Immunity

Edited (by me) image above; original image source

Let us take as given, finally in and by 2016, that PV-10 (chemical small [but heavy] molecule and halogenated xanthene Rose Bengal) is an immunotherapy

That is, the investigational compound is potentially capable of inducing the human body's immune system to mount a possibly worthy defense against solid tumor cancer after first being injected into lesions and tumors of said cancer.

That PV-10 has been independently shown to be that immunotherapy, at arms length from Provectus separately by both Moffitt Cancer Center (Moffitt) and the University of Illinois at Chicago (UIC) — reproducibility, the hallmark of Western Science— should make the claims about and data on PV-10 that much more veracious. See footnotes 1-5 below.

But let's now examine the notion, and growing preclinical and clinical evidence, that PV-10 treatment — singularly or in combination — potentially may lead to something greater and more profound than just the inducement or generation of an immune response....immunity.

AACR 2016 (April), Moffitt: T cell mediated immunity after combination therapy with intralesional PV-10 and co-inhibitory blockade in a melanoma model

2016 (May) peer-reviewed publication, Moffitt: Intralesional rose bengal in melanoma elicits tumor immunity via activation of dendritic cells by the release of high mobility group box 1

SITC 2016 (November), Moffitt: Intralesional injection with Rose Bengal and systemic chemotherapy induces anti-tumor immunity in a murine model of pancreatic cancer



Footnotes of non-Provectus biomedical research for "PV-10 is an immunotherapy:"

1. SSO 2012, Moffitt: Intralesional Injection of Melanoma with Rose Bengal Induces Regression of Untreated Synchronous Melanoma In a Murine Model,
2. 2013 peer-reviewed publication, Moffitt: Intralesional Injection of Rose Bengal Induces a Systemic Tumor-Specific Immune Response in Murine Models of Melanoma and Breast Cancer,
3. SSO 2015, UIC: Intralesional Injection of Rose Bengal Induces an Anti-tumor Immune Response and Potent Tumor Regressions in a Murine Model of Colon Cancer,
4. 2015 peer-reviewed publication, UIC: The Potential of Intralesional Rose Bengal to Stimulate T-Cell Mediated Anti-Tumor Responses, and
5. ASO 2016, UIC: PV-10 Induces Potent Immunogenic Apoptosis in Colon Cancer Cells,

April 2016: Grant McArthur Discusses the Memory of Our Immune System

Could PV-10 (Rose Bengal) be implicated in different kinds of cell death?

Image source

Reference article: Garg et al., Immunogenic versus tolerogenic phagocytosis during anticancer therapy: mechanisms and clinical translation, Cell Death and Differentiation (2016) 23, 938–951.

N.B. There is no reference to Rose Bengal (generic name) or PV-10 (proprietary name) in the February 2015 Garg et al. article.

Article Abstract: Phagocytosis of dying cells is a major homeostatic process that represents the final stage of cell death in a tissue context. Under basal conditions, in a diseased tissue (such as cancer) or after treatment with cytotoxic therapies (such as anticancer therapies), phagocytosis has a major role in avoiding toxic accumulation of cellular corpses. Recognition and phagocytosis of dying cancer cells dictate the eventual immunological consequences (i.e., tolerogenic, inflammatory or immunogenic) depending on a series of factors, including the type of ‘eat me’ signals. Homeostatic clearance of dying cancer cells (i.e., tolerogenic phagocytosis) tends to facilitate pro-tumorigenic processes and actively suppress antitumour immunity. Conversely, cancer cells killed by immunogenic anticancer therapies may stimulate non-homeostatic clearance by antigen-presenting cells and drive cancer antigen-directed immunity. On the other hand, (a general) inflammatory clearance of dying cancer cells could have pro-tumorigenic or antitumorigenic consequences depending on the context. Interestingly, the immunosuppressive consequences that accompany tolerogenic phagocytosis can be reversed through immune-checkpoint therapies. In the present review, we discuss the pivotal role of phagocytosis in regulating responses to anticancer therapy. We give particular attention to the role of phagocytosis following treatment with immunogenic or immune-checkpoint therapies, the clinical prognostic and predictive significance of phagocytic signals for cancer patients and the therapeutic strategies that can be employed for direct targeting of phagocytic determinants.

Provectus says PV-10/Rose Bengal:

• Does not rely on a single pathway to work [I assume 'signalling pathway'],

• Does not focus on a single receptor to work [I assume 'cell receptor'], and

• Has no known resistance [I assume little no cancer drug resistance].

A highly specific compound in its targeting of only diseased (cancerous) tumors/lesions, tissue and cells, sparing healthy ones in the process, might the veracity of PV-10/Rose Bengal's "multiplicity" be based in its lack of "specificity" in regards to cell death?

That is, might Provectus' investigational compound's apparent implication in different kinds of cell death help explain why PV-10/Rose Bengal does not rely on a single pathway or focus on a single receptor to work, and has no known resistance?

Of note in Garg et al.'s article are (a) a table describing "major cell death pathways and their immunobiological" profiles and (b) a figure illustrating "therapeutic exploitation of phagocytosis of dying cancer cells for T-cell-mediated cancer cell elimination."

The table of major cell death pathways includes (i) apoptosis, (ii) autophagy (autophagic cell death) and (iii) immunogenic cell death.

Click to enlarge. Image source

The illustration appears to draw three paths to anti-tumor immunity; one that is direct (e.g., DAMPs like HMGB1), and two that additionally employ co-stimulatory signals such as TLR agonists or co-inhibitory signals such as immune checkpoint therapy.

Click to enlarge. Image source

A sampling of Provectus and independent medical researcher work implicates PV-10 in apoptosis, autophagy, necrosis, and immunogenic cell death:

• Provectus: "The patient who had chemotherapy radioresistant recurrent disease in the head and neck area had rapid necrosis of the injected metastasis and this as well as 3 uninjected sc metastases underwent complete remission over a period of several months," "Rose Bengal induces dual modes of cell death in melanoma cells and has clinical activity against melanoma," Melanoma Research 16(Suppl 1):S8, Sep 2006

• Provectus: "PV-10 accumulation elicits acute autophagy of cancer cells," "Chemoablation of Metastatic Melanoma with PV-10," Presentation at 4th Interdisciplinary Melanoma & Skin Cancer Centres Meeting, Sydney, AU, Nov 2010

• University of Illinois at Chicago: "Treatment of colon cancer cells with PV-10 induced cell cycle arrest, apoptosis, autophagy, and significant ER stress; consistent with immunogenic apoptosis," "PV-10 Induces Potent Immunogenic Apoptosis in Colon Cancer Cells," ASC 2016

• Quote by Provectus about Moffitt Cancer Center work: "This paper is a watershed event in the development of PV-10, walking the reader through all the steps of immune activation after PV-10 injection, from immunogenic cell death and signaling via release of HMGB1, dendritic cell recruitment and infiltration into draining lymph nodes, activation of tumor-specific T cells, and killing of uninjected tumors upon infiltration by these T cells." "Intralesional Rose Bengal in Melanoma Elicits Tumor Immunity Via Activation of Dendritic Cells by the Release of High Mobility Group Box 1," Oncotarget 2016.

Garg et al. observe "The mechanisms of cancer cell death elicited by anticancer therapy and the type of phagocytes (e.g., tumour-resident versus therapy-recruited) interacting with dying cells are decisive factors in making a difference between anti-inflammatory or pro-inflammatory responses."

Some cell death via PV-10 occurs in the injected lesion or tumor (i.e., tumor-resident), which is the upstream trigger of subsequent cell death via a tumor-specific immune response (i.e., therapy-recruited).

Moffitt: IL RB in melanoma elicits tumor immunity via activation of DCs by the release of HMGB1

Updated below, again.

Article link: Intralesional rose bengal in melanoma elicits tumor immunity via activation of dendritic cells by the release of high mobility group box 1

H. Lee Moffitt Cancer Center and Research Institute departments & facilities: Immunology, Flow Cytometry, Translational Science, Cutaneous Oncology, Pathology, and Cutaneous Data Management

Click to enlarge.

Takeaways:

These data/results:

• "...support the role of IL [intralesional] RB to activate dendritic cells at the site of tumor necrosis for the induction of a systemic anti-tumor immune response,"

• "...suggest that IL PV-10 can induce tumor-specific T cells with memory characteristics in M05 melanoma-bearing mice,"

• "...show that CD8+ T cells are crucial for the tumor-specific immune response induced by IL injection of PV-10,"

• "...support that IL injection of PV-10 can boost T cell infiltration in tumors,"

• "...support a role for IL PV-10 to induce DCs [dendritic cells] to take up antigens at the tumor site, infiltrate into the DLN [draining lymph node], and become functionally mature,"

• "...suggest that PV-10-treated tumors may release factors that activate DCs,"

• "...suggest that PV-10 can kill tumor cells at a dose that is not toxic to non-tumor cells,"

• "...support the role of IL PV-10 treatment to induce a systemic anti-tumor immune response in patients with metastatic melanoma," and

• "...support the design of additional clinical studies to measure anti-tumor immune responses after IL injection of PV-10 in patients with melanoma."

The Cancer Immunity Cycle & PV-10

Click to enlarge.

ABSTRACT

Intralesional (IL) therapy is under investigation to treat dermal and subcutaneous metastatic cancer. Rose Bengal (RB) is a staining agent that was originally used by ophthalmologists and in liver function studies. IL injection of RB has been shown to induce regression of injected and uninjected tumors in murine models and clinical trials. In this study, we have shown a mechanism of tumor-specific immune response induced by IL RB. In melanoma-bearing mice, IL RB induced regression of injected tumor and inhibited the growth of bystander lesions mediated by CD8+ T cells. IL RB resulted in necrosis of tumor cells and the release of High Mobility Group Box 1 (HMGB1), with increased dendritic cell (DC) infiltration into draining lymph nodes and the activation of tumor-specific T cells. Treatment of DC with tumor supernatants increased the ability of DCs to stimulate T cell proliferation, and blockade of HMGB1 in the supernatants suppressed DC activity. Additionally, increased HMGB1 levels were measured in the sera of melanoma patients treated with IL RB. These results support the role of IL RB to activate dendritic cells at the site of tumor necrosis for the induction of a systemic anti-tumor immune response.

RESULTS, summary of article subtitles

• IL PV-10 elicits a tumor-specific immune response
• IL PV-10 leads to DC activation
• PV-10 treatment increases DC activation via HMGB1
• IL PV-10 leads to HMGB1 increase in the sera of melanoma patients

DISCUSSION

Melanoma incidence rates have increased rapidly in the United States over the past 30 years and is the fifth most common cancer in men and the seventh most common cancer in women [38]. IL therapy is a promising treatment modality for patients with dermal and/or subcutaneous metastatic melanoma. Importantly, it may induce not only local tumor regression but also a systemic anti-tumor immune response. In a recent clinical trial in metastatic melanoma patients, IL PV-10 led to a
50% objective response rate with mild to moderate side effects [17]. In treated patients, 8% had no evidence of disease after 52 weeks and 26% experienced complete regression in bystander lesions. However, the mechanism by which IL PV-10 leads to systemic anti-tumor immunity is unknown.

In this study, we showed that IL PV-10 led to the necrosis of melanoma cells and the release of HMGB1. These data are consistent with the observation that HMGB1 was passively released from photosensitized HeLa cells treated with a Rose Bengal analog [39]. Pretreatment with Rose Bengal acetate led to apoptosis and autophagy and the secretion of HSP70, HSP90 and HMGB1. In contrast, our results showed that PV-10 treatment induced necrosis in melanoma cells and the secretion of HMGB1, but not HSP70, while the amount of HSP90 was unchanged. This discrepancy may be explained by differences in response to RB and its acetate analog, dose of test article, differences in the cell lines used, or mechanisms of ablative and photodynamic therapies. Moreover, HMGB1 levels in the sera of patients were increased after IL PV-10. This is in line with another study that showed increased HMGB1 levels in the serum of cancer patients after chemoradiation; notably, HMGB1
levels were increased in patients with antigen-specific T cell responses and higher expression of HMGB1 in resected tumor samples was correlated with better survival [40].

Maturation of DCs is crucial for priming CD8+ T cells [41]. HMGB1 has been shown to be important for activation of myeloid and plasmacytoid DCs [25, 31, 42–46]. In our model, DC maturation with up-regulation of CD40, CD80 and CD86 was measured in tumor draining LN after IL PV-10. Furthermore, our study showed that HMGB1 in the supernatant of tumor cells treated with PV-10 was responsible for the up-regulation of CD40 expression on BM-derived DCs and for the increased ability of DC to stimulate T cell activation. It has been shown that short-term CD40 signaling augments DC migration to tumor-draining LNs and induced protective immunity. Moreover, HMGB1 has been shown to enhance DC responses to CCL9 and CXCL12 [47]. Interactions between HMGB1 and RAGE can induce the migration of s.c. injected DCs into DLNs [48]. In our study, IL PV-10
increased the number of DCs migrating from the tumor site into the draining LNs.

In this study, we have shown a mechanism of tumor-specific immune response induced by IL PV-10.
In melanoma-bearing mice, IL PV-10 induced necrosis of tumor cells leading to the release of HMGB1, which is crucial for DC activation. This resulted in DC maturation and infiltration into draining LNs for the activation of tumor-specific T cells. Additionally, increased HMGB1 levels measured in sera of patients treated with IL PV10 suggests that HMGB1 may be involved in eliciting a systemic immune response in patients. We have shown that circulating T cell populations and tumor-specific CD8+ T cells are increased in melanoma patients after IL PV-10 therapy. Together these results support the design of additional clinical studies to measure anti-tumor immune responses after IL injection of PV-10 in patients with melanoma.

MATERIALS AND METHODS, Incl. Human subjects

Fifteen patients with dermal and/or subcutaneous metastatic melanoma were enrolled in a pilot study
(NCT01760499). Peripheral blood and serum were collected prior to biopsy, 7-14 days after IL PV-10 injection into a single melanoma tumor, and 21-28 days after IL PV-10 injection. PBMCs were isolated by Ficoll–Paque Plus (GE healthcare). Blood samples were sent for HLA typing to determinate HLA-matched tumor and HLA mismatched tumor for each patient. Serum was prepared by collecting the supernatant after incubation of blood at room temperature for 1 hour and centrifugation at 1,000 g. Two tumor lesions in each patient were sampled by biopsy pre-treatment; one of the two lesions was injected with IL PV-10 7 days after biopsy, then both residual sites were completely excised 7-14 days later. Biopsy specimens were fixed in formalin and embed in paraffin. The specimens were stained with hematoxylin and eosin stains for determination of pathologic complete response. Immunohistochemistry for melanin A (mel A) was performed. Flow cytometry was performed to detect CD3, CD4, CD8, and CD56 staining on PBMC.

ACKNOWLEDGMENTS

We thank Dr. Dmitry Gabrilovich for valuable comments during the preparation of this manuscript. This work was supported in part by the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the Moffitt Cancer Center, and in part by the Cancer Center Support Grant P30 CA076292 from the National Cancer Institute. This work was also supported by NCI-5K23CA178083-02 (AAS). PV-10 was provided by Provectus Biopharmaceuticals.

Updated (5/13/16): Provectus issued a press release and made an associated 8-K filing today related to Moffitt's PV-10 mechanism of action paper, "Announces Publication of Article in Oncotarget Detailing PV-10's Immuno-Ablative Mechanism of Action" -- with the company's CTO Dr. Eric Wachter, PhD calling the paper's publication "a a watershed event in the development of PV-10."

I really liked the press release, which is rare praise for an aspect of the company — corporate communications — that has been woeful and woefully lacking dating back to when I began due diligence on Provectus. I found the PR crisp, cogent, insightful and nuanced.

Key takeaway: In my view, the upshot of the release, and more importantly the Oncotarget paper, stemming from Moffitt's initial mouse work first presented at AACR in April 2013 — "Intralesional Injection with PV-10 Induces a Systemic Anti-tumor Immune Response in Murine Models of Breast Cancer and Melanoma" — is that PV-10 is an immunotherapy, or an immuno-ablative as Provectus has labelled its lead, advanced, investigational oncology drug that should focus attention on PV-10's physical chemistry properties (i.e., ablative, and e.g., ablation, chemoablation, etc.) rather than the biological chemistry properties of immune checkpoint inhibitors, oncolytic viruses, and certain other classes of immunotherapies. Keep in mind that folks more recently are wondering about the potential immunotherapeutic properties of chemotherapy and radiotherapy, which are "non-biologics."

Mouse-to-man-to-mouse: I'd venture, in my limited experience as a biotechnology or pharmaceutical industry investor, that Moffitt's work might be the epitome of a translational study, going from mouse to human, and back to mice before returning to human, as the cancer center team confirmed and/or discovered new things in their work. As Eric said in the PR {underlined emphasis below is mine}:

"The Moffitt researchers have systematically documented each of the key steps in the immuno-oncology cycle described by Chen and Mellman in their landmark review article (Oncology Meets Immunology: the Cancer-Immunity Cycle. Immunity 2013; 39: 1-10). In an exemplary demonstration of translational medicine, this team identified important immunologic markers in model systems and verified key facets of these in clinical trial participants, and similarly identified other markers in clinical trial participants and substantiated these in mouse models. While a number of their main observations were previously reported at scientific meetings, these are presented here in detailed, integrated fashion for the first time."

Moffitt team leader Dr. Shari Pilon-Thomas also broached this mouse-to-man-to mouse approach:

"Concordance of tumor-specific T cells in peripheral blood of clinical trial participants and mice led us to look for triggers of T cell activation. Working back from these observations, we found that HMGB1 release was common in mouse and man after tumor ablation with PV-10. These results support PV-10 ablation and the resulting tumor necrosis as the upstream trigger for systemic anti-tumor response." {concordance = agreement}

PV-10 is an immunotherapy: With Moffitt's work, presentation as Eric noted in a "detailed, integrated fashion for the first time," I'm hard pressed to understand anyone saying, in an intellectually honest fashion of course, that PV-10 is anything but an immunotherapy. As Eric further noted:

"This paper is a watershed event in the development of PV-10, walking the reader through all the steps of immune activation after PV-10 injection, from immunogenic cell death and signaling via release of HMGB1, dendritic cell recruitment and infiltration into draining lymph nodes, activation of tumor-specific T cells, and killing of uninjected tumors upon infiltration by these T cells."

Additionally, Eric underscored the immunotherapeutic role PV-10 plays as a single agent or monotherapy, and in combination with other therapies and therapeutics {bolded emphasis is mine, too}:

"This mechanism of action informed the design of the two active PV-10 clinical trials: NCT02288897 in patients with locally advanced cutaneous melanoma (melanoma limited to the skin) to test the hypothesis that PV-10 alone can produce a systemic immune response that translates to longer progression free survival (PFS); and NCT02557321 in patients with later stage melanoma to test whether combination of PV-10 with the recently approved systemic immunotherapy, pembrolizumab, can 'induce and boost' an immune response against melanoma."

Updated (5/13/16): I discussed choice of medical journal with Eric. He said New England Journal of Medicine (NEJM), Journal of Clinical Oncology (JCO) and the like focus on relatively large clinical trials. Moffitt's topic and study were not a good match for NEJM, JCO, etc. Oncotarget is a high-impact journal specializing in oncology mechanism and therapeutics (i.e., translational medicine), having a 2014 impact factor of 6.4. For comparison*, for example:

• NEJM's 2014 impact factor was 55.9,
• Lancet, 45.2,
• JCO, 18.4,
• Cancer Research 9.3,
• Clinical Cancer Research, 8.7,
• Oncotarget, 6.4,
• Cancer, 4.9,
• Journal of Immunotherapy, 4.0
• Immunology 3.8, and
• Melanoma Research, 2.2.

The NEJM and the Lancet cover all diseases, while Melanoma Research only covers melanoma. As such, readership naturally is very different, as are resulting citations.

* The information above can be downloaded from this file.

Burning Down The House

Wikipedia's tumor infiltrating lymphocytes (TILs) page describes TILs as "a type of white blood cell found in tumors." It goes on to say that "TILs are implicated in killing tumor cells. The presence of lymphocytes in tumors is often associated with better clinical outcomes." The National Cancer Institute's Dr. Steve Rosenberg, MD, PhD pioneered the approach of using TILs via adoptive cell transfer (ACT) to treat cancer patients. The American Cancer Society notes that TILs are "immune system cells deep inside some tumors...These T cells can be removed from tumor samples taken from patients and multiplied in the lab by treating them with IL-2. When injected back into the patient, these cells can be active cancer fighters." One company taking this approach of ACT via TILs is Lion Biotechnologies (NASDAQ: LBIO).

Another variation on this concept of ACT is where immune cells originating from a patient's blood (as opposed to his or her tumor) are extracted, altered and put back "with the goal of transferring improved immune functionality and characteristics along with the cells." Peripheral blood T cells are genetically engineered to express tumor-antigen specific T-cell receptors. Companies using this approach include Bluebird Bio (BLUE), Juno Therapeutics (JUNO) and Kite Pharma (KITE).

Autologous (below left) and genetically engineered (below right) ACT are illustrated below from W. Joost Lesterhuis and Cornelis J. A. Punt, “Harnessing the immune system to combat cancer,” 2012, Nature Reviews/Drug Discovery, supplement to Nature Publishing Group Journals.

Click to enlarge.

Harnessing the immune system to combat cancer, in the context of intralesional (IL) or intratumoral (IT) compounds like Bacillus Calmette–Guérin (BCG), Interleukin-2 (IL-2), talimogene laherparepvec (T-Vec, Imylgic), velimogene aliplasmid (Allovectin-7), Rose Bengal (PV-10), CAVATAK, Newcastle Virus Disease, HF-10, etc., means more than just destroying the lesion or tumor into which these agents are directly injected —it also means, more critically, the potential to generate a robust immune response, to activate, educate, train and thus enable (collectively, "harness") the immune system to attack cancer elsewhere in circulation.

Mechanism of action (MOA) would explain how an IL or IT agent destroys an injected lesion or tumor. Immune mechanism of action (IMOA?) or mechanism of immune action (MOIA?) would explain how the IL/IT agent harnesses the immune system to destroy uninjected, distant or so-called bystander lesions or tumors. PV-10's IMOA/MOIA also would be very relevant in the context of combining the IL/IT agent with other immunotherapies.

Provectus' CTO Dr. Eric Wachter, PhD analogizes PV-10's systemic response to fire, smoke and ash —where there's smoke and ash, there also is fire. T cells in the [peripheral] blood is the smoke. Regressed tumors are the ash. TILs are what interested observers want to see; that is, the fire.

From here, travel back to February 2013's Cancer Watch article about PV-10 and Moffitt Cancer Center's IMOA/MOIA work (which began in December 2012 [protocol first received date]) in this regard. The article is entitled "Back to Phase 1: Understanding Systemic Effects of PV-10;" Moffitt's work is entitled Detection of Immune Cell Infiltration Into Melanomas Treated by PV-10, a Feasibility Study (lead investigators: Dr. Amod Sarnaik, MD and Dr. Shari Pilon-Thomas, PhD).

Moffitt's work in mice in 2012, and presented and published in 2013, concluded that IL/IT PV-10 treatment led (after lesions/tumors were injected and then destroyed or shrunk) to a systemic response. The Cancer Watch article noted:

"Seeking an immune cell infiltrate 

To find direct evidence of such a systemic immune response is part of the motive behind heading back to the bench—although this time involving human subjects. “A further impetus toward teasing out the precise mechanism of how PV-10 can exert a systemic immune response in patients,” said Dr. Sarnaik in an interview, “is to allow us to rationally combine PV-10 treatment with some of the exciting emerging immunotherapies for metastatic melanoma.” 

The focus at Moffitt, Dr. Sarnaik continued, is on discerning the presence of immune cell infiltrate in untreated tumors after PV-10 injections into other lesions. “We are really interested in harnessing immune cell infiltrate as a form of treatment,” he said, noting also that while creating cancer vaccines has been thought of traditionally as one of the Holy Grails of cancer research, cancer vaccines have turned out to be not strong enough to generate an adequate immune response."

The article then went on to note {underlined emphasis and inserted commentary is mine}:

"Adoptive cell transfer 

The strategy of adoptive cell transfer potentially overcomes the weak vaccine response. With adoptive cell transfer, antigen-specific effector cells are taken from the patient’s tumor and expanded ex vivo under laboratory conditions favoring growth of T-lymphocytes and then re-infused to the patient. This precludes the need to provide antigens or to activate antigen-presenting cells. 

ACT via TILs: In melanoma, T-cells from the tumor are cultured from tumor resection specimens in the presence of interleukin-2. ACT via T cell re-engineering: A second strategy infuses peripheral blood T-cells that have been genetically engineered to express tumor-antigen specific T-cell receptors. 

While adoptive cell transfer offers the advantage that enough T cells can be obtained for infusion in all patients, the T-cell receptors transfected into the T cells have a limited antigen-specificity. The strategy works, Dr. Sarnaik said, only about half the time. “We generate large numbers of T-lymphocytes, but we don’t have control over their quality. 

We think one of the limitations is that the T cells you get out of the tumor just aren’t good enough.” PV-10, however, does cause an immune response, suggesting that a combination treatment may improve the quality of the T-lymphocytes and have a greater impact on the disease. 

When Shari Pilon-Thomas, PhD, also a Moffitt researcher, demonstrated that T-lymphocytes recovered from mice treated with PV-10 do appear to be of a higher quality, as evidenced by stronger tumor reactivity, the stage was set for Dr. Sarnaik’s current 15-patient pilot study. In it, one of two resectable melanoma tumors is injected with PV-10. Both are removed several weeks later. Serum is assessed before and after treatment to look for changes in the infiltration of immune cells. In patients with an immune response, PV-10 therapy can be continued."

Unfortunately, Moffitt's IMOA/MOIA/combination therapy relevancy work was waylaid because PV-10 worked too well — both injected and uninjected lesions or tumors were destroyed too well (pathologic complete response [pCR]) and too quickly (sooner than the study protocol patient biopsy period of 7-14 days post-PV-10 injection).

In April 2014 at AACR, Dr. Pilon-Thomas noted about their human work up to that point (the poster was not released by either Moffitt or Provectus, see the company's press release here):

Too quickly, and "smoke:" "These data are exciting and illustrate successful translation of our pre-clinical work in mice to clinical results in melanoma patients. With only 8 patients we've been able to clearly observe statistically significant increases in beneficial T cell populations in peripheral blood. Ironically, the original aim of the trial to assess tumor-infiltrating lymphocytes was thwarted when biopsies of patient tumors collected just 7-14 days after PV-10 injection no longer contained viable tumor tissue. We are following up both the human data and continuing to design more experiments in mice to better explain the systemic immune effects elicited by PV-10 ablation."

In June 2014 at ASCO, Moffitt further noted:

Too well, and "ash:" "Treatment with IL PV-10 led to pCR in the post-treatment biopsies of both PV10-injected and uninjected study lesions in 4 of the 8 patients, and all 8 exhibited at least partial regression of the injected lesion."

Really, ironically, some of the injected and uninjected going away too quickly in Moffitt's work is reminiscent of Provectus' metastatic melanoma Phase 2 trial, and Eric's argument to the FDA to grant PV-10 breakthrough therapy designation for PV-10 in patients (who would have all of their disease treated) with locally advanced cutaneous melanoma:

"Because of the lack of requirements for patients to have pain symptoms upon enrollment, only a small fraction of patients had clinically significant pain at baseline. So, we analyzed those patients, uh, and presented them that analysis of those data in context of the objective response data. We found that there was a strong relationship between the two types of data, that there was simply not enough of the symptomatic, or symptomology data to show a statistical function. I have to conclude that that’s the principal basis for the rejection of the application. I'd say that it was our assumption going into the application that improvement in symptoms, if we made the patient’s symptoms go away was tantamount to -- I’m sorry -- if we make the patient’s lesions go away that’s tantamount to making the patient’s symptoms of that disease go away. We don’t seem to have been successful in convincing the Agency of that."

Nevertheless, Moffitt found, in humans, smoke — T cells in peripheral blood — and ash — regressed tumors.

The Cancer Watch article went on to note:

"“This is a straightforward study that will give a yes or no answer,” Dr. Sarnaik said. 

If the hypothesis that PV-10 will produce a better immune cell infiltrate is borne out, that would justify testing of combination treatments, Dr. Sarniak said. Likely candidates are adoptive cell therapy, approved drugs like ipilimumab that boost immune response, or PD-1- blocking antibodies (none approved yet)."

Moffitt should have found, in humans, the fire, presumably through the following 7 patients of their originally planned 15-patient study. In November 2015 at SITC they showed they found more smoke: "Increased tumor-specific response was found from those circulating T cells of 5 out of 7 tested patients after IL RB treatment."

The Cancer Watch article concluded:

"What kind of therapy is PV-10? 

Echoing Dr. Sarnaik, Eric Wachter, PhD, Provectus chief technology officer, said that he hopes that the findings of Dr. Sarnaik’s study will point toward rational judgments about combining PV-10 with other documented therapies. “We then might want to try two or more orthogonal therapies to stress tumor cells from several different angles simultaneously, for example an immune therapy plus a metabolic therapy (e.g., a kinase inhibitor), or in a rationally designed sequence.” In a hepatocellular carcinoma model, he added, PV-10 showed significant potential for synergy with 5-fluorouracil. Provectus recently initiated clinical testing of PV-10 with the multikinase inhibitor sorafenib, again bringing in two therapies with divergent mechanisms of action. 

Which category does PV-10 fall into? “I think we are getting a clearer picture of how it might be classified, but it has features of several previously unrelated categories, such as of adoptive cell transfer and vaccination,” Dr. Wachter said. “PV-10 initially reduces tumor burden through chemoablation—but then activates the immune system bringing in capacities completely orthogonal to the ablative tumor destruction,” he added." 

“Amod Sarnaik’s work may give us the molecular basis for closing the loop on one of the founding concepts for going into the clinic in the first place,” Dr. Wachter commented. “Back in the preclinical days at Provectus, Craig Dees, PhD, theorized that ablation of tumors with PV-10 might lead to unmasking of tumor antigenic material. I don’t think he anticipated that it would work as well as it does.”

PV-10 at AACR 2016: T cell mediated immunity after combination therapy with intralesional PV-10

Presentation Title: T cell mediated immunity after combination therapy with intralesional PV-10 and co-inhibitory blockade in a melanoma model

Author Block: Amy M. Weber, Hao Liu, Krithika N. Kodumudi, Amod A. Sarnaik, Shari Pilon-Thomas. H. Lee Moffitt Cancer Center, Tampa, FL

Abstract:

PV-10 is a 10% solution of Rose Bengal, a xanthene dye that was originally developed for ophthalmic use and later used in liver function studies. PV-10 was formulated for intralesional (IL) injection and is currently being investigated as a novel cancer therapeutic. In murine studies, we have previously shown that intralesional (IL) injection of PV-10 leads to a regression of both injected tumors and untreated bystander tumors. We have also shown that combination therapy of IL PV-10 with blockade of PD-1 and PDL-1 leads to increased anti-tumor immunity. In this study, we have examined the role played by specific immune cell populations in eliciting this response in a murine melanoma model. We first investigated the antigen specificity of CD8+ T cells in the spleens of mice treated with the combination therapy of IL PV-10 and systemic co-inhibitory blockade. We found that splenocytes from mice treated with the combination of IL PV-10 and anti PD-1 antibody have an increased mean percentage of OVA antigen-specific CD8+ T cells (5.77%) compared to single treatment with anti PD-1 antibody (3.8%) or IL PV-10 (3.60%) alone in OVA-expressing B16 tumor bearing mice. To investigate the role of T cell subsets in mediating an immune response, OVA-expressing B16 tumor bearing mice were treated with IL PV-10 followed by intraperitoneal injection of anti-PD-1 antibody. In addition, mice were given either 2.43 antibody to deplete CD8+ T cells, GK1.5 antibody to deplete CD4+ T cells, or PC61 to deplete regulatory T cells (Tregs). We found that depletion of CD4+ T cells in combination with IL PV-10 and anti-PD-1 antibody treatment resulted in an enhanced anti-tumor effect, with an average tumor size of 52.8 mm2 on day 25 compared to the control group (173.7 mm2), and depletion of Tregs resulted in an even greater anti-tumor effect, with an average tumor size of 1.2 mm2 on day 25 (p<0.05). In contrast, mice treated with the CD8+ depleting antibody exhibited diminished anti-tumor immunity compared to the control group, with an average tumor size of 200 mm2 on day 25. Together, these studies indicate that the effect of combination therapy with IL PV-10 and co-inhibitory blockade is mediated by CD8+ T cells, and that depletion of both CD4+ T cells and CD25+ Tregs significantly enhances anti-tumor immunity in a melanoma model.

{my bolded and underlined emphasis above}

PV-10 is an immunotherapy

Click to enlarge. Original image source (Google search of "immunotherapy").
Colored editing above is mine.

I think the upshot of the University of Illinois at Chicago's (UIC's) abstract at the 2016 annual meeting of the Academic Surgical Congress -- PV-10 Induces Potent Immunogenic Apoptosis in Colon Cancer Cells -- is its contribution to the growing mound of preclinical and clinical data that:

• First, PV-10 (Rose Bengal) ablates all solid tumors (UIC: "We have previously demonstrated that human and murine colon cancer cells undergo near complete cell death in vitro and in vivo upon direct exposure to PV-10, a synthetic dye currently in clinical trails for intralesional therapy of in-transit melanoma", and

• Second, PV-10 primes the immune system in all solid tumors (UIC: "Therefore, based on these results, further evaluation of PV-10 as a potential agent to stimulate immunologic cell death in solid tumors is warranted.").

Said differently, and perhaps with the benefit of more clinical data, particularly that from Provectus' Phase 1b/2 study PV-10 in Combination With Pembrolizumab for Treatment of Metastatic Melanoma, PV-10 should be called an immunotherapy. This small molecule that generates clinical benefit for and positive outcomes in cancer patients from its physical chemistry has feelings too.

Moffitt Cancer Center in its SITC 2015 poster entitled Intralesional Rose Bengal in Melanoma Elicits Tumor Immunity via High Mobility Group Box 1, noted:

"IL RB therapy also increased antigen-specific T cell proliferation and enhanced tumor regression. In addition, IL RB facilitated dendritic cells (DCs) infiltrating lymph nodes draining from tumor. Incubation of melanoma cells with RB led to necrosis and the release of High Mobility Group Box 1 (HMGB1), which activated DCs. The blockade of HMGB1 significantly reduced the antigen-presenting ability of DCs."

UIC noted in their ASC 2016 abstract:

"Treatment of colon cancer cells with PV-10 induced cell cycle arrest, apoptosis, autophagy, and significant ER stress; consistent with immunogenic apoptosis. In order for cytotoxic agents to act as potential immunotherapeutic strategies in the treatment of solid tumors, immunogenic cell death targeting the endoplasmic reticulum (ER), leading to ER stress may be critical."

Underlined emphasis above and below is mine. 

Playing scientist using Google, note for example abstract comments from Zhu et al. in Endoplasmic reticulum stress and its regulator XBP-1 contributes to dendritic cell maturation and activation induced by high mobility group box-1 protein (Int J Biochem Cell Biol. 2012 Jul;44(7):1097-105):

"High mobility group box-1 protein (HMGB1) had been proved to induce maturation and activation of dendritic cell (DC), however, the endogenous changes and mechanisms underlying are unknown. Since endoplasmic reticulum stress (ERS) activates an adaptive unfolded protein response (UPR) that facilitates cellular survival and repair, we hypothesized that HMGB1 may regulate the function of DC by modulating ERS. In our study, HMGB1 stimulation induced significant ERS responses in DCs in a time- and dose-dependent manner, demonstrated by the up-regulation of a number of ERS markers."

Note: CGMP = Current Good Manufacturing Practice (CGMP). See for example Drug Applications and Current Good Manufacturing Practice (CGMP) Regulations.

SITC 2015: Intralesional Rose Bengal in Melanoma Elicits Tumor Immunity via HMGB1

Updated again below.

Abstract: Intralesional (IL) therapy is under investigation to treat dermal and subcutaneous metastatic cancer. Rose Bengal (RB) is a staining agent that was originally used by ophthalmologists and in liver function studies. Previously, IL injection of RB induced regression of injected and uninjected tumors in murine models. However, the relevant mechanism is yet unknown. In this study, we used an OVA-expressing B16 melanoma murine model and found that IL RB treatment led to increased tumor-specific T cells with memory characteristics. CD8+ T cell are crucial for tumor-specific response elicited by IL RB. IL RB therapy also increased antigen-specific T cell proliferation and enhanced tumor regression. In addition, IL RB facilitated dendritic cells (DCs) infiltrating lymph nodes draining from tumor. Incubation of melanoma cells with RB led to necrosis and the release of High Mobility Group Box 1 (HMGB1), which activated DCs via up-regulation of CD40 expression. The blockade of HMGB1 significantly reduced the antigen-presenting ability of DCs. To determine whether this mechanism was relevant in patients treated with IL RB, we performed a pilot clinical study in melanoma patients (NCT01760499). IL RB led to tumor regression in both RB-injected and uninjected lesions, associated with an increase in circulating T cells. Increased tumor-specific response was found from those circulating T cells of 5 out of 7 tested patients after IL RB treatment. HMGB1 levels in patient sera were also elevated. Together, these results reveal a clinically relevant immunoadjuvant pathway triggered by tumor cell death secondary to ablation with RB.

Click to enlarge

Updated (11/3/15): Items in the abstract of note [to me]:

• "However, the relevant mechanism is yet unknown."

• Clinical (human) data: "Increased tumor-specific response was found from those circulating T cells of 5 out of 7 tested patients after IL RB treatment." {Underlined emphasisis is mine}

• "Together, these results reveal a clinically relevant immunoadjuvant pathway triggered by tumor cell death secondary to ablation with RB."

• "clinically relevant"
• "immunoadjuvant pathway"

Updated (11/3/15): "Clinically relevant" {Underlined emphasis is mine}

"In clinical research is not only important to assess the significance of the differences between the evaluated groups but also it is recommended, if possible, to measure how meaningful the outcome is (for instance, to evaluate the effectiveness and efficacy of an intervention). Statistical significance does not provide information about the effect size or the clinical relevance. Because of that, researchers often misinterpret statistically significance as clinical one. On one hand, a large sample size study may have a statistically significant result but a small effect size. Outcomes with small p-values are often misunderstood as having strong effect sizes. On the other hand, another misinterpretation is present when non statistical significant difference could lead to a large effect size but a small sample may not have enough power to reveal that effect." (Source)

"Ideally, a clinical trial should be able to demonstrate not only a statistically significant improvement in the primary efficacy endpoint, but also that the magnitude of the effect is clinically relevant...However, it is not only important to assess statistical significance, but also to assess the clinical relevance of the effect, and the assessment of clinical relevance has received much less attention in the statistical literature." (Source)

"Immunoadjuvant pathway triggered by tumor cell death:" {Underlined emphasis is mine}

"Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death." (Source: Apetoh et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat. Med. 13, 1050–1059)

"Incubation of melanoma cells with RB led to necrosis and the release of High Mobility Group Box 1 (HMGB1), which activated DCs via up-regulation of CD40 expression."

"CD40 was initially characterized as a co-stimulatory molecule expressed on APCs that played a central role in B and T cell activation. However, this molecular pair functions in the regulation of both APCs and effector lymphocytes (Fig. 1). As we understand more about the number of different DC and T cell subsets, we are likely to find that CD40-CD40L interactions play important and distinct roles in regulating these novel subsets. In addition, as we continue to understand how innate immunity cells directly regulate B cells and antibody responses, the influence of CD40 and CD40L in these interactions should be further clarified. Further insights into the functions of CD40-CD40L interactions will advance our understanding of immune cell crosstalk and interdependent regulation of the immune system." (Source: Ma DY, Clark EA. The role of CD40 and CD40L in Dendritic Cells. Seminars in immunology. 2009;21(5):265-272.)

Potential Catalysts & "Catalysts"

Caveat: I have been hilariously off-base in the past. See, for example, my August 31, 2014 blog post Potential Catalysts.

Updated (8/9/15): To reflect a longer period of pivotal melanoma Phase 3 site activation, and to include as a catalyst the potential approval of Amgen's intralesional agent for metastatic melanoma talimogene laherparepvec (T-Vec).

Updated (8/9/15): To reflect a year-end start to a Phase 1b trial combining PV-10 and an immune checkpoint inhibitor in patients with advanced melanoma.

Click to enlarge.

Click to enlarge.

Why I Remain Long Provectus Biopharmaceuticals

§  My investment thesis is intact. Up until this point FDA regulatory clarity had been lacking and therefore commercial validation was absent despite compelling clinical and business value propositions of Provectus’ Rose Bengal-based investigational oncology and dermatology compounds PV-10 and PH-10, respectively. Company management achieved regulatory clarity. Commercial validation should follow. Thus, I remain long the stock. 

§  The first pathway to oncology approval for ablative immunotherapy PV-10 is the pivotal Phase 3 trial of PV-10 as a monotherapy versus systemic chemotherapy for Stage IIIB and Stage IIIC patients with unresectable locally advanced cutaneous melanoma. If successful, PV-10 would become the standard of care for this patient population. 

§  The oncology clinical development program should expand this year with first, a Phase 1b/2 trial of PV-10 in combination with immune checkpoint inhibition for patients with advanced melanoma, and second,a domestic pivotal trial of PV-10 as a monotherapy versus sorafenib for patients with hepatocellular carcinoma and cancer metastatic to the liver. The program’s expansion asserts PV-10’s multi-indication viability, utility across disease stage spectrum, and functionality as a monotherapy as well as in combination with other treatments. 

§  Regulatory clarity from U.S. and local international regulators should facilitate commercial validation, such as geographic PV-10 licenses in China, India and/or other countries. A key element of these relationships should be joint undertakings with commercial partners of Phase 1b/2 trials of PV-10 as a monotherapy versus each region’s standard of care for patients with hepatocellular carcinoma and cancer metastatic to the liver, thus further expanding the clinical development program in 2016. 

§  Inflammatory dermatoses therapeutic PH-10 finally should garner more public attention. A Phase 2 mechanism of action study began in January. The issuance of a key patent followed in March, and interestingly may suggest non-dermatology applications of PH-10. Together with several completed preclinical toxicity studies, clinical mechanism data due by year-end should facilitate FDA discussions about consensus pivotal Phase 3 trial designs for psoriasis and/or atopic dermatitis. The protocol(s) and regulatory clarity may help the Company secure a global or regional license deal for PH-10 in 2016. 

§  More data should be presented, published and available for due diligence in 2015, such as liver cancer (clinical), PV-10’s mechanism (clinical), PV-10 in combination with immune checkpoint inhibition (preclinical), and PV-10 in combination with external beam radiation therapy (clinical). Mechanism, overall survival-focused liver data and interim melanoma Phase 3 data would affirm that complete response of cancerous tumors after PV-10 injection results in the disease staying away. 

§  Favorable pharmaceutical industry trends should further improve Provectus’ M&A valuation prospects. Big Pharma oncology franchises are separating into those with immune checkpoint inhibition that understand the need for an immune system primer to assist their inhibitors, those with inhibitors that don’t fully embrace this need, and those who have a stimulatory compound, an inhibitor or neither and know they need a matching set to remain relevant.

My investment thesis remains intact

The value proposition of Provectus’ two Rose Bengal-based drugs — intratumorally injected PV-10 for oncology (a 10% solution of Rose Bengal) and topically applied PH-10 for dermatology (a 0.001% to 0.01% gel of the active pharmaceutical ingredient) — is compelling: pristine safety profiles, well tolerated treatments easily administered to and compliable for patients, ready-made products with probable high gross margins inexpensively manufactured at scale, and the potential to fully serve vast addressable markets of unmet needs very profitably over time. Increasing amounts of medical and scientific data as well as independent validation of results and claims confirm the Company’s intrinsic value.

From inception through this year Knoxville, Tennessee-based Provectus Biopharmaceuticals, Inc.[1] (“Provectus” or the “Company”) (NYSE MKT: PVCT) was best described as an early-stage drug development company. Provectus was founded in 2002 by three scientists from nearby Oak Ridge National Laboratory, a U.S Department of Energy (“DOE”) multi-program science and technology facility with a rich history of discovery and innovation (and a member of the DOE network of laboratories that includes Lawrence Berkeley, Lawrence Livermore, Los Alamos, and Sandia). Up until now the Company had been developing PV-10 and PH-10 with no regulatory clarity (i.e., discernible, initial pathways to approval).

Rose Bengal is a unique compound with an extensive and very long clinical history. A water-soluble dye originally created by Gnehm in 1882[2], it has a molecular weight of approximately 1,000 Daltons, which effectively makes it a small molecule. Rose Bengal’s century of prior clinical use includes being added to Safranin Victoria Yellow for treating ocular pneumococcal infection (Römer, 1914)[3], a stain for visualizing corneal ulcers (Kleefeld, 1919)[4], and a marker for observing impaired liver function (Delprat, 1925)[5]. It also has an established FDA safety profile as an intravenous hepatic diagnostic (Robengatope®) as well as a topical ophthalmic diagnostic (Rosettes® and Minims®). To date there are 3,696 medical literature citations of Rose Bengal[6], 233 related to cancer[7]. Rose Bengal’s therapeutic benefits remained hidden until the 1980s when the FDA and the Japanese Ministry of Health and Welfare began scrutinizing artificial food colorings. Japanese researchers evaluated the tumorigenicity of red food dye No. 105 (also made from Rose Bengal), and observed dose-dependent survival increased in the mice receiving it.[8]

In 1998 Provectus’ multi-disciplinary team of founders — a molecular virologist (Dr. Craig Dees, Ph.D.), a chemical engineer (Dr. Timothy Scott, Ph.D.) and an organic chemist (Dr. Eric Wachter, Ph.D.) — looking for drug candidates having antineoplastic activity also happened to come across Rose Bengal.[9] A commercial data search by the former Oak Ridge scientists identified numerous candidates. Subsequent proprietary screening promptly zeroed in on Rose Bengal and other members of the halogenated xanthene family. Preclinical tests with bacterial and cancer cell lines quickly demonstrated Rose Bengal’s impressive cytotoxic activity. Subsequent animal and human studies confirmed Rose Bengal delivered directly into tumors by injection was a selective and potent agent for ablating cancers and harnessing the immune system.

PV-10’s therapeutic value, established preclinically and clinically by Provectus, has been independently repeated and reproduced by affiliated and unaffiliated investigators and researchers around the world:

• Dual ablative and immunotherapeutic mechanisms of action: Primary ablation in 1 to 2 hours that kills cancer tumors into which PV-10 is injected by selectively passing through cell membranes and accumulating in lysosomes to force cell death with no biochemical action or effect, and secondary immunomodulation in 1 to 2 weeks where cell death resulting from local tumor injection attracts tumor-specific T-cells and causes an up-regulating immune effect, which leads to a systemic, tumor-specific immune response in untreated tumors and distant disease[10],

• Simple to store, handle, and use and reuse[11],

• Modest local toxicity predominantly confined to the injection site, and minimal to no systemic toxicity^{11, [12], [13]},

• Rapid, durable and complete tumor destruction, as well as induction of antigen release in injected lesions^{11, 13},

• Prompt, complete healing of injected lesion sites after tumor destruction¹¹,

• Reliable, reproducible induction of loco-regional and systemic immune effects capable of destroying occult tumor cells, untreated lesions and distant metastatic lesions regardless of prior therapies and treatments ^{11, [14]},

• Multi-indication viability: Up to 260 cancer patients treated with PV-10 — 12 in a recurrent breast cancer Phase 1 trial, 6 in a liver cancer Phase 1 trial, 100 in melanoma Phase 1 and 2 trials, 15 in a melanoma Phase 1 mechanism study, 100 patients having cutaneous and subcutaneous tumors irrespective of indication treated through 2013 in Provectus’ compassionate use program (“CUP”), and up to 25 in an investigator-initiated melanoma study of PV-10 in combination with external beam radiation therapy (“XRT”)^{14, [15]}. The Company has not yet reported patient numbers for the expanded liver cancer Phase 1 trial and in the CUP in 2014,

• Orthogonality (i.e., low clinically relevant drug-drug interaction)[16] and synergy with other therapies and therapeutics: targeted therapy¹⁶, systemic chemotherapy[17], XRT[18], and immune checkpoint inhibition[19]^{, [20]}, and

• Third-party validation[21]: Apoptosis, tissue and cell selectivity, clinically relevant loco-regional and systemic effects, tumor-specific immunity, T-cell engagement, immune-mediated response, multi-indication viability.

The initial pathway to approval is set

Provectus’ pivotal melanoma Phase 3 trial should begin treating patients in April. The trial tests the hypothesis that complete response (“CR”) of melanoma lesions after PV-10 injection results in melanoma staying away.^[22] The trial’s patient population should be the same as the patient subgroup in the Company’s Phase 2 trial that received PV-10 injection of all existing melanoma. The subgroup achieved progression-free survival (“PFS”) under modified RECIST of more than 9.8 months, a complete response rate (“CRR”) of 50% and loco-regional disease control (complete response [“CR”] + partial response [“PR”] + stable disease) of 82% despite receiving a limited number of injections over a limited period of time. An initial injection and up to three more [if needed] were delivered to a patient’s lesion during the first 16 weeks of the trial. The majority of lesions in the subgroup achieved CR with only 1 or 2 injections.[23]^{, [24]}

By accepting the registration study’s design and protocol[25] — 225 patients, a primary endpoint of PFS, a comparator of systemic chemotherapy (dacarbazine), and secondary endpoints of CRR, overall survival (“OS”) and the number of adverse events — the FDA acknowledged this intratumorally delivered agent may have a meaningful role to play in the treatment of the systemic disease that is melanoma. The Phase 3 trial will have no restriction on the number of injections a patient’s lesions may receive (which benefits PFS and CRR), the number of lesions a patient may have treated (which benefits OS) or the time over which injections are provided (which benefits PFS, CRR and OS). There is an imperfect trial inclusion criterion: Patients must have failed or not be a candidate for at least one systemic immunotherapy (Bristol-Myers’ ipilimumab, Merck’s pembrolizumab and Bristol-Myers’ nivolumab).

The Agency’s recognizes the severity of loco-regional melanoma and need for better patient treatment options. In October 2014 the FDA requested public comment on “[m]elanoma, specifically unresectable loco-regional disease.”[26] The Agency also accepted Amgen’s biologics license application for intralesional agent and oncolytic virus talimogene laherparepvec (referred to as T-Vec), setting a Prescription Drug User Fee Act date of October 27, 2015. An April 29, 2015 meeting of FDA advisory committees should elucidate the Agency’s views about T-Vec, especially given questions surrounding its pivotal melanoma Phase 3 trial’s comparator (GM-CSF) and endpoints (durable response rate), and the potential deleterious effects of viral shedding[27].

Management has endeavored to build value in Provectus’ fully owned cancer and dermatology assets (unsuccessfully thus far when measured by the Company’s current market capitalization) by differentiating PV-10 and PH-10’s product profiles, innovating unique mechanisms of action (outsourced to Moffitt Cancer Center [“Moffitt”] and rumored Rockefeller University for independent explanation of oncology and dermatology, respectively), demonstrating best-in-class efficacy, persevering in their discussions with the Agency to establish new standards of care (management acknowledged they did not fully listen to the FDA in the past), demonstrating negligible side-effect profiles as well as easy physician and compliable patient administration modes, and targeting therapeutic areas with unmet clinical and commercial needs.[28]

Provectus faced two obstacles during its existence as an early-stage drug development company. First, there was regulator and pharmaceutical industry reservation about using locally delivered agents to treat melanoma. This bias resulted from a lack of material, long-lasting clinical success, illustrated by the initial promise but ultimate failure of Bacille Calmette-Guérin (“BCG”) for melanoma in the 1970s (serious adverse events and no significant clinical benefit[29]) and Vical’s Allovectin-7 for melanoma in 2013 (no clinical benefit[30]^{, [31]}), as well as comparatively lackluster OS in melanoma for T-Vec in 2014[32]. Allovectin-7’s CRR in its Phase 2 trial for metastatic melanoma was far lower than T-Vec’s, which was much lower than PV-10’s[33]. Second, Provectus originally sought consideration of PV-10 from the FDA for an earlier stage of melanoma when all of it is accessible for injection and overall disease burden is lower (Stage III patients), instead of pursuing advanced melanoma where not all disease is injectable (Stage IV patients) like BCG, Allovectin-7, T-Vec and the systemic immunotherapies.

Whether separately from or together with T-Vec, PV-10 may be the first (or one of the first) locally delivered therapeutic agent to be approved for melanoma. Systemically administered high-dose interleukin-2 was approved in 1998 for advanced melanoma.[34] Against the backdrop of a finalized melanoma Phase 3 protocol and evolving FDA and Big Pharma acceptance of intralesional therapies, there is a meaningful probability of PV-10’s melanoma Phase 3 trial being terminated early for efficacy of the treatment arm over the control. Historical data can be honestly used to determine the likelihood of success. Clear wins on trial endpoints should be acknowledged as former biases fall away.

Clinical development should expand substantially

If early-stage drug development company described Provectus from 2002 to 2015, 2015 marks the beginning of the Company’s transition into a regulatory-focused and data-driven organization. The knowledge base developed and consensus achieved by the FDA’s Division of Oncology Products 2 (“DOP2”)[35] and Provectus of what is necessary and sufficient to approve PV-10 as a monotherapy for loco-regional Stage III melanoma patients should enable the parties to design trial protocols and establish prospective pathways to approval in the future (with the FDA better understanding and having more experience with PV-10’s mechanism, utility and functionality) for a PV-10/immune checkpoint inhibition combination for patients with advanced melanoma, and monotherapy use in patients with hepatocellular carcinoma (“HCC”) and cancer metastatic to the liver.

Combination therapy for advanced melanoma: The second trial of Provectus’ oncology clinical development program that may start in 2015 could be a modest-sized (up to 20 patients), single-arm Phase 1b trial combining PV-10 and Merck’s immune checkpoint inhibitor pembrolizumab for patients with unresectable Stage IIIB to Stage IV advanced melanoma. Provectus may elect to go it alone and use the reimbursable pembrolizumab because of Moffitt’s familiarity with it. Alternatively, the Company could enter into a co-development relationship with this Big Pharma or another one (and its approved or investigational inhibitor). Endpoints would comprise acute safety (of the combination regimen, with PV-10 given first followed by the inhibitor) and objective response rate (“ORR”) (CR + PR) measured at 3 to 4 months.[36] This trial protocol would be agnostic to the inhibitor. Moffitt has completed preclinical combination studies of PV-10 and co-inhibitory blockade (anti-CTLA-4, anti-PD-1, anti-PD-L-1). There should be low or no clinically relevant drug-drug interactions. It is rumored Merck has done due diligence on Rose Bengal through its own murine model work.

Phase 1b/2 studies may streamline drug development and rapidly advance a promising agent demonstrating notable efficacy and sufficient safety through the regulatory process. The initial step (Phase 1b) collects safety and response data from a small number of patients. A successful outcome facilitates subsequent discussions with the FDA about a pivotal trial design for the second step (i.e., a randomized Phase 2 or other aptly named late-stage trial).

Amgen and Bristol-Myers already conducted a Phase 1b trial combining T-Vec and ipilimumab in 19 patients with advanced melanoma.[37] No dose-limiting toxicities and a predominance of Grade 3/4 adverse events were reported. In addition to evidence of immunological signaling, preliminary data suggested higher CRR and ORR than either T-Vec or ipilimumab alone, and earlier responses after ipilimumab initiation during the combination regimen than with ipilimumab alone. In contrast, Moffitt described a greater breadth and depth of T-cell response (CD8+, CD4+, CD3+ and NK) from a single injection of PV-10 in a Phase 1 mechanism study, compared to arguably weaker signaling from repeated, prolonged treatments of T-Vec and ipilimumab. Nevertheless, the results of the T-Vec plus ipilimumab trial inform how an immune system primer (T-Vec) enhances the effectiveness of an immune checkpoint inhibitor (ipilimumab).

Monotherapy for HCC/ metastatic liver cancer (U.S.): A third trial that may be started in 2015 could be a domestic liver cancer one, the design of which is dependent on available data and subsequent guidance from DOP2, the same organization within the FDA responsible for melanoma. Provectus previously completed a liver Phase 1 trial that treated six patients with HCC (five tumors) and a colorectal metastasis from 2009[38] to 2011[39]. The Company has not yet presented or published results from this trial. Presentation is expected this summer. Management previously said tumors were substantially ablated with sustained regression and no disease per positron emission tomography–computed tomography at the patients’ 9- to 15-month check-ups.

In 2012[40] Provectus commenced an expanded Phase 1. The number of treated patients is not yet public. Per the trial’s protocol[41] patients were divided into two cohorts; one received PV-10, and the other both PV-10 and sorafenib. Safety was the primary outcome measure for both liver trials. The second trial had a secondary outcome measure (among others) of ORR of injected and measurable untreated lesions (if present). Sorafenib is the standard of care for patients with more advanced liver cancer or who failed loco-regional therapy.[42] Prior to treating patients in the PV-10-plus-sorafenib cohort, however, Provectus conducted an in vitro study that demonstrated a low risk of clinically relevant drug-drug interaction between Rose Bengal and sorafenib derivatives[43]. This orthogonality assigns the efficacy difference between the two cohorts in the second liver trial solely to PV-10’s clinical benefit. Provectus may seek an expedited pathway to approval via a pivotal trial based on (but not limited to) safety from both liver trials, overall survival observations from the first, measures of ORR from the second, and PV-10/sorafenib orthogonality.

Company management recently said they are assessing potential clinical study of other indications like breast cancer and pancreatic cancer[44]. It is unlikely such work would commence this year given management’s potentially taxed mental bandwidth (management made deliberate choices from the outset regarding operational roles and responsibilities, and these remain unchanged). Provectus’ drug development program also comprises on-going third party research that is rumored to include work on breast, pancreatic and prostate cancer, and renal cell carcinoma. Provectus’ Chief Technology Officer Dr. Wachter commented on this during the Company’s last quarterly conference call:

Dr. Wachter: “I cannot comment on other third-party work that may or may not be underway with regard to non-clinical work with PV-10. Obviously that is something that may be of interest and if it is, and we haven't disclosed this, it’s probably a sensitive nature.”[45]

The oncology clinical development program’s expansion in two key areas — primary and metastatic liver cancer as another approvable indication, and combination therapy for advanced melanoma as an approvable indication expansion — establishes PV-10’s multi-indication viability, broader utility across disease stage spectrum, and greater functionality as a monotherapy and in combination with other treatments as an immune system primer.

Regulatory clarity should facilitate commercial validation

Management has pursued international commercial validation for a couple of years, and over time said licensure discussions have taken place for China, India, Brazil, Russia, Korea, Japan, and the Middle East and North Africa. Completing these deals supplement but should not interfere with a worldwide oncology license for the drug or the eventual sale of the Company to Big Pharma. With an FDA-accepted melanoma Phase 3 protocol finally in hand, a deal or deals may be had with a dominant pharmaceutical company in the respective geography. Provectus currently has a memorandum of understanding for China and its territories with two Sinopharm Group Company subsidiaries, China State Institute of Pharmaceutical Industry and A-THINK Pharmaceutical Co.[46]

If a deal is transacted, a key element of the relationship likely would be the joint undertaking (with the geographic partner) of a Phase 1b/2 trial of PV-10 as a monotherapy versus the particular region’s [loco-regional ablation therapy] standard of care for patients with HCC and metastatic liver cancer. This should require the acceptance of the locally focused protocol by the local regulator, and may be the case with the China Food and Drug Administration (“SFDA” or “CFDA”). Dr. Wachter discussed his perspective of the regulatory pathway in China and the Company’s likely approach to it on the last two Provectus’ quarterly conference calls:

November 2014: “What we have learned in the relatively recent past is with our experience in Asia that it will be necessary going into China to do some additional Phase 1 work and that has colored our design parameters for the next Phase of HCC work to use this 1b-2 approach that I might point out from a prepared comments has been so successful with Amgen and a number of other sponsors in recent years as a way to expedite development of the drug from the Phase 1 safety into a very robust randomized Phase 2 study in a quick fashion.”[47]

March 2015: “So in the part of the world like China which is an interesting case, we might be able to access a large number of patients that would be eligible for the study but for instance the approval process to get the study going, not technical review of the protocol and modifications, I have gone to the agency but just the fundamental review process for conducting a clinical trial where an investigation drug can be very prolonged, often times 12 to 18 months.”⁴⁵

Monotherapy for HCC/ metastatic liver cancer (China): PV-10’s Chinese pathway to approval for liver cancer thus might be the first step of a modest-sized, single-arm Phase 1b trial in 2016 combining PV-10 and a Chinese-favored loco-regional ablation therapy (e.g., transarterial chemoembolization, percutaneous ethanol injection)[48]. Endpoints may comprise acute safety of the combination regimen and hepatic ORR measured at some single digit number of months. A successful outcome could facilitate subsequent discussions with the SFDA/CFDA about a pivotal trial design for a randomized late-stage trial comparing PV-10 and the chosen ablation therapy for OS, among other primary and secondary endpoints.

PH-10 should garner more attention as a very valuable therapeutic asset

By launching a Phase 2 mechanism of action study that started recruiting patients in January and should complete data collection by year-end, existing and prospective investors in the Company are able to better value Provectus’ investigational dermatology compound PH-10. The drug may have an addressable market of all inflammatory dermatoses that could be larger than that of PV-10. Company management has tried to license the drug since at least 2010[49] without success because regulatory clarity had been lacking. This was very likely due to a paucity of data for the regulator and prospective commercial partners, particularly as it relates to near complete absence of toxicity for PH-10. Multiple preclinical toxicity studies should be done:

• Genotoxicity (Ames, Micronucleus and Comet assays),
• Reproductive toxicology (rat DART testing),
• Dermal toxicity and phototoxicity (minipig),
• Dermal carcinogenicity, and
• Chronic toxicity (minipig, rat).

In addition, a key PH-10 patent was issued in March (Topical medicaments and methods for photodynamic treatment of disease[50]), which protects use of the drug for non-dermatology applications (e.g., bladder cancer, oral disease, post-surgery). Observations and conclusions from the toxicity studies, clinical mechanism data and existing clinical data from previous trials should facilitate Agency discussions about consensus pivotal Phase 3 trial designs for psoriasis and/or atopic dermatitis. With regulatory clarity in hand, Company management should be in a much better position to finally achieve a global or regional dermatology license for this asset.

Much more data should be presented or published

Provectus last published new clinical data (about its melanoma Phase 2 trial) at the 2010 annual meeting of the Society for Melanoma Research[51] and new preclinical data (about PV-10 in combination with immune checkpoint inhibition [anti-CTLA-4]) at the 2013 annual meeting of the American Association for Cancer Research (“AACR”).[52] Phase 2 trial results and exploratory analyses were presented at annual meetings of the American Society of Clinical Oncology (“ASCO”), European Society for Medical Oncology (“ESMO”), European Association of Dermato Oncology (“EADO”) and European CanCer Organisation (“ECCO”): ASCO 2009[53], ASCO 2010[54], ESMO 2012[55], ECCO 2013[56], EADO 2014[57], ASCO 2014[58] and ESMO 2014[59].

Moffitt published preclinical data at the 2012 annual meeting of the Society of Surgical Oncology[60], AACR 2013[61] and 2014 annual meeting of the Society for Immunotherapy of Cancer (“SITC”)[62], and clinical data at AACR 2014[63] and ASCO 2014[64]. This cumulative work independently repeated and reproduced the Company’s preclinical and clinical results and claims. Moffitt’s recent work has focused on PV-10’s mechanism. According to Moffitt’s disclosures, Provectus provided only PV-10 to the cancer center for its experiments (a non-author is a paid consultant to the Company).

Additional preclinical and clinical data should be forthcoming this year and next:

• Liver (clinical): Company management said they expect to report data at one or more international conferences this summer, possibly the World Congress on Gastrointestinal Cancer in Barcelona, Spain (April 21 abstract notification) and/or the Annual Conference of the International Liver Cancer Association in Paris, France (late-June abstract notification),

• PV-10’s mechanism (clinical): It’s unclear when and where Moffitt would present and/or publish additional mechanism results. Previous study results were presented at AACR and ASCO 2014,

• PV-10 in combination with immune checkpoint inhibition (preclinical): It’s also unclear when and where Moffitt would present and/or publish additional results of combining PV-10 and co-inhibitory blockade (anti-CTLA-4, anti-PD-1, anti-PD-L-1). Cursory results were presented at SITC 2014, and

• PV-10 in combination with XRT (clinical): Australian researchers should disclose interim clinical data of an investigator-initiated study of 25 patients who received a single injection of PV-10 per lesion followed by XRT. Preliminary study of 3 patients in 2010[65] yielded complete responses without a significant increase in acute radiation reaction.

Mechanism (melanoma [clinical], breast [preclinical]), combination with other treatments (XRT and melanoma [clinical]) and therapies (systemic chemotherapy and liver [preclinical], immune checkpoint inhibition/melanoma [preclinical]), overall survival-focused (liver [clinical]) and interim melanoma Phase 3 (clinical) data affirms PV-10’s proposition that complete response of cancerous tumors after PV-10 injection results in the disease staying away, irrespective of solid tumor indication or disease stage. It’s very likely that late-stage disease, where patients may be overwhelmed by inaccessible tumor burden, requires the additional use of XRT or immune checkpoint inhibition to reduce this burden before the PV-10-stimulated immune system finishes the job.

Favorable pharmaceutical industry trends should provide valuation tailwinds

Provectus achieved a sizeable measure of regulatory clarity it previously lacked. I framed this necessary step in my September 2013 investment letter[66]:

“When regulatory clarity is achieved, it would be FDA validation of the local agent's systemic properties and benefit. When more clinical trials are conducted and regulatory clarity is achieved for other indications, regional and/or worldwide license transactions are consummated, and the investment community is more aware of the drug and the Company, Provectus' market capitalization should substantially increase from its current valuation. The share price should enjoy significantly more upside from there when acquisitive global pharmaceutical companies, having fully embraced PV-10's immense oncology value proposition for their respective business franchises, pay up for this unique, fully owned, cancer treatment asset.”

Key clinical trends have overtaken the pharmaceutical industry in regards to treating cancer: the tumor microenvironment as the route of delivery, supercharging T-cells to better harness the immune system, and de facto combination therapy for late-stage disease.

Immune checkpoint inhibition (so called “releasing the brakes”) no longer is the panacea it initially was believed to be for harnessing the immune system to treat late-stage cancer, the pharmaceutical industry’s historic and current focus. After well more than a decade of development and hundreds of millions if not billions of cumulative dollars in expenditure by Big Pharma, the FDA approved anti-CTLA4 therapeutic ipilimumab in 2011 for advanced melanoma, and next generation anti-PD-1 therapeutics pembrolizumab and nivolumab burst onto the scene at ASCO 2013 and were first approved in 2014 for advanced melanoma. Non-specific immunotherapies require a dynamic tumor antigen expression process — front-end radiation therapy, targeted chemotherapies or intralesional therapies like T-VEC and PV-10 — to optimally potentiate the body’s T-cells against the inciting cancer. That is, they require the “engine to be started” and/or the “gas pedal to be stepped on” because the car itself may be unable to move or is moving very slowly (i.e., make non-immunogenic tumors immunogenic, and immunogenic ones more so). Without a viable front-end (immune system primer), the back-end (immune checkpoint inhibition) is limited.

Big Pharma oncology franchises are separating into three categories: those with immune checkpoint inhibition that admit the need for a primer (e.g., Bristol-Myers, Merck, Novartis, Roche), those who do not fully admit such need (e.g., AstraZeneca), and those who have a stimulatory compound (e.g., Amgen), an inhibitor (e.g., Pfizer) or neither (e.g., J&J) and know they require a matching set to remain relevant in oncology. Outside of PV-10 there is an inventory of various co-stimulatory and agonist agents (e.g., anti-CTLA-4, anti-CD137, anti-OX40, anti-CD27, IL-2, IL-12, vaccines, IFN-α, GM-CSF, anti-CD40, Toll-like receptors[67]) that can start the engine and/or step on the gas pedal. Each has advantages and disadvantages with regard to cost, safety and level of antigen expression individually and in combination with a co-inhibitory or antagonist partner. PV-10’s unique functional and approvable blend of low cost, high safety, high expression, and orthogonality may offer the perfect way to prime the immune system[68].

Other relevant Big Pharma trends include obviously bulging balance sheet cash amounts and emptying drug pipelines.

Provectus is on the cusp of meriting a “Phase 3 badge.”[69] Small biotechnology companies whose drug candidates present compelling value propositions based on well-defined patient populations, notable clinical value and advantageous economic value[70]  — and have achieved regulatory clarity — should see dramatically increased market capitalizations, and have presented to them lucrative buy-out options commensurate with the breadth and depth of their propositions. I believe Provectus will be one of these companies:

• The Company’s drug compounds have compelling clinical and business value propositions. Regulatory clarity has been achieved. Commercial validation should follow,

• The first pathway to oncology approval is set,

• The oncology clinical development program should expand this year,

• A regional geographic license for PV-10 should follow in China and/or India, along with a local liver trial involving PV-10,

• Inflammatory dermatoses therapeutic PH-10 finally should garner more attention,

• More data should be presented and published this year, and

• Favorable pharmaceutical industry trends should further improve Provectus’ M&A valuation prospects.

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[1] www.pvct.com

[2] Gnehm R.Ueber Tetrachlorphtalsäure. Justus Liebigs Annalen der Chemie 1887; 238:318–338

[3] Feenstra RPG and Tseng CG. What is actually stained by rose bengal? Arch Ophthalmol 1992; 110:984-993.

[4] Sjögren H. (1933) Zur Kenntnis der Keratoconjunctivitis sicca (Keratitis filiformisbeiHypofunktion der Tränendrüsen). Acta Ophthalmol 1933; 11 suppl. 2:1-151.

[5] Delprat GD. Studies on liver function: Rose Bengal elimination from the blood as influenced by liver injury. Archives of Internal Medicine 1923; vol. 32:401-410.

[6] http://www.ncbi.nlm.nih.gov/pubmed/?term=%22rose+bengal%22

[7] http://www.ncbi.nlm.nih.gov/pubmed/?term=%22rose+bengal%22+cancer

[8] Ito A, Watanabe H, Naito M, Aoyama H, Nakagawa Y, Fujimoto N. Induction of thyroid tumors in (C57BL/6N x C3H/N)F1 mice by oral administration of 9-3',4',5',6'-tetrachloro-o-carboxyphenyl-6-hydroxy-2,4,5,7-tetraiodo-3-isoxanthone sodium (Food Red 105, Rose Bengal B). J Natl Cancer Inst 1986 Jul; 77(1):277-81.

[9] http://www.pvct.com/news/PT-August-2010.pdf

[10] http://www.pvct.com/presentation/corporate/img6.html

[11] Moffitt, 4th European Post-Chicago Melanoma/Skin Cancer Meeting (2014)

[12] Rose Bengal, PV-10 and PH-10’s active pharmaceutical ingredient, has an established FDA safety profile for prior human use as an intravenous hepatic diagnostic (Robengatope®) and topical ophthalmic diagnostic (Rosettes® and Minims®)

[13] Thompson, Agarwala et al., Phase 2 Study of Intralesional PV-10 in Refractory Metastatic Melanoma, Annals of Surgical Oncology (2014)

[14] http://www.pvct.com/publications/SPTAACR2013-Final.pdf

[15] Provectus website, page: Clinical Trials and Compassionate Use/PV-10 for Melanoma

[16] http://eorder.sheridan.com/3_0/display/index.php?flashprint=3356

[17] http://www.pvct.com/publications/SITC-Poster-23Oct12.pdf

[18] http://www.pvct.com/news/MelanomaRes-v20-n1/index.html

[19] http://www.pvct.com/publications/AACR-Poster-10-Apr-2013.pdf

[20] http://www.pvct.com/publications/SITCposter2014.pdf

[21] Illustrative examples: Moffitt Cancer Center, Various; Zamani Taghizadeh Rabe et al., Rose Bengal suppresses gastric cancer cell proliferation via apoptosis and inhibits nitric oxide formation in macrophages, Journal of Immunotoxicology (2014); Tan et al., Novel use of Rose Bengal (PV-10) in two cases of refractory scalp sarcoma, ANZ Journal of Surgery (2013); Koevary, Selective toxicity of rose bengal to ovarian cancer cells in vitro, International Journal of Physiology, Pathophysiology and Pharmacology (2012)

[22] http://www.pvct.com/news/PVCT-TRANS-CONF-CALL-3-12-2015.pdf

[23] http://www.pvct.com/publications/ASCO-Poster-2014.pdf

[24] http://www.pvct.com/publications/Provectus-ESMO-2014-09-28.pdf

[25] https://clinicaltrials.gov/archive/NCT02288897/2015_03_12

[26] https://www.federalregister.gov/articles/2014/10/08/2014-23965/prescription-drug-user-fee-act-patient-focused-drug-development-request-for-comments

[27] http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/10/WC500005222.pdf

[28] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571061/

[29] http://www.skincancer.org/publications/the-melanoma-letter/spring-2012-vol-30-no-1/PV-10

[30] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323546/

[31] http://www.fiercebiotech.com/press-releases/vical-phase-3-trial-allovectinr-fails-meet-efficacy-endpoints

[32] http://www.fiercebiotech.com/story/amgens-viral-cancer-vaccine-misses-its-survival-goal-melanoma-phiii/2014-04-04

[33] See table, http://provectuspharmaceuticalsinc.blogspot.com/p/why-im-long-provectus.html

[34] http://www.cancer.gov/cancertopics/pdq/treatment/melanoma/HealthProfessional/Page9#_693

[35] http://www.fda.gov/AboutFDA/CentersOffices/OfficeofMedicalProductsandTobacco/CDER/ucm093907.htm

[36] http://seekingalpha.com/article/2999576-provectus-biopharmaceuticals-pvct-q4-2014-results-earnings-call-transcript

[37] http://meetinglibrary.asco.org/content/128142-144

[38] http://www.pvct.com/pressrelease.html?article=20091001

[39] http://www.pvct.com/pressrelease.html?article=20110127

[40] http://www.pvct.com/pressrelease.html?article=20120927

[41] https://clinicaltrials.gov/ct2/show/NCT00986661?term=Provectus&rank=4

[42] http://theoncologist.alphamedpress.org/content/15/suppl_4/42.full

[43] http://eorder.sheridan.com/3_0/app/orders/3356/article.php

[44] http://www.sec.gov/Archives/edgar/data/315545/000119312515088039/d862762d10k.htm

[45] http://seekingalpha.com/article/2999576-provectus-biopharmaceuticals-pvct-q4-2014-results-earnings-call-transcript

[46] http://www.pvct.com/pressrelease.html?article=20141114

[47] http://seekingalpha.com/article/2667675-provectus-biopharmaceuticals-pvct-q3-2014-results-earnings-call-transcript

[48] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1422461/

[49] http://www.pvct.com/pressrelease.html?article=20100107

[50] http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PTXT&s1=provectus&OS=provectus&RS=provectus

[51] https://www.pvct.com/news/PV-10_Presentation_Sydney-Nov-2010.pdf

[52] http://www.pvct.com/publications/AACR-Poster-10-Apr-2013.pdf

[53] http://www.pvct.com/publications/ASCO_Poster-2009.pdf

[54] http://www.pvct.com/publications/ASCO_Poster-2010.pdf

[55] http://www.pvct.com/publications/PVCT-ESMO-2012-Poster.pdf

[56] http://www.pvct.com/publications/Provectus-ECCO-2013.pdf

[57] https://www.pvct.com/news/PV-10-EADO-2014-May.pdf

[58] http://www.pvct.com/publications/ASCO-Poster-2014.pdf

[59] http://www.pvct.com/publications/Provectus-ESMO-2014-09-28.pdf

[60] http://www.pvct.com/publications/Paul-Toomey-PV10-SSO-2012.pdf

[61] http://www.pvct.com/publications/SPTAACR2013-Final.pdf

[62] http://www.pvct.com/publications/SITCposter2014.pdf

[63] http://www.pvct.com/pressrelease.html?article=20140306

[64] http://meetinglibrary.asco.org/content/132288-144

[65] http://www.pvct.com/news/MelanomaRes-v20-n1/index.html

[66] http://seekingalpha.com/article/1710182-why-im-long-provectus-pharmaceuticals

[67] http://www.sciencedirect.com/science/article/pii/S1074761313002963

[68] http://ecancer.org/news/5790-provectus-outlines-path-forward-for-pv-10.php

[69] http://www.thestreet.com/story/12696926/1/the-feuerstein-ratain-rule-update-still-perfect-predicting-small-cap-cancer-drug-failure.html

[70] http://obroncology.com/obrgreen/article/Ensuring-a-Compelling-Value-Proposition-in-Oncology