Supplementary Figure Legends

PDF file - 44K, Irradiated whole cell sarcoma-pulsed APCs do not protect mice from a challenge with non-homologous sarcomas. Kaplan-Meier survival curves of mice vaccinated with irradiated whole cell sarcoma-pulsed APCs or autophagosome-pulsed APCs. Autophagosome vaccination was performed as in Figure 2. Subcutaneous whole cell vaccinations consisted of 3x106 antigen-presenting cells pulsed with 5x106 irradiated tumor cells injected into the lower right flank of the mouse. Mice were challenged with 30,000 viable MCA-304 sarcomas. Tumor growth was monitored at least twice weekly. Each survival plot represents 2 independent experiments with 5 mice per group (n=10). Survival curves denoted with represent statistically significant (p>0.05) protection from a tumor challenge compared to no vaccine.

Supplementary Figure from Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity

Fluorescence-conjugated antibody used for flow cytometry

BACKGROUND: Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that plays a key role in the crosstalk between innate (dendritic, macrophage, and natural killer) and adaptive (T and B) cells promoting anti-tumor immune responses. DNA plasmid-based IL-12 delivered to accessible tumors by intratumoral injection and electroporation (TAVO-EP) has been shown to induce activation of innate and adaptive tumor-infiltrating and peripheral immune cells, regression of treated and distant untreated lesions (abscopal effect), and expression of PD-L1 in patients with melanoma or triple-negative breast cancer (TNBC), without the systemic toxicity that has limited therapeutic use of IL-12 historically. The combination of TAVO-EP and pembrolizumab has demonstrated durable responses in melanoma patients with immunologically “cold” tumors or with prior progression on anti-PD1 therapy. Early clinical data indicate similar potential for eliciting strengthened immunogenic responses in TNBC. KEYNOTE-890 (OMS-I141) is a Phase 2 study in patients with metastatic TNBC to evaluate the safety and efficacy of TAVO-EP + pembrolizumab in the second-line or later (2L+) treatment setting (Cohort 1) or TAVO-EP + pembrolizumab + chemotherapy in the first-line (1L) setting (Cohort 2). Cohort 1 data are presented in a separate abstract. Enrollment in Cohort 2 is ongoing. METHODS: Cohort 2 of this Phase 2, open-label, multicenter study will be assessing the safety and efficacy of TAVO-EP in combination with pembrolizumab and chemotherapy as a first-line treatment for metastatic TNBC. Eligible patients are adults with metastatic TNBC (ER and PR staining <10%, HER2 0 to 1+ or [F]ISH-negative), no prior systemic therapy for advanced disease (neo/adjuvant therapy allowed if at least 6-month disease-free interval from last treatment), measurable disease by RECIST v1.1, at least one lesion accessible for TAVO-EP treatment, and biopsy tissue available for post-hoc central determination of PD-L1 expression. Patients will receive pembrolizumab (200 mg IV) every 3 weeks, TAVO-EP (0.5 mg/mL at dose volume of ~1/4 lesion volume) on Days 1, 5, and 8 every 6 weeks, and nab-paclitaxel (100 mg/m2 IV) on Days 1, 8, and 15 every 4 weeks. Additional chemotherapy options may be introduced in future protocol amendments. Tumor assessments will be performed every 12 weeks. On-study biopsies will be collected approximately 3 weeks after start of treatment and at disease progression. The primary endpoint will be ORR assessed by blinded independent review per RECIST v1.1. Additional endpoints will include safety and tolerability, duration of response, immune ORR, progression-free survival (PFS), immune PFS, disease control rate, and overall survival. Planned enrollment in Cohort 2 is 40 patients. Based on positive efficacy data in Cohort 1, additional cohorts are being planned and will be presented. ClinicalTrials.gov: NCT03567720 Citation Format: Melinda Telli, Bianca Devitt, Katharine Cuff, Shaveta Vinayak, Rita Nanda, Alberto J. Montero, Rina Hui, David A. Canton, Christopher Twitty, Sunny Xie, Donna Bannavong, Bridget O'Keeffe, Sandra Aung, Rohit Joshi. Trial in progress: Phase 2 study of intratumoral plasmid interleukin-12 (tavokinogene telseplasmid; TAVO™) plus electroporation in combination with pembrolizumab with or without chemotherapy in patients with inoperable locally advanced or metastatic triple-negative breast cancer (KEYNOTE-890/OMS-I141) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-01-03.

AbstractIntratumoral delivery of plasmid IL12 via electroporation (IT-tavo-EP) induces localized expression of IL12 leading to regression of treated and distant tumors with durable responses and minimal toxicity. A key driver in amplifying this local therapy into a systemic response is the magnitude and composition of immune infiltrate in the treated tumor. While intratumoral IL12 typically increases the density of CD3+ tumor-infiltrating lymphocytes (TIL), this infiltrate is composed of a broad range of T-cell subsets, including activated tumor-specific T cells, less functional bystander T cells, as well as suppressive T regulatory cells. To encourage a more favorable on-treatment tumor microenvironment (TME), we explored combining this IL12 therapy with an intratumoral polyclonal T-cell stimulator membrane-anchored anti-CD3 to productively engage a diverse subset of lymphocytes including the nonreactive and suppressive T cells. This study highlighted that combined intratumoral electroporation of IL12 and membrane-anchored anti-CD3 plasmids can enhance cytokine production, T-cell cytotoxicity, and proliferation while limiting the suppressive capacity within the TME. These collective antitumor effects not only improve regression of treated tumors but drive systemic immunity with control of nontreated contralateral tumors in vivo. Moreover, combination of IL12 and anti-CD3 restored the function of TIL isolated from a patient with melanoma actively progressing on programmed cell death protein 1 (PD-1) checkpoint inhibitor therapy.Implications:This DNA-encodable polyclonal T-cell stimulator (membrane-anchored anti-CD3 plasmid) may represent a key addition to intratumoral IL12 therapies in the clinic.

, ABSTRACT Purpose: IL12 promotes adaptive type I immunity and has demonstrated antitumor ef fi cacy, but systemic administration leads to severe adverse events (AE), including death. This pilot trial investigated safety, ef fi cacy, and immunologic activity of intratumoral delivery of IL12 plasmid DNA (tavo) via in vivo electroporation (i.t.-tavo-EP) in patients with Merkel cell carcinoma (MCC), an aggressive virus-associated skin cancer. Patients and Methods: FifteenpatientswithMCCwithsuper fi cial injectable tumor(s) received i.t.-tavo-EP on days 1, 5, and 8 of each cycle. Patients with locoregional MCC (cohort A, N ¼ 3) received one cyclebeforede fi nitivesurgeryinweek4.PatientswithmetastaticMCC (cohortB, N ¼ 12)receiveduptofourcyclestotal,administeredatleast 6 weeks apart. Serial tumor and blood samples were collected. Results: All patients successfully completed at least one cycle with transient, mild (grades 1 and 2) AEs and without signif-icant systemic toxicity. Sustained (day 22) intratumoral expression of IL12 protein was observed along with local in fl ammation and increased tumor-speci fi c CD8 þ T-cell in fi ltration, which led to systemic immunologic and clinical responses. The overall response rate was 25% (3/12) in cohort B, with 2 patients experiencing durable clinical bene fi t (16 and 55 þ months, respectively). Two cohort A patients (1 with pathologic complete remission) were recurrence-free at 44 þ and 75 þ months, respectively. Conclusions: I.t.-tavo-EP was safe and feasible without systemic toxicity. Sustained local expression of IL12 protein and local in fl ammation led to systemic immune responses and clinically meaningful bene fi t in some patients. Gene electrotransfer, speci fi cally i.t.-tavo-EP, warrants further investigation for immunotherapy of cancer.

Abstract Despite extensive clinical evidence on the efficacy and safety of SARS-CoV-2 vaccines, there remains a paucity of data on their effectiveness in cancer patients who are actively receiving antineoplastic therapeutics. A recent study demonstrated only ~30% of cancer patients had positive serologic test following 2 doses of FDA-authorized SARS-CoV-2 vaccines, in contrast to ~80% positivity rate in healthy individuals, regardless of the age. Therefore, further investigation into novel approaches to boost immune response to SARS-CoV-2 vaccines in cancer patients is required. Our previous preclinical and clinical studies have established intratumoral IL-12 plasmid (TAVO) electroporation (EP) induces localized expression of IL-12p70, converting immune-excluded tumors into inflamed immunogenic lesions, thereby generating objective responses in both treated and untreated, distant tumors. Based on the enhancement of immunotherapy efficacy by IL-12, we leveraged the flexibility of our DNA plasmid-EP platform to express SARS-CoV-2 spike protein in addition to IL-12 (CORVax12) as an intratumoral vaccine candidate which we hypothesized could not only drive anti-SARS-CoV-2 immune responses but also generate a productive anti-tumor response. Naïve mice were vaccinated via intradermal injection of SARS-CoV-2 spike plasmid followed immediately by EP with or without plasmid-encoded mIL-12 on days 1 and 21. Longitudinal serum samples were collected to interrogate virus-specific cellular responses as well anti-spike IgG antibody. A surrogate viral neutralization test (sVNT) assessed serum blockade of soluble human ACE2 binding to immobilized SARS-CoV-2 spike. Our data demonstrated that intradermally electroporated CORVax12 elicits significantly higher anti-SARS-CoV-2 spike IgG antibodies and neutralization when compared with EP of SARS-CoV-2 spike alone. Next, we asked if improved SARS-CoV-2 immune response may be observed when CORVax12 is incorporated into intratumoral EP in single-tumor bearing mice. CORVax12 robustly inhibited tumor growth, induced high percentages of germinal-center B cells and class switched B cells in tumor draining lymph nodes, and generated high of anti-spike IgG and neutralization antibodies. To further investigate systemic effects of this combination, we continued with contralateral tumor mice models. In both CT26 and B16-F10 tumor models, CORVax12 intratumoral EP induced strong systemic anti-tumor responses similar to IL-12 EP alone while also producing high serum levels of anti-SARS-CoV-2 spike IgG and neutralization antibodies. Critically, this anti-viral immunity did not limit this IL-12-based intratumoral anti-tumor therapy. In summary, our preclinical data indicates that intratumoral EP of CORVax12 can induce IgG responses to SARS-CoV-2 spike as well as apparent viral neutralizing activity all while maintaining local and systemic anti-tumor effects expected from TAVO Treatment. This combined intratumoral therapy represents a novel strategy to address both tumor burden and anti-SARS-CoV-2 immunity in patients with cancer. Citation Format: Mia Han, Jack Y. Lee, Vincent Wu, Kurt Sakurada, Bianca Nguyen, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of IL-12 and SARS-Cov-2 spike plasmids drives a coordinated vaccine response and elicits robust anti-tumor immunity [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P006.

BACKGROUND: Electroporated plasmid interleukin-12 (tavokinogene telseplasmid; TAVO-EP) delivered to accessible tumors by intratumoral injection induces sustained local expression of IL-12. IL-12 is a potent immunoregulatory cytokine that plays a key role in the crosstalk between innate (dendritic, macrophage, and natural killer) and adaptive (T and B) cells, promoting anti-tumor immune responses. TAVO-EP has been shown to induce activation of innate and adaptive tumor-infiltrating and peripheral immune cells, regression of treated and distant untreated lesions (abscopal effect), and expression of PD-L1 in patients with melanoma or triple-negative breast cancer (TNBC), without the systemic toxicity that limited therapeutic use of IL-12 historically. The combination of TAVO-EP and pembrolizumab has demonstrated durable responses in melanoma patients with immunologically “cold” tumors or with prior progression on anti-PD1 therapy. Anti-PD1 monotherapy has just over 5% overall response rate (ORR) in the second-line or later (2L+) treatment setting for advanced TNBC. New antibody-directed conjugate (ADC) chemotherapy has increased rates of responses compared with prior standard chemotherapy in 2L+ advanced TNBC; however, short duration of response (DOR), and toxicity are issues of concern. Therapies that can induce durable responses with limited toxicity are needed. METHODS: Cohort 1 of this Phase 2, open-label, multicenter study assessed the safety and efficacy of TAVO-EP in combination with pembrolizumab as 2L+ treatment for advanced TNBC. Eligible patients had at least 1 line of prior systemic therapy for advanced or metastatic disease, measurable disease by RECIST v1.1, and ≥1 lesion accessible for TAVO-EP treatment. Patients received pembrolizumab (200 mg IV) every 3 weeks and TAVO-EP (0.5 mg/mL at dose volume of ~1/4 lesion volume) on Days 1, 5, and 8 every 6 weeks. Tumor assessments were performed every 12 weeks. The primary endpoint was RECIST v.1.1 ORR by investigator review. Secondary endpoints included safety and tolerability, DOR, progression-free survival (PFS), immune-related RECIST (iRECIST) ORR and PFS, disease control rate, and overall survival (OS). ClinicalTrials.gov: NCT03567720. RESULTS: Between 01Nov2018 and 30Jan2020, 26 patients were enrolled and received at least one dose of study treatment (median follow up of 11.1 months). Patients had a median of 2 prior lines of systemic therapy for advanced disease (range 1-5). Among 23 patients evaluable for response, the ORR was 17.4% (4 with partial response [PR]). One responder with centrally confirmed PD-L1-negative disease and chest wall and bulky liver metastases had a sustained PR and an iRECIST complete response (CR). One responder had near complete regression of a large fungating chest wall skin lesion. The median DOR was 16.6 months. Median OS was 11.0 months (range 0.6-27.5+). The most common treatment-related adverse events (TRAEs) (all grades) were administration site pain and fatigue. Grade 3 TRAEs were reported in 6 patients (23%) including fatigue (11.5%); acute kidney injury, enterocolitis, and myocarditis (3.8% each). There were no Grade 4 or 5 TRAEs. CONCLUSIONS: The combination of TAVO-EP and pembrolizumab in pretreated patients with advanced TNBC resulted in durable RECIST v1.1 responses, including in PD-L1-negative disease, and was well tolerated. This novel immunotherapeutic regimen warrants further evaluation in 2L+ advanced TNBC. Cohort 2 exploring TAVO-EP + pembrolizumab + chemotherapy in frontline TNBC is currently enrolling. Citation Format: Melinda L. Telli, Irene Wapnir, Bianca Devitt, Katharine Cuff, Hatem Soliman, Shaveta Vinayak, David A. Canton, Christopher G. Twitty, Sunny Xie, Ying Lu, Donna Bannavong, Bridget O'Keeffe, Sandra Aung, Rohit Joshi. Durable responses with intratumoral electroporation of plasmid interleukin-12 plus pembrolizumab in patients with advanced triple-negative breast cancer: Cohort 1 update from KEYNOTE-890 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-06.

Clinical studies have demonstrated that local expression of the cytokine IL-12 drives interferon-gamma expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our late-stage melanoma trials identified a significant on-treatment increase of intratumoral CXCR3 transcripts that was restricted to responding patients, underscoring the clinical relevance of tumor-infiltrating CXCR3(+) immune cells. In this study, we sought to understand if the addition of DNA-encodable CXCL9 could augment the anti-tumor immune responses driven by intratumoral IL-12. We show that localized IL-12 and CXCL9 treatment reshapes the tumor microenvironment to promote dendritic cell licensing and CD8(+) T cell activation. Additionally, this combination treatment results in a significant abscopal anti-tumor response and provides a concomitant benefit to anti-PD-1 therapies. Collectively, these data demonstrate that a functional tumoral CXCR3/CXCL9 axis is critical for IL-12 anti-tumor efficacy. Furthermore, restoring or amplifying the CXCL9 gradient in the tumors via intratumoral electroporation of plasmid CXCL9 can not only result in efficient trafficking of cytotoxic CD8(+) T cells into the tumor but can also reshape the microenvironment to promote systemic immune response.

The global SARS-CoV-2 (CoV2) pandemic has focused efforts to generate effective vaccines that induce potent and persistent immunity. Recently, we described a novel vaccine approach using electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax) plus plasmid Interleukin-12 (IL-12). IL-12 is an effective vaccine adjuvant that increases Th1 and Th2 antibodies in the serum. Here we examine the persistence of anti-Spike antibodies present in the serum of mice one year following CORVax vaccination. C57BL/6 and BALB/c were vaccinated intramuscular (IM) and/or intradermal (ID) with a plasmid encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12. Mice received plasmid EP immediately following injection. Splenocytes and serum were harvested at various time points to interrogate virus-specific cellular responses as well anti-spike antibody titers. Anti-Spike IgG antibodies were elicited by EP of CORVax (IC50 = 1/2112), as well as EP of CORVax combined with IL-12 (IC50 = 1/4214) approximately 40 days after the booster vaccination. These anti-Spike IgG titers decayed over time but were still present 1 year after vaccination: CORVax (IC50 = 1/351 day 146, IC50 = 1/208 day 383); CORVax + IL12 (IC50 = 1/590 day 146, IC50 = 1/266 day 383). Our data shows that EP of CORVax induces IgG responses to CoV2 Spike and the CoV2 Spike receptor binding domain. At one year following vaccination the anti-Spike IgG titers were higher in mice that received CORVax plus IL-12, however the rate at which the titers waned from their initial peak was comparable whether the mice received IL-12 or not. Additional studies are ongoing to determine whether the addition of IL-12 will enhance an anti-Spike memory response. Generous support from The Chiles Foundation, Nancy Lematta, and the Providence Medical Foundation.

Abstract BACKGROUND: Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that plays a key role in the crosstalk between innate (dendritic, macrophage, and natural killer) and adaptive (T and B) cells promoting anti-tumor immune responses. DNA plasmid-based IL-12 delivered to accessible tumors by intratumoral injection and electroporation (TAVO-EP) has been shown to induce activation of innate and adaptive tumor-infiltrating and peripheral immune cells, regression of treated and distant untreated lesions (abscopal effect), and expression of PD-L1 in patients with melanoma or triple-negative breast cancer (TNBC), without the systemic toxicity that has limited therapeutic use of IL-12 historically. The combination of TAVO-EP and pembrolizumab has demonstrated durable responses in melanoma patients with immunologically “cold” tumors or with prior progression on anti-PD1 therapy. Early clinical data indicate similar potential for eliciting strengthened immunogenic responses in TNBC. KEYNOTE-890 (OMS-I141) is a Phase 2 study in patients with metastatic TNBC to evaluate the safety and efficacy of TAVO-EP + pembrolizumab in the second-line or later (2L+) treatment setting (Cohort 1) or TAVO-EP + pembrolizumab + chemotherapy in the first-line (1L) setting (Cohort 2). Cohort 1 data are presented in a separate abstract. Enrollment in Cohort 2 is ongoing. METHODS: Cohort 2 of this Phase 2, open-label, multicenter study will be assessing the safety and efficacy of TAVO-EP in combination with pembrolizumab and chemotherapy as a first-line treatment for metastatic TNBC. Eligible patients are adults with metastatic TNBC (ER and PR staining <10%, HER2 0 to 1+ or [F]ISH-negative), no prior systemic therapy for advanced disease (neo/adjuvant therapy allowed if at least 6-month disease-free interval from last treatment), measurable disease by RECIST v1.1, at least one lesion accessible for TAVO-EP treatment, and biopsy tissue available for post-hoc central determination of PD-L1 expression. Patients will receive pembrolizumab (200 mg IV) every 3 weeks, TAVO-EP (0.5 mg/mL at dose volume of ~1/4 lesion volume) on Days 1, 5, and 8 every 6 weeks, and nab-paclitaxel (100 mg/m2 IV) on Days 1, 8, and 15 every 4 weeks. Additional chemotherapy options may be introduced in future protocol amendments. Tumor assessments will be performed every 12 weeks. On-study biopsies will be collected approximately 3 weeks after start of treatment and at disease progression. The primary endpoint will be ORR assessed by blinded independent review per RECIST v1.1. Additional endpoints will include safety and tolerability, duration of response, immune ORR, progression-free survival (PFS), immune PFS, disease control rate, and overall survival. Planned enrollment in Cohort 2 is 40 patients. Based on positive efficacy data in Cohort 1, additional cohorts are being planned and will be presented. ClinicalTrials.gov: NCT03567720 Citation Format: Melinda Telli, Bianca Devitt, Katharine Cuff, Shaveta Vinayak, Rita Nanda, Alberto J. Montero, Rina Hui, David A. Canton, Christopher Twitty, Sunny Xie, Donna Bannavong, Bridget O'Keeffe, Sandra Aung, Rohit Joshi. Trial in progress: Phase 2 study of intratumoral plasmid interleukin-12 (tavokinogene telseplasmid; TAVO™) plus electroporation in combination with pembrolizumab with or without chemotherapy in patients with inoperable locally advanced or metastatic triple-negative breast cancer (KEYNOTE-890/OMS-I141) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-01-03.

AbstractPurpose:Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed.Patients and Methods:Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets.Results:Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti–PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti–PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response.Conclusions:These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.

BackgroundElectroporated plasmid interleukin-12 (pIL-12-EP; tavokinogene telseplasmid; TAVO) induces sustained intratumoral expression of IL-12, a cytokine that is integral for response to anti-PD-1 antibodies. Here, we present updated safety and response duration data from KEYNOTE 695, a Phase 2, multicenter, open-label trial of pIL-12-EP in combination with pembrolizumab in patients with stage III/IV melanoma immediately following confirmed progression on an anti-PD-1 antibody.MethodsPatients with confirmed disease progression after ≥12 weeks‘ treatment with an anti-PD-1 antibody alone or in combination were eligible. Patients received intratumoral pIL-12-EP on days 1, 5 and 8 every 6 weeks and pembrolizumab 200 mg every 3 weeks. Responses were assessed by the investigator at 12-week intervals using RECIST v1.1; overall survival (OS) and duration of response (DoR) assessments were conducted using the Kaplan-Meier method.ResultsOf the first 56 patients treated, 50% had visceral disease (M1b-d), 80% had received 1–2 and 20% ≥3 prior lines of therapy, 27% had prior ipilimumab and 21% prior BRAF/MEK inhibitors. 61% of patients were primary refractory to anti-PD-1. 54 patients were efficacy evaluable, defined as patients who had at least one post-treatment scan. The investigator-assessed objective response rate (ORR) per RECIST was 27.8% (4 CR, 11 PR); ORR per iRECIST was 29.6%. In patients with M1b-d staging, ORR was 33.3% (n=9/27), and in those receiving prior ipilimumab, ORR was 33.3% (n=5/15). Seven patients had 100% reduction in target lesions, and regression was observed in non-injected lesions. The median DoR had not been reached. With a median follow up of 19.3 months, the median OS (95% CI) was 24.5 (14.4, NR) months (figure 1). The study is now fully enrolled. In 105 patients with safety data, there were no Grade 4/5 treatment-related adverse events (TRAEs) reported. Grade 3 TRAEs occurred in 5.7% and comprised cellulitis in two patients and arthralgia, pneumonitis, enteritis, keratoacanthoma, lichen planus and musculoskeletal chest pain in one patient each. The Grade 1/2 TRAEs in ≥10% patients were fatigue (27.6%), procedural pain (20.0%), diarrhea (17.1%), nausea (10.5%) and pruritus (10.5%). ORR by blinded independent central review has commenced and a global phase 3 trial is planned.Abstract 383 Figure 1Overall survival in patients treated with pIL-12-EP in combination with pembrolizumab. Dark grey bars: time on study treatment, light grey bars: end of treatment to death or censoringConclusionsPatients with anti-PD-1 therapy refractory advanced melanoma can achieve deep, durable responses in both injected and non-injected lesions with pIL-12-EP plus pembrolizumab. Intratumoral pIL-12-EP in combination with pembrolizumab was generally well tolerated, with minimal Grade 3 and no Grade 4/5 TRAEs.Trial RegistrationNCT03132675Ethics ApprovalThe study was approved by a central IRB and/or local institutional IRB/Ethics Committee as required for each participating institution.ConsentWritten informed consent was obtained from the patients participating in the trial; the current abstract does not include information requiring additional consent

Abstract Interleukin-12 (IL-12) is a pro-inflammatory cytokine involved in the generation of an inflammatory tumor microenvironment and is critical in eliciting a productive anti-tumor immune response. It has been investigated as an anti-cancer therapeutic using various delivery routes, but intratumoral injection of plasmid IL-12 (tavokinogene telseplasmid; TAVO) followed by electroporation is a gene therapy approach that results in more sustained production of IL-12 locally with minimal systemic immune-related toxicity. Here we show that TAVO not only provides protection in the treated triple-negative breast cancer (TNBC) lesion, but also induces a systemic, abscopal effect. Single cell RNAsequencing (scRNAseq) of infiltrating immune cells shows a significant increase in both CD4 and CD8 T cells as well as dendritic cells within the treated lesions, while simultaneously decreasing a granulocytic myeloid derived suppressor population. scRNAseq allows for a detailed look into not only the overall pathway enrichment caused by TAVO treatment, but also the specific receptor-ligand interactions occurring between cell types. A combination of these analyses revealed an enrichment in the IFN-gamma induced PDL1 pathway by TAVO, typified by an increase in the interaction between PDL1 on dendritic cells and PD1 on CD8 T cells. Further, dramatic enrichment of the CXCL9/10/11/CXCR3 axis was observed, consistent with previous studies in melanoma. Analysis of paired TCR alpha and beta chains on T cells additionally demonstrated a dramatic shift in tumor infiltrating T cell (TIL) clonality and frequency. In sum, these preclinical studies identify a signature of increased antigen presentation, T cell infiltration and expansion, and a decrease in the number of granulocytes but also a particular enhancement of the PDL1 immunosuppressive pathway following TAVO treatment. Using this signature, we focus on an in-depth analysis of 2 patients from a single arm, prospective clinical trial of TAVO monotherapy (OMS-I140) in pre-treated advanced TNBC that went on to receive anti-PD-1 as their immediate next therapy with clinical anti-tumor response. Together these data support the combination of TAVO with PD1/PDL1 inhibitors while also identifying other key pathways that may enhance responsiveness in TNBC patients for whom treatment options remain limited. Citation Format: Erika J Crosby, Hiroshi Nagata, Melinda L Telli, Chaitanya R Acharya, Irene Wapnir, Kaitlin Zablotsky, Erica Browning, Reneta Hermiz, Lauren Svenson, Donna Bannavong, Kellie Malloy, David A Canton, Chris G Twitty, Takuya Osada, Herbert Kim Lyerly. Intratumoral delivery of tavokinogene telseplasmid (plasmid IL-12) and electroporation induces an immune signature that predicts successful combination in patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-22.