James: I would like to add to Dr. Petit's statement. On July 25, 2018, the journal, VACCINES published an article on LM vaccines and the current thought about them. Advaxis is mentioned throughout the article and also appears to be the standard by which others are measured. There is a disclosure at the bottom of the article stating that Advaxis did pay for a part of the article but the author emphasized that Advaxis had nothing to do with the writing of the manuscript.
I've included the link for anyone who wants to read the entire article but have clipped some excerpts. I think that we are all understanding that finding treatments to allay or cure cancer is an overwhelming project and the fate of Advaxis now hangs in a very precipitous way. Perhaps Advaxis is going to continue to be a going concern by way of RS and dilution even in the face of good news - too many sharks want the blood despite the fact that they can kill the host. It's too late to sell and now time just to watch and be a teeny part of a science that may heal.
Review: VACCINES, Published – 25 July 2018
Listeria monocytogenes as a Vector for Cancer Immunotherapy: Current Understanding and Progress https://www.mdpi.com/2076-393X/6/3/48/pdf
p. 5: Both the LmddA and the XFL-7 strain are currently being evaluated in clinical trials by Advaxis Inc., Princeton, NJ, USA.
p. 7: In contrast, Lm vaccines manufactured by Advaxis Inc. are based on either the XFL-7 or the LmddA strains described above.
p. 8: Mostly trials showing ADXS...
p. 9: Most clinical trials studying AXAL have been in patients with cervical cancer. After a Phase I clinical trial demonstrated safety , multiple Phase II trials were initiated. Results from a Phase II trial in India comparing AXAL and AXAL plus cisplatin to a historical control were recently published . In this trial, patients with refractory cervical cancer received either three doses of AXAL or four doses of AXAL with five infusions of cisplatin. While median overall survival (OS) was equivalent, patients receiving AXAL or AXAL plus cisplatin experienced 1.5 to 2-fold increases in overall survival at 12 months (34.9%) and 18 months (24.8%) . In addition to combination with chemotherapy, AXAL is also being tested in combination with immune checkpoint inhibitors. Preclinical models using AXAL with aPD-1 inhibitors have demonstrated a reduction in Treg and MDSC abundance and an increased antitumor response compared to AXAL alone . A Phase I/II trial combining AXAL with the aPD-1 inhibitor durvalumab was initiated but briefly put on hold after a patient died of respiratory failure (ClinicalTrials.gov identifier NCT02291055). After an investigation, the FDA reinstated this trial and it is currently ongoing. Of great interest, AXAL was recently advanced into the double-blind, randomized Phase III AIM2CERV clinical trial, comparing AXAL to a placebo in patients with high-risk, locally-advanced cervical cancer (ClinicalTrias.gov identifier NCT02853604). This is currently the only Phase III trial testing an Lm-based vaccine. Patients in the AIM2CERV trial will receive chemoradiation with curative intent followed by multiple infusions of AXAL or placebo. Dosing of AXAL or the placebo is to occur every 3 weeks for the first 3 months, followed by an infusion every 8 weeks for a total of five doses or until disease recurrence. The primary endpoint will compare disease-free survival (DFS) in patients receiving AXAL or placebo and is expected to be met in June 2020.
While most trials with AXAL have been in cervical cancer, AXAL is also being pursued in patients with HPV-associated oropharyngeal, anal, and lung cancers. In oropharyngeal cancer, a Phase I trial was terminated early after a patient suffered a dose-limiting toxicity (ClinicalTrials.gov identifier NCT01598792). Despite this setback, a Phase II trial testing AXAL as a monotherapy in anal cancer continues to recruit patients (ClinicalTrials.gov identifier NCT02002182). In anal cancer, a Phase I trial studying AXAL in combination with radiation and the chemotherapies mitomycin and 5-FU was recently completed (ClinicalTrials.gov identifier NCT01671488). Of the nine patients who completed treatments, eight were progression-free at a median follow-up time of 42 months . Based on those results, a subsequent Phase II trial assessing AXAL as a monotherapy in anal cancer was initiated (ClinicalTrials.gov identifier NCT2399813). This trial is expected to be completed in March 2022.
In addition to AXAL, Advaxis is also pursuing a second-generation version called ADXS-DUAL in collaboration with Bristol-Myers Squibb. ADXS-DUAL possess E7 from both HPV 16 and HPV 18 serotypes. Although the majority of clinical trials by Advaxis have focused on AXAL, Advaxis is also advancing Lm vaccines against the tumor antigens PSA (prostate-specific antigen) and HER2 (human epidermal growth factor 2).
PSA is a serine protease whose expression is normally confined to the prostate. While its utility as a serum diagnostic marker in prostate cancer remains controversial, it is well-accepted that PSA can act as an immunotherapeutic target in prostate cancer . Early studies piloting tLLO-PSA vaccines were based on the XFL-7 strain of Lm; however, clinical studies use ADXS31-142 based on the LmddA strain. Both versions have been shown to modestly reduce tumor burden and the number of tumor-infiltrating Tregs in mice inoculated with the prostate cancer cell line TPSA23 [33,38]. Based on the hypothesis that radiation therapy may induce immunogenic tumor cell death and promote favorable inflammatory environments for T-cell activation, follow-up studies assessed ADXS31-142 in combination with radiation therapy [77,78]. While ADXS31-142 or radiation alone slowed TPSA23 tumor growth, combination therapy led to multiple instances of complete tumor regression . Synergistic antitumor responses were associated with significant increases in PSA-specific T cells in the spleen and tumor  as well as enhanced intratumoral Th1 responses . Recent reports have
demonstrated an additional therapeutic benefit by combining ADXS31-142 and radiation therapy with aPD-1 or aPD-L1 inhibitors .
Currently, ADXS31-142 is being tested in a Phase I/II trial in patients with metastatic castration-resistant prostate cancer. The trial (ClinicalTrials.gov identifier NCT02325557), also known as the KEYNOTE-046 trial, is assessing progression-free survival following ADXS31-142 alone or in combination with the aPD-1 antibody pembrolizumab. While the trial is not yet completed, an early report found that patients vaccinated with ADXS31-142 generated T-cell responses against multiple prostate antigens beyond PSA through a process known as epitope spreading . In this context, it is believed that PSA-specific T cells inducing prostate cancer cell death and the production of inflammatory cytokines result in the release of additional tumor antigens and induction of immune responses against additional epitopes, ultimately broadening the therapeutic scope of the vaccine.
HER2 is a receptor tyrosine kinase that is frequently overexpressed by cancers, including breast, salivary gland, and bone [79,80]. HER2 expression is often associated with poor prognosis, as HER2 signaling has been shown to directly activate pathways responsible for tumor growth and survival . While antibody-based therapies targeting HER2 have been developed, many patients still experience recurrence after treatment. In addition to antigen escape, antibody-based therapies against HER2 primarily induce antibody-dependent cytotoxicity in the absence of adaptive immune responses. Indeed, poor Th1 responses after anti-HER2 antibody therapy strongly correlated with recurrence, leading to speculation that vaccines boosting Th1 responses could improve the standard of care [81,82].
Due to its large molecular mass, early Lm-based HER2 vaccine designs consisted of five different Lm vaccines, with each expressing tLLO fused to a unique region of the rat HER2 extracellular or intracellular domain . Individually, each Lm strain antagonized the growth of the HER2 expressing NT-2 tumor cell line to a similar extent. Based on this proof of concept, a single Lm vaccine against human HER2, known as ADXS31-164, was subsequently developed [39,83]. ADXS31-164 is derived from the attenuated LmddA strain and is engineered to secrete a chimeric tLLO fusion protein containing immunogenic regions from two extracellular domain sequences and one intracellular domain sequence of HER2 . The chimeric HER2 protein is designed to encompass most of the known human MHC class I epitopes of the HER2 protein. Preclinical studies with this vaccine demonstrated the ability of ADXS31-164 to reduce the number of regulatory T cells in tumors and increase the CD8+ T-cell/Treg ratio . Impressive results with ADXS31-164 in preclinical studies has led to its advancement to clinical trials in canines and humans.
Recently, results from a Phase I trial of ADXS31-164 in canine osteosarcoma were published . In this trial, canines received three doses of ADXS31-64 following amputation and chemotherapy. Out of 18 immunized dogs, 15 developed T-cell responses against HER2. Impressively, this led to a significant reduction in metastatic disease and increase in survival. Canines vaccinated with ADXS31-164 had a median survival time of 956 days, and 56% were still alive three years after chemotherapy, compared to a historical control of 423 days and 22%, respectively . Canines with immune responses to the intracellular HER2 domain had the most robust effects on survival, potentially related to the fact that the intracellular HER2 domain is indispensable for oncogenic cell signaling . In contrast to HER2/neu antibodies, which are encumbered by adverse effects, including cardiotoxicity , ADXS31-164 was not associated with cardiotoxicity in canines . In 2014, Advaxis licensed the use of ADXS31-164 in canines to Aratana Therapeutics Inc. where it was renamed AT-014. The USDA, in early 2018, granted conditional clinical approval for the use of AT-014 in canine osteocarcoma. Currently, AT-014 is the only Lm vaccine to receive any clinical approval. While data from mice and dogs indicate promising potential, results from human trials have yet to be released. Currently, an ongoing Phase I/II trial is examining the use of ADXS31-164 in HER2 expressing solid tumors. This trial is expected to be completed in December of 2018.
Vaccines 2018, 6, 48 11 of 19
p. 12: 5.8. pLADD and ADXS-NEO
During tumorigenesis, cancer cells undergo genetic alterations which can lead to the cumulative production of mutated protein sequences not found in healthy tissues . These mutated proteins, termed neoantigens, may undergo proteasomal degradation and MHC loading, forming unique epitopes presented only by the tumor cells harboring the underlying mutations. In theory, vaccines targeting neoantigens may generate stronger T-cell responses due to the absence of tolerance mechanisms as well as fewer off-target effects . Based on this rationale, two Lm vaccines targeting personalized neoantigens, ADXS-NEO and pLADD, were recently approved for Phase I testing. Proof of concept for ADXS-NEO was demonstrated using CT26 and MC38 colorectal cancer cell lines, and this was presented at the American Association for Cancer Research annual meeting [93,94]. The development of personalized vaccines involved whole-exome sequencing to identify tumor-specific mutations followed by analysis with predictive algorithms to identify potential neoepitopes. After screening to verify immunogenicity, the neoantigens were cloned into Lm and found to induce tumor regression upon immunization [93,94]. A Phase I trial testing ADXS-NEO in patients with metastatic head and neck, colon, and lung cancer is underway (ClinicalTrials.gov identifier NCT03265080). The personalized Lm vaccine pLADD is similarly undergoing Phase I testing in patients with microsatellite stable metastatic colorectal cancer.
(ClinicalTrials.gov identifier NCT03189030). In addition to personalized vaccines, Advaxis is currently developing ADXS-HOT. Because sequencing for neoantigens is a time-consuming and expensive process, ADXS-HOT is designed to be an “off-the-shelf” therapy containing a collection of frequently-mutated somatic, cancer testis, and oncofetal antigens . However, the clinical details concerning this vaccine have not been released.
p. 13: 6. Conclusions and Future Directions
While preclinical models have shown impressive therapeutic benefits of Lm vaccines, currently, no Lm vaccines are FDA-approved. It is important to note that Lm vaccines are still relatively nascent, with most clinical trials only being initiated in recent years. As these trials advance, further improvements to Lm vaccine technology will likely be made in the coming years. Indeed, after initial clinical trials used Lm expressing a single tumor antigen, Lm vaccine technology has evolved to include vaccines expressing multiple tumor antigens. By targeting multiple antigens, it may be possible to induce therapeutic responses that are less susceptible to antigen escape. Yet, even vaccination against multiple tumor antigens may still not be sufficient to induce optimal therapeutic responses. Tumors consist of many cell types and the tumor microenvironment has been hypothesized to be a protective niche that may limit antitumor responses. Thus, many strategies moving forward are likely to combine Lm vaccines with approaches that remodel the tumor microenvironment. One such strategy has been to directly target angiogenesis using Lm vaccines. Indeed, Lm vaccines targeting tumor-associated angiogenic proteins, including CD105 and VEGFR2, have demonstrated inhibited tumor growth [97–100] as well as the ability to induce secondary antitumor immune responses via epitope spreading [98,99]. While exciting, these vaccines have not yet been tested clinically. Other studies are likely to explore combination approaches using Lm vaccines with radiation, chemotherapy, immune checkpoint inhibitors, and other drugs. These studies are expected to firmly establish the utility of Lm vaccines in the emerging immunotherapeutic landscape for the benefit of patients with a variety of malignancies.
Conflicts of Interest: The authors are supported, in part, by sponsored research projects from Advaxis Inc. Advaxis had no role in the preparation of this manuscript.