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

Updated below: 11/8/16 {thrice}.

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Intralesional injection with Rose Bengal and systemic chemotherapy induces anti-tumor immunity in a murine model of pancreatic cancer

Shari Pilon-Thomas, Amy Weber, Jennifer Morse, Krithika Kodumudi, Hao Liu, John Mullinax, Amod A Sarnaik H. Lee Moffitt Cancer Center, Tampa, FL, USA

Journal for ImmunoTherapy of Cancer 2016, 4(Suppl 1):P256

Background
Rose Bengal is a xanthene dye that has been utilized for liver function studies and is currently used topically in ophthalmology. Intralesional (IL) Rose Bengal (PV-10) has been shown in murine models and melanoma clinical trials to induce regression of treated melanoma lesions and uninjected bystander lesions. This study was undertaken to measure whether IL PV-10 can induce systemic anti-tumor effects alone or in combination with gemcitabine (Gem) therapy in a murine model of pancreatic cancer.

Methods
C57BL/6 mice received Panc02 pancreatic tumor cells subcutaneously (SC) on one flank to establish a single tumor. On day 7, tumor was treated with IL PV-10. Control mice received IL phosphate
buffered saline (PBS). Tumor growth was measured. Splenic T cells were collected and co-cultured with Panc02 or irrelevant B16 cells. Supernatants were collected to measure Panc02-specific T cell responses by IFN-gamma ELISA. To measure the effect of IL PV-10 on the growth of an untreated, bystander tumor, mice received Panc02 cells in bilateral flanks. The resulting right tumor was injected IL with PV-10 or PBS. Tumor sizes were measured for both the right (treated) and left (untreated/bystander) tumors. To determine the efficacy of combination therapy with IL PV-10 and systemic Gem, mice bearing a single or bilateral Panc02 tumors were treated with PV-10 alone or in combination with Gem. Mice received 60 mg/kg Gem intraperitoneally (IP) twice per week.

Results
C57BL/6 mice bearing Panc02 tumors treated with IL PV-10 had significantly smaller tumors than mice treated with PBS (p < 0.001). A significant increase in the IFN-gamma production in response to Panc02 was measured in the splenocytes of mice treated with PV-10 as compared to mice treated with PBS (p < 0.05). Mice with bilateral tumors had a significant regression of tumors injected IL with PV- 10 and there was a reduction in the untreated (bystander) flank Panc02 tumor (p < 0.01). Gem therapy in combination with IL PV-10 injection led to enhanced tumor regression (p < 0.05) compared to IL PV-10 or Gem alone in both a single tumor model and a bilateral tumor model.

Conclusions
Regression of untreated pancreatic tumors by IL injection of PV-10 in concomitant tumor supports the induction of a systemic anti-tumor response. Addition of Gem chemotherapy enhances the effects of IL PV-10 therapy. Given that patients with metastatic pancreatic cancer have a dismal prognosis, combination therapy of IL PV-10 combined with Gem may benefit patients with metastatic pancreatic cancer.

Updated (11/8/16).1: My underlined emphasis above. Note PV-10 use in the above murine model work as a monotherapy, and in combination with systemic chemotherapy.

Updated (11/8/16).2: H/t @bradpalm1:

Click to enlarge. Tweet image source

"Gemcitabine reduces MDSCs, tregs and TGFβ-1 while restoring the teff/treg ratio in patients with pancreatic cancer," Eriksson et al., Journal of Translational Medicine 2016 14:282

"Conclusions 

Gemcitabine regulates the immune system in patients with pancreatic cancer including MDSCs, Tregs and molecules such as TGFβ-1 but does not hamper the ability of effector lymphocytes to expand to stimuli. Hence, it may be of high interest to use gemcitabine as a conditioning strategy together with immunotherapy."

Updated (11/8/16).3: Unlike some, I do not read too much into Moffitt's abstract with respect to the additivity or synergism of PV-10 and gemcitabine (systemic chemotherapy) based on the cancer center's murine model work (i.e., p values of PV-10 alone, and in combination with chemo). And, I'd like to see the poster if Provectus facilitates its release (e.g., the company did not facilitate the release of Moffitt's AACR 2016 poster, which I believe may lead to a peer-reviewed publication).

I believe the point of this mousie work, which of course is beyond cell line (in vitro) work but behind clinical studies, is to demonstrate in principle that (a) intralesional (IL) PV-10 could be used to treat pancreatic cancer (i.e., tumor type, leading to a suitable cancer indication) via (i) ablation/destruction of an injected tumor and (ii) the subsequent triggering of the immune response to reduce or destroy an untreated one, and (b) IL PV-10 plus chemo sees enhanced untreated tumor reduction or destruction.

These principles of (x) ablation/destruction by injection and (y) immunologic signalling (immune system harnessing) already have been shown preclinically by Moffitt for PV-10 as a monotherapy in melanoma (also clinically) and breast cancer (AACR 2013), and in combination with checkpoint inhibition for melanoma (SITC 2014).

So, this continues Rose Bengal, PV-10 and Provectus' theme of (A) agnosticism (ablation, immunologic signalling), (B) synergism (in that one therapy enhances another; "induce and boost"), (C) orthogonality (although this would be better shown in clinical work to emphasize no greater toxicity, if not less to far less), and...

(D) PV-10 is an immunotherapy. See January 19, 2016 blog post PV-10 is an immunotherapy.

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

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

Updated below: 9/26/16 {twice}, 9/28/16 and 9/30/16.

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Updated (9/26/16): According to the Pancreatic Cancer Action Network, there are three chemotherapy drugs approved by the United States Food and Drug Administration (FDA) for the treatment of pancreatic cancer: albumin-bound paclitaxel (ABRAXANE®), gemcitabine (Gemzar®) and fluorouracil (5-FU).

"Gemcitabine (Gemzar®) was approved in 1996 for the treatment of unresectable pancreatic cancer. Studies have also shown that there is a benefit to using gemcitabine after surgery for pancreatic cancer. Prior to gemcitabine, fluorouracil (5-FU) was used as the standard treatment for unresectable pancreatic cancer. Both of these drugs are still used today. 

Most recently, in September 2013, albumin-bound paclitaxel (ABRAXANE®) was approved to be used in combination with gemcitabine (Gemzar®) as first-line treatment for metastatic pancreatic adenocarcinoma, the most common type of pancreatic cancer. 

In addition to the three FDA-approved drugs, FOLFIRINOX, a combination of three chemotherapy drugs (5-FU/leucovorin, irinotecan, and oxaliplatin) is commonly used in the treatment of metastatic pancreatic adenocarcinoma. In 2010, a Phase III clinical trial showed positive results for patients treated with FOLFIRINOX. Due to the results of this study, FOLFIRINOX is also considered a standard treatment option for patients with metastatic pancreatic cancer. However, patients treated with FOLFIRINOX may experience more severe side effects than those treated with gemcitabine alone, so this combination is usually given to patients who are healthy enough to tolerate the potential side effects."

Abraxane is made by Celgene. Gemzar is made by Eli Lilly. Provectus previously showed the combination of 5-FU and PV-10 for hepatocellular carcinoma in murine model work (SITC 2012).

Updated (9/26/16): I believe the systemic chemotherapy used in Moffitt's work is gemcitabine (Gemzar®), which by now is produced by generics manufacturers.

Some recent information on the competitive landscape for pancreatic cancer:

• "Eli Lilly's chemotherapy drug Gemzar (gemcitabine) is widely considered the standard of care for pancreatic cancer. Generic gemcitabine is available from numerous companies" {January 2014 source},

• "In the past, companies have combined various targeted treatments with gemcitabine in an attempt to increase their survival benefit. These efforts include Bristol-Myers Squibb/Eli Lilly's Erbitux, Roche's Avastin, Pfizer's Inlyta, and Amgen/Bayer's Nexavar, among others. Unfortunately, none of these drugs has shown a significant survival benefit when combined with gemcitabine" {above source},

• "Evolving Paradigms in Pancreatic Cancer: Frontline Treatment Regimens:"

• "Frontline therapies for patients with metastatic pancreatic cancer have advanced in the past 5 years, with the FDA approval of nab-paclitaxel (Abraxane) plus gemcitabine (Gemzar). These advancements branch off the established treatment paradigm of gemcitabine monotherapy, which showed a significant extension in overall survival (OS) compared with 5-FU alone in a phase III study."

• "Pancreatic cancer is a particularly tough cancer to treat and a number of other companies have failed in their efforts to develop an effective treatment. In February, Incyte halted trials of its drug Jakafi in solid tumors, after a Phase 3 study in pancreatic cancer failed to show efficacy. And this past May saw pancreatic cancer immunotherapies from NewLink Genetics and Aduro Biotech fall short of goal in Phase 3 and Phase 2b trials, respectively" {September 2016 source}, and

• Momenta Pharmaceuticals has decided to end development of necuparanib, an experimental drug for advanced pancreatic cancer and the biotech's lead clinical candidate, according to a regulatory filing submitted Thursday. Enrollment in a Phase 2 trial testing necuparanib in combination with Celgene's Abraxane was halted earlier this month after an independent data safety monitoring board concluded treatment didn't demonstrate a sufficient level of efficacy {above source}.

Updated (9/28/16): In 2011 co-founder and former Chairman and CEO Dr. Craig Dees, PhD presented Provectus' murine model work examining PV-10's "immunologic potential in treating melanoma and other cancer indications including liver, pancreatic and colorectal cancer." See several pertinent slides below.

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Updated (9/30/16): H/t @bradpalm1: Vitamin D + immunotherapy + chemotherapy..."Physicians and Physicists Join Forces to Fight Pancreatic Cancer," The Wall Street Journal, September 25th, Ron Winslow

"The team is planning a clinical trial soon in which patients who are candidates for pancreatic-cancer surgery first will be treated with synthetic vitamin D, chemotherapy and Bristol-Myers Squibb Co.’s cancer immunotherapy Opdivo. Opdivo is one of the new so-called checkpoint inhibitors that are transforming treatment of cancers such as melanoma and lung cancer. The drugs work by releasing brakes on immune-system T cells, enabling them to pursue tumor cells." 

Bristol-Myers also is combining Opdivo with several other companies and drugs to treat pancreatic cancer, such as Celgene (Abraxane/Paclitaxel protein-bound) and Aduro Biotech (CRS-207 and GVAX)*. Opdivo combination trials for pancreatic cancer on CT.gov include at least eight studies.

* CRS-207 and GVAX failed as a regimen for pancreatic cancer.

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.

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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.”

Hypothesis: Intralesional PV10 induces systemic immunity in humans

Provectus issued a press release today and made an associated 8-K filing (that included another item) regarding Moffitt Cancer Center's Dr. Vernon Sondak, MD's presentation in Munich, Germany, Provectus Biopharmaceuticals' Data on PV-10 as Treatment for Melanoma Presented at 5th European Post-Chicago Melanoma / Skin Cancer Meeting. The company made Dr. Sondak's presentation available; the link is here.

I believe the key takeaway comes from the 19th slide in the deck, which clearly and simply states [what I think is] the ultimate goal of Moffitt's research work on PV-10: to prove or disprove the hypothesis that intralesional PV10 induces systemic immunity in humans [for melanoma].

Click to enlarge. Slide no. 19. New 2015 slide (v. 2014)

The other slide of note [to me], and that I was struck by, was the presentation's final slide (of "The Moffitt PV-10 Team").

Click to enlarge. Slide no. 21. New 2015 slide (v. 2014)

Below I compare Sondak's 2014 presentation to his 2015 one, which in the early going are ostensibly the same. A shareholder who attended last year's presentation sent me pictures of it.

Disclosures

Click to enlarge. 2014

Click to enlarge. 2015: Slide no. 2

Potential Applications

Click to enlarge. 2014

Click to enlarge. 2014

Click to enlarge. 2015: Slide no. 3

Click to enlarge. 2015: Slide no. 4

Properties of the Ideal Agent

Click to enlarge. 2014

Click to enlarge. 2015: Slide no. 5

New in 2015: Sondak included a "history of" slide, which speaks to Rose Bengal lying around in plain sight of Big Pharma for about 75 years before Provectus' cofounders "re-discovered" it, and the drug substance/drug product's safety and specificity (i.e., Pharmacology).

Click to enlarge. 2015: Slide no. 6

Properties of PV-10

Click to enlarge. 2014 

Click to enlarge. 2014

Click to enlarge. 2015: Slide no. 7

Click to enlarge. 2015: Slide no. 8

Click to enlarge. 2015: Slide no. 9

New in 2015: A picture of Melanoma Institute Australia and the University of Sydney's Dr./Prof. John Thompson, MD with PV-10 (Rose Bengal) on his face?

Click to enlarge. 2015: Slide no. 10 

Click to enlarge. 2014

Click to enlarge. 2014

Click to enlarge. 2014

Click to enlarge. 2014

Click to enlarge. 2015: Slide no. 12

Click to enlarge. 2015: Slide no. 13

Click to enlarge. 2015: Slide no. 11

Click to enlarge. 2015: Slide no. 14

New in 2015: Summary/overview slides of Moffitt's work to assess systemic immunity from intralesional therapy, in mice and humans (noting whether and where such work was or was not published in a peer-reviewed journal).

Click to enlarge. 2015: Slide no. 15

Click to enlarge. 2015: Slide no. 16

Click to enlarge. 2015: Slide no. 17

Click to enlarge. 2015: Slide no. 18

Click to enlarge. 2015: Slide no. 19. Notable, as discussed above

Conclusions

Click to enlarge. 2014

Click to enlarge. 2015: Slide no. 20

Final Slide

Sondak's final June 2014 presentation slide was telling, given the FDA's May 2014 denial of Provectus' application for breakthrough therapy designation for PV-10 (in locally advanced cutaneous melanoma), and that he made that presentation well before the Agency's April 2015's advisory committees overwhelming vote in favor of Amgen's talimogene laherparepvec ("T-Vec") having an overall favorable benefit-risk profile for the treatment of injectable regionally or distantly metastatic melanoma (for the latter, see blog post The first guy through the wall).

Click to enlarge. 2014

In June 2015, however, there are smiling faces:

Click to enlarge. 2015: Slide no. 21

Recall Eric said on Provectus' May 7th 1Q15 conference call:

"We really can't speculate on the direct implications of the T-VEC decision other than to say that clearly the regulatory environment is continuing to change what the agency identified deficiencies in the T-VEC package. And this may come as significant review questions when the agency finalizes the review of potential decision to approve or not approve the BLA, the application for approval for that my logic may have relevance for us going forward. That being said, the end points that were used for the T-VEC pivotal study were different than the ones that we used in Phase 2 study. And more importantly, the T-VEC Phase 3 study, pivotal study was larger, so have more patients than we have in the Phase 2 study. I would speculate that we can see that what was clearly very strong headwinds a year ago are maybe abating here in Washington and that presumably bodes well for future success with PV-10." {Underlined emphasis is mine}

N.B. Provectus did not make Dr. Sondak's 2014 presentation at the 4th European Post-Chicago Melanoma / Skin Cancer Meeting available last year. For whatever reason (e.g., perhaps Eric is more comfortable and confident now than he was then*), Provectus' Chief Technology Officer Dr. Eric Wachter, PhD appears to have allowed the company's Chief Operating Officer/Chief Financial Officer Peter Culepper to press release the 2015 presentation.

* If true and in the absence of more explanation directly from him on this, then my reaction is that such feeling(s) is(are) are disrespectful to Provectus shareholders and representative of an intellectually inconsistent sub-process.

Revolutionize, Perfect, Quintessential

In regards to PV-10, Moffitt Cancer has said over the last couple of years:

• Single Injection May Revolutionize Melanoma Treatment, Moffitt Study Shows (Moffitt press release, August 2013),

• PV-10 might offer the perfect way to prime the immune system (interview of Moffitt's Dr. Jeffrey Weber, MD, PhD, ASCO 2014, May-June 2014), and

• [PV-10 injection]...stimulated release of interferon-gamma, the “quintessential antitumor cytokine.” (interview of Moffitt's Dr. Amod Sarnaik, Dermatology Times, May 2015).

Speaking of cytokines...

Image and article source

Cytokines are the messengers of the immune system. Cytokines are substances, either proteins or glycoproteins, secreted by immune cells. They have autocrine and paracrine functions, so that they function locally or at a distance to enhance or suppress immunity. In cancer therapy, we generally use cytokines to enhance immunity. 

Cytokines regulate the innate immune system: natural killer (NK) cells, macrophages, and neutrophils. They also regulate the adaptive immune system, the T and B cell immune responses. In the immune system, cytokines function in cascades. Thus clinical trials of individual cytokines are rarely useful, since cytokines tend not to work individually. Some of the individual cytokines that have been tested and found ineffective for cancer treatment include interleukin 1 beta (IL-1 beta), although it may be useful because it helps to mediate the severe toxicity of interleukin 2 (IL-2). Tumor necrosis factor (TNF) certainly sounded promising, but in fact caused severe hypotension when used systemically. Interleukin 4 (IL-4) showed minimal anti-cancer activity and was toxic. Interleukin 6 (IL-6) had some activity against cancer cells, but turned out to be a growth factor for myeloma cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF), used primarily in stem cell transplant to reconstitute the myeloid series, has been studied for melanoma with controversial results. 

Which cytokines are important for cancer? IL-2 and interferon-alfa 2b are two cytokines approved by the FDA for treatment of cancer. IL-2 has demonstrated activity against renal cell, melanoma, lymphoma, and leukemia. Interferon has activity in the same histologies but also in Kaposi's sarcoma, chronic myelogenous leukemia, and hairy cell leukemia. Overall, cytokines are substances that appear to have application in the treatment of hematologic malignancies or immunogenic tumors. {Underlined emphasis is mine}

"Together, these studies support the induction of increased tumor-specific immunity after co-inhibitory blockade in combination with IL PV-10 therapy."

Moffitt Cancer Center's PV-10-related abstract from the 29th annual meeting of the Society for Immunotherapy of Cancer ("SITC") was made available today by SITC's Journal for ImmunoTherapy of Cancer. The company issued a press release and filed an associated 8-K, and the stock also was halted because of Moffitt and the abstract.

Efficacy of intralesional injection with PV-10 in combination with co-inhibitory blockade in a murine model of melanoma

PV-10 is a 10% solution of Rose Bengal that is currently being examined as a novel cancer therapeutic. We have previously shown that intralesional (IL) injection of PV-10 into a single subcutaneous B16 melanoma tumor led to regression of both the injected tumor and uninjected B16 lung lesions. Tumor regression correlated with the induction of systemic anti-melanoma T cell immunity. In melanoma patients, IL injection of PV-10 has led to regression of treated lesions as well as untreated bystander lesions. In this study, we have examined whether IL PV-10 and co-inhibitory blockade could improve anti-tumor immunity and regression of melanoma. B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor. Treatment of this lesion with a single IL injection of PV-10 alone led to partial regression of the injected B16 lesion. Systemic administration of anti-CTLA-4 or anti-PD1 antibodies in combination with IL PV-10 resulted in increased tumor regression and improved survival in this model. Treatment with PV-10 also led to the induction of T cells that produced IFN-γ (495 ± 198 pg/ml) in response to B16 cells but not to irrelevant MC-38 cells. Combination therapy with IL PV-10 and anti-CTLA-4 led to increased IFN-γ responses to B16 (1235 ± 191 pg/ml, p < 0.05). In another experiment simulating heavy tumor burden using a bilateral model, systemic administration of anti-PD-L1 antibodies in combination with IL PV-10 led to regression of the injected B16 lesion as well as a bystander subcutaneous lesion on the opposite flank (p < 0.01 compared to mice treated with anti-PD-L1 antibodies or IL PV-10 alone). Together, these studies support the induction of increased tumor-specific immunity after co-inhibitory blockade in combination with IL PV-10 therapy.

It strikes me there are two general takeaways:

1. PV-10 works with everything, and
2. Provectus' drug should expand the relevance and use of co-inhibitory blockade agents (i.e., PD-L1s, PD-1s, and CTLA-4s) from (a) late-stage cancer in the U.S. to (b) Stage III and IV disease globally.

In the context of Moffitt's abstract, and the underlying work from which it was drawn and written, PV-10 works with [at a minimum] co-inhibitory blockade agent categories PD-L1, PD-1 and CTLA-4. Reference material on this should be:

• PV-10's so-called orthogonality with other drugs (i.e., the likely minimal risk of PV-10 causing clinically relevant drug-drug interactions is likely minimal). See the paper In vitro inhibition of human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes by rose bengal: system-dependent effects on inhibitory potential and Provectus' associated press release, and

• Provectus' combination therapy patent application with Pfizer (filed in 2012), Combination of Local and Systemic Immunomodulative Therapies for Enhanced Treatment of Cancer, of which Pfizer shares in economics only related to PV-10 & CTLA-4 combinations. The application's claims should cover combinations of PV-10 and {PD-L1s, PD-1s, CTLA-4s, and other drug categories}.

In the context of global and stage-related relevancy and use, challenges of approved and investigational co-inhibitory blockade agents include non-specificity, dose limiting toxicity, and cost:

• PD-L1, PD-1 and CTLA-4 cancer immunotherapies are non-specific immunotherapies that do not achieve sufficient, let alone notable, levels of complete responses,

• Their toxicity and side effects limit the amounts of them that can be given to patients. Side effects have to be, when possible, physician-managed, and

• Their prohibitive cost to research and make, and thus price to sell, very likely will limit their use in the U.S., where the duration of use to achieve longer survival, ultimately ineffective as it is, would be more than $1 million per patient. Their cost/price should largely diminish or prohibit their use elsewhere in the world: "We've kind of maxed out what we're either willing or able to pay for these kinds of drugs, so it's a problem when you start combining them. It can't just keep going exponentially, so that eventually it will be $1 million a year to get treated -- that's crazy." (a quote from Moffitt's Dr. Jeffrey Weber, M.D., Ph.D., Reuters' New cancer therapy comes of age, cost a 'toxic' side effect, September 2014)

Presumably, combining with PV-10 would make these drugs more relevant and increase their use because:

• An effective, long-lasting, sustainable immune response for late-stage patients (where all disease burden is not accessible to PV-10 injection) requires both their use and PV-10's: non-specific and specific immunotherapies, respectively. The human body has specific and non-specific immune system components: the innate and adaptive immune systems, respectively. See my blog post entitled PV-10 is not bigger than Mother Nature, 

• Less of them would be used, potentially reducing or mitigating their dose limiting toxicities and side effects, and

• The duration of their use then should be shorter were their combination with PV-10 to be more effective, reducing overall treatment cost. 

Returning to the detail of the abstract, specific takeaways include:

• Moffitt touching on three keys to cancer treatment using PV-10 and a co-inhibitory blockade agent: (i) tumor regression, (ii) improved survival, and (iii) impact (tumor regression) on both treated and untreated lesions:

• Systemic administration of anti-CTLA-4 or anti-PD1 antibodies in combination with IL PV-10 resulted in increased tumor regression and improved survival in this model.
• In another experiment simulating heavy tumor burden using a bilateral model, systemic administration of anti-PD-L1 antibodies in combination with IL PV-10 led to regression of the injected B16 lesion as well as a bystander subcutaneous lesion on the opposite flank (p < 0.01 compared to mice treated with anti-PD-L1 antibodies or IL PV-10 alone).

• Specificity works, by saying they achieved T-cell responses to B16 cells and not MC 38 cells. The study was focused on melanoma (i.e., B16 cells). MC 38 cells relate to or reference colon cancer.
• Treatment with PV-10 also led to the induction of T cells that produced IFN-γ (495 ± 198 pg/ml) in response to B16 cells but not to irrelevant MC-38 cells.

• Moffitt's previous pre-clinical work, published in 2013, Intralesional Injection with PV-10 Induces a Systemic Anti-tumor Immune Response in Murine Models of Breast Cancer and Melanoma (AACR 2013 poster, PLoS One paper), noted PV-10's ability to destroy tumors (i.e., complete responses). This work, and PV-10's ability to destroy tumors (i.e., complete response), was followed up in the cancer center's human feasibility study (ASCO 2014). The study design underlying their SITC 2014 revelations required Moffitt to wound -- and not destroy -- the tumors. In order to demonstrate the ability of PV-10 in combination with a co-inhibitory blockade agent to achieve tumor regression, improve survival, and impact both treated and untreated lesions, Moffitt had to inject tumors with a sub-optimal amount of PV-10 (i.e., "wound") so as not to achieve a complete response, and be able to show combinations could impact the tumor.

• Reading the abstract, it struck me that Moffitt ordered the effectiveness of the co-inhibitory blockade agent in combination with PV-10 as, first, the PD-L1, and tied for second, the PD-1 and CTLA-4. Given that, which do you think of Big Pharma is more worried (as a non-combo partner, or non-owner of the PV-10 lead in the combo)?

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Co-stimulatory & Co-inhibitory

Moffitt's PV-10 presentation at the 2014 annual meeting of the Society for Immunotherapy of Cancer ("SITC") is entitled Efficacy of intralesional injection with PV-10 in combination with co-inhibitory blockade in a murine model of melanoma (see Moffitt @ SITC (September 19, 2014) on the blog's News page).

Earlier this month, one of the SITC presentation's co-authors and Moffitt assistant professor and researcher, Dr. Shari Pilon-Thomas, Ph.D., co-authored an online OncLive article entitled Immunotherapy Combined With Chemotherapy for Pancreatic Cancer: A Game Changer? (see blog post Treating Cancer). In it Dr. Pilon-Thomas and her fellow authors write:

Of note, the immune system’s involvement in cancer development and progression has sparked much interest in recent years. The model of the cancer-immunity cycle suggests an interplay of immune-suppression and immune-stimulation. In normal individuals, a state of immunosurveillance is in place. However, within the tumor microenvironment, inhibitory signals and immunosuppressive cells are present and tip the scale in favor of immune suppression. {Underlined emphasis is mine}

Continued: The idea of the cancer-immunity cycle proposes that, for a cancer immune response to be generated, the net balance between immune stimulation versus immune suppression must be tipped in favor of the former. Studies in various cancers have suggested that tumors evade the immunogenic process mostly by factors that promote immunosuppression. {Underlined emphasis is mine}

The theory of immune surveillance suggests, according to Peggs et al., "...that the immune system plays a key role in suppressing tumor growth and that the incidence of cancer would be much greater were it not for the ability of the immune system to identify and eliminate nascent tumor cells...While the immune system appears capable of eliminating or containing early tumor growth, some tumor cells escape detection and eventually cause cancer." Said another way, when thinking about the growing potential role and promise of cancer immunotherapy, "...we continually develop malignant cells every day that are consumed by the immune system to prevent tumor development, and the immunotherapy drugs seem to target the failure of immune recognition and immune response" (Dr. Peter Salgo, M.D.).

The balance between co-stimulation and co-inhibition is described by Inman et al.: "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."

So, it would seem to me, generalizing (or simplifying, perhaps too much):

• If co-stimulation > co-inhibition, the immune system can act decisively against cancer,

• If co-inhibition > co-stimulation, cancer overwhelms the immune system and renders it ineffective or useless, and

• If co-stimulation = co-inhibition (that is, some sort of equilibrium state), the immune system wages battles against cancer to varying degrees of success with potentially no ultimate resolution to the war itself.

[Daniel] Chen & Mellman (2013), authors of Oncology Meets Immunology: The Cancer-Immunity Cycle, note stimulatory and inhibitory factors at step of the cycle.

Click to enlarge. Figure 2 (above) of [Daniel] Chen et al.'s article.

The authors write:

Each step of the Cancer-Immunity Cycle requires the coordination of numerous factors, both stimulatory and inhibitory in nature. Stimulatory factors shown in green promote immunity, whereas inhibitors shown in red help keep the process in check and reduce immune activity and/or prevent autoimmunity.

[Daniel] Chen et al. then note "[t]he numerous factors that come into play in the Cancer-Immunity Cycle provide a wide range of potential therapeutic targets."

Figure 3 (below) "...highlights examples of some of the therapies currently under preclinical or clinical evaluation. Key highlights include that vaccines can primarily promote cycle step 2, anti-CTLA4 can primarily promote cycle step 3, and anti-PD-L1 or anti-PD-1 antibodies can primarily promote cycle step 7. Although not developed as immunotherapies, chemotherapy, radiation therapy, and targeted therapies can primarily promote cycle step 1, and inhibitors of VEGF can potentially promote T cell infiltration into tumors—cycle step 5."

Click to enlarge. Figure 3 (above) of [Daniel] Chen et al.'s article.

In my illustration below, found on the blog's PV-10, and the Cancer Immunity Cycle, I endeavored to show Provectus' drug promoted steps 1, 2, 3 and 7. I believe, but with no confirmation of course, Moffitt has shown via primarily their murine model work (and maybe their human study) that PV-10 promotes steps 4, 5 and 6.

Click to enlarge.

I revised my table of combination study deals to reflect [as I think they are] stimulatory and inhibitory compounds is below.

Click to enlarge.

Pilon-Thomas et al. concluded (note the article discussed immunosurveillance in the context of chemotherapy, immunotherapy and pancreatic cancer):

The cancer-immunity cycle is an ideal model to envision how tumor cells evade immuno-surveillance as well as where future modalities may intervene with hopes of potentiating tumor cell death. The cancer-immunity cycle together with the immune-modulating functions of chemotherapies that are used in pancreatic cancer creates a rationale for investigating vaccine-chemotherapy combinations. 

Studies to date have suggested benefits of adding immunotherapies to standard chemotherapy regimens. Additional benefits are also suggested by the indication that immunotherapy may render improved chemosensitivity at later dates. In addition, vaccines are often well tolerated with minimal toxicities, which make them a favorable approach. The hope is that we can identify the appropriate combination of vaccine and immune-modulating chemotherapy that will eradicate the disease. There is also likely to be a role for immune checkpoint therapy with inhibitors of PD-1 and PD-L1. Such phase I single-agent studies are currently in progress for pancreatic cancer. The results of studies so far create hope that the combination of chemotherapy with immunotherapy may be a game changer in the treatment of pancreatic cancer.

PV-10 has some interesting features that cross categories. Provectus management previously had called the drug as a chemoablative immunotherapeutic agent (later revising the descriptor to "immuno-chemoablative"). Underlined portion number one, "chemoablative," described PV-10's chemotherapeutic-like feature of rapid tumor ablation and destruction mechanism of action ("MOA"). Underlined portion number two, immuno," described the drug's MOA whereby it harnessed the immune system to battle cancer locally (at the site of injection) and elsewhere around the body.

While not specifically a vaccine because PV-10 is not antigen-specific, it could be considered vaccine-like because it is minimally or not at all toxic but expresses many, many more than one antigen.

As a side note, immune checkpoint therapy in the above Moffitt comments refers to, I believe, ipilimumab, which is why anti-CTLA4 is in step 3, priming and activation, of the cancer immunity cycle (and why Bristol-Myers is exploring the combination of ipilimumab and anti-PD-1 agent nivolumab.

[Lieping] Chen et al. write, when discussion combination therapies:

Traditional chemotherapy and radiation therapy, together with depleting mAbs or treatment with small-molecule inhibitors, all directly target and kill cancer cells, leading to the destruction of the tumour stroma and the release of tumour antigens. When coupled with these direct killing mechanisms, immunomodulatory biologics promote the priming and expansion of existing tumour-specific T cells and their de novo generation, with a potential to form long-lasting and self-sustained antitumour responses. In recent years, small-molecule inhibitors targeting tumours that harbour mutated BRAF (vemurafenib (Zelboraf; Plexxikon/Roche)) or translocated BCR–ABL (imatinib (Gleevec; Novartis)) have shown high initial response rates in clinical trials165. However, the duration of the antitumour response is limited owing to acquired drug resistance. A combination of these fast-acting small-molecule inhibitors with immune co-inhibitory blockade — for example, with CTLA4-specific or PD1-specific mAbs — could promote the priming and expansion of tumour-specific CTLs against multiple tumour antigens and/or epitopes, prevent the generation of escape variants or drug-resistant mutant cancer cells and induce sustained T cell responses. {Underlined emphasis is mine.}

Circling back to beginning of this post, Moffitt's presumed presentation at SITC potentially entitled Efficacy of intralesional injection with PV-10 in combination with co-inhibitory blockade in a murine model of melanoma, the cancer center previously have described their successful pre-clinical work that combined PV-10 with systemic immunotherapies to mean, I believe, checkpoint inhibitors (e.g., ASCO 2014).

The interplay of co-stimulation and co-inhibition (with the goal of more of the former than the latter), and Moffitt's use of what seems to be the broader term co-inhibitory blockade, I wonder whether their work describes the better therapeutic outcome of PV-10 and inhibitory factors of step 7 of the cancer immunity cycle above (see Figures 2 and 3 of [Daniel] Chen et al.).