March 5, 2014

"...systemic immunity induced by PV-10 tumor ablation"

An abstract of Moffitt Cancer Center's work, led I think by Dr. Amod Sarnaik, MD, a surgical oncologist, and Dr. Shari Pilon-Thomas, Ph.D., a medical researcher, to better understand PV-10's immunological activity through a Phase 1 feasibility study and be presented via poster at the 2014 annual meeting of the American Association for Cancer Research ("AACR"), was revealed today. According to, "[t]he main purpose of this study is to find out more about how PV-10 works in melanoma tumors. Researchers also want to find out if there are changes in the body's immune cells (cells that fight infection and illnesses) after PV-10 is given, both inside the melanoma tumors and circulating in the blood."

At the 2013 AACR annual meeting last April, Moffitt's then abstract related to their murine model work on PV-10 concluded: "In total, these studies support the induction of tumor-specific T cell-mediated immunity after single treatment with IL-PV-10 in multiple histologic subtypes." The upshot of this appeared to me to be:
Multiple histologic subtypes. I don't think melanoma is one disease but rather has multiple histologic subtypes. Courtesy of a fellow shareholder and internist: "Interestingly, Rosenberg’s comments in a recent editorial makes the case for nurturing in vivo adaptive T-cell immunity such as PV-10 triggers. Adaptive as in dynamically adapt to the continuous mutations that occur, not try to identify, adopt and try to clone T-cells responding to some random tumor mutation." Rosenberg is Dr. Steven Rosenberg of the National Cancer Institute.

"All cancers contain multiple unique mutations and future progress in cancer gene therapy will likely result from the immunologic targeting of these mutated proteins. Melanomas contain an unusually large number of mutations probably due to the mutagenic action of ultraviolet light on the skin. The unique ability of TILs from melanoma to cause durable cancer regressions appears to result from attack against these individual cancer mutations. Smoking-induced lung cancers and cancers in patients with mutations in mismatch repair genes also have large numbers of sporadic mutations. The successful attack against unique antigens on melanoma suggests that TILs or gene-modified lymphocytes recognizing unique mutations on other cancers might be effective for use in therapy as well. Most other solid cancers, however, have anywhere from one-fifth to one-tenth the number of mutations compared with those present in melanoma and the targeting of these mutations represents a daunting problem. T cells reacting with sporadic or driver mutations on most cancers are likely to be infrequent though techniques for the identification of unique exomic mutations on individual cancers and the development of methods for obtaining T cells against them are rapidly being developed." Source: Cancer Gene Therapy (2014) 21, 45–47; doi:10.1038/cgt.2014.3, Finding suitable targets is the major obstacle to cancer gene therapy, Steven A Rosenberg).

Moffitt noted last year (2013) their ongoing work was to investigate the immune mechanism of PV-10 ablation in cutaneous melanoma patients (enrolled in the center's Phase 1 feasibility study).

This year (2014), Moffitt concluded: "In sum, these clinical and preclinical results increase our understanding of the cytotoxic and immunological mechanisms that may play a role in systemic immunity induced by PV-10 tumor ablation." The upshot of this appears to me to be:
  • Ablation of tumors following PV-10 injection (mechanism #1 [cytotoxic mechanism] of action #1 [destroy injected tumors]),
  • Leads to immunity around the body (mechanism #2 [immunological mechanism] of action #2 [destroy non-injected tumors]).
My further thoughts related to Moffitt's 2014 abstract:
Immunotherapeutic strategies incorporating intralesional (IL) ablative therapy to elicit a tumor specific immune response are under investigation as a non-surgical option to induce tumor regression of cutaneous neoplasms.
[My] takeaway: "Non-surgical option" is a translational outcome from Moffitt's work. By injecting PV-10 into tumors, turn un- or non-resectable melanoma into resectable. Make larger amounts of resectable disease much less before resecting it. Eliminate lesser amounts of disease without the need for resection or excision (surgery) at all.
Rose Bengal (RB) is a water-soluble xanthene dye that was originally used as an intravenous liver diagnostic and is in use by ophthalmologists to stain damaged cells in the eye. In murine models of breast cancer and melanoma, we have shown that IL injection of PV-10 (10% RB in saline solution) leads to ablation of injected tumors and regression of non-injected bystander tumors. In these models, increased anti-tumor T cell responses were measured, supporting the induction of systemic anti-tumor immunity after tumor ablation with PV-10.
Takeaway: Confirmation of Moffitt's murine model work (for both melanoma and breast cancer) by the cancer center's human study results of local and systemic efficacy and results (for melanoma). This repeatability, from mice to humans, is consistent with Craig, Tim and Eric's ("Provectus'") repeatability experience in cell lines to mice to higher-level animals (like horses and dogs) to human clinical trials and the compassionate use program. It would appear Moffitt fully reproduced Provectus' work. Repeatability.
In our ongoing phase I clinical trial exploring melanoma regression in patients, IL PV-10 has led to a significant decrease of melA positive melanoma cells in the biopsies of both PV10-injected and non-injected lesions.
Takeaway: I think more specific confirmation of local and systemic efficacy by virtue of said cell decreases in injected and non-injected lesions, respectively.
This regression correlated with increased circulating CD3+T cells (p=0.03) in peripheral blood mononuclear cells (PBMC).
Takeaway: I think this confirms a primary immune response derived from PV-10 by virtue of more circulating CD3+ T-cells (naïve T-cells, I believe). I think a high or higher levels of this metric corresponds with a potent (or more potent) immune response. Update 3/6/14: As I later was informed, the CD3+ protein group is associated with the T-cell receptor. "The T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells) that is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules." It's a very specific marker for T-cells.
T cells purified from PBMC from a melanoma patient produced increased IFN-gamma in response to autologous tumor after treatment with PV-10.
Takeaway: I believe this means feasibility study patients had tumor tissue banked from a resection or excision prior to PV-10 treatment. The patient then was treated with PV-10. Blood subsequently taken from him or her showed reactivity to previously resected tumor tissue; that is, Moffitt could specifically target the previously resected tumor tissue by using immune cells isolated from post-PV-10 treatment-drawn blood. I imagine if the human work was consistent with murine model work, blood drawn pre-PV-10 treatment would not have the same effect (i.e., it would not react).
Ex vivo models implemented to investigate this phenomenon indicate that the cytotoxicity induced by PV-10 is not apoptosis-dependent as evidenced by Annexin staining of melanoma cells following PV-10 treatment.
Takeaway: I think this is confirmation that while PV-10's ablation is apoptosis-like, it is not apoptosis-dependent. Management has previously described this cytotoxic mechanism as eliciting cell destruction naturally (a form of cell death that mimics both features of necrosis and apoptosis).
PV-10 directly induced necrosis of melanoma cells at 50 uM, but was not toxic to healthy fibroblasts at the same dose.
Takeaway: I think this is confirmation PV-10 targets diseased tissue only, and spares healthy tissue. Again, aspects of the drug's value proposition management has described for quite a while.
Further preclinical translational testing has shown that treatment of murine B16 cells with PV-10 leads to release of HMGB1, a soluble Damage Associated Molecule Pattern (DAMP) that is important for activation of dendritic cells (DCs). In the murine B16 melanoma model, there is a significant increase in the number of DCs infiltrating the tumor-draining lymph nodes after IL injection of PV-10. These findings suggest that PV-10 treatment leads to the release of DC activating factors and DC recruitment. Further studies to determine the role of PV-10 on T cell activation are ongoing.
Takeaway: Returning to their murine model work not previously presented or detailed, I believe, the potential for DC activation through HMBG1 release. In Moffitt's July 2013 PLoS paper, they hypothesized large amounts of tumor debris created by the rapid ablation that followed PV-10's injection into tumors, debris containing antigens (thus, large amounts of debris should mean large numbers of antigens), are taken up by DCs (see blog post, red underlined portion 4 in paragraph 3). DCs, antigen-presenting cells, present (display) antigens they've taken up to the body's immune system's T-cells. It's all about the T-cells. For example, "Cytotoxic T cells ("CTLs") specific for tumor antigens play a major role in the immunity against cancer." Will Moffitt show PV-10 specifically activates these CTLs?

I would presume this aspect of their human work, the confirmation of DC activation in patients in the feasibility study, will make-up their next presentation somewhere sometime (ASCO 2014?, a journal article?, another medical conference?).
In sum, these clinical and preclinical results increase our understanding of the cytotoxic and immunological mechanisms that may play a role in systemic immunity induced by PV-10 tumor ablation.
Takeaway: Moffitt now knows more (way more?) about PV-10's dual MOAs. PV-10's rapid tumor ablation leads to body-wide (systemic) immunity.

As with the 2013, more data, information and detail should be available on the 2014 poster, which I would expect the company to press release and make available on April 7th (the poster session is on April 6th, a Sunday).

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