2018:e1426519. Two recent studies [29, 30] demonstrated the ad vantage in anti -tumor activity in the use of the bifunctional agent vs. the use of a PD - 1/PD-L1 checkpoint in combination with a systemic TGF- β inhibitor. TGF -β is also known to alter human carcinoma cells from a more epithelial to a more mesenchymal phenotype and such tumor cells are more resistant to a range of therapies (Figure 5). In LT IB studies , TGF -β‒induced mesenchymalization of carcinoma cells was shown [31] to be reversed by M7824, rendering tumor cells more susceptible to chemotherapy and immune - mediated killing [26, 28, 31] The first-in-human tria l of M7824 (anti -PD-L1/TGF -βR2) was conducted at the CCR [32]. Nineteen heavily pretreated patients with solid tumors (non -melanoma) were treated; the MTD was not reached. M7824 was shown to saturate peripheral PD -L1 and sequester all released (acid released) plasma TGFβ1, -β2, and -β3 throughout the dosing period at >1 mg/kg (recommended phase 2 dose is 1200 mg or about 10 mg/kg) . There were signs of clinical efficacy across all dose levels (Figure 6 and Fi gure 7), including one ongoing CR (cervical cancer) , two durable PRs (pancreatic and anal cancers), one near PR (cervical) and two cases of prolonged stable disease. 16 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 5. Functions of M7824. 17 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 6. Phase I trial of M7824, spider plot of tumor responses and reduction of serum levels of TGF β1. 18 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 7. M7824 Phase I, responses in two patient s. In the Phase I study [ 32] 15/43 (34.9%) of patients with checkpoint naïve HPV associated malignancies had radiographic tumor responses according to RECIST 1.1 or iRECIST with M7824 (anti-PD-L1/TGF -βR2) therapy. Prior studies by others have shown approximately a 15% objective response in this population employing checkpoint inhibition therapy. In an ongoing phase II study ( NCT03427411) 3/22 (13.6%) patients with checkpoint refractory advanced HPV associated malignancies have had responses to M7824 which compares well to the less than 5% response rate seen in the literature with further checkpoint therapy. Sufficient peripheral blood mononuclear ce lls w ere available from 12 patients on the phase I trial to test for HPV -specific T -cell responses before and after M7824 treatment. Considering all post - therapy timepoints examined, 7 of 12 patients (58%) had an increase in HPV -specific T cells post - vs pre -M7824 therapy. Immune responses after 1 and 3 cycles of M7824 were compared between patients who developed a clinical response of stable disease or better vs those with progressive disease ( Figure 8). In clinical responders, HPV -specific T cells developed in 3 of 4 patients (75%) after 1 cycle of M7824 and in 4 of 4 patients (100%) after 3 cycles. On the other hand, in patients with progressive disease, HPV -specific T cells developed in only 1 of 5 pa tients (20%) after 1 cycle of M7824 and in 1 of 4 patients (25%) after 3 cycles (Supplementary Table 3). There was also an increased magnitude of HPV -specific T cells generated in clinical responders vs 19 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 nonresponders: clinical responders had on average 12- to 27- fold more HPV -specific T cells producing cytokines or positive for CD107a (LAMP1, a functional marker of T and NK cell activity) after 1 and 3 cycles of M7824 compared with nonresponders ( Figure 3A ). Another observation worth noting is that 2 of the 3 patients with HPV+ cancer who received prior lymphodepleting chemotherapy (e .g., cyclophosphamide, fludarabine) were assessed for immune responses post - vs pre -M7824 therapy, and neither had evidence of an HPV T -cell‒specific immune response. In contrast , in patients with HPV+ cancer who did not receive prior lymphodepleting chemotherapy, 6 of 9 (67%) had a positive T- cell‒specific immune response. Figure 8. Immune responses in HPV+ patients with disease control (BOR of SD, PR, or CR) vs nonresponders (BOR of PD) to M7824 therapy by intracellular cytokine staining following in vitro stimulation with a mixture of overlapping 15- mer peptide pools encoding HPV-16 E6 and E7, as previously described. 21 20 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 NHS -IL12 Immunocytokine (M9241) NHS -IL12 is a tumor t argeting immunocytokine that binds to DNA -histone in necrotic areas of tumor. Studies by others have shown the ability of radiolabeled NHS -IL12 to target murine and human tumors (Figure 9). Prior studies in the LTIB have shown the ability of this agent to elicit anti-tumor activity in multiple murine models , and in combination with anti -PD-L1 and with radiation and chemotherapy [ 33]. Gulley (GMB, CCR) and the L TIB conducted the Phase I dose escalation trial of the NHS -IL12 immunocytokine [ 34]. Due to known prior toxicities using a rec. Figure 9. NHS -IL12 Immunocytokine. (A) NHS76 is a fully human 2 nd generation TNT antibody bound to 2 murine IL -12 (p70) molecules. (B) a: Specific tumor targeting of transplanted lung carcinoma by the MAb NHS -IL12(mu). Control MAb BC1- IL12(mu). b: NHS -IL12 tumor targeting of nuclear DNA histones. IL-12 protein, multiple ascending dose levels were employed and the agent was administered every 4 weeks. Time -dependent increase in serum IFN -γ and subsequent rise in IL -10 increased with dose and varied greatly among patients ( Figure 10A). Increases in TCR diversity and TIL density in the TME following NHS -IL12 dosing were observed in those patients with elevation IFN -γ serum levels (Figure 10B). Toxicity was acceptable with an MTD of 16.8 mcg/kg and this total dose was also found to be safe when given split in half on an every 2 week schedule . Although no objective tumor responses were see n, of 30 patients with measurable disease, 15 had stable disease, with some durable (from 6‒30+ months). 21 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 10. (A) Diversity of IFN -γ spikes in sera among patients 3 days post -NHS -IL12 treatment for cycles 1 (C1) and 2 (C2): as examples, 2 patients with high (pts 1 and 2, red and blue) and low (pts 3 and 4, green and orange) levels are shown. (B) Changes in TCR clonal diversity in the tumor biopsy correlate with serum IFN -g responses post -NHS - IL12 treatment. (C) # of clones in tumor comprising the top 25% of the repertoire of patient 1 pre - and post -NHS -IL12, demonstrating an increase in TCR diversity in a high IFN-g responder after therapy. 22 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Our recent preclinical studies have also shown the enhanced anti -tumor effect of avelumab when used in combination with NHS -IL12 in syngeneic mice bearing an intravesical bladder tumor (Figure 11, left panel) and that this effect is mediated via an IFN -γ‒dependent mechanism. Studies in NSG -β2m‒/‒ mice bearing HTB1, a human bladder carcinoma, and reconstituted with human PBMC, also showed the enhanced anti -tumor effect of the combination that coincided with increased NK and CD8+ in the TME ( Figure 11, right panel ). Figure 11. Anti-tumor a ctivity of NHS -IL12 + anti -PD-L1 in NSG- β2m‒/‒ mice bearing human bladder cancer (HTB1). This further provided the rationale for an ongoing CCR led clinical study of avelumab in combination with NHS -IL12 ( NCT02994953) , currently evaluating safety of the combination. In addition, to the combination being found to be safe and well -tolerated, evidence of radiographic tumor responses has been observed. As an example, a patient with checkpoint refractory metastatic urothelial cancer with a large sacral mass and retroperitoneal adenopathy at enrollment has had an ongoing complete response for 18+ months. Combination Immunot herap ies In addition to harnessing the potential multi- functionality of M7824, we have conducted several combination studies employing this agent with other immune modulating agents . We have previously shown [ 27] enhanced anti -tumor activity when M7824 wa s combined with vaccine in EMT6, a metastatic breast carc inoma murine model ( Figure 12 ). 23 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 12. Combined effect of Ad- Twist vaccine and M7824 in EMT6 breast carcinoma model (Knudson, 2018) Furthermore, combining M7824 with NHS -IL12 also greatly enhanced anti -tumor activity in the EMT6 breast carcinoma model ( Figure 13 ). Tumor challenge studies showed the presence of immune memory in mice that received the combination of M7824 and NHS -IL12, which conferred protection against re -challenge with tumor. Similar results were also seen in the MB38 -CEA murine colorectal carcino ma model. Multimodal therapies were also carried out in mice bearing HPV + tumors. The combination of PDS0101 vaccine and M7824 showed significant anti -tumor efficacy in the NSG β 2m-/- humanized mouse model reconstituted with human PBMC and bearing human cervical HPV+ cancer (SiHa) subcutaneously ( Figure 14). Figure 13. Anti -tumor effect of M7824 + NHS -IL12 combination treatment in EMT6 . 24 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 14. Tumor growth in humanized mice bearing SiHa (human HPV+ cervical cancer) and treated with PBS control, PDS0101 vaccine, M7824, or PDS0101+M7824. In contrast to the previous clinical trials, where we saw anti- tumor efficacy of M7824 or PDS0101 alone in HPV -associated tumors, in this model there was no single therapy effect of M7824 or PDS0101, but the combination significantly decreased tumor burden. Furthermore, in C57BL/6J mice bearing TC -1 (a murine lung carcinoma line transfected wi th HPV16 E6 and E7 oncoproteins), which is a very aggressive model of HPV+ carcinoma, the combination of PDS0101 vaccine, M7824 and NHS -IL12 resulted in the greatest l evel of anti -tumor activity compared to the use of individual agents or dual combinations ( Figure 15 ). The use of the combination of PDS0101 vaccine, M7824 and NHS -IL12 also resulted in an intense influx of CD4+ and CD8+ T cells in tumors ( Figure 16 , upper panels and Figure 17 ), increased activation and proliferation of T-cells ( Figure 16, lower panels ), as well as an enhanced HPV specific T cell response measured by ELISpot not seen with lesser combinations ( Figure 18) . Treatment Tumor volume >200 mm3 PBS Control 6/12 M7824 4/12 PDS0101 5/12 PDS0101+M7824 1/12 25 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 15 A. Tumor growth in C57BL/6J mice bearing TC -1 HPV+ tumors, and treated with combinations of PDS0101, M7824, and NHS- IL12. Figure 15 B. Tumor control (low volume) in C57BL/6J mice bearing TC -1 HPV+ tumors, and treated with combinations of PDS0101, M7824, and NHS-IL12. Treatment Tumor Volume <300mm3 PBS Control 0/16 DOTAP 0/8 M7824 0/16 PDS0101 3/16 NHS 6/16 M7824+NHS 8/16 PDS0101+NHS 10/16 PDS0101+M7824 5/16 PDS0101+M7824+NHS 13/17 26 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 16. Increased T -cell infiltration by tumor weight, and increased T- cell activation and proliferation in combination treated mice. (Rumfield et al; unpublished) 27 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 17. Immunohistochemistry shows increased T -cell infiltration in C57BL/6J mice bearing TC-1 HPV+ tumors, and treated with the combination of PDS0101, M7824, and NHS - IL12. 28 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 Figure 18. PDS0101 generated HPV -specific T -cells, as measured by ELISpot. Table shows a summary of tumor volume and E7- specific IFN γ ELISpot for C57BL/6J mice bearing TC-1 HPV+ tumors, and treated with combinations of PDS0101, M7824, and NHS - IL12 1.2.3 Safety and E fficacy of I/O Combinations in Humans Table 3: Previously evaluated combinations of I/O agents demonstrating an acceptable safety profile. Avelumab (anti- PD-L1) + NHS -IL12 NCT02994953 CEA/ MUC1 vaccine + M7824 NCT03315871 Dose Rational e: In the phase I dose escalation trial of combination Avelumab (anti -PD-L1) + NHS -IL12, escalating doses of NHS-IL12 were evaluated at 8,12, and 16.8 mcg/kg given every 4 weeks in combination with standard dosing of Avelumab. The combination including NHS -IL12 at the highest dose evaluated (16.8 mcg/kg given every 4 weeks) was found to be well tolerated. The only DLT observed on the trial was a transient grade 3 AST/ALT elevation at NHS -IL12 at 12 mcg/kg but otherwise no other DLT was observed and the MTD was not reached. Therefore, Treatment Tumor Volume <300mm3 E7 ELISpot PBS Control 0/16 - DOTAP 0/8 - M7824 0/16 - PDS0101 3/16 + NHS 6/16 - M7824+NHS 8/16 - PDS0101+NHS 10/16 + PDS0101+M7824 5/16 + PDS0101+M7824+NHS 13/17 - IFNγ+ Spots per 2.5 x * * * * 29 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 for this protocol, NHS -IL12 was originally used at a dose of 16.8 mcg/kg every 4 weeks in combination with the standard phase II dose of M7824 of 1200 mg given every two weeks. While we observed no DLTs with the combination of NHSIL12 at 16.8 mcg/kg along with M7824 and PDS0101 in the six participant lead in safety cohort, after treating 19 participants on protocol we observed that the majority of participants (11/19; 57.9%) needed dose reductions of NHSIL12 when given in combination with PDS0101 and M7824 due mostly to grade 2 fever and flu like symptoms. At this point (with Protocol Amendment Version 12.11.2020) we lowered the starting dose of NHS -IL12 in this protocol from 16.8 mcg/kg to 8 mcg/kg to try and minimize tox icity. After evaluating a total of 39 patients on this protocol including 21 with starting dose of 16.8 mcg/kg and 18 patients with starting dose of 8 mcg/kg we observed what looks to be reduced efficacy in patients receiving 8 mcg/kg as a starting dose with only 3/18 patients responding with the 8 mcg/kg starting dose as compared to 11/21 with the 16.8 mcg/kg starting dose. Therefore, based on this observation that the possibility that a lowering starting dose of NHS -IL12 may reduce efficacy we are amending to allow treatment (effective with Protocol Amendment Version 09.20.2021) with a limited number of doses of NHS -IL12 at 16.8 mcg/kg (four doses total), followed by dosing at 8 mcg/kg every 4 weeks thereafter . This is based upon responding patients in the first 21 patients which received anywhere between 1 -4 doses of NHS -IL12 at 16.8 mcg/kg before dose reductions. 1.2.4 Immune Assays Multiple immune assays have been developed in the LTIB to better define the mechanism(s) involved in the use of specific novel agents, as monotherapy or in combination therapies, both for preclinical and clinical studies. In addition to a nalyses of biopsies, analyses of the peripheral immunome can provide valuable information in that multiple samples can be analyzed over the course of a given therapy vs. pre -therapy. The LTIB has now developed and employed [ 15, 16, 31, 35] a flow cytometry‒based assay that can analyze 123 immune cell subsets in human P BMC from one vial of processed PBMC (approximately 107 cells). This assay will be used in this clinical trial to detect multiple (n=32) subsets of CD4+ T cells, CD8+ T cells (n=29), Tregs (n=7), B cells (n=5), NK (n=20), NKT (n=4), DC (n=10), and MDSC (n=16) to better understand the role of each agent. We also plan to evaluate changes in TCR clonal diversity both in biopsies and the periphery. One example involves an ongoing first -in-human trial of the NHS -IL12 tumor -target ing immunocytokine . TCR diversity increased 6- 14‒fold in biopsies of patients with a high or intermediate IF N-γ response but were unchanged or decreased with a low IFN -γ response. These findings also correlated with TIL in biopsies. We are also currently employing NanoString analyses to identify a gene signature in biopsies and PBMC, pre - and post -treatmen t. The GMB will conduct multiplexed, multispectral imaging of FFPE tissue to evaluate multiple immune parameters within the TME before and after treatment in patients with available tissue. 1.2.5 Summary • We are proposing a novel I/O combination to help overcome multiple barriers in an effective anti -tumor immune response. o (1) HPV therapeutic vaccine targeting tumor associated antigens to prim e an anti - tumor immune response o (2) NHS -IL12 and M7824 to enable activated immune cells to work better in the TME 30 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 • Preclinical studies have shown that treatment with the combination of an HPV therapeutic vaccine, M7824 and NHS -IL12 leads to greater anti-tumor activity than any single or double combination of these agents 2 ELIGIBILITY ASSESSMENT AND ENROLLMENT 2.1 ELIGIBILITY CRITERIA 2.1.1 Inclusion Criteria 2.1.1.1 Subjects with cytologically or histologically confirmed locally advanced or metastatic HPV associated malignancies : • Cervical cancers ; • P16+ Oropharyngeal cancers ; • Anal cancers ; • Vulvar, vaginal, penile, and squamous cell rectal cancers; • Other locally advanced or metastatic solid tumors (e.g., lung, esophagus) that are known HPV+. 2.1.1.2 Subjects must have measurable disease, per RECIST 1.1.