6104 Background: Recurrent respiratory papillomatosis (RRP) is a rare, neoplastic disorder caused by chronic human papillomavirus (HPV) type 6 or 11 infection. Significant morbidity can occur due to airway obstruction and transformation into malignant cancer. Repeat surgical debulking has historically been the most common treatment for RRP symptom management. Zopapogene imadenovec-drba (zopa), a novel adenoviral vector-based immunotherapy, is the first and only FDA-approved treatment for adults with RRP. Zopa is now recommended as the first-line treatment for adults with RRP in an RRP Foundation position statement authored by 16 key opinion leaders (Best et al. Laryngoscope 2026). Methods: The pivotal trial (NCT04724980) evaluated zopa in patients with RRP requiring ≥3 clinically indicated interventions 12 months (m) prior to treatment. 12m follow-up data was reported and demonstrated that 4 subcutaneous injections of zopa (5x10 11 particle units per injection; n=35) were well-tolerated, with no serious adverse events, no grade >2 treatment-related adverse events, and no early treatment discontinuations. The most common adverse events were injection-site reaction, fatigue, chills, fever, and myalgia. Robust efficacy was observed following zopa treatment with 51% (34 to 69; 95% CI) of patients achieving a complete response (CR), defined as no requirement for interventions in the 12m following treatment, and 86% (30/35) of patients experiencing a decrease in interventions in the year following treatment as compared to the year prior to treatment. Here we present data up to 51m of follow-up. Results: As of December 15, 2025, 83% (15/18) of patients who achieved a CR at 12m remain in CR with no recurrence of papilloma requiring surgical or medical intervention. The median duration of follow-up for patients in CR was 36m (range: 30-51m), with 3 patients having a response lasting more than 4 years. No new safety events were observed during long-term follow-up. Conclusions: Zopa treatment demonstrated significant clinical benefit with the vast majority of CR patients experiencing ongoing durable complete responses for up to 4 years with excellent long-term safety. Updated follow-up results for all patients achieving CR will be available at the time of presentation. Clinical trial information: NCT04724980 .

Background Recurrent respiratory papillomatosis (RRP) is a rare debilitating condition caused by chronic infection with human papillomavirus (HPV) type 6 or 11. Papillomas develop in the aerodigestive tract, leading to significant voice disturbance and airway obstruction. No systemic treatment currently exists. We aimed to assess the safety and clinical activity of PRGN-2012 in adult patients with RRP treated at the recommended phase 2 dose. Methods This was a single-centre, single-arm, phase 1/2 trial. Adult patients aged 18 years or older with RRP who required three or more interventions in the 1 year before treatment received adjuvant PRGN-2012 on day 1 following surgical debulking of disease, and on days 15, 43, and 85. Primary outcome measure was complete response rate, defined as the percentage of patients who did not require an intervention to control RRP in the 12 months after treatment. Safety outcomes included treatment-related adverse events. This study is registered ClinicalTrials.gov (NCT04724980). Findings From March 16, 2021, to June 1, 2023, 38 patients were enrolled and received the 12-week treatment course. Among the 35 patients treated at the recommended phase 2 dose of 5x1011 particle units, 18 (51%) of 35 patients had a complete response (95% CI 34-69) with the median duration of complete response yet to be reached. Adverse events were mild and included grades 1-2 injection site reaction (34 [97%] of 35), fatigue (28 [80%] of 35), chills (25 [71%] of 35), and fever (24 [69%] of 35). Interpretation PRGN-2012 treatment resulted in complete response in 51% of the patients treated and was safe. Based on these positive pivotal study results, a biologics license application to the US Food and Drug Administration (FDA) is planned, positioning PRGN-2012 to be an FDA-approved medical treatment for adult patients with RRP. Funding National Institutes of Health. Copyright Published by Elsevier Ltd.

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

LBA6015 Background: Recurrent respiratory papillomatosis (RRP) is a rare, neoplastic disorder caused by chronic infection with human papillomavirus (HPV) type 6 or 11. Significant morbidity can occur due to airway obstruction and, although rare, transformation into malignant cancer. There are no approved therapeutics. RRP is currently managed with frequent ablative procedures that can lead to irreversible laryngotracheal scarring and disability. PRGN-2012 is a gorilla adenovirus-based gene therapy/immunotherapy designed to generate HPV6/11-specific T cell immunity. The FDA has granted PRGN-2012 Breakthrough Therapy Designation for the treatment of RRP recognizing that this single arm study can serve as pivotal to support a licensing application. Methods: This pivotal trial (NCT04724980) evaluates the safety and efficacy of PRGN-2012 in patients with RRP requiring a minimum of 3 surgeries in the 12 months prior to treatment. Eligible patients received 4 subcutaneous (SC) injections of PRGN-2012 at dose level (DL) 1 (1x1011 Particle Units (PU) per injection; n=3) or DL2 (5x1011 PU per injection; n=35) over 12 weeks. The primary endpoint was the rate of complete response (CR), defined as no requirement for surgery in the 12 months following completion of treatment. Other key endpoints include duration of response, change in extent of papilloma growth (anatomic Derkay score) and vocal function. Results: PRGN-2012 was well-tolerated, with no serious adverse events, grade > 2 treatment-related adverse events, or early treatment discontinuations. The most common adverse events were injection-site reaction, fatigue, chills, fever, and myalgia. PRGN-2012 treatment at DL2 significantly (p<0.0001) decreased the requirement for surgery, with a median number of surgeries in the 12 month period decreasing from 4 (3-10) prior to treatment to 0 (0-7) surgeries post-treatment. Approximately 90% of patients experienced a decrease in the number of surgeries in the 12 months post-treatment compared to pre-treatment with PRGN-2012. The primary endpoint evaluation demonstrated a confirmed complete response in 17/31 (55%) of evaluable patients. The median duration of complete response has yet to be reached with a median follow up of 15 months (12 - 30) at the time of data cutoff (March 6, 2024). Additionally, PRGN-2012 treatment resulted in a significant reduction in Derkay score, improvement in vocal function and generation of HPV-specific immune responses. Conclusions: These data demonstrate the overall favorable safety profile and significant clinical benefit of PRGN-2012 in adult RRP patients. These findings support PRGN-2012 as a potential therapeutic option for this patient population where no FDA-approved therapeutics exist. Clinical trial information: NCT04724980 .

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.

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 .

This study investigated the role of IL-35 in systemic sclerosis (SSc) patients, focusing on CD4+ T cell response and immunomodulatory cytokine production. By comparing the cytokine levels in healthy donors (HD) and SSc patients using ELISAs, we found a significantly lower plasma IL-35 concentration in the SSc patients (52.1 ± 5.6 vs. 143 ± 11.1, p < 0.001). Notably, the IL-35 levels showed a negative correlation with TGF-β (p < 0.001) and IL-17 (p = 0.04). Assessing the IL-35R expression across cell types in the SSc patients and HDs via flow cytometry, we found higher levels on monocytes (40.7 + 5.7 vs. 20.3 ± 1.9, p < 0.001) and lower levels on CD8+ T cells (61.8 ± 9.2 vs. 83.4 ± 0.8, p < 0.05) in the SSc patients. The addition of recombinant IL-35 to stimulated peripheral blood mononuclear cells reduced the IL-17+CD4+ T cell percentage (9.0 ± 1.5 vs. 4.8 ± 0.7, p < 0.05) and increased the IL-35+CD4+ T percentage (4.1 ± 2.3 vs. 10.2 ± 0.8, p < 0.001). In a Treg:Tresponder cell Sco-culture assay with HD and SSc samples, rIL35 decreased the cell proliferation and levels of IL-17A (178.2 ± 30.5 pg/mL vs. 37.4 ± 6.4 pg/mL, p < 0.001) and TGF-β (4194 ± 777 pg/mL vs. 2413 ± 608 pg/mL, p < 0.01). Furthermore, we observed a positive correlation between the modified Rodnan skin score (mRSS) and TGF-β (p < 0.001), while there was a negative correlation between mRSS and IL-35 (p = 0.004). Interestingly, higher levels of plasmatic IL-35 were detected in individuals with limited disease compared to those with diffuse disease (60.1 ± 8.0 vs. 832.3 ± 4.1, p < 0.05). These findings suggest that IL-35 exhibits anti-inflammatory properties in SSc and it may serve as a marker for disease severity and a therapeutic target.

The UltraCAR-T® platform, with decentralized overnight manufacturing, has shifted the autologous CAR-T manufacturing paradigm. This disruptive approach has been validated in clinical and pre-clinical studies for both hematologic and solid tumors. UltraCAR-T cells, manufactured using an overnight process with minimal ex vivo manipulation of autologous T cells. UltraCAR-T cells are engineered to co-express CAR, membrane bound IL-15, and kill switch genes using non-viral gene transfer via high-throughput UltraPorator™ system. Multigenic design and overnight manufacturing renders UltraCAR-T cells with enhanced in vivo expansion and persistence for durable antitumor activity. Chronic antigen stimulation from the tumor can lead to CAR T having an exhausted phenotype contributing to treatment failures. Next generation UltraCAR-T cells also incorporate a novel cell intrinsic blockade of PD-1, superseding the need for combination therapy with checkpoint inhibitors and mitigate classic T cell exhaustion that occurs from chronic stimulation, thereby expanding the therapeutic window for efficacy. Using mesothelin (MSLN) as an exemplar from the CAR-target library, we developed a robust in vitro and in vivo model of continuous tumor antigen exposure to evaluate antitumor activity of the UltraCAR-T cells. MSLN UltraCAR-T cells were generated with and without intrinsic PD-1 blockade. In vitro, UltraCAR-T cells with intrinsic PD-1 blockade demonstrated enhanced inflammatory cytokine expression and cytotoxicity at low effector:target ratios. In long-term co-culture assays with recurring challenge of MSLN+ PD-L1+ tumor cells every 2-3 days, UltraCAR-T cells with intrinsic PD-1 blockade showed sustained potent cytotoxicity which was superior to CAR-T cells lacking PD-1 blockade and in combination with αPD-1 antibody. Intrinsic PD-1 blockade markedly enhanced polyfunctionality of UltraCAR-T cells in the presence of MSLN+ PD-L1+ tumor. In xenograft solid tumor model, a single administration of MSLN UltraCAR-T cells manufactured using UltraPorator demonstrated significant antitumor efficacy. Blood analysis showed sustained downregulation of PD-1, robust antigen specific expansion and durable persistence with central memory/stem-cell memory as the dominant phenotype of UltraCAR-T in vivo. Rechallenging the mice, who became tumor-free after a single UltraCAR-T infusion, with tumor for a second time to simulate tumor relapse led to the significant reduction in tumor burden without additional UltraCAR-T treatment. Collectively, these pre-clinical data highlight the improved efficacy of intrinsic PD-1 blockade in next generation UltraCAR-T cells using non-viral gene delivery and an established rapid, decentralized manufacturing process. Citation Format: Giorgio Zenere, Carol Poortman, Afreen Sayed, Devi Gunasekera, Cheryl Bolinger, Pooja Chauhan, Jacques Plummer, Shamim Ahmad, Rutul R. Shah, Helen Sabzevari, Douglas E. Brough, Tim Chan. Next generation UltraCAR-T® cells with intrinsic checkpoint inhibition and overnight manufacturing overcome suppressive tumor microenvironment leading to sustained antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1791.

Recurrent respiratory papillomatosis (RRP) is a rare, debilitating neoplastic disorder caused by chronic infection with human papillomavirus (HPV) type 6 or 11 and characterized by growth of papillomas in the upper aerodigestive tract. There is no approved medical therapy, and patients require repeated debulking procedures to maintain voice and airway function. PRGN-2012 is a gorilla adenovirus immune-therapeutic capable of enhancing HPV 6/11–specific T cell immunity. This first-in-human, phase 1 study (NCT04724980) of adjuvant PRGN-2012 treatment in adult patients with severe, aggressive RRP demonstrates the overall safety and clinically meaningful benefit observed with PRGN-2012, with a 50% complete response rate in patients treated at the highest dose. Responders demonstrate greater expansion of peripheral HPV-specific T cells compared with nonresponders. Additional correlative studies identify an association between reduced baseline papilloma HPV gene expression, greater interferon responses and expression of CXCL9 and CXCL10 , and greater papilloma T cell infiltration in responders. Conversely, nonresponders were characterized by greater HPV and CXCL8 gene expression, increased neutrophilic cell infiltration, and reduced T cell papilloma infiltration. These results suggest that papilloma HPV gene expression may regulate interferon signaling and chemokine expression profiles within the tumor microenvironment that cooperate to govern clinical response to therapeutic HPV vaccination in patients with respiratory papillomatosis.

Background: Survival and integrity of the spiral ganglion is vital for hearing in background noise and for optimal functioning of cochlear implants. Numerous studies have demonstrated that supplementation of supraphysiologic levels of the neurotrophins BDNF and NT-3 by pumps or gene therapy strategies supports spiral ganglion survival. The endogenous physiological levels of growth factors within the inner ear, although difficult to determine, are likely extremely low within the normal inner ear. Thus, novel approaches for the long-term low-level delivery of neurotrophins may be advantageous. Objectives: This study aimed to evaluate the long-term effects of gene therapy-based low-level neurotrophin supplementation on spiral ganglion survival. Using an adenovirus serotype 28-derived adenovector delivery system, the herpes latency promoter, a weak, long expressing promoter system, has been used to deliver the BDNF or NTF3 genes to the inner ear after neomycin-induced ototoxic injury in mice. Results: Treatment of the adult mouse inner ear with neomycin resulted in acute and chronic changes in endogenous neurotrophic factor gene expression and led to a degeneration of spiral ganglion cells. Increased survival of spiral ganglion cells after adenoviral delivery of BDNF or NTF3 to the inner ear was observed. Expression of BDNF and NT-3 could be demonstrated in the damaged organ of Corti after gene delivery. Hearing loss due to overexpression of neurotrophins in the normal hearing ear was avoided when using this novel vector–promoter combination. Conclusion: Combining supporting cell-specific gene delivery via the adenovirus serotype 28 vector with a low-strength long expressing promoter potentially can provide long-term neurotrophin delivery to the damaged inner ear.

Pulmonary fibrosis involves various types of immune cells and soluble mediators, including TGF-β and IL-35, a recently identified heterodimeric cytokine that belongs to the IL-12 cytokine family. However, the effect of regulatory IL-35 may play an important role in fibrotic diseases. The aim of this paper is to explore the immunoregulatory role of IL-35 in the development of fibrosis in interstitial lung disease (ILD). To gain a better understanding of this issue, the concentrations of IL-35 and different profibrotic cytokines in fibrotic (F-ILD) and non-fibrotic (NF-ILD) patients by ELISA were compared to that of intracellular IL-35 and IL-17 on CD4+ T cells stimulated in the presence of BAL or with different ratios of recombinant IL-35 (rIL-35) and TGF-β (rTGF-β), which were evaluated by flow cytometry. We observed that BAL concentration of IL-35 was lower in F patients (p < 0.001) and was negatively correlated with concentrations of TGF-β (p < 0.001) and IL-17 (p < 0.001). In supplemented cell cultures, BAL from NF but not F patients enhanced the percentage of IL-35 + CD4+ T (p < 0.001) cells and decreased the percentage of IL-17 + CD4+ T cells (p < 0.001). The percentage of IL-35 + CD4+ T cells correlated positively with BAL concentration of IL-35 (p = 0.02), but correlated negatively with BAL concentrations of IL-17 (p = 0.007) and TGF-β (p = 0.01). After adjusting the concentrations of recombinant cytokines to establish a TGF-β: IL-35 ratio of 1:4, an enhanced percentage of IL-35 + CD4+ T cells (p < 0.001) but a decreased percentage of IL-17 + CD4+ T cells (p < 0.001) was observed. After adding recombinant IL-35 to the BAL from F patients until a 1:4 ratio of TGF-β: IL-35 was reached, a significantly increased percentage of IL-35 + CD4+ T cells (p < 0.001) and a decreased percentage of IL-17 + CD4+ T cells (p = 0.003) was found. These results suggest that IL-35 may induce an anti-fibrotic response, regulating the effect of TGF-β and the inflammatory response on CD4+ T cells. In addition, the TGF-β: IL-35 ratio in BAL has been shown to be a potential biomarker to predict the outcome of F patients with ILD.

Current chimeric antigen receptor (CAR)-T manufacturing utilizes viral vectors and extensive ex vivo expansion at large central facilities leading to an exhausted CAR-T phenotype, high costs and long vein-to-vein times. While allogeneic CAR-T can reduce delays in patient treatment, they require extensive manipulation of donor cells, severe lymphodepletion and demonstrate short persistence limiting their therapeutic window. The UltraCAR-T® platform is designed to overcome these limitations by utilizing non-viral multigene delivery and a rapid, decentralized manufacturing process without ex vivo activation or expansion of T cells. Patient’s own T cells are collected and manufactured at the medical center and re-infused one day after gene transfer. Here we describe the next generation UltraCAR-T platform that addresses the inhibitory tumor microenvironment by incorporating a novel mechanism for intrinsic downregulation of one or more checkpoint inhibitor (CPI) genes. Our design achieves intrinsic CPI blockade without gene editing and is aimed at avoiding systemic toxicity and the high cost of combining CPI antibodies. Next generation UltraCAR-T cells simultaneously express CAR, membrane bound IL-15 (mbIL15), kill switch, and incorporate intrinsic CPI blockade. To illustrate the ability of this platform, we designed exemplary non-viral transposons to generate UltraCAR-T cells against multiple tumor targets incorporating intrinsic blockade of either one (PD-1) or two (PD-1 and TIGIT) CPI genes. Healthy donor T cells were transfected using the UltraPorator™ electroporation system to manufacture UltraCAR-T cells without ex vivo activation or expansion. The co-expression of CAR, mbIL15 and kill switch was confirmed by flow cytometry and western blot. Activated UltraCAR-T showed significant reduction in CPI gene expression compared to control CAR-T cells lacking the CPI blockade and did not show unintended off-target activity. Downregulation of CPI gene(s) on UltraCAR-T enhanced cytotoxicity and inflammatory cytokine production, especially at low effector to target (E:T) cell ratios, when co-cultured with PD-L1+/CD155+ tumor cells. Single-cell cytokine proteomics showed significant increase in polyfunctionality of UltraCAR-T with intrinsic downregulation of CPI gene(s). In vivo, a single infusion of receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific UltraCAR-T with intrinsic PD-1 blockade resulted in rapid expansion, an increase in preferred T cell memory (TSCM/TCM) populations, and significantly improved overall survival of ROR1+ PD-L1+ tumor bearing mice. These preclinical data highlight the improved efficacy of incorporating intrinsic CPI blockade in UltraCAR-T cells using non-viral gene delivery and an established rapid, decentralized manufacturing process. Citation Format: Tim Chan, Cheryl Bolinger, Sean Scott, Mengyan Du, Carol Poortman, Byron Koenitzer, Taranjit Athwal, Lindsey Shepard, R. Daniel Slone, Shourik Dutta, Steven Zilko, James M. Dunleavey, Giorgio Zenere, Jacques Plummer, Bernward Klocke, Christian Zinser, Shamim Ahmad, Douglas E. Brough, Rutul R. Shah, Helen Sabzevari. Incorporation of intrinsic checkpoint blockade enhances functionality of multigenic autologous UltraCAR-T® cells manufactured using non-viral gene delivery and rapid manufacturing process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2821.

Traditional methods for chimeric antigen receptor (CAR) T manufacturing utilize viral vectors, ex vivo activation and expansion of T cells to achieve clinically relevant cell numbers, which leads to an exhausted T cell phenotype, high manufacturing costs, and treatment delays. The UltraCAR-T platform is designed to overcome these limitations using our advanced non-viral gene delivery system and a rapid, overnight manufacturing process (Blood 2019 134 (Supplement_1):2660; Blood 2020 136 (Supplement 1):17); Cancer Research 2020 80 (16Suppl):6593). UltraCAR-T cells, which express antigen specific CAR, membrane-bound IL-15 (mbIL15), and kill switch genes, are manufactured at the medical center's cGMP facility using autologous T cells and administered back to the patient only one day after gene transfer. UltraCAR-T cells are currently under clinical investigation for hematological (NCT03927261) and solid tumors (NCT03907527).

TPS6092 Background: R/M HPVC (cervical, anal, oropharyngeal, etc.) are incurable by current therapies. For newly diagnosed LA HPV-OPSCC standard-of-care (SOC) is radiotherapy ± chemotherapy (C/RT) or surgery ± adjuvant C/RT, with considerable risk of relapse. Newly diagnosed LA SNSCC treatment follows the OPSCC paradigm, and detection of HPV appears to confer improved prognosis. Neoadjuvant PD-1 immune checkpoint blockade (ICB) before surgery may improve RFS and is being evaluated in a multicenter phase III clinical trial (Keynote-689). PRGN-2009 (P) is a novel gorilla adenovirus vaccine containing 35 non-HLA-restricted epitopes of HPV 16 and 18 shown to induce HPV specific responses (preclinical models). Bintrafusp alfa (BA) is a bifunctional fusion protein targeting TGF-β and PD-L1 with promising activity in HPVC. This trial will evaluate the safety and activity of P/ P + BA in patients with previously treated R/M HPVC and as neoadjuvant/induction therapy before SOC surgery or C/RT in newly diagnosed LA HPV-OPSCC and HPV-SNSCC. Methods: This is a first-in-human, investigator-initiated, single-center phase I/II trial. Pts with previously treated (incl. ICB) R/M HPVC are eligible for Phase I: P dose escalation arm (3+3 design, 6-12 patients) testing 2 dose levels (1x1011, 5x1011 viral particle units, SC Q2W three times, then Q4W), and combination arm (10 patients) testing P (recommended phase 2 dose (RP2D), same schedule) + BA (1200 mg IV Q2W). Treatment (both arms) will continue until disease progression, unacceptable toxicity, decision to withdraw. Primary endpoint is safety. Secondary endpoints include ORR (RECIST 1.1), PFS, and OS. For Phase II, patients with newly diagnosed stage II/III (AJCC Cancer Staging Manual, 8th ed.) HPV-OPSCC and stage II/III/IVA/IVB HPV-SNSCC planned for SOC C/RT or surgery will be eligible for two treatment arms of 20+2 patients each (sequential): P arm and P + BA, to evaluate the treatment activity. All patients will have pre-treatment biopsy, receive two cycles of the study treatment at the NCI Clinical Center two weeks apart, followed by post-treatment biopsy and SOC treatment (at the referring institution) 4 weeks after the first study treatment. Primary endpoint is post-treatment ≥2-fold increase in tumor-infiltrating CD3+ cells. Secondary endpoints include RFS, OS. Exploratory endpoints for both arms include analyses of immune subsets, soluble factors, and HPV-specific immune responses in peripheral blood and tissue where available, and in Phase II sequencing (exome, scRNA), immune spatial profiling with multiplex immunofluorescence, and salivary HPV DNA. Clinical trial registry: NCT04432597. Clinical trial information: NCT04432597.

There are approximately 44,000 cases of human papillomavirus-associated (HPV-associated) cancer each year in the United States, most commonly caused by HPV types 16 and 18. Prophylactic vaccines successfully prevent healthy people from acquiring HPV infections via HPV-specific antibodies. In order to treat established HPV-associated malignancies, however, new therapies are necessary. Multiple recombinant gorilla adenovirus HPV vaccine constructs were evaluated in NSG-β2m-/- peripheral blood mononuclear cell-humanized mice bearing SiHa, a human HPV16+ cervical tumor, and/or in the syngeneic HPV16+ TC-1 model. PRGN-2009 is a therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T cell enhancer agonist epitopes. PRGN-2009 treatment reduced tumor volume and increased CD8+ and CD4+ T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6-specific T cells, and increased multifunctional CD8+ and CD4+ T cells in the tumor microenvironment. These studies provide the first evaluation to our knowledge of a therapeutic gorilla adenovirus HPV vaccine, PRGN-2009, showing promising preclinical antitumor efficacy and induction of HPV-specific T cells, along with the rationale for its evaluation in clinical trials.

BackgroundPRGN-2009 is a novel gorilla adenovirus vaccine containing 35 non-HLA-restricted epitopes of HPV 16 and 18 which is being tested in an open-label, NCI-sponsored, single-center Phase I/II study alone and combined with the bifunctional TGF-β ”trap”/anti-PD-L1 fusion protein bintrafusp alfa (BA) (NCT04432597).MethodsFor the Phase I of the trial, eligible patients are adults with previously treated (checkpoint blockade allowed) recurrent/metastatic HPV-associated cancers. Objectives are to assess the safety and determine the recommended phase 2 dose (RP2D) of PRGN-2009 alone and combined with BA. Treatment followed a single-agent 3+3 dose escalation at two dose levels of PRGN-2009 (dose level 1: 1x1011 viral particle units (VPU), dose level 2: 5x1011 VPU) subcutaneously Q2W for 3 times, then Q4W for up to one year in total. After determination of RP2D, a combination cohort of 10 patients treated with PRGN-2009 at the RP2D combined with BA (1200 mg IV Q2W for 52 weeks) opened. Peripheral blood mononuclear cells collected from patients before and after vaccination with PRGN-2009 were stimulated with overlapping peptide pools and assessed by intracellular cytokine staining to identify HPV-16 and HPV-18 specific T-cells, as well as T-cells targeting cascade antigens not encoded by the vaccine.ResultsSix patients were enrolled in the single-agent PRGN-2009 dose-escalation phase (3 with cervical cancer, 2 with anal cancer, 1 with vaginal cancer). Observed adverse events were Grade 1-2 flu-like syndrome (headache, body aches), injection site reactions (erythema, pruritus, soreness, localized edema), fatigue, and rash. There were no dose limiting toxicities, and 5x1011 VPU was selected as RP2D. Four patients had stable disease as best response, (one ongoing, 10 months on treatment).T-cells targeting HPV-16 and/or HPV-18 were increased after vaccination in 100% of patients, with 3/6 (50%) developing HPV-16 T cells and 5/6 (83%) developing HPV-18 T cells. In some patients, the magnitude and breadth of HPV-16 and HPV-18 specific T cells were notably increased after repeated vaccination. T cells that target the cascade antigens brachyury and MUC1 were also increased in all patients evaluated. Multifunctional T-cell responses against all these antigens were also developed after vaccination in the majority of patients. No differences in immunogenicity were noted between the two dose levels. Enrollment is underway in combination with BA. Updated data will be presented.ConclusionsThe Phase 1 results demonstrate the safety of single-agent PRGN-2009 and induction of anti-HPV T-cell immune responses, supporting the hypothesis that PRGN-2009 could potentially induce anti-tumor effects in HPV-associated cancers.AcknowledgementsThis research was supported in part by the Intramural Research Program of the NIH, NCI.Trial RegistrationNCT04432597Ethics ApprovalApproved by the NIH IRB (Ref No 543876). All participants have given informed consent before taking part in the study.

Activation of antigen-specific T-lymphocyte responses may be needed to cure disorders caused by chronic infection with low-risk human papillomavirus (lrHPV). Safe and effective adjuvant therapies for such disorders are needed. The safety and efficacy of a novel gorilla adenovirus vaccine expressing a protein designed to elicit immune responses directed against HPV6 and HPV11, PRGN-2012, was studied using in vitro stimulation of T lymphocytes from patients with recurrent respiratory papillomatosis, in vivo vaccination studies, and therapeutic studies in mice bearing tumors expressing lrHPV antigen. PRGN-2012 treatment induces lrHPV antigen-specific responses in patient T lymphocytes. Vaccination of wild-type mice induces E6-specific T-lymphocyte responses without toxicity. In vivo therapeutic vaccination of mice bearing established HPV6 E6 expressing tumors results in HPV6 E6-specific CD8+ T-lymphocyte immunity of sufficient magnitude to induce tumor growth delay. The clinical study of PRGN-2012 in patients with disorders caused by chronic infection with lrHPV is warranted.

ABSTRACTFactors influencing the damage to the inner ear can include the surgical approach used for vector delivery, the volume of vector used, the buffer that the vector is suspended in as well as the host response to the vector capsid and vector genes that are transferred. We evaluated the effect of Ad5 capsid adenovectors on hearing function after delivery to the perilymph of adult C57Bl/6 mice. Hearing was evaluated before surgery and 3 days post‐surgery by auditory brain stem response (ABR) and distortion product otoacoustic emissions (DPOAE). A second group of mice underwent repeated delivery of adenovector two times to determine if a preliminary exposure to an Ad vector could induce an inflammatory response leading to further loss. The first adenovector (Ad.11D.LacZ) was delivered to the posterior semicircular canal or via round window. In the second surgery, a second adenovector (Ad.11D.gfp) was delivered to the horizontal semicircular canal. The functional outcome was tested prior, 7 days post first vector delivery, and 3 days post second vector delivery via ABR and DPOAE. Dual delivery via the semicircular canals resulted in preservation of hearing suggesting that pre‐exposure to Ad5 capsid does not predispose to hearing loss. Delivery to the round window resulted in hearing loss that was worsened after the second vector delivery, suggesting that delivery route and prior injury to the inner ear rather than the repetition of delivery predisposes to further hearing loss. Anat Rec, 303:600–607, 2020. © 2019 American Association for Anatomy

In the aggregate, our mouse studies suggest that GAd is a promising gene therapy vector that utilizes lung ECs as a source of therapeutic payload production and a highly desirable toxicity profile. Further genetic engineering of the GAd capsid holds the promise of in vivo vector tropism modification and targeting.