Safety and Efficacy of TGF-β1/COX-2 Silencing Therapeutic in Adults With Cutaneous Squamous Cell Carcinoma In Situ

Nonmelanoma skin cancer (NMSC) is a common cancer with an annual incidence in the United States of over 3 million new diagnoses.^1,2 There are 2 major subtypes: basal cell carcinoma (BCC, which rarely metastasize) and squamous cell carcinoma (SCC).³ Recent analysis of skin cancer data suggests that malignant SCCs, which develop from hair follicle stem cells, account for 20%-50% of all skin cancers.⁴ Surgical excision or destruction by various means are recommended for NMSCs, but residual, NMSC can carry an increased risk of deep tumor progression, metastasis, and recurrence.^1,5 Patients undergoing surgery also risk infection, hematoma, and scarring. There is a limited number of approved nonsurgical pharmacotherapy options for local treatment of BCC and SCC, and so, alternative modalities to treat NMSC are thus needed.^1,6-8 Overexpressed transforming growth factor-beta1 (TGF-β1) and cyclooxygenase-2 (COX-2) are strongly associated with SCC development. TGF-β1 has a prominent role in epithelial cell proliferation and tumor progression.^9,10 Overexpressed TGF-β1 signaling, mediated by mutated Smad proteins and/or their impaired phosphorylation process, is observed in tumors and impairs angiogenesis and extracellular matrix remodeling and causes immune evasion, further promoting tumor development.^11-15 As the cancer progresses, TGF-β1 signaling promotes epithelial-to-mesenchymal transition, leading to eventual metastasis.^11,12 Silencing COX-2 expression downregulates the production of prostaglandin E2 and inhibits tumor cell proliferation.^16,17 Similar to TGF-β1, inhibiting COX-2 expression downregulates epithelial-mesenchymal transition, inhibits migration of tumor cells, and promotes their apoptosis by reducing the anti-apoptotic regulator protein B-cell lymphoma 2.^16-18 COX-2 inhibitors, such as celecoxib, have been demonstrated to be effective in decreasing the risk of NMSC and SCC.^16,19 The use of RNA interference (RNAi) therapeutics to silence TGF-β1 and COX-2 in NMSC deserves further attention as a noninvasive alternative to conventional surgery. The objectives of this study were to evaluate the safety and efficacy of various doses of intralesional injections of TGF-β1/COX-2 combined with histidine-lysine polypeptide (siRNA/HKP) nanoparticle

therapeutic in patients with cutaneous SCC in situ (isSCC).

Study Design This open label, dose escalation cohort study took place at the Center for Clinical and Cosmetic Research (Aventura, FL). Subject recruitment with rolling enrollment, treatment, follow-up, and data collection took place from March 21, 2019, to October 21, 2020. Subjects with isSCC were equally divided into 5 cohorts of different dose levels of TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic to determine its optimal dose for isSCC treatment.The primary efficacy endpoint was the proportion of subjects with histological clearance, defined as the absence of detectable evidence of isSCC tumor cell nests, of treated isSCC lesion at the end of treatment visit. Safety endpoints included the incidence and severity of treatment emergent adverse events (TEAEs, an event with a start date on or after the treatment start date) and serious adverse events (SAEs), the incidence of TEAEs and SAEs leading to discontinuation of trial medication, and the incidence and severity of Local Skin Response (LSR). Clinical judgement determined that the sample size would be 25 subjects (5 subjects per cohort). Adults with isSCC provided informed consent to have a biopsy performed for baseline immunohistochemistry analysis and were then screened for study eligibility based on the inclusion and exclusion criteria listed in Table 1. During the screening visit, the provider recorded demographic information, medical and medication history, vital signs, and physical examination findings; and digitally

photographed and assessed the lesion using the LSR grading scale. The LSR grading scale assigns a score (0-4) based on the severity of skin reactions in terms of erythema, flaking/scaling, crusting, swelling, vesiculation/pustulation, and erosion/ ulceration. A composite LSR score was summed. Eligible patients who provided their written informed consent were consecutively allocated into 5 dosing cohorts of 5 subjects each, with the first 5 subjects enrolled assigned to the 10 μg/ treatment group, then the 20 μg/treatment group, 30 μg/ treatment group, 60 μg/treatment group, and finally the 120 μg/ treatment group. Each cohort was treated and assessed before the dose escalation and treatment of the next cohort began. Intralesional injections of TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic were given once weekly for up to 6 weeks to each successive cohort based on the absence of Dose Limiting Toxicities in the previous cohort. The Advarra Institutional Review Board (Columbia, MD) approved the study protocol, which is listed on clinicaltrials.gov (NCT04293679). Investigational Product, Treatment, and Follow-up STP705 (Sirnaomics, Inc., Gaithersburg, MD) was the investigational product. It is composed of 2 siRNA oligonucleotides targeting TGF-β1 and COX-2 (0.16 mg each) and is formulated in nanoparticles with 1.44 mg of histidine-lysine co-polymer (HKP) peptide at a ratio of 1:4.5 in mass weight. It is then formulated into a sterile, lyophilized, dry powder and refrigerated at 2°C to 8°C. Reconstitution with water transforms it into a colorless and transparent solution. Within 4 weeks of enrollment, subjects, beginning with the 10 μg/treatment group and ending with the 120 μg/treatment cohort, received the TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic injection into their isSCC lesions weekly for 6 weeks. Prior to receiving the injection at each treatment visit, the provider recorded medical and medication history (at week 1 visit only), concomitant medications, vital signs, physical examination findings, and LSR; performed anti-drug antibody (ADA) analysis; and photographed the lesion. After the injection was administered, the provider recorded vital signs, adverse

events, concomitant medications assessment, and LSR, and photographed the lesion. At the end of treatment visit, the same assessments were performed, and data recorded. Additionally, the provider surgically excised the tumor area to assess for histological clearance. A dermatopathologist blinded to clinical study information examined at least 10 microscopic sections stained with hematoxylin and eosin of each excised specimen to ensure that there was no residual isSCC. There was a 4-week follow-up period of 2 visits 2 weeks apart. During follow-up, the provider recorded vital signs, adverse events, and concomitant medications; performed a physical examination and ADA analysis (first visit only); and photographed the wound. Statistical Analysis The statisticians (Amarex Clinical Research, Germantown, MD) performed all statistical analysis using SAS for Windows, version 9.3 or later (SAS, Cary, NC). Efficacy analysis was performed on the intention-to-treat population, comprised of all enrolled subjects. Safety analyses were performed on the safety population, comprised of all subjects who received at least 1 dose of the investigational product. Descriptive statistics were used for continuous variables; frequencies and percentages were used for categorical variables. Adverse events were coded using the Medical Dictionary for Regulatory Activities. Missing data were not imputed.

Twenty-seven subjects provided initial informed consent to participate in the screening process; 2 (7%) withdrew their consent. Twenty-five subjects were enrolled and assigned to each group of 5. Their mean age was 67 years (SD: 10; median: 68; range: 44-83). There were 13 men (52%). All subjects were White. Table 2 summarizes patient demographics and baseline characteristics by cohort. Concomitant medications taken by the subjects were comparable across groups. Fifteen subjects (60%) took medication affecting the cardiovascular system, 10 (40%) took anti-infectives for systemic use, and 8 (32%) took medications affecting the nervous system. All subjects received the treatment during weeks 1-4. The investigator determined that no injection should be administered to 2 subjects (8%) during week 5 and 3 subjects (12%) in the 120 μg/treatment cohort during week 6. All subjects completed the study and underwent surgical excision of the isSCC lesion site at the end of treatment visit. Efficacy Data The majority of subjects (19/25, 76%) achieved histological clearance of lesion by week 6: 60% (3/5) of subjects in both the 10 μg/treatment and 20 μg/treatment groups, 80% (4/5) in both the 30 μg/treatment and 120 μg/treatment groups, and 100% (5/5) in the 60 μg/treatment group. Figures 1A and 1B provide a before and after example of a lesion site on a 45-year old, White female patient in the 60 μg/treatment group that was cleared of isSCC after 6 weeks of treatment with TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic. Safety Data There were 7 TEAEs reported for 5 subjects: 2 in the 20 μg/ treatment group (40%), 1 in the 60 μg/treatment group (20%), and 2 in the 120 μg/treatment group (40%; Table 3). The incidence of TEAEs did not vary in a dose-dependent pattern. One subject had a moderate TEAE; 4 had mild TEAEs. There were no severe TAEs, SAEs, or events that led to treatment discontinuation or study interruption. None of the TEAEs appeared to be related to the investigational product. Table 4 summarizes skin response data. At week 1, the mean predose scores were 3.6, 3.2, 3.6, 4.2, and 2.8 for the 10 μg/ treatment, 20 μg/treatment, 30 μg/treatment, 60 μg/treatment, and 120 μg/treatment dose groups, respectively. By end of treatment, a decrease in the mean scores was observed in the 10 μg/treatment, 20 μg/treatment, 30 μg/treatment, and 60 μg/treatment groups. There were 9 incidences of a Grade 4 erythema: 8 in 1 subject in the 60 μg/treatment group at the preand postdose time points during the week 3-6 visits and 1 in the 30 μg/treatment group at the postdose time point during the week 1 visit (after a predose grade of 3). By end of treatment,

the subject had Grade 2 erythema. There were 3 incidences of Grade 4 flaking/scaling in the 120 μg/treatment dose group that occurred at pre-and postdose time points during the week 6 visit and predose during the end of treatment visit. No crusting above grade 3 was reported. No swelling above grade 2 was reported. No vesiculation/pustulation or erosion/ulceration was reported.

Efficacy data demonstrated that the TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic is effective in treating isSCC lesions. The majority of subjects in each dose cohort and the majority of subjects overall in this study (76%, 19/25) achieved histological clearance of lesion by 6 weeks. The TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic has an acceptable safety profile. The safety parameters did not vary in a dose-dependent pattern. No TEAEs appeared related to the investigational product; the majority (6/7) were mild. There were no events leading to treatment interruption. The injection was generally well-tolerated, with a majority of subjects experiencing no or only low-grade LSRs following injection and

a decrease in the mean scores observed in the 10 μg/treatment, 20 μg/treatment, 30 μg/treatment, and 60 μg/treatment groups (Table 4). The 30 μg/treatment and 60 μg/treatment doses were determined to be the recommended doses for future research involving intralesional TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic, based on the higher rates of histological clearance (80% an 100%, respectively) and lower LSR scores. The HKP delivery system of TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic is an additional therapeutic benefit. Gene therapeutics are often hindered by poor delivery systems, which inefficiently deliver nucleic acids to the tumor.²⁰ The HKP polymer represents a unique biodegradable siRNA carrier that targets oncogenes with tumor-suppressing proteins.^20,21 The histidine and lysine amino acids in the HKP can alter their linear and branched structure and amino acid sequence to better carry the siRNA into tumor cells, so that the nucleic acid therapeutic is more effective in tumor suppression. The major limitations of this study are the small sample size, lack of power calculation for the primary endpoint, and lack of randomization and blinding of providers and patients. Efficacy of theTGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic appeared to be dose related, although there were not enough subjects to have any statistical significance to demonstrate treatment effect. However, the dose response findings of this exploratory, prospective study have been used for the design of randomized controlled trials (RCTs) analyzing the safety and efficacy of the TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic injection on patients with cancers. Intralesional TGF-β1/COX-2 siRNA/HKP nanoparticle therapeutic is simple, noninvasive, safe, and efficacious in treating is SCC. The combination siRNA therapeutic appears to promote tumor suppression by silencing the expression of oncogenes, decreasing the risk of tumor progression and recurrence. Investigation of this novel therapeutic in a RCT is ongoing.

MN performed research funding and is a Consultant to Sirnaomics; BB performed research funding and is a Consultant to Sirnaomics; PL is CEO of Sirnaomics; MM is an employee of Sirnaomics. Funding/Support and Role of Funder/Sponsor: Sirnaomics, Inc. (Gaithersburg, MD) sponsored and funded this study; was responsible for STP705 manufacturing and quality control; and had a role in the study design and conduct, preparation, review, and approval of the manuscript, and the decision to submit the manuscript.

The authors would like to thank Kristen Eckert (Strategic Solutions, Inc, Bozeman, MT) for her assistance in writing and editing the manuscript. Amarex Clinical Research (Germantown, MD) provided clinical study monitoring and oversight. Dr. Vivek Shah (Maven Pharma Solutions, Inc, Bartlett, IL) coordinated manuscript development. Dr. Marc Lemaitre (ML Consult LLC, Cincinnati, OH) for his contribution to the CMC (chemistry, manufacturing, and controls) work. Dr Zhifeng Long, PhD (Sirnaomics, Inc, Gaithersburg, MD) managed stability studies of the investigational product. David M. Evans, PhD (Sirnaomics, Inc) contributed to the development of the investigational product.

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Michael Molyneaux MD michaelmolyneaux@sirnaomics.com