Advaxis

Precision for Medicine (Precision) developed and qualified two 17 color flow immunophenotyping assays to be used as pharmacodynamic biomarkers for Advaxis clinical studies in patients with Metastatic Non-Small Cell Lung Cancer treated with ADXS-503 alone and in combination with Pembrolizumab (Pembro). ADXS-503 (A503) is an off-the-shelf, attenuated Listeria monocytogenes (Lm)-based immunotherapy bioengineered to elicit potent T-cell responses against 22 tumor antigens commonly found in NSCLC. Pembrolizumab (Pembro) is a programmed death receptor-1 (PD-1)-blocking antibody approved for the treatment of advanced lung cancer. A503 and Pembro have complementary mechanisms of immune activation and reversal of immune tolerance. Here, we qualified two multi-color flow cytometry assays to quantify total PD-1 expression in cryopreserved peripheral blood mononuclear cells (PBMCs) from individuals that were treated either with A503 only or with A503 in combination with Pembro. The detection of free PD-1 and Pembro-bound PD-1 was achieved by co-staining a partially competing αPD-1 antibody (clone PD1.3.1.3) with a biotinylated αHu-IgG4 antibody. The robustness of the assay was demonstrated using a nine-point half-log serial dilution of Pembro, where the highest concentration was 10µg/mL and the lowest concentration was 0.001µg/mL, including a no drug control. The assay conditions were optimized for sensitivity, optimal signal:noise ratio, detection of free and drug bound receptor by titrating and testing various commercial αPD-1 antibody clones and tertiary reagents to detect biotinylated αHu-IgG4. The Pembro bound receptor was detected using a biotinylated αHu-IgG4 antibody, while the free receptors were quantified using a commercial αPD-1 antibody. The assay was able to quantify free and drug bound PD-1 in the intended immune cell types without compromising the staining of other cell surface and intra-nuclear markers. Majority of the evaluable patients, 6 out of 8, had increased counts of NK, CD4+ and CD8+ T-cells, including TCM, TEM and memory stem cells after the administration of ADXS-503 ± Pembro. PD1 expression on circulating CD4+, CD8+ and NK T-cells was also increased while PD-L1 expression was elevated in on-therapy tumor biopsies in some of these patients. Measuring total PD-1 in T-cells can be more challenging in patients on Pembrolizumab therapy as no known commercial non-competing αPD-1 antibody clones are available. This novel assay will facilitate the evaluation of total PD-1 expression as a pharmacodynamic biomarker in T-cells when PD-1 blockade is being used. These results also support that combination of ADXS-503 with PD-1 blockade could lead to enhancement of efficacy of anti-tumor immunotherapy. Citation Format: Venkat Mohanram, Natalya Belkina, Angelina R. Bisconte, Jonathan W. Goldman, Gregory J. Gerstner, Missak Haigentz, Thomas Stinchcombe, Balazs Halmos, Surya Vangala, Victor Kabala, Dinesh Simkhada, Cristiane Metran, Darren Davis, Megan Parsi, Andres A. Gutierrez, Deborah Phippard, Suresh S. Ramalingam. Evaluation of total PD-1 expression using multi-color flow cytometry in metastatic non-small Cell lung cancer patients treated with multi-neoantigen vector (ADXS-503) alone and in combination of pembrolizumab to assess T-cell & T-cell memory subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1671.

e21682 Background: ADXS-503 (A503) is an off-the-shelf, attenuated Listeria monocytogenes (Lm)-based immunotherapy bioengineered to elicit potent T cell responses against 22 tumor antigens commonly found in NSCLC (i.e., 11 hotspot mutations and 11 tumor-associated antigens, TAAs). Pembrolizumab (Pembro) is a programed death receptor-1 (PD-1)- blocking antibody with proven efficacy in NSCLC. A503 and Pembro have complementary mechanisms of immune activation and reversal of immune tolerance. Methods: This is a phase I study of A503 ± Pembro in patients (pts) with metastatic squamous or non-squamous NSCLC. Dose escalation with A503 alone has been tested at two dose levels (i.e., 1 and 5 x108 CFU) in Part A in pts refractory or intolerant to prior systemic therapy. In dose escalation Part B, A503 is being evaluated at the same dose levels (DLs) in combination with Pembro in pts with disease progression on Pembro. Part C will be a dose expansion cohort with A503 + Pembro as first-line treatment in the metastatic setting. A503 ± Pembro (200 mg) will be infused intravenously every 3 weeks until disease progression or limiting toxicity. Main endpoints include safety, tolerability and immune-correlative data. Results: Nine patients have been treated: 7 with A503-alone in Part A and two pts with A503+ Pembro in Part B-DL1. No pts in Part A experienced dose-limiting toxicities at the 2 DLs tested. Two Grade 3 adverse events occurred in one pt related to the infusion: hypertension and flu-like syndrome. A transient and manageable SAE (Grade 2 acute kidney injury) was possibly related to A503 alone at 5 x108 CFU. A503+ Pembro have been well tolerated in 2 pts in Part B-DL1. Four pts achieved a best overall response of stable disease, three in Part A and one in Part B. A503 alone induced immune responses in pts so far evaluated in Part A. Conclusions: ADXS-503 alone has demonstrated a manageable safety profile and immune responses in Part A. The 1x108 CFU was identified as the recommended phase II dose. Dose escalation with A503+ Pembro is ongoing and dose expansion in first line treatment is due to start shortly. Clinical trial information: NCT03847519.

2616 Background: ADXS-503 (A503) is an off-the-shelf, attenuated Listeria monocytogenes (Lm)-based immunotherapy bioengineered to elicit potent T cell responses against 22 tumor antigens commonly found in NSCLC (i.e., 11 hotspot mutations and 11 tumor-associated antigens, TAAs). Pembrolizumab (Pembro) is a programmed death receptor-1 (PD-1)- blocking antibody approved for the treatment of advanced lung cancer. A503 and Pembro have complementary mechanisms of immune activation and reversal of immune tolerance. Methods: A phase 1 study of A503 ± Pembro has been conducted in patients (pts) with metastatic squamous or non-squamous NSCLC. In dose-escalation part B, A503 was added-on to Pembro within 12 weeks of the first scan showing disease progression per RECIST criteria v1.1. Both, A503 (1 x10 8 CFU) and Pembro (200 mg) were infused by IV every 3 weeks until disease progression or limiting toxicity. The dose-escalation cohort has established safety, tolerability and immunogenicity of the combination therapy and it has been further expanded to evaluate efficacy (Goldman JW et.al., SITC 2020). Results: Nine pts have been treated and evaluated in Part B. Pembro + A503 combo has been well tolerated and without immune related AEs. Of the nine evaluable pts, one has achieved partial response (PR) and 3 stable disease (SD), yielding an overall response rate (ORR) of 11% and disease control rate (DCR) of 44%. Two patients have had clinical benefit for over 12 months (i.e., one PR and one SD) and both of them had been on Pembro therapy for 2 years before enrollment. The two other pts with SD have sustained it for almost 6 months thus far. Seven pts have been evaluated for immunogenicity. In all pts there was a transient release of pro-inflammatory cytokines and proliferation of cytotoxic- and memory-CD8+ T cells. Seven evaluable pts had antigen-specific T cells within 1-2 weeks after starting therapy and 4/7 showed antigen spreading. Conclusions: ADXS-503 as an add-on therapy to Pembro at disease progression has been well tolerated and it has induced antigen specific-T cell responses and durable disease control in 44% of pts. Part B cohort is currently enrolling additional pts to further explore the potential reversal of Pembro resistance with ADXS-503. Clinical trial information: NCT03847519.

Objective: Our goal was to evaluate the safety, tolerability, and efficacy of PD-L1 immune checkpoint blockade (durvalumab) alone or in combination with a tumor-selective vaccination (ADXS11-001) in patients with persistent-recurrent or metastatic cervical cancer (PRmCC) and metastatic HPV+ SCCHN in part A (dose escalation) or with PRmCC in part B (dose-expansion). Part B results are reported here.

Background Recent advances in the field of cancer immunotherapy have identified CD8 + T cell responses against tumor-specific mutations as a key driver of tumor regression and overall survival. ADXS-NEO is a personalized Listeria monocytogenes ( Lm )-based immunotherapy designed to target a patient’s mutation-derived tumor-specific neoantigens. The objective of this study is to demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific point mutations and control tumor growth by generating neoantigen-specific T cell responses using a pre-clinical mouse tumor model. Methods Whole-exome sequencing of the MC38 mouse tumor cell line identified 2870 unique non-synonymous mutations. The netMHCcons algorithm was used to predict 137 potential neoantigens. We validated 20 immunogenic neoantigens either by peptide immunization followed by ELISPOT or by the presence of CD8 + T cells recognizing the neoantigen peptide following checkpoint inhibitor treatment. Two ADXS-NEO vectors were constructed; Lm20, targeting 20 validated immunogenic neoantigens, and Lm19, targeting most of the non-validated NSMs. Results Both Lm19 & Lm20 significantly slowed tumor growth in C57BL/6 mice compared to control. An accumulation of ADXS-NEO-specific TILs was observed in tumor bearing mice treated with either Lm19 or Lm20. Examination of the tumor microenvironment in Lm19 or Lm20 treated mice revealed a decrease in the frequency and absolute number of Tregs, TAMs, MDSCs, and PD1 high exhausted CD8 + T cells as well as an increase in the frequency and absolute number of effector CD8 + T cells, relative to control. Conclusion ADXS-NEO is a potent immunotherapy capable of driving immune responses against tumor-specific mutations and leading to tumor control in mice.