SARS-CoV-2 (CoV2) has precipitated a global pandemic and the effectiveness of standard vaccine strategies to induce potent and persistent immunity to CoV2 is in question, particularly for the elderly. This problem is not dissimilar to what we have struggled with in our quest to induce immunity to cancer antigens, where vaccine-induced anti-cancer immune responses can be weak. Here, we describe a novel vaccine approach which leverages electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax). As IL-12 has been shown to augment the efficacy of immunotherapy in aged mice,1 we have initiated studies to evaluate if plasmid IL-12 (TAVO™) can similarly augment anti-CoV2 immune responses in young mice and have planned studies in aged animals.A prefusion stabilized CoV2 spike plasmid expression vector was constructed, a master cell bank generated and clinical-grade plasmid manufactured. C57BL/6 and BALB/c were vaccinated via intramuscular (IM) and/or intradermal (ID) injection followed immediately by EP of plasmids encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12 on days 1 and 14 or 21. Mice were followed for >120 days to assess safety. Splenocytes and serum were harvested at different time points to interrogate virus-specific cellular responses as well anti-spike IgG1/IgG2 antibody titers. A surrogate viral neutralization test (sVNT) assessed serum blockade of soluble hACE2R binding to immobilized CoV2 spike.Preliminary data shows that EP of CORVax alone or combined with IL-12 was safe. EP of CORVax was able to elicit anti-Spike IgG antibodies (IC50 = 1/2112), as well as IgG antibodies targeting the receptor binding domain of the Spike protein (IC50 = 1/965) approximately 40 days after the booster vaccination. In 2 of 2 experiments, CORVax combined with IL-12 significantly (P<0.0001) increased the sVNT titers at 2 months, but this benefit was lost by 3 months.Early preclinical data shows that EP of CORVax can induce IgG responses to CoV2 Spike and the receptor binding domain (RBD) as well as apparent viral neutralizing activity. The addition of IL-12, at least transiently, increased sVNT titer. We plan to investigate alternate vaccine boosting strategies while extending these studies into aged animals and initiate a clinical trial in the near future.Ruby CE, Weinberg AD. OX40-Enhanced tumor rejection and effector T cell differentiation decreases with age. J Immunol2009;182:1481–9. https://doi.org/10.4049/jimmunol.182.3.1481.

Background Electroporated plasmid IL-12 (TAVO or tavokinogene telseplasmid) is a novel pro-inflammatory intratumoral therapy with substantial single agent activity in melanoma, which has been shown to synergize with anti-PD-1 antibodies in patients predicted as non-responders to anti-PD-1.1 2 Interim data from patients with stage III/IV melanoma actively progressing on anti-PD-1 antibody are presented herein. Methods Patients with confirmed disease progression by RECIST v1.1 after at least 12 weeks of treatment on pembrolizumab or nivolumab (or combination checkpoint blockade) and within 12 weeks of last dose (with no intervening therapies) were enrolled. There was no limit on the number of prior lines of therapy. At least one accessible lesion was electroporated with plasmid IL-12 (pIL-12-EP) on days 1, 5 and 8 every 6 weeks and pembrolizumab was administered every 3 weeks. Tumor response in treated and untreated lesions was assessed by RECIST v1.1 every 12 weeks. Endpoints include ORR, safety, PFS, OS, and DOR. Results The first 56 patients treated of 100 planned were included in this interim analysis. Of these, 84% had Stage IV disease, 30% had M1c or M1d disease, and 27% had prior exposure to ipilimumab. In 54 efficacy evaluable patients the investigator-assessed ORR was 30% (3 CR/13 PR), 5 patients had 100% reduction of target lesions. All responses have been confirmed, only two responding patient progressed while on study, 2 patients completed the study with ongoing responses (figures 1 and 2). In patients with M1c/M1d disease, the ORR was 35.2% (n=6/17). Tumor reduction was observed in untreated lesions in 12 of 12 patients who had unaccessible lesions or accessible untreated lesions. The median overall survival (mOS) and duration of response (mDOR) has not been reached, with a median follow-up time of 13 months. Grade 3 treatment-related adverse events (TRAEs) were seen in 5.4% of patients, and there were no grade 4/5 TRAEs. The rate of grade 3 treatment-emergent (TEAEs) regardless of cause was 23.2%. The median time for pIL-12-EP treatment was 10 minutes (range 2,46). Consistent with prior studies of single-agent pIL-12-EP, tumor IHC, and transcriptomic assessments revealed hallmarks of antigen-specific antitumor immunity in this study. Additional analyses including microbiome, TCR clonality, and peripheral blood biomarker assays will be presented. Conclusions In this rigorously defined PD-1 antibody refractory patient population, the addition of pIL-12-EP to PD-1 antibody therapy induced deep, durable, systemic response in local treated and distant visceral metastatic untreated lesions with nominal systemic toxicity. Trial Registration Trial Registration: NCT#03132675 Ethics Approval The study was approved by a central IRB and/or local institutional IRBs/Ethics Committees as required for each participating institution. Consent Written informed consent was obtained from the patients participating within the trial, the current abstract does not contain sensitive or identifiable information requiring an additional consent from patients. References Algazi A, Bhatia S, Agarwala S, et al. Intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients. Annals of Oncology 2019;31:532–540. Algazi A, Twitty C, Tsai K, et al. Phase II trial for IL-12 plasmid transfection and PD-1 blockade in immunologically quiescent melanoma. Clinical Cancer Research 2020;26:2827-2837.

Preclinical and clinical studies have demonstrated that plasmid IL-12 (tavokinogene telseplasmid) delivered intratumorally via electroporation (TAVO) induces local expression of IL-12p70, converting immunologically excluded tumors into inflamed immunogenic lesions, which is fundamental to generating objective responses in both treated and untreated distant tumors. Recent optimization of electroporation parameters and plasmid design has yielded a significant increase in intratumoral expression and anti-tumor efficacy in preclinical models. To further develop this platform, longitudinal biomarker data from TAVO clinical trials was interrogated to identify key immunological components associated with an effective therapy. This analysis revealed that the composition of TAVO-inflamed lesions, specifically the frequency of CD8+ TIL and the chemokines necessary for their maintenance relative to suppressive intratumoral immune subsets, coincided with clinical responses. We therefore developed a plasmid that encodes both IL-12 and the effector T cell chemokine, CXCL9, which, when transfected into a tumor, are functionally active both in vitro as well as in vivo, yielding abscopal responses and increased survival. To further amplify this technology, a strategy was developed to engage the highly relevant and recently described bystander non-tumor reactive T cells in the tumor microenvironment (TME). To accomplish this pan-T cell amplification, a separate therapeutic plasmid encoding membrane-bound anti-CD3 scFv (145-2C11) was used to decorate transfected tumors with the potent polyclonal T cell stimulator. Membrane expression of anti-CD3 in vivo was confirmed by immunoblot and flow cytometry. Combination of IL-12/CXCL9 with anti-CD3 delivered by electroporation (collectively, “SPARK”) increased both antigen-specific and polyclonal T cells responses, demonstrated by in vivo CTL and proliferation assays. Finally, SPARK upregulated the transcription of key immunological genes, leading to regression of both treated and untreated tumors, which heightens its ability to reshape the TME and drive systemic anti-tumor immunity. In conclusion, this data supports a model whereby IL-12 in concert with CXCL9 inflames the lesion, leading to a brisk T cell infiltrate, which in sequence with anti-CD3 stimulation, drives a broad yet robust systemic T cell response. SPARK represents a significant advancement in cytokine-based immunotherapy. Note: This abstract was not presented at the meeting. Citation Format: Mia Han, Anandaroop Mukhopadhyay, Bianca Nguyen, Kurt Sakurada, Jack Lee, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of plasmid IL-12 and CXCL9 with membrane-bound anti-CD3 elicits robust anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3195.