IMPORTANCE Patients who experience progression of advanced human papillomavirus (HPV)-associated cancers and who have previously received first-line systemic treatment have a poor prognosis and limited therapeutic options. OBJECTIVE To assess the clinical activity of the combination of the HPV type 16 therapeutic vaccine PDS0101, the tumor-targeting interleukin 12 antibody-drug conjugate PDS01ADC, and the bifunctional anti-programmed cell death ligand 1 (PD-L1)/transforming growth factor beta (TGF-beta) bintrafusp alfa in advanced HPV-associated cancers. DESIGN, Setting, and Participants This nonrandomized clinical trial was phase 1/2 and investigator initiated, and was conducted at a single US cancer research center between June 2020 and July 2022. Patients with advanced or metastatic HPV-associated cancers were eligible, including patients who were both immune checkpoint blockade (ICB) naive and ICB resistant. The cutoff date for data analysis was May 13, 2024. INTERVENTION Patients received 1 mL of PDS0101 subcutaneously every 4 weeks for 6 doses then every 12 weeks for 2 additional doses, PDS01ADC, 16.8 mu g/kg, subcutaneously every 4 weeks or PDS01ADC, 8 mu g/kg, subcutaneously every 2 weeks, and bintrafusp alfa, 1200 mg, intravenously every 2 weeks. MAIN OUTCOMES AND MEASURES Objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors version 1.1 in ICB-naive patients. RESULTS Of the 50 eligible patients, 26 (52%) were men and the median age was 56 years (range, 28-80 years). The median (IQR) follow-up was 37.7 (30.6-42.0) months. Fourteen patients (28%) were ICB naive, with an ORR of 35.7% (95% CI, 12.8%-64.9%), and median overall survival (OS) 42.4 months (95% CI, 8.3 months-not estimable); in ICB-resistant patients, the ORR was 16.7% (6 of 36 patients; 95% CI, 6.4%-32.8%) and median OS was 15.8 months (95% CI, 9.0-21.3 months). Among patients with HPV-16-positive tumors (37 patients [74%]), in the ICB-naive group (8 patients [21.6%]) the ORR was 62.5% (95% CI, 24.5%-91.5%) and a median OS measure was not reached. Grade 3 and 4 treatment-related adverse events occurred in 26 of 50 patients (52%). There were no treatment-related deaths. CONCLUSIONS AND RELEVANCE In this trial, the combination of PDS0101, PDS01ADC, and bintrafusp alfa showed an acceptable safety profile and promising antitumor activity and improved OS in patients with HPV-16-positive cancers, in both ICB-naive and ICB-resistant patients, warranting further evaluation of the combination of PDS0101 and PDS01ADC with simultaneous PD-L1/TGF-beta inhibition in these populations.

IMPORTANCE Patients who experience progression of advanced human papillomavirus (HPV)–associated cancers and who have previously received first-line systemic treatment have a poor prognosis and limited therapeutic options. OBJECTIVE To assess the clinical activity of the combination of the HPV type 16 therapeutic vaccine PDS0101, the tumor-targeting interleukin 12 antibody-drug conjugate PDS01ADC, and the bifunctional anti–programmed cell death ligand 1 (PD-L1)/transforming growth factor β (TGF-β) bintrafusp alfa in advanced HPV-associated cancers. DESIGN, Setting, and Participants This nonrandomized clinical trial was phase 1/2 and investigator initiated, and was conducted at a single US cancer research center between June 2020 and July 2022. Patients with advanced or metastatic HPV-associated cancers were eligible, including patients who were both immune checkpoint blockade (ICB) naive and ICB resistant. The cutoff date for data analysis was May 13, 2024. INTERVENTION Patients received 1 mL of PDS0101 subcutaneously every 4 weeks for 6 doses then every 12 weeks for 2 additional doses, PDS01ADC, 16.8 µg/kg, subcutaneously every 4 weeks or PDS01ADC, 8 µg/kg, subcutaneously every 2 weeks, and bintrafusp alfa, 1200 mg, intravenously every 2 weeks. MAIN OUTCOMES AND MEASURES Objective response rate (ORR) by Response Evaluation Criteria in Solid Tumors version 1.1 in ICB-naive patients. RESULTS Of the 50 eligible patients, 26 (52%) were men and the median age was 56 years (range, 28-80 years). The median (IQR) follow-up was 37.7 (30.6-42.0) months. Fourteen patients (28%) were ICB naive, with an ORR of 35.7% (95% CI, 12.8%-64.9%), and median overall survival (OS) 42.4 months (95% CI, 8.3 months-not estimable); in ICB-resistant patients, the ORR was 16.7% (6 of 36 patients; 95% CI, 6.4%-32.8%) and median OS was 15.8 months (95% CI, 9.0-21.3 months). Among patients with HPV-16–positive tumors (37 patients [74%]), in the ICB-naive group (8 patients [21.6%]) the ORR was 62.5% (95% CI, 24.5%-91.5%) and a median OS measure was not reached. Grade 3 and 4 treatment-related adverse events occurred in 26 of 50 patients (52%). There were no treatment-related deaths. CONCLUSIONS AND RELEVANCE In this trial, the combination of PDS0101, PDS01ADC, and bintrafusp alfa showed an acceptable safety profile and promising antitumor activity and improved OS in patients with HPV-16–positive cancers, in both ICB-naive and ICB-resistant patients, warranting further evaluation of the combination of PDS0101 and PDS01ADC with simultaneous PD-L1/TGF-β inhibition in these populations. Trial Registration ClinicalTrials.gov Identifier: NCT04287868

Background Human papillomavirus (HPV)-associated malignancies continue to present a major health concern despite the development of prophylactic vaccines. Standard therapies offer limited benefit to patients with advanced-stage disease. Despite improved outcomes with programmed cell death protein-1 (PD-1) targeted therapies, treatment resistance and modest response rates highlight a significant unmet need to develop novel therapies for these patients. PDS0101 (designated HPV vaccine) is a liposomal nanoparticle HPV16-specific therapeutic vaccine that has been shown to generate strong HPV-specific responses in preclinical and clinical studies. Here we assess the efficacy of this HPV vaccine in combination with the tumor-targeting immunocytokine NHS-IL12 (PDS01ADC), plus either αPD-1 or the class I histone deacetylase inhibitor Entinostat.Methods Mice bearing HPV16+, αPD-1 refractory TC-1 and mEER tumors were treated with HPV vaccine, NHS-IL12, and either αPD-1 or Entinostat to determine antitumor efficacy and survival benefits. A comprehensive analysis of the tumor microenvironment was performed using flow cytometry, multiplex immunofluorescence, chemokine and cytokine assessment, and single-cell RNA sequencing with T-cell receptor (TCR) enrichment.Results Combination of HPV vaccine and NHS-IL12 with either Entinostat or αPD-1 yielded significant antitumor activity and prolonged survival in αPD-1 refractory models of HPV16+ cancer, with superior activity employing Entinostat versus αPD-1 combination. Entinostat triple therapy increased overall and HPV16-specific tumor CD8+ T-cell infiltration with heightened cytotoxicity. TCR sequencing revealed a CD8+ T-cell clone unique to vaccine-treated cohorts, which displayed an enriched cytotoxic transcriptional profile with triple therapy. These effects were paralleled by strong differentiation of tumor-associated macrophages (TAMs) towards pro-inflammatory, antitumor M1-like cell states. Single-cell transcriptomic analysis indicated all three agents were required for highest modulation of both CD8+ T cells and TAMs conducive to tumor control. A biomarker signature reflecting the preclinical findings was found to be associated with improved survival in patients with HPV-associated malignancies.Conclusion Together, these findings provide a rationale for the combination of HPV vaccine, NHS-IL12, and Entinostat in the clinical setting for patients with HPV16-associated malignancies.

This first-in-human phase 1 study (NCT04432597) evaluated the safety and recommended phase 2 dose (RP2D) of PRGN-2009, a gorilla adenoviral-vector targeting oncoproteins E6, E7 (human papillomavirus (HPV)16/18) and E5 (HPV16), as monotherapy (Arm 1A) and combined with the bifunctional TGF-β “trap”/anti-PD-L1 fusion protein bintrafusp alfa (BA; Arm 1B), in patients with recurrent/metastatic HPV-associated cancer. Patients with ≥ 1 prior treatment (immunotherapy allowed) received PRGN-2009 (1 × 1011 particle units or 5 × 1011 particle units, subcutaneously) every 2 weeks for 3 doses, then every 4 weeks (Arm 1A), or PRGN-2009 (RP2D, schedule per Arm 1A) and BA (1200 mg, intravenously) every 2 weeks (Arm 1B). Primary endpoints were safety and RP2D of PRGN-2009; secondary objectives included overall response rate (ORR) and overall survival (OS). Seventeen patients were treated. In Arm 1A (n = 6) there were no dose limiting toxicities or grade 3/4 treatment-related adverse events (TRAEs), 5 × 1011 PU was selected as RP2D, no responses were observed, and median OS (mOS) was 7.4 months (95

Abstract Background: PRGN-2009 is a novel gorilla adenovirus vaccine targeting HPV16/18, and bintrafusp alfa (BA) is a bifunctional anti-PD-L1/TGFβ fusion protein. Patients with pretreated advanced HPV-associated malignancies received PRGN-2009 vaccine (5 × 1011 particle units, subcutaneous administration) q2 weeks for 3 administrations and biweekly 1200mg BA followed by q4 weeks vaccine and biweekly BA (NCT04432597), with an overall response rate of 30%. We report here correlative analyses from this first-in-human Phase I study using peripheral blood from 10 patients with HPV16+ (n=8), HPV18+ (n=1), or HPV45+ (n=1) disease to identify immune correlates associated with clinical response. Methods: Peripheral HPV-specific T cell responses, HPV circulating tumor DNA (ctDNA) from plasma, serum cytokines and soluble factors, and peripheral immune cell subsets were assessed to evaluate changes induced with treatment and peripheral correlates associated with clinical activity. Patients with complete response (CR, n=1), partial response (PR, n=2; 1 confirmed) or stable disease (SD, n=1) (n=4 total) were compared to patients with progressive disease (PD, n=6). Results: Eighty percent (8/10) of patients developed either HPV16 or HPV18-specific T-cell responses after repeat administration of PRGN-2009 with BA. Eight of 10 patients had detectable HPV16 or 18 ctDNA at baseline and changes in ctDNA load with treatment generally correlated with outcomes. Early transient increases in IL-8, TNFα, and IFNγ occurred, with greater increases in IL-8 at 2 weeks and TNFα at 4 weeks in patients with PD compared to CR/PR/SD. The patient who achieved a durable CR had HPV45+ disease, and had a) a 50% increase in CD8+ T cell frequency at 2 weeks, b) increases in frequencies of CD8+ naïve T cells expressing Ki67+ and central and effector memory T cells expressing 4-1BB, PD-1, or TIM-3, and c) considerable increases in HPV18-specific T cell responses through 484 days after treatment start, indicating durable antigen-specific T cell activity. Conclusions: The majority of patients developed HPV16/18 specific T-cell responses after repeat administration of PRGN-2009 in combination with BA. Transient increases in IL-8 and TNFα 2-4 weeks after start of treatment were associated with worse outcomes, suggesting that early changes in cytokines may reflect disease course. Furthermore, the early expansion of CD8+ T cell subsets in the patient developing a CR with HPV45+ disease after receiving a vaccine targeting HPV16 and HPV18 epitopes suggest a broadening and boosting of T cell responses that enabled potent anti-tumor activity. A high degree of homology between HPV18 and HPV45 may have contributed to the expansion of HPV18 specific T cells in the patient with CR. These data highlight the importance of assessing peripheral blood in immunotherapy trials to reveal important immunological phenomena. Citation Format: Meghali Goswami, Charalampos S. Floudas, Julius Strauss, Douglas E. Brough, Amy R. Lankford, Caroline Jochems, James L. Gulley, Jeffrey Schlom, Renee N. Donahue. Peripheral immune analyses from phase I trial of HPV vaccine PRGN-2009 in combination with bintrafusp alfa in patients with HPV-associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1193.

Chemotherapy and immune checkpoint inhibitors have a role in the post-neoadjuvant setting in patients with triple-negative breast cancer (TNBC). However, the effects of nivolumab, a checkpoint inhibitor, capecitabine, or the combination in changing peripheral immunoscore (PIS) remains unclear. This open-label randomized phase II OXEL study (NCT03487666) aimed to assess the immunologic effects of nivolumab, capecitabine, or the combination in terms of the change in PIS (primary endpoint). Secondary endpoints include the presence of ctDNA, toxicity, clinical outcomes at 2-years and association of ctDNA and PIS with clinical outcomes. Forty-five women with TNBC and residual invasive disease after standard neoadjuvant chemotherapy were randomized to nivolumab, capecitabine, or the combination. Here we show that a combination of nivolumab plus capecitabine leads to a greater increase in PIS from baseline to week 6 (91%) compared with nivolumab (47%) or capecitabine (53%) alone (log-rank p = 0.08), meeting the pre-specified primary endpoint. In addition, the presence of circulating tumor DNA (ctDNA) was associated with disease recurrence, with no new safety signals in the combination arm. Our results provide efficacy and safety data on this combination in TNBC and support further development of PIS and ctDNA analyses to identify patients at high risk of recurrence.

2628 Background: Combinatorial treatments may improve efficacy in checkpoint blockade resistant (CBR) R/M HPV-C. PRGN-2009 is a gorilla adenovirus immunotherapy vaccine containing 35 non-HLA-restricted epitopes of HPV-16 and 18. We conducted a first-in-human Phase I study of PRGN-2009 alone and combined with bintrafusp alfa (BA), a bifunctional TGF-β “trap”/anti-PD-L1 fusion protein in adult pts with pretreated R/M HPV-C. We report safety and efficacy results. Methods: Pts received PRGN-2009 1x10 11 or 5x10 11 particle units (PU) SC Q2W for 3 times, then Q4W (dose escalation part, DEP) or the recommended phase 2 dose (RP2D) of PRGN-2009 plus BA 1200 mg IV Q2W (combination part, CP) until progressive disease, unacceptable toxicity or patient withdrawal. Primary endpoints were safety and RP2D; secondary endpoints included overall response rate (RECIST 1.1). Exploratory endpoints included the induction of HPV-16/18 specific T-cell responses in peripheral blood mononuclear cells collected pre- and post-PRGN-2009 treatment. Results: Between August 11, 2020, and May 5, 2022, 17 pts were enrolled. Median follow-up at data cutoff was 7.4 months and 18.6 months for the DEP and CP, respectively. In the DEP, 6 pts received PRGN-2009, for a median duration of 3.0 months (range 1.8-17.9). Five pts had ≥2 prior lines of anticancer therapy. There were no dose limiting toxicities. Adverse events (AE) related to treatment (TRAE) were Grade 1-2 flu-like syndrome, injection site reactions, fatigue, rash. 5x10 11 PU was selected as RP2D. Stable disease was noted in 4 pts, longest duration being 14.9 months. In the CP, 11 high-risk HPV positive pts received PRGN-2009 with BA for a median duration of 10.0 months (range 0.5-23.0). Four pts (36.3%) had received ≥3 prior treatment lines; 10 pts (90.9%) were CBR. Grade 3 TRAEs occurred in 1 pt (9.1%; duodenal hemorrhage [DH] attributable to BA); 2 pts (18.1%) had grade 4 TRAEs (DH, pharyngeal mucositis [n=1 each], attributable to BA). Both pts with DH were concurrently receiving NSAIDs. No treatment-related deaths occurred; one pt with DH died after refusing core standard medical management. Of 10 pts evaluable for response one CBR pt had a complete response (duration 15.3 months); partial responses were seen in two pts (one CBR). Overall response rate was 30.0% (95% CI 6.7-65.3%). Two pts were treated beyond progression without delayed response. Median overall survival was 7.4 months (95% CI, 2.9-26.8 months) for DEP and 12.5 months (95% CI, 9.6-inestimable) for CP. Post vaccination 14/16 (88%) pts developed T-cell responses to HPV-16 and/or HPV-18, with 6/6 in DEP and 8/10 in CP. Conclusions: PRGN-2009 is safe, well-tolerated, and induces HPV-specific T-cell immune responses. Further, PRGN-2009 combined with BA demonstrated clinical activity in pts with pretreated HPV-associated cancers, naïve or resistant to checkpoint blockade. Clinical trial information: NCT04432597 .

BACKGROUND:NHS-IL12 is a first-in-class, recombinant fusion protein composed of the human monoclonal antibody NHS76 (binds exposed DNA/histones at sites of intratumoral necrosis) fused to 2 IL-12 heterodimers. The maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of NHS-IL12 monotherapy given subcutaneously (SC) every 4 weeks was previously reported. The study was expanded to include a high-exposure cohort with NHS-IL12 SC every 2 weeks (q2w).METHODS:This single-arm, phase I trial evaluated NHS-IL12 12 µg/kg SC q2w or 16.8µg/kg SC q2w in patients with metastatic solid tumors. The primary endpoint was safety.RESULTS:Using a 3+3 design, 13 patients with advanced cancer were enrolled and 12 were dose-limiting toxicity (DLT) evaluable. There was 1 DLT (Grade 3 aspartate transaminase/alanine transaminase [AST/ALT] elevation). Other grade 3 toxicities included: flu-like symptoms 1/13 (8%), decreased absolute lymphocyte count (ALC) 1/13 (8%), decreased white blood cell count (WBC) 1/13 (8%), but most adverse events reported were low grade and self-limiting grade. Fifty percent of evaluable patients (6/12) experienced stable disease (SD) with 42% (5/12) developing progressive disease (PD) at the first restaging.CONCLUSION:Biweekly NHS-IL12 was well tolerated in this small phase I study. Additional studies incorporating NHS-IL12 with other immunomodulating agents are underway. (ClinicalTrials.gov Identifier: NCT01417546).

PDF file - 264K, Tumor growth rates following vaccine.

Dose response curves for avelumab-mediated ADCC assays of 3 human cancer cell lines using purified NK cells from the same healthy donor as effectors at an effector:target ratio of 25:1.

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PDF file - 62K, IL-5 following vaccine.

PDF file - 90K, Consort diagram.

Supplementary Figures and Tables

Background: Poor clinical outcomes are noted in pts with TNBC who do not achieve a pathologic complete response (pCR). We characterized peripheral immune subsets and the role of minimal residual disease (MRD) detection via ctDNA in pts who participated in the OXEL study. Methods: OXEL (Opdivo® -XELoda ®) is a recently completed phase II open-label 3-arm randomized study of nivolumab (nivo), capecitabine (cape) or the combination as adjuvant therapy (tx) for pts with residual TNBC after appropriate neoadjuvant chemo. Residual disease was defined as ≥ 1.0 cm of primary tumor and/or nodal involvement. Eligible pts had completed definitive local tx. Pts were randomly assigned to nivo 360 mg iv q3wks x 6 (arm A); cape 1250mg/m2 po bid D1-D14 q3 wks x 6 (arm B); nivo 360mg iv q3wks + cape 1250mg/m2 po bid D1-D14 q3 wks x 6 (arm C). Peripheral blood mononuclear cells (PBMCs) and ctDNA were assessed at baseline (D1 of cycle 1), 6, and 12 wks and at time of recurrence, if applicable. PBMCs were stained with 30 markers and analyzed by flow cytometry to identify changes in 158 immune cell subsets at 6 wks, as a percent of total PBMCs. RaDaRTM, a deep sequencing based, tumor-informed personalized assay was utilized to detect the presence of ctDNA in plasma. Distant disease-free survival (DDFS) and overall survival (OS) were analyzed by the Kaplan-Meier method and Log-Rank test was used to compare DDFS and OS according to baseline MRD results. All pts will be followed for distant recurrence and survival for 3 yrs. Here we report the translational endpoints of the OXEL study. Clinical endpoints according to treatment received will be reported in a future analysis. Results: 45 pts were enrolled between 8/2018 and 6/2021. 29 (64%) were Caucasian and 14 (31%) were African American. Mean age at enrollment was 51 [+/- 12]. 93% of pts received a taxane-anthracycline containing neoadjuvant tx. 15 pts were randomized to each arm. DDFS probability at 1-yr and 2-yrs was 0.71 (+/- 0.07) and 0.66 (+/- 0.08) respectively. At 12 mos of median follow up, 13/45 pts (29%) experienced distant recurrence, none had local recurrence. 43 pts were evaluated for PBMC subsets. Changes in PBMC subsets at 6 wks were different amongst the arms; in arm A, reductions in NK subsets, including a 33% reduction in CD56dimCD16- cells, were observed, while in arm B, increases in naïve CD4+ T cells (+45%) and CD73+CD8+ T cells (+12%) and reductions in ki67+CD8+ T cells (-48%) were noted. In arm C, increases were observed in conventional dendritic cells (+36%), effector memory ki67+CD4+ T cells (+46%), and CD56dimCD16- NK cells (+29%). 33 pts underwent successful MRD analysis. 12/33 (36%) pts were MRD+ at baseline. 2/12 pts MRD+ at baseline subsequently cleared MRD, with undetectable ctDNA on future time points; neither patient has had recurrence to date. The remaining 10/12 MRD+ pts (83%) have experienced distance recurrence. 21/33 (64%) pts were ctDNA negative at baseline; 20/33 remained negative for all follow up timepoints. 10/11 pts experiencing distant recurrence were MRD+ at baseline, compared to 1/11 pt who became MRD+ at wk 6 post initiation of tx. At 12 mos of median follow-up, baseline MRD+ testing was significantly associated with an inferior DDFS ( p<0.0001 Log-rank test, median DDFS 4.0 mos vs. not reached) and OS (p=0.02 Log-rank test, median OS not reached for both groups). Results will be updated at the time of abstract presentation. Conclusions: Changes in PBMC subsets were associated with receipt of chemo and/or immunotherapy. Our results suggest that baseline MRD+ in pts without pCR is a poor prognostic factor. Future trials aiming to optimize adjuvant treatment with chemo and/or immunotherapy in residual TNBC should consider incorporating ctDNA as a selection marker of pts at higher risk of recurrence. Citation Format: Filipa Lynce, Candace Mainor, Xue Geng, Greg Jones, Ilana Schlam, Hongkun Wang, Ute Feger, Renee Donahue, Nicole Toney, Caroline Jochems, Jeffrey Schlom, Christopher Gallagher, Rita Nanda, Deena Graham, Erica M Stringer-Reasor, Neelima Denduluri, Julie Collins, Asma A Dilawari, Ami Chitalia, Shruti Tiwari, Raquel Nunes, Rebecca Kaltman, Katia Khoury, Margaret Gatti-Mays, Sandra M Swain, Heather A. Parsons, Paula Pohlmann, Claudine Isaacs. Peripheral immune subsets and circulating tumor DNA (ctDNA) in patients (pts) with residual triple negative breast cancer (TNBC) treated with adjuvant immunotherapy and/or chemotherapy (chemo): The OXEL study [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 PD9-02.

Background Globally more than 600,000 cases of HPV-associated cancers occur annually. Approximately 15-20% of cases respond to PD-(L)1 inhibitors, and approximately 30%, including 10% of immune checkpoint inhibitor (ICI) refractory patients, respond to bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1. Thus, for most patients who are ICI refractory, there is no effective therapy. Preclinical studies have shown that the triple combination of bintrafusp alfa, M9241, a tumor-targeting IL-12 immunocytokine, and PDS0101, a therapeutic vaccine targeting HPV-16, resulted in maximal tumor reduction. A phase II trial (NCT04287868) evaluating this triple therapy has shown a manageable safety profile and preliminary evidence of clinical activity in ICI refractory HPV-associated cancers, with 45% of patients having disease reduction, including 27% with objective responses. Methods Peripheral blood from patients with ICI refractory HPV-associated malignancies (n=27) treated with the triple therapy was analyzed prior to and 2 weeks post first treatment (a timepoint prior to restaging) for multiple serum cytokines and soluble factors, complete blood counts, and 158 immune cell subsets. HPV-16 specific T-cells were assessed before and during treatment in a subset of patients (n=14). Immune parameters were evaluated for changes with therapy and compared between patients deriving clinical benefit (with a best overall response of stable disease, partial response, or complete response) versus those with progressive disease (PD). Results The triple therapy promoted a pro-inflammatory serum cytokine and factor milieu, and significantly increased NK cells (p=0.002) and decreased conventional dendritic cells (cDCs, p=0.001), plasmacytoid DCs (p=0.011), CD4+ T-cells (p=0.008), CD8+ T-cells (p=0.004), T-regulatory-cells (p=0.002), and B-cells (p=0.042). HPV-16 specific T-cells were increased >2 fold after therapy in 11/14 patients evaluated. Before therapy, patients developing clinical benefit from the triple therapy had significantly higher levels of CD8+ naïve T-cells (p=0.037), trends of higher CD8:MDSC ratios (p=0.098), and significantly lower levels of cDCs (p=0.019) and classical monocytes (p=0.049), than patients developing PD. A greater early increase (2 weeks after one treatment cycle) in soluble granzyme B (p=0.004), TNFa (p=0.013), and monocytes (p=0.025), and less of a decrease in cDCs (p=0.006) associated positively with clinical benefit, while trends of an increase in the neutrophil to lymphocyte ratio (p=0.073) associated inversely. Conclusions These studies interrogating the peripheral immunome add insight into the combined mechanism of action of bintrafusp alfa, M9241, and PDS0101 in patients with HPV-associated cancers, and provide valuable information to identify ICI refractory patients potentially more likely to benefit from immunotherapy. Ethics Approval All patients gave written informed consent for participation. This study was approved by the National Cancer Institute's Institutional Review Board. The trial registration number is NCT04287868.

Background More than 630,000 cases of HPV related cancer (e.g. cervical, oropharyngeal, anal) occur worldwide annually. Unfortunately, only about 15-20% of cases respond to PD-(L)1 inhibitors alone. Recent data with the triple combination of PDS0101 (a therapeutic vaccine targeting HPV 16 E6/E7), M9241 (a tumor-targeting IL-12 immunocytokine), and bintrafusp alfa (a bifunctional fusion protein targeting TGF-β and PD-L1) have shown early clinical activity in advanced checkpoint refractory HPV related cancers, with 45% of patients having disease reduction, including 27% with objective responses.1 In addition, preclinical studies have shown that the triple combination of entinostat (histone deacetylase inhibitor), M9241, and bintrafusp alfa may also have promising anti-tumor activity in checkpoint refractory disease. Methods 7 patients with advanced checkpoint refractory HPV positive cancers who had been previously treated with PDS0101, M9241 and bintrafusp alfa (NCT04287868) and progressed went on to be treated with the combination of entinostat, M9241, and bintrafusp alfa (NCT04708470). These patients received bintrafusp alfa at 300 mg IV q 2weeks, entinostat 3 mg po weekly including a 1 week lead in of entinostat alone, and M9241 at either 4 mcg/kg SC q 2 weeks or 8 mcg/kg SC q 4weeks (based on dose escalation cohort). Pts receiving M9241 at 8 mcg/kg did not receive entinostat on the week of M9241 treatment but did on all other weeks. Results 7 patients with advanced checkpoint refractory HPV positive cancers (5 oropharyngeal, 1 anal, 1 neuroendocrine rectal) who had progressed on PDS0101, M9241 and bintrafusp alfa went on to receive entinostat, M9241, and bintrafusp alfa. After switching to the entinostat based triple combination, 2/7 patients had grade 3 treatment related AEs including grade 3 anemia in one patient and grade 3 leukopenia/lymphopenia in another. Otherwise, there were no grade 3 or greater treatment related AEs observed in patients switching to the entinostat based triple combination. 3/7 (43%) patients (2 oropharyngeal, 1 anal) have had tumor reduction of 28.8%, 38.3% and 42.6% by RECIST after switching to the entinostat based triple combination. Conclusions Escalating doses of the triple combination of entinostat, M9241 and bintrafusp alfa continue to be evaluated in an ongoing phase I/II trial (NCT04708470). To date the triple combination appears to have a manageable safety profile along with encouraging clinical activity in patients with advanced checkpoint refractory HPV related cancers including in pts who have previously progressed on PD(L)1 inhibitor then on PDS0101, M9241 and bintrafusp alfa. Trial Registration NCT04708470 Reference Strauss J, et al. Phase II evaluation of the combination of PDS0101, M9241, and bintrafusp alfa in patients with HPV 16+ malignancies. J Clin Oncol. 2022;40(suppl 16; abstr 2518). Ethics Approval Ethics approval for NCT04708470 was obtained from the National Institutes of Health IRB (Ref # 551458). Participants gave informed consent prior to participating.

2501 Background: There are more than 630,000 cases of HPV associated malignancies including cervical, oropharyngeal and anal cancer worldwide annually. HPV 16 is responsible for the majority of these cases. About 15-20% of HPV associated malignancies respond to PD-(L)1 inhibitors, but for the overwhelming majority of patients who progress on these immunotherapies there is no effective standard of care therapy. Preclinical studies have shown that the triple combination of PDS0101 (Versamune-HPV), a liposomal multipeptide therapeutic vaccine targeting HPV 16 E6/E7, M9241, a tumor-targeting immunocytokine composed of IL-12 heterodimers fused to a monoclonal antibody targeting free DNA in necrotic tumor regions, and bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, resulted in maximum HPV-specific T cell responses, T cell tumor infiltration and tumor reduction as compared to any one or two of these agents alone. Methods: Fourteen pts with HPV 16+ relapsed or refractory advanced cancer were enrolled to the triple combination of PDS0101, M9241 and bintrafusp alfa (NCT04287868). Pts received bintrafusp alfa at 1200 mg flat dose i.v. every 2 weeks, M9241 at 16.8 mcg/kg s.c. every 4 weeks and PDS0101 given as two separate 0.5 ml s.c. injections every 4 weeks. Dose reductions of M9241 to 8 mcg/kg were allowed as well as skipped doses of any agent for ongoing toxicities. Results: Fourteen pts with advanced HPV 16+ cancers (5 cervical, 2 vaginal/vulvar, 4 anal, 3 oropharyngeal) were treated. 4/14 (28.6%) pts had a grade 3 treatment related toxicity including grade 3 hematuria in 2 pts with cervical ca and prior pelvic radiation and grade 3 AST/ALT elevation in 2 pts, one with anal ca and one with vaginal ca. For one patient with grade 3 AST/ALT elevation dose reduction of M9241 from 16.8 to 8 mcg/kg allowed for continued treatment with AST/ALT remaining at grade 1 or less. One additional patient had transient asymptomatic grade 4 neutropenia. No other treatment related grade 3 or greater toxicities were noted. 10/14 (71%) pts have had objective responses: 1 CR (anal ca) and 9 PRs (3 cervical, 2 vulvar/vaginal, 2 anal, 2 oropharyngeal) with 9/10 of these responses ongoing after a median 5 month of follow up. Of the 14 pts, 6 pts have checkpoint naïve disease and 8 pts have checkpoint refractory disease. 5/6 (83%) pts with checkpoint naïve disease and 5/8 (63%) pts with checkpoint refractory disease have had objective responses. Analyses of immune responses and other immune correlates are ongoing. Conclusions: Triple combination of PDS0101, M9241 and bintrafusp alfa appears to have a manageable safety profile along with early evidence of notable clinical activity for pts with both checkpoint naïve as well as checkpoint refractory HPV 16+ advanced malignancies. Clinical trial information: NCT04287868.

BACKGROUND:FOLFOX plus bevacizumab is a standard of care (SOC) for first-line treatment of microsatellite-stable metastatic colorectal cancer (MSS mCRC). This study randomized patients to SOC or SOC plus avelumab (anti-PD-L1) plus CEA-targeted vaccine.METHODS:Patients with untreated MSS mCRC enrolled to a lead-in arm assessing safety of SOC + immuno-oncology agents (IO). Next, patients were randomized to SOC or SOC + IO. The primary endpoint was progression-free survival (PFS). Multiple immune parameters were analyzed.RESULTS:Six patients enrolled to safety lead-in, 10 randomized to SOC, and 10 to SOC + IO. There was no difference in median PFS comparing SOC versus SOC + IO (8.8 months (95% CI: 3.3-17.0 months) versus 10.1 months (95% CI: 3.6-16.1 months), respectively; hazard ratio 1.061 [P = .91; 95% CI: 0.380-2.966]). The objective response rate was 50% in both arms. Of patients analyzed, most (8/11) who received SOC + IO developed multifunctional CD4+/CD8+ T-cell responses to cascade antigens MUC1 and/or brachyury, compared to 1/8 who received SOC alone (P = .020). We detected post-treatment changes in immune parameters that were distinct to the SOC and SOC + IO treatment arms. Accrual closed after an unplanned analysis predicted a low likelihood of meeting the primary endpoint.CONCLUSIONS:SOC + IO generated multifunctional MUC1- and brachyury-specific CD4+/CD8+ T cells despite concurrent chemotherapy. Although a tumor-directed immune response is necessary for T-cell-mediated antitumor activity, it was not sufficient to improve PFS. Adding agents that increase the number and function of effector cells may be required for clinical benefit.