Treatment of skin tumors with intratumoral interleukin 12 gene electrotransfer in the head and neck region: a first-in-human clinical trial protocol

Immune therapies are currently under intensive investigation. Immune checkpoint inhibitors became standard of care for variety of tumor types, providing in many cases excellent responses in patients with advanced or progressive disease, unsuitable for treatment with local therapies. However, there are still patients that are non-responders and the reasons for that are still not well understood.¹

The other category of immune stimulators are cytokines. These have been extensively investigated, predominantly in combined treatment schemes. The IL-12 is a soluble cytokine with anti-tumor effectiveness. It stimulates adoptive and natural immunity in the organism, predominantly through production of interferon gamma (IFN-γ).² Besides immunostimulatory effectiveness it also has antiangiogenic mode of action.² Treatment with recombinant IL-12 has confirmed its anti-tumor effectiveness, but due to the high drug concentrations required to induce effect also severe toxicity were observed, which eventually lead to abrogation of IL-12 use.³

Gene therapy provides a new technical approach for more localized delivery of the therapeutic proteins to tissues, therefore reducing their eventual systemic toxicity. In most of the clinical trials, genes are delivered by viral vectors; however, non-viral gene delivery systems like gene electrotransfer (GET) also provide a safe approach for naked plasmid DNA delivery to tumors.^4,5 GET is based on electroporation that uses electric pulses to destabilize the cell membrane for delivery of large and non-permeant molecules into the cells.⁶ The transport of plasmids through the cell membrane and into the nucleus for transcription is not well understood.⁷ However, there are several clinical studies providing evidence of feasibility, safety, and effectiveness of this non-viral gene delivery.^4,5,8,9

Studies on murine tumor models have shown that GET with plasmid DNA encoding interleukin 12 (IL-12 GET) is especially successful in the treatment of skin tumors and their metastases.¹⁰ Furthermore, the safety and efficacy data of such treatment on skin melanoma metastases were already published.⁴ In a human clinical study with IL-12 GET of melanoma metastases, local and also systemic effectiveness on the distant non-treated tumors were demonstrated. The study included 24 patients with skin metastatic melanoma.⁴ The plasmid encoding human IL-12 under the control of the CMV promoter and with resistance to kanamycin, as the selection gene, was used. Gene therapy was performed three times on each tumor, resulting in promoted local clinical response of treated tumors and in systemic anti-tumor effect on distant non-treated nodules in 53% of patients. This treatment approach is currently being investigated in the treatment of melanoma, Merkel cell carcinoma, breast cancer^8,9,11,12, and also in combination with immune checkpoint inhibitors.^13,14

However, despite these encouraging results, IL-12 plasmid used in the described studies contain antibiotic resistance gene serving as a selection marker for the production of plasmid DNA. As the presence of antibiotic-resistance genes raises safety concerns, European Union regulatory requirements endorse the use of plasmids without the selective genes. Therefore, we developed the phIL12 plasmid and its clinical grade production process in our previous studies.^15,16 In the proposed first-in-human study (EudraCT: 2021-000852-21, ISRCTN15479959, ClinicalTrials NCT05077033) we intend to study the safety and tolerability of phIL12 GET in treatment of basal cell carcinomas in patients with operable tumors in the head and neck region. The study is designed as an exploratory, dose escalating study with the aim to determine the safety and tolerability of the treatment and to identify the dose of plasmid phIL12 that is safe and elicits its biological activity.

Basal cell carcinoma accounts for 80% of all non-melanoma skin cancers and is the most common malignant tumor that occurs on the skin. It grows slowly and rarely metastasizes. However, basal cell carcinoma comprises heterogeneous histologic variants, from highly biologically benevolent, well-limited and superficially growing tumors to aggressive, infiltratively growing or deeply invasive lesions. Due to complex anatomical conditions in some parts of the head (nose, orbital area, ears) combined with a possibly more aggressive form of growth, an inadequate first treatment may present a serious therapeutic problem. The basic therapeutic options in basal cell carcinoma are surgery, radiotherapy and electrochemotherapy, that result in comparable local control when early lesions are treated.¹⁷ Since the baseline tumor assessment could be inadequate and the disease underestimated, relapses at the treatment site are not uncommon. Repeated treatment, whether surgical or radiotherapeutic, is associated with reduced efficacy and/or functional or cosmetic impairment in the treated area. Therefore, from clinical perspective, searching for new therapeutic alternatives is of high importance. Recently, immunotherapy became important for the treatment of locally advanced or metastatic basal cell carcinoma, with first approved immune checkpoint inhibitor cemiplimab, which has set the stage for investigation of other immunotherapies.¹⁸

Basal cell carcinoma has the highest mutational burden compared to other skin cancers resulting in the activation of the local immune response that prevents rapid tumor growth and metastasis.¹⁹ Indeed, clinical studies confirmed that electrochemotherapy is more effective in basal cell carcinoma than in other histological tumor types.^20,21 This can be explained by a higher mutational load and an increased presence of tumor antigens or neoantigens that are triggered after electrochemotherapy and are required for effective stimulation of immune system and elimination of tumor cells.²²

In accordance with these findings we have designed the proposed clinical study. It is based on GET of plasmid DNA encoding IL 12 into the tumor, aiming to subsequently stimulate local immune response that would result in tumor eradication. If this therapeutic approach with IL-12 will prove to be safe and effective, it will be the first “proof of principle” of its kind in clinic and a valuable addition to the existing therapeutic armamentarium. However, in the case that our study will confirm the safety of the proposed therapy, but will not yield expected therapeutic response, the tumors will be treated with standard treatment, i.e. surgery. The prolonged interval to surgery should not affect prognosis of treatment outcome, due to slow course of basal cell carcinoma growth.²³

This is a clinical, interventional, open label, single arm, Phase I trial for intratumoral phIL12 GET. It is intended to treat basal cell carcinomas in patients with operable tumors in the head and neck region. The aim of the study is to evaluate the safety and tolerability of phIL12 GET. The study is designed as an exploratory, dose escalating study with the aim to determine the dose that produces IL-12 expression in the tumors with best biological activity, infiltration of the immune cells and no toxicity. Study hypothesis: Intratumoral phIL12 GET is safe and tolerable in treatment of skin tumors (Figure 1).

Figure 1

The aim will be achieved with i) controlled application of the treatment, ii) evaluation of trial and obtained clinical results, iii) presentation of the results by providing written and audio-visual material, participation at and organization of expert meetings and scientific meetings.

In the study 3–6 patients per IL-12 dose level will be included; 3 doses, for a total estimated number of 9 patients (depending on the course of the study, from 3 and up to 18 patients will be included). The enrollment of the patients will be staggered: the waiting period will be 30 days after the treatment of previous patient, based on expected duration of acute and subacute toxicity. Consecutive cohorts of 3 to 6 patients will be treated with increasing doses of phIL12 at three dose levels (0.5 mg/ml, 1 mg/ml and 2 mg/ml) according to an adapted 3 + 3 design as described below. There will be no intra-patient dose escalation. The clinical study will be conducted in accordance with this protocol and GCP guidelines and in accordance with the regulations.

The study is conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Institute of Oncology Ljubljana (protocol code ERIDEK-0086/2020, date of approval 25 November 2020). The study was also approved by the National Ethics Committee of the Republic of Slovenia (0120-524/2020-12) and the Agency for Medicinal Products and Medical Devices of the Republic of Slovenia. The study was registered in the ISRCTN (ISRCTN15479959) and Clinical Trials (NCT05077033) database.

Primary and secondary objective of the study are presented in Table 1 and Table 2, respectively.

The current trial is designed to evaluate the safety and tolerability of the intratumoral phIL12 plasmid DNA gene electrotransfer. The drug dosage that will be established as safe and capable to elicits local immune response will be used for design of next clinical trials.

The gene electrotransfer utilizing IL-12 plasmid DNA was tested extensively in preclinical models and was also used in clinical trials before. To our knowledge, one Phase 1 clinical study (NCT00323206)⁴ and four Phase 2 clinical studies have been conducted in USA in patients with malignant melanoma, cutaneous lymphoma, squamous cell carcinoma of the head and neck, and Merkel cell cancer (NCT01502293, NCT01579318, NCT02345330, NCT01440816).^9,28 Further, four new studies (NCT03132675, NCT04526730, NCT03567720, NCT03823131) are active, also in mucosal head and neck squamous cell carcinoma patients, to evaluate a combination of GET of plasmid DNA encoding IL-12 in combination with pembrolizumab or nivolumab.

The above-mentioned Phase 2 clinical studies were performed with tavokinogene telseplasmid (TAVO^TM), a plasmid encoding IL-12 produced by OncoSec Medical Incorporated (USA). It is a plasmid that encodes genes for the p35 and p40 subunits of the heterodimeric human IL-12 protein separated by an internal ribosome entry site. Constitutive expression of the subunits is driven by a single cytomegalovirus promoter. Once the plasmid is introduced into a mammalian cell, a functional IL-12 p70 protein is expressed and secreted into the tumor microenvironment. Intratumoral GET of accessible lesions is performed using an in situ EP device.⁸ The TAVO^TM plasmid contains a kanamycin resistance gene as the selection gene for the production process, a feature that is discouraged by EMA.

Plasmid phIL12, like TAVO^TM, is a plasmid DNA, encoding for p35 and p40 subunits of the heterodimeric human IL-12 protein. However, the plasmid backbone of phIL12 is different, since we aimed for the plasmid devoid of antibiotic resistance marker and for the treatment-inducible, tumor-specific promoter, whereas the transgene product is the same in both plasmids, a functional IL-12 p70 protein. In phIL12 plasmid, operator-repressor titration ORT^® technology, which is based on providing the titration of repressor of essential gene for propagation of bacteria in plasmid, was used. Multiple operators present on the plasmid titrate the repressor leading to expression of essential gene. In our case, the essential gene in bacteria was dapD, which was under transcriptional control of lac operator/promotor (lacO/P). Genome encoded Lacl repressor prevents bacterial growth, unless transformed with plasmid containing the lac operator (lacO) that titrates the Lacl repressor from the operator.²⁹ In addition, since the use of phIL12 GET is foreseen as an adjuvant treatment to local ablative therapies, to increase their local and elicit a systemic response, the IL12 coding sequence was placed under the transcriptional control of an inducible p21 (or cyclin dependent kinase inhibitor 1A (CDKN1A)) promoter that can be activated by the hypoxic tumor microenvironment and/or by the genotoxic stress induced by local ablative therapies, such as tumor irradiation of electrochemotherapy.¹⁶

Based on preclinical data for TAVO^TM that are adequately comparable to phIL12 and since the expression product and a mode of action are the same (i.e. protein IL-12), we decided to use the same doses as in the above clinical studies as a basis for selecting a starting dose in non-clinical study and in this first-in-human phase I study.

The treatment protocol with TAVO^TM was designed as a repetitive protocol at days 1, 5 and 8, with possible repetitive cycles at 6- or 12-weeks intervals. However, our intent is to use phIL12 as adjuvant immunotherapeutic to the established local ablative therapies. As already reported, ablation of tumors induces local immune response with immunogenic cell death.^30,31 Therefore, studies combining local ablative therapies like radiotherapy or electrochemotherapy can be boosted from local to locoregional or even systemic response by immune checkpoint inhibitors. The recent study combining electrochemotherapy with pembrolizumab, demonstrated that this approach is feasible. The patients treated with pembrolizumab and ECT experienced lower disease progression rates and longer survival than those who received pembrolizumab alone.³² Similar approach could be by immunostimulation using phIL12 intratumoral GET. Further, different studies combining tumor irradiation and immune checkpoint inhibitors demonstrated the same.^{33, 34, 35, 36} Therefore, in the future clinical trials phIL12 is envisioned as an immunostimulating component to in situ vaccinating effect of local ablative therapies such as electrochemotherapy or radiotherapy. We believe that in the frame of such treatment combination, a single treatment with IL-12 GET would be sufficient to elicit pronounced antitumor effect.

The designed first-in-human clinical trial is aimed at evaluation of safety and tolerability of phIL12 GET, thus setting the stage for the use of phIL12 GET as an adjuvant treatment to local ablative therapies, to increase their local and elicit a systemic response.