or about 950 μΐ_^.

[0060] Some injectable compositions for use in the present invention comprise ethanol.

In one embodiment, the composition comprises ethanol that does not contain any water or denaturant, for example, Dehydrated Alcohol, USP grade. Ethanol can be present in an amount of from about 1 %w/w to about 9 %w/w, such as from about 3 %w/w to about 8 %w/w, from about 5 %w/w to about 7.5 %(w/w), from about 1 %w/w to about 3 %w/w, from about 3 %w/w to about 4 %w/w, from about 4 %w/w to about 5 %w/w, from about 5 %w/w to about 6 %w/w, from about 6 %w/w to about 7 %w/w, from about 6.5 %w/w to about 7.5 %w/w, or from about 8 %w/w to about 9 %w/w. In a particular embodiment, the

composition comprises about 7.5 %w/w ethanol.

[0061] In some embodiments, the composition comprises an excess of ethanol (i.e., a greater amount than is soluble in sesame oil), for example, at least 10 %w/w ethanol, at least 12 %w/w ethanol, at least 14 %w/w ethanol, which is used to dissolve greater amounts of the IRM compound. In such cases, when the IRM-ethanol solution is added to the sesame oil, the IRM dissolves much more quickly than simply adding the IRM to a premixed sesame oil- ethanol solution; the excess ethanol (that is present beyond the solubility limits in sesame oil) is then evaporated off to produce the final formulation (containing 9 %w/w ethanol or less).

[0062] Other pharmaceutically acceptable co-solvents can be used instead of or in

addition to ethanol.

[0063] The injectable compositions used in the invention can also include a

pharmaceutically acceptable sesame oil, such as Sesame Oil, NF. In some embodiments, the sesame oil may be present in an amount of from about 90 %w/w to about 99 %w/w, such as from about 91%w/w to about 98%w/w, from about 92%w/w to about 97%w/w, from about 93%w/w to about 96%w/w, from about 94%w/w to about 95%, in further example from about 92%w/w to about 93%w/w, from about 92.1%w/w to about 92.9%w/w, from about 92.2% w/w to about 92.8%w/w, from about 92.3 %w/w to about 92.7%w/w, from about 92.4%w/w to about 92.6%w/w, from about 92.4%w/w to 92.5%w/w; and more specifically about 92.40%w/w or about 92.47%w/w. In some embodiments, the sesame oil may be refined such that one or more polar compounds have been substantially removed from the sesame oil or reduced in content without substantially altering the fatty acid profile of the sesame oil. For example, the sesame oil may have a fatty acid profile that includes palmitic acid, stearic acid, oleic acid, and linoleic acid. Other fatty acids may also be present at lower levels, typically less than 1 %w/w. Polar compounds present in sesame oil can include but are not limited to compounds such as monoglycerides, diglycerides, free fatty acids, plant sterols, coloring matter (chlorophyll, carotene), sesamin, sesamolin, products resulting from oxidation, and environmental chemicals. Polar compounds in sesame oil can be

quantitatively measured using standard tests such as the acid value test, hydroxyl value test, peroxide value test, and trace nitrogen value test. Standard chromatography methods can be used to remove or substantially reduce the content of at least one polar compound from sesame oil to provide a refined sesame oil. Suitable chromatographic methods that are well known in the art include gravity based column chromatography, flash column

chromatography, medium pressure liquid chromatography, or high pressure chromatography.

[0064] In some embodiments, the sesame oil has a hydroxyl value less than or equal to 2.

The hydroxyl value of sesame oil can be determined according to the published procedure described in USP 36 <401> Fats and Fixed Oils, Hydroxyl Value. In some embodiments, the acid value of the sesame oil is less than or equal to 0.1. The acid value of sesame oil can be determined according to the published procedure described in USP 36 <401>Fats and Fixed Oils, Acid Value. In some embodiments, the peroxide value of the sesame oil is less than or equal to 1. The peroxide value of sesame oil can be determined according to the published procedure described in USP 36 <401> Fats and Fixed Oils, Peroxide Value. In some embodiments, the total nitrogen content of the sesame oil is less than or equal to 1 ppm. The trace nitrogen value of sesame oil can be determined according to the published method described in ASTM D5762-12. In some embodiments, the sesame oil contains no more than 0.05 %w/w of sesamin. In some embodiments, the sesame oil contains no more than 0.05 %w/w of sesamolin. The levels of sesamin and sesamolin can be determined according to the published sesamin/sesamolin assay described by T. Tashiro, Y. Fukuda. T. Osawa and M. Namiki in Journal of the American Oil Chemists' Society, 67, 508 (1990).

[0065] Other pharmaceutically acceptable lipids, including but not limited to castor oil, cottonseed oil, soybean oil, safflower oil, and medium chain triglycerides, can be used instead of or in addition to sesame oil.

[0066] In some embodiments, the pharmaceutical formulations may further include one or more additives including, but not limited, to, antioxidants, antimicrobials, adjuvants, preservatives, thickeners, suspending agents, surfactants, and/or dispersing agents.

Prevention of presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. In some embodiments, the formulation can include an antioxidant such as butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like. A skilled artisan can choose a suitable concentration of a given additive, depending upon its identity and function. For instance, the added antioxidant concentration in the formulation can be at least about 10 ppm, 50 ppm, 100 ppm, 200 ppm, 300 ppm or 400 ppm. In a preferred embodiment, the composition comprises about 300 ppm (0.03% w/w) BHA. In another preferred embodiment the composition comprises about 200 ppm (0.02% w/w) BHA. In another preferred embodiment the composition comprises about 400 ppm (0.04%w/w) BHA.

[0067] The pH of the composition administered to a subject is, generally, about 5.5 to about 8.5, preferably about 6.0 to about 7.8, which are suitable pH levels for injection into a mammal. The pH of the composition can be adjusted by any suitable acid or base, such as hydrochloric acid or sodium hydroxide.

III. Methods of Use

[0068] IRMs, such as MEDI9197, can induce the production of certain cytokines, and are useful as immune response modifiers that can modulate the immune response in a number of different ways, rendering them useful in the treatment of a variety of disorders.

[0069] In addition to the ability to give rise to cytokine induction, IRMs can bring about an effect on other aspects of the innate immune response. For example, natural killer cell activity can be stimulated, perhaps due to cytokine induction. IRMs can also bring about activation of macrophages, which in turn stimulate secretion of nitric oxide and the production of additional cytokines. Further, IRMs can bring about proliferation and differentiation of B -lymphocytes.

[0070] IRMs can also bring about an effect on the acquired immune response. For

example, the production of the T helper type 1 (T_HI) cytokine IFN-γ may be induced indirectly, and the production of the T helper type 2 (T_H2) cytokines IL-4, IL-5, and IL-13 may be inhibited upon administration of an IRM such as MEDI9197.

[0071] To those ends, the invention provides, in one embodiment, a method of increasing immune activation in a human subject by administering via injection a composition comprising 0.005-0.037 mg/mL MEDI9197. Increased circulating levels of one or more of IFN-γ, CXCL10, or CXCL11 within 24 hours of administration, compared with circulating levels prior to administration, are indicative of increased immune activation in the subject. [0072] The invention also provides a method of increasing inflammation in a human subject by administering via injection a composition comprising 0.005-0.037 mg/mL

MEDI9197. Increased infiltration, at the injection site, of immune cells expressing one or more of CD40, CD8, CD56, or PD-L1 within 3 weeks of administration, compared with expression prior to administration, indicates increased inflammation in the subject.

[0073] Another embodiment of the invention is a method of trafficking immune cells to a site of inflammation in a human subject by administering via injection a composition comprising 0.005-0.037 mg/mL MEDI9197. An increase in THI signature expression, an increase in Type 1 IFN signature expression, or a decrease in CD8a transcript expression in a blood sample from the subject within 24 hours of administration, compared with expression prior to administration, indicates trafficking of T cells and/or NK cells to a site of

inflammation.

[0074] The subject to which the injectable composition comprising MEDI9197 is

administered may have a disease (e.g., a viral or neoplastic disease); administration of the composition may provide therapeutic treatment. For example, the subject may have a) a neoplastic disease such as melanoma, leukemia (e.g., myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, B-cell lymphoma, and hairy cell leukemia), breast cancer, lung cancer, prostate cancer, colon cancer, head or neck cancer, bladder cancer, or other cancer; (b) a viral disease such as a disease resulting from infection by a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), or a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts); (c) a disease associated with wound repair, such as inhibition of keloid formation and other types of scarring (e.g., the composition may enhance wound healing, including chronic wounds).

[0075] Accordingly, the invention can include methods of treating a disease in a human subject, such as a neoplastic disease or a viral disease. In some embodiments, the subject has a solid tumor, such as a head/neck tumor, a breast tumor, lymphoma, melanoma, or a bladder tumor. Preferably, the method comprises injection of the composition into a tumor mass, i.e., intratumoral injection. One embodiment includes a method for treating cutaneous T cell lymphoma. Another embodiment includes a method of treating a cutaneous or subcutaneous tumor in a human subject. In a preferred embodiment, the method comprises administering to a human subject via intratumoral injection a composition comprising 0.005-0.037 mg/mL MEDI9197, 92.47% (w/w) sesame oil, 7.5% (w/w) dehydrated alcohol, and 0.03% (w/w) BHA. In some instances, the peak plasma concentration of MED 19197 in the subject is <100 pg/mL.

[0076] The composition comprising MEDI9197 can be administered as a single dose or multiple doses. The composition can be administered as many times (i.e., cycles) as needed to achieve a targeted endpoint. Injection intervals may vary. For example, the composition can be administered every 1, 2, 3, or 4 weeks, or every 1, 2, 3, 4, 5, or 6 months. In one embodiment, the composition is administered once every four weeks for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 cycles. Dosage regimens can be adjusted to provide the optimum desired response.

EXAMPLES

[0077] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe a Phase 1, first-time-in-human (FTIH), multicenter, unblinded, dose escalation study of the TLR 7/8 agonist MEDI9197, delivered by IT injection. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.

Example 1. Materials and Methods

Subjects and Dosing

[0078] Twenty three male and female patients, 18 years of age or older, with

subcutaneous/cutaneous tumors were enrolled. Screening procedures including medical histories, physical examinations, standard clinical laboratory safety tests, and

electrocardiograms were performed during the screening period and at various times during the treatment period.

[0079] The formulation is a sterile solution consisting of MEDI9197 dissolved in a

mixture of highly refined sesame oil (92.47% w/w) formulated with dehydrated alcohol (7.5% w/w) and 300 ppm (0.03% w/w) butylated hydroxyanisole (BHA) as antioxidant. (See US 2016/0271259.) The MEDI9197 active solution is supplied as 3 mL formulation in 5-mL amber glass serum vials containing a sterile solution of 0.6 mg/mL MEDI9197 in 92.40% Sesame Oil, NF (%w/w), 7.5% Dehydrated Alcohol USP (%w/w), and 0.03% Butylated Hydroxyanisole NF (%w/w). The diluent is also supplied as 3 mL of formulation in 5-mL amber glass serum vials, with sesame oil (92.47% w/w) formulated with dehydrated alcohol (7.5% w/w) and 300 ppm (0.03% w/w) butylated hydroxyanisole (BHA) as antioxidant.

[0080] For dosing, the 0.6 mg/ml active solution was diluted with the diluent to the

desired strength, and 1 mL was administered to each cutaneous/subcutaneous lesion between 1.5 and 5 cm that was easily accessible (where easily accessible is defined as a cutaneous or subcutaneous mass that is palpable and/or visualizable by ultrasound). Fixed-volume intratumoral injections were performed in the same tumor at each visit (Q28d) using imaging guidance or visual inspection.

[0081] Subjects were observed and vital signs monitored for a minimum of 6 hours after each dose of MEDI9197 for immediate adverse events (AEs).

Sample Collection

[0082] Plasma samples for PK evaluation were collected in tubes with potassium

ethylene diamine tetraacetic acid (K2 EDTA) at various study visits for the determination of MEDI9197 concentration using liquid chromatography and mass spectrometry (LC/MS/MS). Exploratory blood samples were collected for analysis of circulating levels of soluble factors, such as C-reactive protein, cytokines, and chemokines. Whole blood was collected for PBMC isolations. For gene expression analysis, whole blood samples were collected in PAXgene tubes and stored frozen for RNA sample preparation. Pre/post treatment changes in mPvNA levels of inflammatory/immune cytokines and factors associated with Thl and IFN pathways were analyzed.

[0083] Image-guided core needle tumor biopsies were performed according to

institutional practice. The biopsies were taken pre-injection and post-injection on day 22 (+/- 1 day). Biopsy samples were placed in formalin and processed for formalin-fixed paraffin- embedding, or were immediately frozen in liquid nitrogen and stored at -80°C. Biopsy samples were used to evaluate biomarkers by immunohistochemical and pharmacogenomic analysis, including the expression level and localization of a number of immune related proteins. Example 2. Study Design and Safety Results

[0084] The original study design was a 3 + 3 dose escalation, shown in Table 1 and FIG.

2, to evaluate a range of doses from 0.005 mg to 2.4 mg/tumor to determine the maximum tolerated dose (MTD)/ maximum assessed dose per protocol (MAD). The MTD is the highest dose level administered that does not exceed the toxicity criteria of > 1/3 or > 2/6 dose-limiting toxicities (DLTs). A schematic of the study schedule is shown in FIG. 3.

Table 1. Dose Calculation for MEDI9197

[0085] In mice, a MEDI9197 dose of about 0.13 mg/cm³ tumor consistently demonstrated statistically significant efficacy in the B 16-OVA model. The starting clinical dose for small human tumors (0.037 mg/1.8 cm³ tumor) is about 0.02 mg/cm³, 6-fold lower than the efficacious dose used in the mouse tumor model. The lowest effective concentration to induce in vitro cytokine production in human and monkey PBMCs and in mouse splenocytes was 59 ng/mL (100 nM); this concentration was selected as the minimal anticipated biological effect level (MABEL) for systemic effect.

[0086] In the monkey toxicology study (WIL 180037), serum cytokine levels increased at all dose levels tested (1.25 to 5 mg/kg). Median serum MED 19197 C_max ranged from 1.8 to 4.3 ng/mL, which was lower than the lowest effective concentration to induce in vitro cytokine production (59 ng/mL), indicating cytokine increase observed in monkey blood in vivo was likely due to spillover from local production, not systemic production of cytokine.

[0087] While mice express both TLR7 and 8, published literature suggests that mouse

TLR8 has an altered binding domain that makes it less responsive to TLR8-selective agonists (Jurk et al, 2002; Hemmi et al, 2002; Colak et al, 2014; Gorden et al, 2006). Therefore, data generated in experiments with MEDI9197 using mice likely represents preferential activation of TLR7 as compared to TLR8. However, both TLR7 and TLR8 are responsive to

TLR7-selective and TLR8-selective agonists in humans and monkeys (Wagner et al, 1999; Ketloy et al, 2008; Wille-Reece et al, 2006; Philbin et al, 2012). Therefore, the monkey toxicology data is more relevant to human than the mouse toxicology data. The monkey HNSTD was 5 mg/kg (about 60 mg/m²) following SC injection, which was the highest dose tested in Study WIL- 180037. Per the International Conference on Harmonisation (ICH) S9 Guidance, the allowable starting clinical dose is 1/6 of the HNSTD (10 mg/m²) which is equivalent to 16 mg/injection.

[0088] Injections were given every four weeks (Q4W). Dose escalation began at

0.037 mg/mL (0.023 mg/m²); one patient received dose level 1.5 (0.055 mg/mL). Our starting dose provides a significant safety margin: 438-fold based on the ICH S9 Guidance allowable starting dose or 2,631-fold based on monkey HNSTD. Two DLTs were observed: Grade 3 cytokine release syndrome (CRS) at 0.037 mg and Grade 4 CRS at 0.055 mg. The MTD was determined to be 0.037 mg/mL/tumor every four weeks. In light of the observed DLT at the 0.037 mg dose, two lower doses of 0.0012 mg/mL and 0.0005 mg/mL were added to the study design.

[0089] The mean duration of exposure was 12 weeks, with a range of 3-52 weeks. The mean relative dose intensity was 94%, with a range of 50-127%.

[0090] The most common (>20%) drug related AEs are pyrexia, fatigue, chills, decreased lymphocyte count, nausea, and injection site pain. One patient discontinued MEDI9197 as a result of a drug-related AE (Grade 4 CRS); there were no Grade 5 drug-related AEs.

Fever/CRS generally occurs within 24 hours of injection. Fever usually resolves within 48 hours; CRS lasts approximately one week, with counts taking approximately one week to recover, although some patients do not recover until week three. For most patients, the fever/CRS is absent or decreased with subsequent injections. A summary is shown in Table

2.

Table 2. Treatment- Related Adverse Events (Occurring in >4 Patients in Total Population

Example 3. Pharmacodynamic and Pharmacokinetic Effects

[0091] Tumoral and peripheral PD effects of MEDI9197 were assessed from patients treated with 0.037 (n=6), 0.012 (n=8), and 0.005 (n=8) mg. Local PD effects were assessed by immunohistochemistry (IHC) in longitudinal biopsies.

[0092] Peak plasma levels of MEDI9197 were extremely low (<0.1 ng/mL) (FIG. 4A-

4D). These levels are about 600-fold below the minimum effective concentration (59 ng/mL) that induced cytokines in vitro in human PBMCs.

[0093] The majority of patients treated with 0.037 mg demonstrated an increase in CD8

(T cells), CD40 (myeloid and B cells), CD56 (NK cells), or PD-Ll (tumor and immune cells) markers 3 -weeks after treatment initiation, based on quantitative image analysis (FIG.5A-

5C).

[0094] RNAseq analysis of paired tumor biopsies showed an increase in TLR7/8- downstream regulated genes (FIG. X) and innate and adaptive immune activation signatures such as Type 1 IFN, IFNy and T effector signatures (>1.5 fold) consistent with IHC and indicating increased inflammation(FIG. 6). Analysis of whole blood microarray data demonstrated increases (>2 fold) in Type 1 IFN and T_HI gene expression signatures and a transient decrease (>1.5 fold) in CD8A transcript and NK cell signature expression at 24 hours post-dose, suggesting trafficking of T and NK cells in patients treated with 0.005, 0.012 and 0.037 mg of MEDI9197 (FIG. 7A-7D).

[0095] Increased peripheral levels of IFN-γ, CXCLIO, and CXCLl 1 were observed for all cohorts (FIG. 8A-8C). Median peak values of IFN-γ, CXCLIO, and CXCLl l in Cohort 1 were 236, 9,286, and 558 pg/mL, respectively, within 24 hours of IT administration of 0.037 mg of MEDI9197 (FIG. 8G-8I). This fold-change increase ranged from 4.5-43, 30-132, and 3.7-52 versus baseline for IFN-γ, CXCLIO, and CXCLl 1, respectively. Median peak values of IFN-γ, CXCLIO, and CXCLl l in Cohort -1 were 125, 6,684, and 808 pg/mL,

respectively, within 24 hours of IT administration of 0.012 mg of MEDI9197. This fold- change increase ranged from 1.1-35, 2.6-72, and 1.9-24 versus baseline for IFN-γ, CXCLIO, and CXCLl 1, respectively. Median peak values of IFN-γ, CXCLIO, and CXCLl 1 in Cohort -2 were 58, 5,120, and 344 pg/mL, respectively, within 24 hours of IT administration of 0.005 mg of MEDI9197. This fold-change increase ranged from 0.9-6.4, 0.9-27, and 0.8-7.3 versus baseline for IFN-γ, CXCLIO, and CXCLl l, respectively.

[0096] Interestingly, a second IT injection into the same lesion resulted in a blunted

elevation in peak IFN-γ, CXCLIO, and CXCLl l levels in 5/6 Cohort 1 patients by 51.1%, 67.2%, and 58.2%, respectively, versus post-first dose (FIG. 9A-9C). A downward trend in cytokine elevations was observed 24 hours post-dose 2 compared to dose 1 (FIG. 9G-9I). These results are consistent with tolerance or tachyphylaxis.

[0097] Metabolic response was assessed in a Cohort -1 patient with rhabdomyosarcoma receiving repeated MEDI9197 ΓΤ injections into a single lesion and then palliative radiation to the injected lesion. PET scans at Baseline and day 197 demonstrated a decrease in FDG uptake in both injected and uninjected lesions (FIG. 10A). Tumor size decreased by 36% in both lesions, compared to baseline (FIG. 10B). Changes in tumor size and SUV values observed in the injected lesion on Days 141 and 197 were confounded by palliative radiation to the injected lesion (FIG. 10B), the effect of which is unknown.

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Witt PL, Ritch PS, Reding D, McAuliVe TL, Westrick L, Grossberg SE, et al. Phase I trial of an oral immunomodulator and interferon inducer in cancer patients. Cancer Res. 1993;53:5176-80. 8] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. The present invention is further described by the following claims.

Claims

1. A method of increasing immune activation in a human subject, the method comprising administering to the subject a composition comprising 0.005-0.037 mg/mL

MEDI9197, wherein the composition is administered via injection into a tumor; thereby increasing immune activation in the subject.

2. The method of claim 1, wherein the subject has increased circulating levels of one or more of IFN-γ, CXCL10, or CXCL11 within 24 hours of administration, compared with circulating levels prior to administration.

3. A method of increasing inflammation in a human subject, the method comprising administering to the subject a composition comprising 0.005-0.037 mg/mL MEDI9197, wherein the composition is administered via injection into a tumor; and wherein the subject has increased infiltration in the tumor of immune cells expressing one or more of CD40, CD8, CD56, or PD- Ll within 3 weeks of administration, compared with expression prior to administration, thereby increasing inflammation in the subject.

4. A method of trafficking immune cells to a site of inflammation in a human subject, the method comprising administering to the subject a composition comprising 0.005- 0.037 mg/mL MEDI9197, wherein the composition is administered via injection into a tumor; thereby trafficking T cells to the site of inflammation.

5. Use of an injectable composition comprising 0.005-0.037 mg/mL MEDI9197 to increase immune activation in a human subject, wherein the subject has increased circulating levels of one or more of IFN-γ, CXCL10, or CXCL11 within 24 hours of administration, compared with circulating levels prior to administration.

6. Use of an injectable composition comprising 0.005-0.037 mg/mL MEDI9197 to increase inflammation in a human subject having a tumor, wherein the subject has increased infiltration in the tumor of immune cells expressing one or more of CD40, CD8, CD56, or PD- Ll within 3 weeks of administration, compared with expression prior to administration.

7. Use of an injectable composition comprising 0.005-0.037 mg/mL MEDI9197 to traffic immune cells to a site of inflammation in a human subject.

8. The method or use of any one of claims 1 to 7, wherein the concentration of MEDI9197 in the composition is 0.037 mg/mL.

9. The method or use of any one of claims 1 to 7, wherein the concentration of MEDI9197 in the composition is 0.012 mg/mL.

10. The method or use of any one of claims 1 to 7, wherein the concentration of MEDI9197 in the composition is 0.005 mg/mL.

11. The method or use of any preceding claim, wherein the composition comprises sesame oil and dehydrated alcohol.

12. The method or use of claiml 1, wherein the composition comprises at least 92.47% (w/w) sesame oil and at least 7.5% (w/w) dehydrated alcohol.

13. The method or use of any preceding claim, wherein the composition comprises butylated hydroxyanisole (BHA).

14. The method or use of claiml3, wherein the composition comprises 0.03% (w/w)

BHA.

15. The method or use of any preceding claim, wherein the subject has a cutaneous or subcutaneous tumor.

16. A method of treating a cutaneous or subcutaneous tumor in a human subject, the method comprising administering to the subject via intratumoral injection a composition comprising 0.005-0.037 mg/mL MEDI9197, 92.47% (w/w) sesame oil, 7.5% (w/w) dehydrated alcohol, and 0.03% (w/w) BHA.

17. The method of claim 16, wherein the concentration of MEDI9197 in the composition is 0.005 mg/mL.

18. The method of claim 16, wherein the concentration of MEDI9197 in the composition is 0.012 mg/mL.

19. The method of claim 16, wherein the concentration of MEDI9197 in the composition is 0.037 mg/mL.

20. The method of claim 16, wherein the peak plasma concentration of MEDI9197 in the subject is <100 pg/mL.

21. The method of claim 4, wherein the immune cells are T cells.

22. The method of claim 4, wherein the immune cells are NK cells.

23. The method of claim 7, wherein the immune cells are T cells.

24. The method of claim 7, wherein the immune cells are NK cells.