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}

The Early Obsolescence of Checkpoint Inhibitors

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Take as a starting point Inman et al.’s 2007 article entitled Costimulation, coinhibition and cancer, and their statement therein: (underlined emphasis is mine):

“If sufficient co-stimulation is provided in the presence of adequate tumor-associated antigenic stimulation, the immune system will act against tumor antigen and, thus, destroy early tumors before they become fully established. Contrarily, if co-inhibitory signaling dominates, the immune system will be tolerized to tumor antigens, and the tumor will be permitted to grow unfettered and unmolested by the immune system. If neither co-stimulatory nor co-inhibitory signals dominate, the adaptive immune system may remain in a tenuous state of equilibrium, militating against tumor outgrowth with varying degrees of success.”

The essence of the authors’ view might be that the immune system is capable of decisively acting against cancer only in the situation where or circumstance that co-stimulation dominates co-inhibition. Take also as context to this starting point, however, that what we don’t know about the immune system probably dwarfs what we know about it.

The notion of “releasing the brakes” in the medical literature and mainstream press describes the approach of inhibiting cancer’s ability to suppress or block the body’s immune system from acting, and thus to evade attack. Although possibly coined in the early-2000s (see, for example, Tirapu et al.’s 2002 article entitled Effective tumor immunotherapy: start the engine, release the brakes, step on the gas pedal,...and get ready to face autoimmunity), use of the releasing-the-brakes phrase may have grown more widespread starting in the late-2000s and around the time of Dr. James Allison, Ph.D’s seminal work of blocking (inhibiting) the CTLA-4 protein receptor (using Bristol-Myers’ ipilimumab) and, later, the follow-up scientific exploration of blocking (inhibiting) PD-1 and PD-L1 ligands too (and associated PD-1 therapeutics pembrolizumab and nivolumab, for example, from Merck and Bristol-Myers, respectively).

Medical literature has more sparsely touched on, and mainstream press much less so, the other two components of the get-the-car-moving analogy (where the car is the immune system), “starting the engine” and “stepping on the gas pedal,” where these phrases relate to different aspects of stimulating the body’s immune system.

Possibly over-using the car analogy further, with the potential risk of over-simplifying it inappropriately, consider T cell immunity as a car at rest. More immunogenic tumors and their associated cancers like melanoma are like a car sitting on a slight incline. Release its brakes by treating the tumors (and thus the cancer) with checkpoint inhibitors, and the car may roll forward move some distance, notable or otherwise. With other less or non-immunogenic cancers, think of the car as sitting on a flat surface. Releasing the brakes does not enable the car to move any meaningful distance, if at all.

If you want to get the car to really move, you have to start its engine, and then step on its gas pedal. Releasing the brakes might help the car move farther and faster, but it also is quite possible the car may be able to move sufficiently without the need for further action other than to start its engine and/or stepping on its gas pedal.

The continued use of get-the-car-moving analogy of course requires the assumption the car can drive by itself; that is, the immune system can handle its own business once it has been started, and is appropriately up and running from stepping on the gas pedal.

Now consider Winograd et al.’s 2015 article entitled Induction of T cell immunity overcomes complete resistance to PD-1 and CTLA-4 blockade and improves survival in pancreatic carcinoma. In particular, note the underlined sentences (my emphasis) from the article’s abstract:

“Disabling the function of immune checkpoint molecules can unlock T cell immunity against cancer, yet despite remarkable clinical success with monoclonal antibodies (mAb) that block PD-1 or CTLA-4 resistance remains common and essentially unexplained. To date, pancreatic carcinoma is fully refractory to these antibodies. Here, using a genetically engineered mouse model of pancreatic ductal adenocarcinoma in which spontaneous immunity is minimal, we found that PD-L1 is prominent in the tumor microenvironment, a phenotype confirmed in patients; however, tumor PD-L1 was found to be independent of IFN-γin this model. Tumor T cells expressed PD-1 as prominently as T cells from chronically infected mice, but treatment with PD-1 mAb, with or without CTLA-4 mAb, failed in well-established tumors, recapitulating clinical results. Agonist CD40 mAb with chemotherapy induced T cell immunity and reversed the complete resistance of pancreatic tumors to PD-1 and CTLA-4. The combination of αCD40/chemotherapy plus PD-1 and/or CTLA-4 induced regression of subcutaneous tumors, improved overall survival, and confered curative protection from multiple rechallenges, consistent with immune memory not otherwise achievable. Combinatorial treatment nearly doubled survival of mice with spontaneous pancreatic cancers although no cures were observed. Our findings suggest that in pancreatic carcinoma, a non-immunogenic tumor, baseline refractoriness to checkpoint inhibitors can be rescued by the priming of a T cell response with αCD40/chemotherapy.”

CD40 is a co-stimulatory protein, while chemotherapy has been understood to act in a stimulatory fashion through the subsequent release of cancer antigens.

Achieving T cell immunity almost if not actually by definition should mean overcoming resistance to cancer, thus overcoming checkpoint blockade and mitigating the need to artificially release the brakes.

Should stimulation via stimulatory therapeutics and therapies start the engine and enables the gas pedal to be stepped on sufficiently and appropriately (i.e., with minimal or manageable side effects or adverse events) so as to achieve T cell immunity, brakes may not be necessary once the car is moving (in context, and given the car [the immune system] can drive itself and not careen off the road because it then should know what it is doing).

Over time, however, road friction may start slowing the car down to the point where waning immunosurveillance (the immune system recognizing and eliminating continuously arising cancerous cells) no longer can protect the patient from relapse (analogous to how waning varicella zoster antibody titers may result in a bout of shingles). Keeping the brakes disengaged, especially with non-immunogenic tumors, should have some role going forward, making Merck, Bristol-Myers, Roche, AstraZeneca, Pfizer and other companies’ checkpoint inhibitors not necessarily obsolete as much as persnickety.

"These murine studies support combination therapy with IL PV-10 and co-inhibitory blockade."

Provectus issued a press release, filed an associated 8-K and made available Moffitt Cancer Center's PV-10-related poster from the 29th annual meeting of the Society for Immunotherapy of Cancer ("SITC") Monday. Of the conclusions provided by Moffitt, it struck me the key one was the first:

Moffitt affirmed there is a clinical rationale and value proposition, based on pre-clinical murine model work, to undertake a study (studies) combining PV-10 and each/any of the anti-PD-L1, -PD1 and -CTLA4 therapeutic agents.

❐ Moffitt's initial murine model work investigating PV-10 as a monotherapy -- "Intralesional Injection of Melanoma with Rose Bengal Induces Regression of Untreated Synchronous Melanoma In a Murine Model," Society of Surgical Oncology Annual Meeting, March 2012, and "Intralesional Injection with PV-10 Induces a Systemic Anti-tumor Immune Response in Murine Models of Breast Cancer and Melanoma," American Association for Cancer Research Annual Meeting, April 2013 -- demonstrated:

• Regression in both injected and un-injected melanoma tumors,
• Anti-tumor immunity (T-cell generation & activity), and
• Increased survival (in mice).

❐ Moffitt followed up their mousie work with a human feasibility study of PV-10 as a monotherapy -- "Assessment of immune and clinical efficacy after intralesional PV-10 in injected and uninjected metastatic melanoma lesions," American Society of Clinical Oncology Annual Meeting, June 2014 -- that demonstrated:

• Regression in both injected and un-injected melanoma tumors, and
• Anti-tumor immunity (T-cell generation & activity).

❐ Moffitt continued their murine model work investigating PV-10 in combination with each of three categories of checkpoint inhibitors (anti-PD-L1, -PD1, -CTLA4) -- "Efficacy of Intralesional Injection with PV-10 in Combination with Co-Inhibitory Blockade in a Murine Model of Melanoma," Society for Immunotherapy of Cancer Annual Meeting, November 2014 -- that demonstrated:

• Regression in both injected and un-injected melanoma tumors,
• Anti-tumor immunity (T-cell generation & activity), and
• Increased survival (in mice).

All of Moffitt's work, murine and human, involved a single intralesional injection of PV-10 per injected lesion. It would appear what Moffitt is doing with PV-10 the way they are doing it is procedural, meaning the cancer center is trying to better understand PV-10's tumor-specific immunity with scientific experimental methods, rather than with specific clinical or clinically translational approaches at this time. Ultimately, it seems Moffitt wanted to know if PV-10 worked, found out it did, then wanted to know how well it worked, and then found out how much it did -- as a single agent, and in combination with other agents.

All Moffitt posters -- SSO 2012, AACR 2013, ASCO 2014, SITC 2014 -- have been exclusively co-authored by Moffitt researchers/employees. It is interesting to note Provectus, it appears, freely allowed the cancer center to undertake this work without, it would seem, involvement or interference. I imagine the company, in addition to providing PV-10 drug product to Moffitt, compensates or pays or contributes funding to the cancer center and/or researchers in some form or fashion, like other biopharmaceutical companies do.

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The Methods section of Moffitt's SITC 2014 poster was sparse on experimental method detail, as was the Methods section of their AACR 2013 poster. As such, we will have to wait for an/the associated peer-reviewed paper that would describe the method(s) of the SITC work (as their 2013 PLOS One paper did for the AACR poster and work); specifically, the modifications the cancer center made to their PV-10 injection approach (contrasted with the AACR work's approach) to elicit and elucidate the value of PV-10 in combination with checkpoint inhibitors.

The 2013 "monotherapy" poster/paper appeared to show relatively greater interferon gamma production relative to control than the 2014 "combination therapy" poster did. PV-10's propensity to completely destroy and/or dramatically reduce injected and un-injected tumors -- the subject of Moffitt's August 2013 Single Injection May Revolutionize Melanoma Treatment, Moffitt Study Shows press release-- must have required modifications to the experimental design in order to facilitate the combination therapy murine model work (i.e., don't kill the tumor completely but "partially kill" it so as to observe and measure the subsequent effect of a checkpoint inhibitor on PV-10-damaged-but-not-destroyed-tumor), such as treating part of a large tumor, reducing PV-10 dose per volume, or diluting the concentration of the drug.

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Peter noted in the company's third quarter 10-Q:

We also have begun to consider co-development transactions with one or more pharmaceutical or biotech companies to combine PV-10 with immunology agents such as those referred to as immune checkpoint inhibitors...Furthermore, the strategy of the Company for the benefit of stockholders is a series of partnerships followed by an acquisition of the Company along the lines of Celgene-Abraxis, although there can be no assurance that such partnerships or acquisition will occur. An interim transaction could be a co-development deal like Roche-NewLink, Bristol-Celldex or AstraZeneca-Incyte. {Underlined emphasis is mine.}

NewLink published preclinical combination study work (their drug + anti-PD1/PD-L1 antibodies) at AACR 2014 (April)*, co-conducted by NewLink employees and Georgia Regents University Research Institute researchers/employees.

• The company licensed their drug to Genentech/Roche in October 2014.

* "The current preclinical studies suggest a mechanistic rationale for a combining IDO pathway

inhibitors with agents targeting the PD-1/PD-L1/PD-L2 pathway."

Celldex published preclinical combo work (their drug + checkpoint blockade therapies) at SITC 2013 (November)*, conducted by Celldex employees.

• The company announced a combination collaboration of their drug with Bristol-Myers' PD-1 agent in May 2014.

* "These studies...support the initiation of combination trials with conventional and immune-based therapies."

Incyte filed a protocol for combining its subject drug with Bristol-Myers' anti-CTLA-4 agent Yervoy in 2012. From what I can gather (and I may be wrong) the trial of the combination therapy was initiated alone by Incyte (I cannot find any publication of preclinical work that may have preceded this trial). At an ASCO 2014 presentation of a Phase 1/2 melanoma study, principal investigators (that included Moffitt Cancer Center's Dr. Jeffrey Weber, M.D., Ph.D.) noted "[p]reclinical data support antitumor synergy for INCB024360 when administered with an antibody antagonist to checkpoint receptors," referencing a October 2013 (submitted)/February 2014 (published) SITC journal paper* (the paper, however, does not present preclinical combo work on the subject drug but another related Incyte compound), co-conducted by Celldex employees and the University of Chicago researchers/employees

• The company announced combination collaborations of their drug with Merck's PD-1 agent in February 2014, AstraZeneca's PD-L1 agent in May 2014, and Bristol-Myers' PD-1 agent also in May 2014.

* "These three combinations are attractive to pursue clinically..."

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Provectus published preclinical combination study work (PV-10 + anti-CTLA-4 mouse antibodies) in April at AACR 2013. Moffitt published their preclinical combination study work (PV-10 + anti-PD-L1, -PD1, and -CTLA4) in November at SITC 2014, of course.

The company should have sufficient data to establish the rationale for a combination study with a Big Pharma partner:

• Preclinical from Moffitt's poster, and additional material not included on it,

• Clinical, perhaps, and in context, from Moffitt's human feasibility study ("Six of 8 patients had metastatic disease refractory to previous ipilimumab, anti-PD-1 and/or vemurafenib therapy"), and

• Clinical, possibly, from Provectus' compassionate use program ("CUP"), where patients previously on checkpoint inhibitors who recurred from these may have sought help from PV-10 (Phase 2 patients were refractory to a median of 6 previous interventions, including immunotherapy (21%)).

Arriving at an agreement on business terms of a so-called co-development deal is/will be another story; however, this information should be the hard data of PV-10's immunological activity (most of it generated independently of Provectus by Moffitt) that may facilitate a discussion, or two, with Big Pharma.

Immune Surveillance

Following up on my Juxtaposition blog post, there is the potential for a melanoma combination therapy Phase 1 (or 1b)/2 trial of PV-10 and a checkpoint inhibitor, which management briefly discussed on their 2Q14 conference call.

Eric: As Pete mentioned earlier, we're also looking beyond single-agent therapy with PV-10 to address the needs of patients with more extensive disease, particularly those with visceral tumors that are not injectable. One attractive and complementary approach may be to combine PV-10 with a systemic immunotherapy, such as an immune checkpoint protein inhibitor. Immune checkpoint protein inhibitors, such as anti-CTLA4, anti-PD-1, and anti-PD-L1 agents, are an important advance in the treatment of melanoma and other cancers, another subject of intense development in our industry. 

However, while these drugs represent an important step forward, like any drug they are not perfect and they might be improved. As was clearly presented by the medical oncology community earlier this summer at ASCO, using an agent like PV-10 to prime the immune system could be synergistic in combination with such a systemic agent. 

Our patent application on this strategy was published in 2012 and we've been vigorous by pursuing this approach since. The nonclinical research we first presented at the Society for Immunotherapy of Cancer Annual Meeting in 2012, together with ongoing translational clinical research on PV-10's mechanism of action that we were sponsoring at Moffitt Cancer Center and our own Phase 2 data, provide a rationale for combination testing of PV-10. 

This development track, separate from the Phase 3 study I discussed earlier, could represent a path forward for patients with significant disease burden not amenable to intralesional injection, and is a possible candidate for co-development with one or more pharmaceutical or biotech companies. {Underlined emphasis is mine}

Eric wouldn't insert statements about this topic into his comments until and unless at least minimally substantive progress has been made towards this end. He first addressed a combination study via St. Luke’s Cancer Center and Temple University's and principal investigator Dr. Sanjiv Agarwala, M.D.'s presentation at the 10th European Association of Dermato-Oncology (EADO) congress in May ("Commencement of combination studies in Stage IV disease in second half of 2014"). The reality of a combination study/trial probably is dependent on the partnering pharmaceutical company's, the one with the checkpoint protein inhibitor to be combined with PV-10, interest in and willingness to:

• At a minimum, run and shoulder the cost of a trial, aside from Provectus's minimal expense to provide PV-10 drug product,
• At a minimum (more than likely) make a one-time payment to Provectus, such as was the case in Bristol-Meyers' collaboration with Celldex Therapeutics, and
• Forego meaningful rights of first somethings, unless the partner fully compensates Provectus for this ask.

I summarized a number of previously announced and/or conducted combination studies in my Combinations (July 24, 2014) news items under the blog's News tab.

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Although Provectus management has understood and communicated the potential for combination therapy involving PV-10 for the treatment of metastatic melanoma, noting this on the 2Q14 conference call (i.e., the Provectus-Pfizer patent application, the SITC 2012 poster presentation), they historically have not prioritized this potential regulatory pathway, and have been disinterested in and unwilling to allocate resources to conduct a typical combination study where companies share trial costs and the "non-CTLA-4/PD-1" (i.e., non-Bristol-Myers or Merck) partner conducts (sponsors) the trial.

There is no certainty a trial comes together, but it would appear Provectus is engaged in the kind of basic or fundamental conversations, data sharing and expectation setting necessary to contemplate a combination study and develop the associated relationship between the parties involved.

The consensus of melanoma key opinion leaders appears to be that “[t]he future lies in combinational approaches with drugs from the new melanoma landscape” (e.g., intralesional therapies, BRAF inhibitors, ipilimumab, anti-PD-1 antibodies). Moffitt Cancer Center's pre-clinical work combining PV-10 with other approved and investigational immunotherapeutic agents more than likely would be, and would have to be, the driving force behind any clinical combination study focused on metastatic melanoma patients with visceral disease (i.e., Stage IV M1b-c). The cancer center already has alluded to the completion and results of this pre-clinical work, which should form the rationale for a future clinical trial or trials, at ASCO 2014 by concluding "IL PV-10 may be rationally combined with systemic immunotherapy for the treatment of metastatic melanoma," and again at the 4th European Post-Chicago Melanoma & Skin Cancer Meeting 2014 by concluding "[p]reclinical data suggest PV-10 would be a good candidate to evaluate in conjunction with available systemic therapies and new agents in development."

I previously wrote about historical pre-clinical and some clinical work on PV-10 in combination with other treatments (systemic chemotherapy [pre-clinical], systemic immunotherapy [pre-clinical], radiotherapy [clinical]) in my "IL PV-10 may be rationally combined with systemic immunotherapy for the treatment of metastatic melanoma" blog post.

A potential clinical trial combining PV-10 with a checkpoint inhibitor should be viewed in the context of other clinical trials and studies Provectus expects  hopes to commence in 2014 and/or 2015:

• Melanoma, PV-10: Phase 3 trial, locally advanced unresectable/unresected cutaneous melanoma, 2H2014,

• Dermatology, PH-10: Phase 1 trial, mechanism of action/feasibility study, 2H2014 (my estimate),

• Liver cancer, PV-10: Phase 2/3 trial, locally advanced, unresectable/unresected liver cancer (my speculation of the trial title), 1H2015 (my estimate); the trial also should include liver tumors caused by primary non-liver cancers (i.e., cancers metastatic to the liver), and

• Melanoma, PV-10: Phase 1 (or 1b)/2 trial, metastatic melanoma, 1H2015 (my estimate, if a deal is consummated in 4Q14); with a trial population similar to that of the ipilimumab+talimogene laherparepvec of mostly Stage IV M1b-c patients

I do not believe a deal between Provectus and a pharmaceutical company (and its checkpoint inhibitor), the first step towards an eventual clinical trial, would be consummated until Moffitt's preclinical combination study data is more broadly communicated. Meaning, neither Bristol-Myers nor Merck, for example, would even contemplate acceding to Provectus deal expectations until and unless Moffitt substantiates their above mentioned conclusions with very compelling data (i.e., together PV-10 makes your drug much better than your drug alone).

I think the venue for the cancer center's communication will be the 29th Annual Meeting of the Society of Immunotherapy for Cancer, November 6-9, 2014. Provectus first addressed combination treatment at the 2012 annual SITC meeting concluding the co-administration of PV-10 immuno-chemoablation with other systemic therapy could yield potent synergy in uninjected tumors.

But, more than just the pre-clinical drug combination study results, I also am very interested in other aspects of Moffitt's work, and Dr. Jeffrey Weber, M.D., Ph.D.'s comprehensive views on the utility and use of PV-10.

First, what is the basis for Weber's comment "PV-10 might offer the perfect way to prime the immune system?" Dr. Weber has said (paraphrasing) the utility of a primer is simply its ability to synergize with the immune agent in question in terms of clinical effect when given prior to the second agent. Moffitt data showing the strength of the systemic responses PV-10 can stimulate (i.e., efficacy of PV-10 plus a checkpoint inhibitor >> efficacy of the checkpoint inhibitor alone) should make this/his point.

Second, what is PV-10's role and participation in each step of the cancer immunity cycle (Chen & Mellman, 2013)? Under the blog's PV-10, and the Cancer Immunity Cycle tab I illustrate PV-10's involvement in steps 1 (release of cancer cell antigens), 2 (cancer antigen presentation), 3 (priming and activation) and 7 (killing of cancer cells). And although Dr. Weber has said (paraphrasing) one should prioritize which steps are the most important (presumably in order to determine how to utilize what agents in combination and why; for example, impacting on endothelial cells have been less promising as a therapeutic than many of the other steps), I'd like to understand Moffitt's view on PV-10's involvement in steps 4 (trafficking of T cells to tumors), 5 (infiltration of T cells into tumors) and 6 (recognition of cancer cells by T cells).

And third, what are Weber's thoughts about PV-10 in the context of or in regards to immune surveillance? Immune surveillance theory is:

The concept that the immune system protects the host against cancer was first posited by Ehrlich in 1909 (1) and modified in the 1950s by Burnet and Thomas (2, 3), who proposed that it was instrumental in eliminating precancerous or cancerous cells, through a “surveillance” function. However, the concept fell out of favor when studies in the 1980s indicated that tumors failed to develop more rapidly in nude mice (which lack T cells and B cells, but not NK cells) than in wild-type mice. It was resurrected in the 1990s, when a body of evidence emerged indicating that immunodeficient mice were at greater risk for spontaneous tumor development (4). These studies led to further refinement of the theory, now referred to as “cancer immunoediting,” encompassing three phases: elimination, equilibrium, and escape. (J Clin Invest. 2007;117(5):1130–1136. doi:10.1172/JCI32136)

In a February article of The American Journal of Managed Care, Weber said:

"Well, the Holy Grail of cancer immunology is to create a cancer vaccine. Provenge was the first and only cancer vaccine that was ever approved. But that is truly the mantra, that is, the immune system is the ultimate way to perform targeted therapy. So immunotherapy is targeted therapy, and its hallmark is memory." {Underlined emphasis is mine} 

And: "There are certainly data to suggest that this idea of immune surveillance is indeed valid. On the other hand, people on immunosuppressants don’t always present with 30 different types of solid tumors. Transplant patients often develop squamous skin cancers, especially virally related squamous skin cancers. So there are data to suggest that we always have immune surveillance to prevent cancer from developing." 

And: "You can divide immunotherapies into 4 or 5 categories: these include chemicals like cytokines; antibodies…cells, which are not really well developed; vaccines, which is always the Holy Grail to try to vaccinate someone against his or her own cancer. You’ve got 1 approved vaccine. One of the antibodies, which are the most exciting and promising, is approved. That’s ipilimumab, the anti-CTLA4 antibody. In terms of the cytokines, in 1996 and 1998 IL-2, interleukin-2, was approved for kidney cancer and for melanoma. The cell therapy is immature, and you will hear a lot more about the antibodies coming up in the next couple of years." {Underlined emphasis is mine} 

Recall that Moffitt said, following their initial murine model work, that:

"These murine studies confirm that PV-10 chemoablation results in both a direct effect on injected lesions as well as a systemic response that leads to regression of uninjected subcutaneous and lung lesions. Intralesional PV-10 treatment leads to the induction of tumor-specific immunity." (AACR 2013) {Underlined emphasis is mine} 

And that Provectus concluded at SITC 2012, by virtue of murine model work:

Chemoablation with PV-10 results in the induction of tumor-specific immunity. Immunodeficient mice exhibited no anti-tumor effect on re-challenge. Adoptive transfer of immunity only occurred following chemoablation in donor mice. Tumor-specific, long-term immunity and adoptive transfer implicate immune cell mediation. This immuno-chemoablative response to PV-10 is tantamount to “in situ vaccination.” (SITC 2012) {Underlined emphasis is mine} 

"IL PV-10 may be rationally combined with systemic immunotherapy for the treatment of metastatic melanoma"

PV-10 can be a significant resource for oncologists treating earlier stages of disease (i.e., locally cutaneous advanced melanoma, for one). The drug also may play a key role for physicians in combination therapies for patients with late to very late stage disease (i.e., metastatic melanoma).

Key opinion leaders in melanoma, like Europe's Dr. Axel Hauschild, M.D., Ph.D., see a role for intralesional agents in combination with other agents to treat metastatic melanoma. Hauschild, a member of OncoSec's Melanoma Advisory Board (OncoSec [OTCQB: ONCS] treats melanoma tumors using intratumoral electroporation of plasmid interleukin-12), led a poster highlights session for melanoma/skin cancers at ASCO 2014. In regard to all intralesional agents he noted (paraphrasing) they have no systemic toxicity, have high CR rates, and are good candidates for combinatorial use due to immune priming {underlined emphasis is mine}.

A very interesting conclusion from Moffitt's Assessment of immune and clinical efficacy after intralesional PV-10 in injected and uninjected metastatic melanoma lesions ASCO poster was "IL PV-10 may be rationally combined with systemic immunotherapy for the treatment of metastatic melanoma" (where IL means intralesional).

Moffitt's Dr. Vernon Sondak, M.D. will speak further about PV-10 at a symposium (see below) of the 4th European Post-Chicago Melanoma Meeting: Interdisciplinary Global Conference on News in Melanoma.

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Provectus has considered the role of PV-10 in combination therapies for some time.

Ex. #1-P (2011) The company began working with strategic advisory board member Dr. Craig Eagle, M.D. (Pfizer) as early as sometime in 2011 (I think) on joint Provectus-Pfizer patent application Combination of Local and Systemic Immunomodulative Therapies for Enhanced Treatment of Cancer. Although the patent was filed in March 2012, its priority data date was October 3, 2011, which coincidentally is the same date as AstraZeneca's MedImmune's MedImmune in-licenses cancer immunotherapy tremelimumab from Pfizer press release: "Under the terms of this agreement, MedImmune will assume global development rights to tremelimumab and Pfizer will retain the rights to use tremelimumab with specified types of combination therapies."

Ex. #2-P (2012) The company presented murine model data on PV-10 combination therapy -- PV-10 and systemic chemotherapy 5-fluorouracil -- applied to hepatocellular carcinoma and melanoma tumors at the October 2012 Society for Immunotherapy of Cancer annual meeting (see Provectus Presents Nonclinical Data on Antitumor Immune Response to PV-10 Immuno-Chemoablation): "Treatment of mice with systemic chemotherapy (i.e., 5-fluorouracil, "5-FU") had minimal effect on either tumor, while combination of intralesional PV-10 with systemic 5-FU elicited maximal response in uninjected tumors."

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Generation of an antitumor response and immunity using a small molecule drug (PV-10)

Ex. #3-P (2013) Provectus presented murine model data on PV-10 combination therapy -- PV-10 and a systemic immunotherapy anti-CTLA-4 antibody at the April 2013 American Association for Cancer Research ("AACR") annual meeting (see Provectus Presents Data on PV-10 Combination Therapy at American Association of Cancer Research Annual Meeting): "PV-10 and PV-10 plus 9H10 exhibited robust response in both treated and untreated tumors. The combination therapy was most effective in the low dose model, where advantages in tumor growth and survival benefit were most pronounced. Increased apparent toxicity of 9H10 at the higher dose levels highlighted the need for establishment of an optimal dose in future mechanism studies and clinical trials...This work shows that, as hypothesized, addition of the immunologic effects of an anti-CTLA-4 agent augments the benefits of PV-10. For visceral or other inaccessible disease, combination of PV-10 with CTLA-4 blockade offers important potential for synergy...PV-10's ability to reduce tumor burden and elicit tumor-specific immunologic stimulation make it a logical potential complement to anti-CTLA-4 agents, such as ipilimumab."

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Combination of PV-10 Immuno-chemoablation and Systemic Anti-CTLA-4 Antibody Therapy in Murine Models of Melanoma

Ex. #4-P (2013) The company again discussed important contexts for PV-10 combination, such as "While more frequent dosing could potentially improve outcomes, for patients with extensive tumor burden inaccessible to PV-10 injections a combination therapy strategy may be attractive" (see June 2013's PV-10 Moves Forward): "In phase 2 and 3 clinical trials, Dr. Wachter pointed out, the higher doses of ipilimumab were associated with an increase in immune-related adverse effects, presumably mirroring the dose response seen in murine models with 9H10. Dr. Wachter concluded, “Examples of successful treatment of established B16 tumors in murine models are rare. These results demonstrate that when all existing tumor is accessible for injection, PV-10 is highly effective both in animal models and clinically in cancer patients. Given that tumor ablation with PV-10 induces tumor-specific immunity, the combination of PV-10 with CTLA-4 blockade has important potential for synergy.” Speaking further in an interview, he said, “I think the case has been made successfully for PV-10’s role as a potent stimulator of specific anti-tumor activity. This is evident in clinical data from Phase 1 and 2 testing, where regression of untreated bystander tumors correlated with ablation of tumors, and in these nonclinical mechanism studies. And, our recent murine studies show that this stimulation works robustly in combination with CTLA-4 blockade.”"

Ex. #5-P&M (2013-2014) Moffitt's Dr. Jeffrey Weber, in addition to his history of regulatory approval contributions to ipilimumab (Yervoy) and vemurafenib (Zelboraf) as monotherapies for metastatic melanoma, has successfully contributed to the approval of combination therapies (see Moffitt Cancer Center reports key role in FDA approval of Mekinist/Tafinlar for melanoma): "“This new combination therapy is a huge step in the right direction for the treatment of melanoma, and our researchers played a large role in bringing this treatment option to patients,” Jeffrey S. Weber, MD, PhD, director of Moffitt’s Melanoma Research Center of Excellence, Tampa, Fla., said in a press release...“A clinical trial in which Moffitt was the major contributor showed a 76% success rate for patients treated with the Mekinist and Tafinlar combination,” Weber said. “We also found this therapy reduced the incidence and severity of some of the toxic effects patients experienced when the drugs were used alone.”"

In the same Provectus-sponsored white paper referenced in Ex. #4-P (2013) above, Eric is further quoted: "Further studies designed to confirm the apparent synergy are underway, including one with only the low 9H10 dose/ PV-10 combination. A phase 1/2 anti-CTLA-4 dose escalation trial with PV-10 is warranted, Dr. Wachter said. Similarly, models for kinase inhibitors and an analogue for vemurafenib are being sought. Vemurafenib, like PV-10, rapidly reduces tumor burden. PV-10 murine research demonstrated unambiguously, Dr. Wachter noted, that tumor burden is a critical variable in predicting response to a combination therapy. It has been suggested that earlier research into therapeutic melanoma vaccines faltered because tumor burden grew beyond the immune system’s capacity for control before the vaccine could develop its full effect. “We think that the combination of PV-10 with something like a kinase-inhibitor has the potential to dial back or reduce tumor burden even better than an anti-CTLA-4 agent while the systemic PV-10 immunologic effect is developing. The kinase inhibitor would do the early work against visceral disease until PV-10 can catch up and take the baton across the finish line.” While the PD-1 And PD-L1 drugs will be interesting candidates for combinations, because none are approved, testing is currently impractical."

Recall Eric's comments regarding Moffitt from the May 23rd conference call presented in my post "Why did it take four years...to arrive at this point?": "We began a dialogue with researchers from Moffitt Cancer Center early in 2011." This dialogue resulted in work conducted by Moffitt that was presented at:

• The March 2012 annual meeting of the Society of Surgical Oncology (see Intralesional Injection of Melanoma with Rose Bengal Induces Regression of Untreated Synchronous Melanoma in a Murine Model),
• The April 2013 AACR annual meeting (see Intralesional Injection with PV-10 Induces a Systemic Anti-tumor Immune Response in Murine Models of Breast Cancer and Melanoma), 
• The April 2014 AACR annual meeting (see Induction of anti-melanoma immunity after intralesional ablative therapy [poster not yet released by Moffitt]), and
• The April 2014 American Society of Clinical Oncology ("ASCO") (see Assessment of immune and clinical efficacy after intralesional PV-10 in injected and uninjected metastatic melanoma lesions). 

Moffitt authors of the first three above mentioned posters include (i) P. Toomey, K. Kodumudi, L. Martin, A. Mackay, A. Sarnaik and S. Pilon-Thomas, (ii) S. Pilon-Thomas, A. Weber, K. Kodumudi, L. Kuhn, P. Toomey, and A. Sarnaik, and (iii) H. Liu, K. Kodumudi, A. Weber, A. Sarnaik and S. Pilon-Thomas. Moffitt's PV-10 ASCO poster [not yet released] was authored by A. Sarnaik, G. Crago, H. Liu, K. Kodumudi, A. Weber, T. McCardle, J. Weber and S. Pilon-Thomas {underlined emphasis is mine}.

• Co-author: Jeffrey S. Weber
• Poster abstract: "Further studies are ongoing to determine the mechanism by which PV-10 increases tumor-specific T cell responses as well as to establish the interaction of intralesional PV-10 with combination checkpoint protein inhibition" {underlined emphasis is mine}
• Poster conclusion: "IL PV-10 may be rationally combined with systemic immunotherapy for the treatment of metastatic melanoma."

The focus of Moffitt's ASCO poster was the center's work with PV-10 in its human feasibility study. As such, I presume Moffitt/Weber's combination exploration and work (presumably murine model studies) will be presented at a later date.

Provectus: "“We think that the combination of PV-10 with something like a kinase-inhibitor has the potential to dial back or reduce tumor burden even better than an anti-CTLA-4 agent while the systemic PV-10 immunologic effect is developing. The kinase inhibitor would do the early work against visceral disease until PV-10 can catch up and take the baton across the finish line.”"

"Whenever T cells and B cells are activated, some become "memory" cells. The next time that an individual encounters that same antigen, the immune system is primed to destroy it quickly. This is active immunity because the body's immune system prepares itself for future challenges" (Source material: Understanding Cancer Series: The Immune System, National Cancer Institute).

In elucidating PV-10's mechanism of immune response (the second of the drug's two-step mechanism of action, the first step being ablation) Provectus noted Moffitt researchers, in their AACR 2014 poster (human feasibility study), showed "...significant decreases in melanoma cells in injected tumors and uninjected bystander tumors 7-14 days after PV-10 injection as evidenced by pathologic evaluation confirmed with immunohistochemical staining of biopsy specimens for melA (a marker of melanoma)... were accompanied by increased populations of CD3+, CD4+ and CD8+ T cells along with NKT cells in peripheral blood." Moffitt concluded in their ASCO 2014 poster that "IL PV-10 can enhance tumor-specific reactivity in circulating T-cells."

It seems to me chemoablation via PV-10 ablation causes antigenization, antigenization causes immunization. PV-10 causes antigenization. Antigenization causes immunization. Antigenization is the expression of antigens, in a tumor into which PV-10 has been injected, in context. Immunization is "the process by which...[the] immune system becomes fortified against an agent." PV-10 facilitates the relationship between antigenization and immunization.

ASCO 2013 Immunotherapy Highlights Covered by $PVCT-$PFE's Combination Therapy Patent Application

Sally Church, writer of the Pharma Strategy Blog, notes ASCO 2013 will bring new data on immunotherapy agents called checkpoint regulatory inhibitors.

All of these groups of compounds are covered by Provectus' joint patent application with Pfizer for the combination of PV-10 and other therapies. All of the above drugs have been combined with PV-10 by Moffitt.

The "Immuno-Oncology" Market That Encompasses $PVCT & PV-10

On May 15, a Wall Street Journal article highlighted middle market investment bank Leerink Swann's of immuno-oncology addressable market as an annual $20 billion figure. A week later, Citigroup analyst Andrew Baum was quoted by Reuters sizing the market as $35 billion a year over the next 10 years for, where this category of drugs would be used in some way in the management of up to 60% of all cancers. I think the immuno-oncology category includes immuntherapies broadly, but it's possible the above estimates are inclusive only of anti-CTLA-4, anti-PD1 and anti-PDL-1 (and the like) agents.

I Think it's a Good Time to be a $PVCT Shareholder

Shareholders are closer to the end-game for the stock than to their journey's beginning. The company's  life sciences technology is head and shoulders above its peers. The lead compound presents a compelling clinical value proposition capable, perhaps, of indeed shifting the paradigm of cancer treatment (and treatment of inflammatory skin disorders). The drug already is productized, with a product business proposition to match that of the underlying technology. Provectus appears to be nearing transparency regarding regulatory clarity for oncology. The company also appears to be nearing certain license deals that will provide initial market validation. In sum, management, Provectus and its shareholders are nearing the end-game.

Provectus' technology is a game changer, productized to effect the change, and can be delivered and scaled to profit from the change to come.

The technology, PV-10 for oncology, is an effective local treatment capable of as effectively treating distant disease and visceral metastases. The drug, through expression of antigens in context in tumors into which PV-10 is injected fortifies the immune system against cancerous agents. Immunization occurs because PV-10's chemoablation causes rapid, complete, durable necrosis of tumor lesions.

"Scientists have been trying for decades to understand why the body's immune system didn't see cancer cells as the enemy and attack them. Recent discoveries revealed that tumors are adept at cloaking themselves by hijacking the body's own mechanism for preventing the system's T-cells, the infection- and disease-fighting cells of the immune system, from running amok against healthy tissue."

The above quote from Wall Street Journal (WSJ) article New Cancer Drugs Harness Power of Immune System (5/15/13) is not quite on-point. Rather, the quote below from Bloomberg article Human Immune-Boosting Cancer Drugs Seen Extending Lives (5/12/13) is more to the point.

"Now many believe that by strengthening the immune system’s ability to identify and kill cancer cells, they can broaden the attack so it will fight any dangerous malignancy. “You’re setting up a fair fight” with the disease, said Nils Lonberg, a senior vice president at Bristol-Myers, in a telephone interview. “The immune system is just as adaptable as the cancer.”"

How do you strengthen the immune system? Medical science has done it before with infectious diseases. Immune systems can be overwhelmed and, thus, are unable to help the human body fight back. Antibiotics themselves do not cure patients of infectious diseases that have afflicted them. Rather, the immune system cures the hosts with the help of antibiotics. Treating infectious diseases requires that antibiotics should have near absolute specificity for the infectious agent. Tough infections require multiple agents.

I think it's a good time to be a Provectus shareholder.

The volume of the conversations about immunotherapy has grown much, much louder. Take Xconomy's Luke Timmerman's article Genentech Follows Fast at ASCO as Cancer Immunotherapy Picks Up (5/15/13) for example.

"Genentech made its name in cancer by creating targeted antibody drugs that zero in on tumor cells while mostly sparing healthy tissue. Now it’s seeking to compete in the next wave of cancer immunotherapies, which are designed to spark the immune system to attack tumors like a virus."

Or take NBC's Nightly News report Immunotherapy targets cancer cells with remarkable results (5/15/13).

"Cancer cells typically put up a chemical shield to protect against the body’s disease-fighting T cells. But immunotherapy can break down the shield and let the T cells get to work."

The more the biopharmaceuticals industry, investing community and general public talk about immunotherapy, the better the environment or atmosphere for PV-10 and Provectus to flourish.

From the same WSJ article above: "More broadly, Leerink [Swann, a middle market investment bank] believes the "immuno-oncology" drug market could amount to a $20 billion category annually, putting it on a par with the cholesterol-lowering statin market at its height."

From dendritic cells (Dendreon's Provenge) to antitumor antibodies (Bristol-Myers' anti-CTLA-4 agent Yervoy) to immune checkpoint blockades (ASCO 2013's anti-PD1 and anti-PDL-1 agents):

"The immunotherapy of cancer has made significant strides in the past few years due to improved understanding of the underlying principles of tumor biology and immunology...[t]he pillars of human cancer therapy have historically been surgery, radiotherapy, and chemotherapy, but a fourth modality of immunotherapy has been well documented since 1890..." (Kirkwood et al.).

Big Pharma is tacking closer to the ultimate goal, but they mostly still are sailing wide of the objective.

"If the new generation of immune therapies lives up to its promise, “this is going to be a paradigm shift for treating cancer,” said Merck senior vice president Gary Gilliland in an interview. “We are pretty good at shrinking tumors, but not good at getting rid of them. Immune therapy is a way to begin to approach that.”"

Yes, Big Pharma is pretty good at shrinking cancerous tumors, but not yet good enough at making them go away.

Craig's postulation that you treat cancer like you treat an infectious disease requires a/the drug treatment to possess near absolute specificity for diseased tissue (spare normal tissue and clear rapidly from it) and induce a host response (combined apoptosis-autophagy of treated targets because intralesional delivery produces a “vaccine-like” systemic effect that enhances normal human body defenses) over a very wide range of activity (broad spectrum antibiotic-like)


PV-10 chemoablation causes rapid, complete, durable necrosis of tumor lesions.

I think it's a good time to be a Provectus shareholder.

The general discourse at the moment about immunotherapies, however, is far from well understood or a consensus. Take last week's exchange on Adam Feuerstein's Twitter feed @adamfeuerstein below.

Feuerstein had been explaining the fundamental bear case for Vical's Allovectin-7, but aside from perhaps some valid trial design questions, his "red flags" included a lack of positive statements by melanoma experts and institutional healthcare investors as well as a lack of immune-related toxicity by Allovectin-7. Church is not a fan of immune-related toxicity because of the long-term dangers of altering the immune system in order to use it. All of this despite the on-point replies of other respondents: directing T-cells to attack tumors rather than a broad uncontrolled response, the beauty of local treatment is limited toxicity, revolutionary if the local treatment works on visceral mets, etc.

Many monoclonal immunotherapies do cause terrible toxicities, which are related not just related to "taking the brakes off" the immune system. Their target antigens are not only on/in the immune cells they wish to suppress. Just like chemotherapy agents, immunotherapy agents also can cause collateral damage with more adverse side effects like cancer and death.

The nature of the conversation Feuerstein, Church and the others need to have has to be deeper and more substantive. It's too simplistic to say Allovectin-7 does or will not work. Whether it's Allovectin-7 or talimogene laherparepvecone (T-Vec, formerly OncoVEX), or PV-10 itself, one has to balance positive effects with side effects. Think of it as a biological signal-to-noise ratio. One can titer up dosages until one sees bad things happen.

I think Pfizer probably refused to do this with its anti-CTLA-4 agent tremelimumab, which is why it has a greater failure record in clinical trials than it's Bristol-Myers' ipilimumab. Bristol-Myers probably had a higher tolerance for "bad things to happen" in order to suss out the optimal or near-optimal signal-to-noise or benefit-to-side-effect ratio.

Remind yourself of Provectus' combination therapy work at AACR 2013 utilizing PV-10 and a systemic anti-CTLA-4 antibody. The highest dose of the latter killed the murine subjects more quickly. Lower doses were less effective but also less dangerous. Interestingly, the results the company concluded that while PV-10 is highly effective when all existing tumor is accessible for injection, for visceral or other inaccessible disease the combination of PV-10 with CTLA-4 blockade has important potential synergy. Nevertheless, the apparent toxicity at high doses of the anti-CTLA-4 antibody markedly reduced the effect of the antibody alone and the combination of PV-10 and the antibody.

If an immunotherapy is working, there has to be some response (e.g., inflamation, redness, blistering, itching, etc.). These "side effects" come with the territory, and merely are signs the immune system is now in the game. So, while one indeed is generating a toxic response by recruiting the immune system, the key is focusing this toxicity as much as possible.

The problem with Yervoy, and perhaps immunotherapies like anti-PD-1, anti-PDL-1 agents and other treatments, is the collateral damage they cause to the body. PV-10's great strength has been and is its ability to the target only what it kills and kill only what it targets. Another strength is the drug's ability to generate a very specific, targeted, immune response with very little collateral damage to anything but the tumor itself. So, some blistering occurs, body temperature rises, etc. Nothing more.

I think it's a good time to be a Provectus shareholder.

Still, regulatory clarity has to become transparent. Is it an MM Phase 3 trial under SPA powered for for a 6-month progressional free survival for PV-10 vs. 2-month plus PFS for the comparator DTIC?Or is it breakthrough designation therapy and accelerated approval or another accelerated pathway?

Further still, commercial validation through one or more meaningful license transactions (global, regional, dermatology) is required. For example, will it be Hisun-Pfizer Pharmaceuticals in China? Or another company or entity? The Hisun-Pfizer joint venture was established in 2012 to sell off-patent medicines in China and other emerging markets. But economics are economics. Pfizer's 49% equity ownership and thus 49% ownership of any PV-10 sales in China would be a strong inducement to widening the entity's mandate through a license deal with Provectus.

Who will it be in India? In Japan? When will PH-10 be licensed, and to whom?

Provectus is squarely in the eye of key cancer patient advocates. The company participated in Melanoma Research Alliances' (MRA's) Fifth Annual Scientific Retreat in February. In April Peter joined MRA's President and CEO Wendy Selig, LiveSTRONG's President and CEO and Provectus corporate advisory board member Doug Ulman, Moffitt Cancer Research Center's Dr. Shari Pilon-Thomas and some media for dinner.

Key opinion leaders like Moffitt's Dr. Jeff Weber and Dr. Vernon Sondak and Pfizer's Dr. Craig Eagle are enthused about PV-10 as both a monotherapy and combination therapy.

More data is on the way. And data is driving and will drive the deals.

Yes, I think it's a good time to be a Provectus shareholder.

$PVCT #AACR2013: Combination of PV-10 immuno-chemoablation and systemic anti-CTLA-4 antibody therapy in murine models of #melanoma

Presentation Title: Combination of PV-10 immuno-chemoablation and systemic anti-CTLA-4 antibody therapy in murine models of melanoma

Abstract Body: Rose bengal disodium (PV-10) is an investigational small molecule ablative agent currently entering pivotal phase 3 clinical testing as a monotherapy for locoregional control of cutaneous metastatic melanoma. Upon intralesional (IL) administration, PV-10 localizes to the injected tumor tissues while clearing rapidly from healthy tissue. Tumor infiltration with PV-10 leads to rapid necrosis of the injected lesion, with complete resolution common within 2-8 weeks. In phase 2 testing in 80 patients with Stage IIIB-IV(M1c) melanoma, IL PV-10 elicited an objective response in injected tumors in 51% of patients (CR:25%, PR:26%) after 1-4 treatment cycles. In addition to this direct ablative effect on injected tumors, some patients achieved an objective response in their monitored untreated tumors (CR:26%, PR:7% in 42 subjects with monitored untreated lesions) in an apparent immune-mediated bystander response that highly correlated with successful ablation of their injected tumors. Treatment was generally well tolerated, with adverse events confined mainly to the injection site and no grade 4 or 5 adverse events associated with use of PV-10. Recent nonclinical testing in the B16-F10 murine melanoma tumor line has confirmed that PV-10 ablation induces tumor-specific immunity, resulting in marked suppression of synchronous lung metastases upon ablation of a flank tumor and tumor-specific IFN-γ production. In this study we assess potential benefit of combination of PV-10 immuno-chemoablation with the hamster anti-murine CTLA-4 antibody 9H10 in bilateral flank and lung metastasis models (B16-F10 melanoma in C57BL/6 mice). Results from these models will be reported and could support clinical development of combination therapy in advanced melanoma patients, such as stage IV patients with substantial tumor burden in locations inaccessible to PV-10 injection. The rapid reduction in tumor burden and tumor specific immunologic stimulation provided by PV-10 may complement the immune stimulation of anti-CTLA-4 antibodies such as ipilimumab without increased toxicity.