Safety and efficacy of p 62 DNA vaccine ELENAGEN in a first-inhuman trial in patients with advanced solid tumors

Elenagen is a plasmid encoding p62/SQSTM1, the first DNA vaccine possessing two mutually complementing mechanisms of action: it elicits immune response against p62 and mitigates systemic chronic inflammation. Previously, Elenagen demonstrated anti-tumor efficacy and safety in rodent tumor models and spontaneous tumors in dogs. This multicenter I/IIa trial evaluated safety and clinical activity of Elenagen in patients with advanced solid tumors. Fifteen patients were treated with escalating doses of Elenagen (15 mg per doses, 5 times weekly) and additional 12 patients received 1 mg dose. Ten patients with breast and ovary cancers that progressed after Elenagen were then treated with conventional chemotherapy. Adverse events (AE) were of Grade 1; no severe AE were observed. Cumulatively twelve patients (44%) with breast, ovary, lung, renal cancer and melanoma achieved stable disease for at least 8 wks, with 4 of them (15%) had tumor control for more than 24 wks, with a Clinical Research Paper


INTRODUCTION
Two mechanisms contributing to cancer have been extensively studied, inefficient anti-cancer immune response and chronic inflammation. Thus, two approaches to cancer therapy, stimulating anti-cancer immunity and eliminating chronic inflammation, are under active development. A novel biological agent, plasmid (DNA vaccine) encoding p62 (SQSTM1), acts through both of these mechanisms at the same time. Previously, it was demonstrated that anti-cancer agents may possess more than one activity, e.g. chemo-therapeutic agents inducing cancer cell death and modulating immune system at the same time.
Comparing to traditional (protein/peptide) vaccines, DNA-based vaccines have several advantages: they can induce both humoral and cellular immunity, they are safe, inexpensive and stable, and can be easily modified to enhance immune response [1][2][3][4][5]. There is already approved anti-melanoma vaccine for dogs (Oncept), and a number of ongoing phase I-II trials of DNA vaccines for various solid tumors in humans [2,6]. However, being efficient in small animals, DNA vaccines often are not strong enough to control tumors in humans. Besides immunosuppressive tumor microenvironment, another apparent reason for vaccine inefficiency is that they exert selective pressure on cancer cells, which leads to the loss of the vaccineencoded antigens (immune escape) and results in the tumor relapse [7,8]. To avoid this problem, an effective antigen for a vaccine should be, besides evoking a robust immune response: 1) essential for cancer cells to avoid its loss through immunoediting; 2) dispensable for normal tissues to reduce the risk of toxicity, and 3) highly expressed in tumors as compared to the normal tissues [9].
We have chosen p62 (SQSTM1) protein as the antigen for a novel DNA vaccine (Elenagen). p62 is multifunctional protein which participates in selective autophagy and signal transduction, in particular, inflammatory response [10] [11]. p62 satisfies all the above requirements: 1) its levels are elevated in almost all human tumors tested so far; 2) tumors require p62 for growth and metastases, 3) p62 is dispensable for normal cells [3]. We have employed a plasmid DNA vaccine as a platform for p62 expression.
In our preclinical studies of Elenagen we found that: 1) it's intramuscular injection caused a strong antitumor effect in various animal models of breast cancer, melanoma, sarcoma and lung carcinoma [12]; 2) along with inhibition of growth of primary tumors, it also suppressed metastases [12], which are responsible for 90% of cancer-related deaths;3) importantly, beyond immune attack on tumors, Elenagen, unlike other vaccines, can also suppress chronic inflammation [13], a common feature of cancer that accelerates progression of the disease and worsens its outcome [14]; 4) apart from most of anti-cancer vaccines tested so far, it demonstrated efficiency not only in transplantable tumors in rodents, but also in spontaneous tumors of dogs that are very similar to humans [15].
Although DNA vaccines are generally considered safe enough [16], we performed broad-range preclinical safety studies of Elenagen in laboratory animals. The Elenagen administration was well-tolerated, without acute or chronic toxicity, allergic activity, and it did not cause embryonic toxicity and teratogenicity (Supplementary  Table S1). There was no hematotoxic, hepatotoxic and nephrotoxic effects, as well as effects on carbohydrate and lipid metabolism. Furthermore, histological examination of brain, heart, lung, liver, kidney, spleen, thyroid and thymus did not find any anomalies. Overall, administration of Elenagen was not accompanied by any significant side effects (Supplementary Table S1).
Based on these efficiency and safety data in animals, we started the first clinical trial of Elenagen in patient with solid tumors.

Patients
The study included 27 patients: 15 patients in dose-escalation cohorts, and 12 in extension cohort; 2 patients have failed screening. Patient's characteristics are presented in Table 1. All patients had progressive metastatic solid tumors of one of 6 localizations: breast, colon, kidney, lung, skin (melanoma), and ovary. The most common tumors studied were breast (33.3%) and ovary (22.2%) cancers, and melanoma (22.2%). Patients enrolled had several (up to 8) chemotherapy schemes but were at the tumor progression state at the time of enrollment (Table 1).

Safety
All patients included in the study completed at least one course (5 injections) of Elenagen vaccinations, and no discontinuation of the treatment occurred. No serious AEs caused by vaccination in dose range of 1-5 maximum of 32 wks. The patients with breast and ovary cancers achieved additional tumor stabilization for 12-28 wks when treated with chemotherapy following Elenagen treatment. Therefore, Elenagen demonstrated good safety profile and antitumor activity in advanced solid tumors. Especially encouraging is its ability to restore tumor sensitivity to chemotherapy. mg were observed in any of 27 patient of the study. There was no any acute toxicity or manifestations of cardiotoxicity. Autoimmunity (dsDNA antibody) was also within a normal range (not shown). The most common treatment-related grade 1 AE were injection site erythema or swelling, nausea, fatigue and fever; these events were independent on dose, transient, and resolved without treatment (Table 2). Thus, based on these data we concluded that Elenagen is safe and without dose-limited toxicity in the range of 1-5 mg per injection per week.

Dose-escalation study
In the first part of the study, patients with different solid tumors were subjected to escalated doses of Elenagen and their tumor response was monitored by RESIST 1.1 criteria every 8 wks until tumor progression. The best overall response observed was SD (stable disease): it happened in 3 of 5 patients in 1 st cohort (1 mg), 1/5 patients in 2 nd cohort (2.5 mg), and 3/5 patients in 3 rd cohort (5 mg) ( Table 3). The maximal duration of SD observed in the 1 st cohort was 32 wk, in the 2 nd cohort -24 wks, and in 3 rd cohort -8 wks. Although small size of the cohorts does not allow to make reliable conclusion about dose-response effect, there is no apparent dependence at given Elenagen doses which is common for DNA vaccines [17,18].
Patient's tumor localization in the first 3 cohorts was selected randomly according to protocol inclusion/ exclusion criteria (Supplementary Table S2) and patient's availability. SD was observed in different tumors: in 4 of 8 patients with breast cancer (PFS from 8 to 32 wks), in 2 of 2 patients with ovary cancer (PFS 8 and 24 wk), in 1 patients of 2 with lung cancer (8 wk); and in none of 3 with colon cancer (Table 3), demonstrating potentially broad anti-tumor activity of Elenagen. Furthermore, among major subtypes of breast cancer, SD was observed in 2 of 4 patients with triple-negative, in 1 of 2 with Her2 positive, and in 1 of 2 ER + PR + Her2tumors (Table 3), indicating that all major breast subtypes are sensitive to Elenagen.

Extended cohort study
Based on these pilot data on Elenagen efficiency, we extended our clinical study with 12 patients using  (Table 3). The efficiency of Elenagen in all 27 patients (combined 1 st and 2 nd parts of the study) is presented in Table 4. Besides breast cancer, SD was also observed in 4 from 6 patients with ovary cancer, in 2 from 6 patients with melanoma, and 1 patient with neuroendocrine renal cancer; in total, 12 patients from 27 (44%) demonstrated SD for at least 8 wks. Remarkably, some patients with breast, ovary and renal cancer achieved tumor control for more prolonged period, from 24 to 32 wks (4 of 27 patients) thus reaching clinical benefit rate of 15%. Based on these data, we concluded that Elenagen as a single agent can evoke tumor control in a range of solid tumors: breast, ovary, renal, lung and melanoma, but without apparent activity in colon cancer.

Effect of chemotherapy on tumor response following Elenagen treatment
Seven breast cancer patients and three ovarian cancer patients who progressed after one or several rounds of Elenagen treatment were administered conventional chemotherapy on compassionate basis. The rational for this was that immunotherapy can synergize with chemoand radiotherapy via several mechanisms (see refs [19][20][21] for review and Discussion below). We found that patients with breast and ovarian cancers who failed several rounds of previous chemotherapy and were considered treatment-   (Table 5). Accordingly, a patient with hormone-sensitive breast cancer (05-014) who stopped to respond to tamoxifen, restored sensitivity to it (Table 5).
Ovarian cancer patients who failed platinum-and taxanbased treatments restored sensitivity to carboplatin (05-008, 016, Table 6). Some patients with breast and ovarian cancer also responded to new therapy regimens: eribulin in breast cancer and irinotecan for ovarian cancer ( Table  5, 6). Therefore, even if Elenagen did not stop tumor progression as a monotherapy or when tumors progressed after multiple rounds of Elenagen treatment, the disease still could be controlled by chemotherapy even if it stopped to work before. This may indicate that Elenagen could become the first agent restoring tumor sensitivity to chemotherapy. So far, this can be applied to breast and ovary cancers and several anti-neoplastic drugs such as cyclophosphamide, adriamycin, platinum and taxan derivatives, but it may be a wider phenomenon.

DISCUSSION
Among new methods to treat cancer, immunotherapy looks especially promising. Here we conducted a firstin-human trial of Elenagen, a DNA vaccine targeting p62. Although p62 is not oncogene per se under normal conditions, its overexpression is a common feature of   [23]. It was also shown that upon oncogenic transformation p62 becomes necessary for tumor initiation and progression: Her2--induced formation of breast cancer and RAS-induced formation of lung cancer are hampered in p62 knockout animals [24,25]. Furthermore, fully transformed cells and tumors do not lose their dependence on p62, i.e. they become "addicted" to p62, a phenomenon known as nononcogene addiction [26]. Accordingly, depletion of p62 suppressed growth of cancer cells in vitro and tumors in vivo in [27,28]. Importantly, unlike tumor cells, p62 is dispensable for normal cells since p62 knockout animals are normally developed and demonstrate only minor anomalies (lateronset obesity) [29]. This indicates that, at least under normal conditions, other proteins can substitute some functions of p62 (e.g., p62 homolog NBR1 in autophagy) [30]. All these properties make p62 an excellent target for anticancer drug development. However, to our knowledge, there is only one study on chemically targeting p62 using XRK3F2 compound for treatment of myeloma [31]. The problem with targeting p62 by a drug is that it is multidomain protein having at least 8 putative domains, and their roles in cancer are far from clear. In case of myeloma it is ZZ domain targeted by XRK3F2 appear to be important for their growth [31].
We have created a p62-encoding DNA vaccine. We found that p62 vaccine can generate immune response to p62 antigen [12], and anti-tumor response requires intact immune system since it lack in immunodeficient SCID mice (in preparation). Accordingly, in our study in dogs with spontaneous breast cancer we observed accumulation of T-lymphocytes within tumors [15]. We found that p62 vaccine has a broad range of anticancer activity in preclinical models of breast, lung carcinoma, melanoma and sarcoma [12]. Importantly, we also demonstrated p62 activity in spontaneous breast tumor in dogs [15]. Besides suppressing growth of primary tumors, Elenagen was able to markedly decreased development of metastasis [12].
We serendipitously uncovered another property of p62 plasmid -its ability to suppress chronic inflammation. We found that in a model of ovariectomy-induced osteoporosis in mice p62 plasmid injection prevented generation of pro-inflammatory cytokines and suppressed osteoporosis [13]; similarly, p62 alleviated inflammation and obesity caused by high-fat diets in rats (in preparation). It is well-known that chronic inflammation can play immune-suppressive role [32,33] [34]. Furthermore, similar to p62 levels, elevated levels of inflammatory markers associated with worse survival, e.g., in breast [35] [36] and ovarian cancer [37] [38]. We hypothesize that, besides evoking immune response to a cancer antigen (like other vaccines), Elenagen can also alleviate chronic inflammation within a tumor thus enhancing the response. As a result of such dual action, p62 vaccine can be more potent than traditional vaccines.
The main goal of the present clinical trial was to assess Elenagen safety. Here we report that Elenagen demonstrated good safety profile ( Table 2). Although patients selected for the phase I safety study were so called "terminal patients", Elenagen still showed efficiency in breast cancer, having effect in all 3 subtypes of it (Table  4). Also, it was effective in ovary, lung, renal cancer and melanoma (Table 4). Applied as a monotherapy, Elenagen cased stabilization of disease for up to 32 wks in some patients with breast, ovary and renal cancer, although in most patients tumor stabilization was lasted for only 8 wks.
If we compare the results of this trial with other recent phase I/II trials of other immune-therapeutics (Supplementary Table S3), we can make several  conclusions. First, Elenagen, similar to antibodies and other vaccines, and anti-CTLA4, in contrast to conventional therapeutics, does not evoke objective response (i.e. complete + partial response), but produces tumor control better than most other vaccines, antibodies, or anti-CTLA4 (Supplementary Table S3). Second, anti-PD1/PDL1 antibodies are more effective than other immune-therapeutics, but they however, have more severe side effects (Supplementary Table S3). But regarding specifically breast and ovarian cancers, Elenagen efficacy in tumor control is compatible with anti-PD1/anti-PDL1, but have much better safety (Supplementary Table S3). During the trial we observed a phenomenon which, to our knowledge, has not been described in clinics before. When patients with breast and ovarian cancer were pretreated with Elenagen, they restored, at least partially, their sensitivity to conventional chemotherapy which they previously failed (Table 5, 6). There are several possible explanations for such effect. First, chemoradiotherapy (e.g., doxorubicin) can enhance anti-p62 immune response by killing tumor cells via immunogenic cell death [21,39]. Second, it is currently becoming evident that conventional chemo-radiotherapy, besides killing tumor cells directly, can also modulate immune response. For instance, cyclophosphamide, 5-fluorouracil, cisplatin can target immunosuppressive regulatory T and myeloid-derived cells, just enhancing immune response. Preconditioning vaccine and CAR-T therapies with these "chemotherapeutic" agents is already in clinical use [19][20][21] . Furthermore, some drugs such as gemcitabine and taxanes can directly stimulate immune system [39]. For instance, a vaccine directed to Her2-expressing mouse mammary tumors was potentiated by doxorubicin and paclitaxel [40]. Accordingly, treatment with doxorubicin after vaccination increased immune response to Her2 vaccine in clinics [41], and in patients with other tumors, chemotherapy applied after vaccination was more effective than vaccines or chemotherapy alone [42,43].
Although Elenagen alone demonstrated anti-tumor activity in a range of solid tumors, in future trials we consider its combination with chemo-radiotherapy. We also believe that Elenagen should be synergistic with immune-checkpoint inhibitors. Combining immune checkpoint blockers (ICB) with vaccines targeting specific cancer antigens is an emerging approach [44]. So far, cancer vaccines are mostly failed in clinical trials because immune response they generate was apparently too weak to control tumor growth in patients owing to immuno-suppressive tumor microenvironment (e.g., due to activation of immune checkpoints). If so, one may expect that removing immune checkpoint blockade by ICB would enhance vaccine efficiency. Indeed, in preclinical models of cancer ICB significantly enhanced vaccine efficiency [45] [46]. Partially because of these findings, cancer vaccine field demonstrates resurgence [47], and there are currently several clinical trial of vaccine-ICB combination, e.g. with Prostvac, GVAX, CEA/MUC etc [44]. We believe that p62-encoding DNA vaccine Elenagen is a promising candidate within this booming area of research.

Ethics
The clinical trial followed Declaration of Helsinki and Good Clinical Practice Guidelines. It was approved by Ethics Committee and registered by Russian Ministry of Health on 09.09.2014 (Clinical trial #506, Protocol E001).
It was also approved by local ethic committee of each participating institutions and written informed consent for participation in the study was obtained from all participants.

Patients and study design
This multi-center, non-randomized, open-label, phase I/IIa trial was conducted in cohorts of patients with advanced metastatic solid tumors whose diagnosis was confirmed histologically. Inclusion/exclusion criteria for the patients in the study are presented in Supplementary  Table S2. Only patients with radiologically measurable diseases after exhaustion of standard treatment options and in progression were enrolled into the study. The study consisted of two parts. The first part was doseescalation study: 3 cohorts of patients with 5 patients in each received doses of 1, 2.5 and 5 mg, respectively. The second part was the expansion study containing 12 patients each receiving 1 mg dose. Each patient has received 5 i.m. injections of Elenagen (at concentration of 2.5 mg/ml in saline) once a week and then followed for 3 weeks before assessment of disease. If a patient demonstrated lack of disease progression, Elenagen injections were continued until tumor/metastases relapse. Standard regimens of chemotherapy were administered to 10 patients with breast or ovarian cancers, who either did not respond to Elenagen or demonstrated disease progression after a stabilization period on Elenagen treatment (s).

Safety assessment
Screening and baseline assessments included evaluation of demographic data, medical and history, prior medications, a complete physical examination, vital signs, Karnofsky performance status, ECG, and clinical laboratory tests (including urinalysis and a serum pregnancy test if applicable), tumor history, primary diagnosis, and previous treatments. Safety assessments were performed after each injection. Adverse events (AE) severity was assessed according to the Common Terminology Criteria for Adverse Events (NCI-CTCAE), version 3.0. Safety assessments consisted of monitoring and recording all AEs; documenting concomitant medications; regular monitoring of hematology, blood chemistry, and urine values; periodic measurement of vital signs, Karnofsky performance status, ECGs; and performance of physical examinations. An ECG was recorded for each patient at screening, then 1 and 3 days after each dosing, and at the final visit. AE screenings and laboratory tests were performed weekly or as clinically indicated. Additionally, to assess inflammation and autoimmune response, CRP levels and antibody to dsDNA were measured, respectively, every week.

Tumor response
Tumor measurements were assessed by computed tomography (CT) and evaluated at the centers using Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Assessments were conducted at baseline and then 8 weeks after the start of the treatments; in case of tumor stabilization, monitoring was continued every 8 weeks (5 weekly injections followed by 3 weeks observation treatment) till tumor progression. Tumor response was defined as complete response (CR), partial response (PR), stable disease (SD), or disease progression (PD). SD was defined as to be maintained for 8 weeks and durable SD for more than 24 weeks. Clinical benefit was defined as CR, PR, and durable SD for more than 24 weeks.

ACKNOWLEDGMENTS
We wish to thank Profs M.Y.Sherman for helpful discussion, and Dr. N. Bezdenezhnykh, K. Romanova, N. Reztsova, and M. Ziser for technical and administrative support.

CONFLICTS OF INTEREST
Vita Sherbinina, Andrey Chursov, Vladimir L Gabai, and Alexander M Shneider are employees of CureLab Oncology Inc. All other authors have no conflicts of interests.

FINANCIAL SUPPORT
Pharma 2020 Technology Transfer Grant, Russian Federation.