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.”

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}

More of Better > More or Less of Worse

In a February 2013 Cancer Watch article entitled Back to Phase 1: Understanding Systemic Effects of PV-10, Moffitt Cancer Center's Dr. Amod Sarnaik, M.D. said of the cancer center's Phase 1 feasibility study, result of which later were presented at AACR 2014 and ASCO 2014:

“A further impetus toward teasing out the precise mechanism of how PV-10 can exert a systemic immune response in patients 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... 

...“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... {Underlined emphasis is mine}

A potent, long-lived systemic immune response to solid tumor cancers should:

(i) Originate at the tumor sites themselves, 

(ii) Require a broader array of tumor antigens to be released and presented, 

(iii) Require this array to be comprised of pristine, un- or non-denatured antigens (i.e., whole tumor antigens, not antigen fragments,), and 

(iv) Result from the subsequent, more comprehensive, T-cell response.

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MD Anderson Cancer Center surgical oncologist and Provectus principal investigator Dr. Merrick Ross, M.D., noted in a recent video, below, while speaking at the setting of ASCO 2014:

"The rapid lysis of the tumor and when the tumor is lysed it does not denature the antigen. So the antigens are expressed in a way where an inflammatory response can occur and therefore antigen presentation probably is up-regulated and enhanced, which could lead to a systemic host response." {My transcription, and underlined emphasis is mine}

The idea is to present as many un-denatured or pristine antigens as possible to dendritic cells ("DCs") and antigen presenting cells ("APCs"). Showing fewer pristine antigens or more denatured ones cannot generate a sustainable, systemic, specific, anti-tumor response. Showing more pristine ones should have the opposite and a much better effect.

Said another way, by a shareholder, blog reader and internist whose patients include those afflicted with cancer:

"Rapid lysosomal-mediated tumor lysis following IL injection of PV-10 uniquely produces pristine, un-denatured antigens that up-regulates antigen presentation to DCs and APCs with a resultant systemic immune response (to paraphrase Merrick Ross). The fidelity of these un-denatured tumor antigens (akin to injecting whole tumor antigens, not antigen fragments, into a patient) is what provokes a very accurate and specific immunological T-cell response that bystander tumors are vulnerable to (presuming they don’t possess too many mutated antigens due to selection pressure from previously therapies)." {My transcription, and underlined emphasis is mine}

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Roche's Genentech's Drs. Daniel Chen, M.D, Ph.D. and Ira Mellman, Ph.D.'s Oncology Meets Immunology: The Cancer-Immunity Cycle provides the opportunity to illustrate the interplay between (a) immune checkpoint blockade [Step #7] and (b) the creation, release and presentation of antigens [Steps #1 and 2], and the subsequent priming and activation of the immune system [Step #3]. I write interplay of these steps because they appear to be what industry thus far is focusing on when it considers combination therapies for late stage diseases, and permutations of treatments and therapeutics from each of these steps in an eventual combination.

The illustration below builds on (is revised by me of) Chen & Mellman's Figure 1:

The generation of immunity to cancer is a cyclic process that can be self propagating, leading to an accumulation of immune-stimulatory factors that in principle should amplify and broaden T cell responses. The cycle is also characterized by inhibitory factors that lead to immune regulatory feedback mechanisms, which can halt the development or limit the immunity. This cycle can be divided into seven major steps, starting with the release of antigens from the cancer cell and ending with the killing of cancer cells. Each step is described above, with the primary cell types involved and the anatomic location of the activity listed. Abbreviations are as follows: APCs, antigen presenting cells; CTLs, cytotoxic T lymphocytes.

Click to enlarge.

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Various types of treatments and/or therapeutics in Steps #1, #2 and #3 may create and release antigens ("Antigen Release"), present them to DCs and APCs ("Antigen Presentation"), and prime the body's T-cells ("T-Cell Priming).

See Chen & Mellman's section entitled Initiating Anticancer Immunity: Antigen Release.

Attempts to activate or introduce cancer antigen-specific T cells, as well as stimulate the proliferation of these cells over the last 20 years, have led to mostly no, minimal or modest appreciable anticancer immune responses. The majority of these efforts involved the use of therapeutic vaccines because vaccines can be easy to deploy and have historically represented an approach that has brought enormous medical benefit (reviewed by Palucka and Banchereau, 2013). Yet, cancer vaccines were limited on two accounts. First, until recently, there was a general lack of understanding of how to immunize human patients to achieve potent cytotoxic T cell responses. This limitation reflects continued uncertainties concerning the identities of antigens to use, their mode of delivery, the types of adjuvants required, and the proximal characteristics of the desired T cell response (Palucka and Banchereau, 2013). Second, the presence of the immunostat in the tumor microenvironment may dampen or disable antitumor immune responses before clinically relevant tumor kill can occur. Thus, as long as these negative signals are in place, the prospects for vaccine-based approaches used alone are likely to be limited. {Underlined emphasis is mine}

Therapeutic vaccination is not the only approach to accelerating and expanding the production of T cell immunity. Because anticancer T cells can be produced spontaneously, there is a growing appreciation that the tumor itself represents a type of endogenous vaccine. Accessing the naturally occurring source of cancer-associated antigens avoids problems associated with selection and delivery (Heo et al., 2013, van den Boorn and Hartmann, 2013). This approach is also convenient, but achieving it requires detailed knowledge around whether standard of care chemotherapy or targeted therapies are compatible with immunotherapies. Some therapies are thought to cause tumor cell death in a fashion that promotes immunity (reviewed in Zitvogel et al., 2013). However, it is unclear whether this effect can be accurately predicted and will, in any event, require empirical study. Chemotherapy, radiation therapy, and targeted therapies must also be evaluated for their effects on the immune system. Although it is assumed that many might be antagonistic, there are some reports that others might promote T cell activity (Demaria et al., 2005, Duraiswamy et al., 2013, Hiniker et al., 2012, Ott et al., 2013, Postow et al., 2012, Stagg et al., 2011, Zitvogel et al., 2013). {Underlined emphasis is mine}

See Chen & Mellman's section entitled Presentation, and T Cell Priming.

Another exciting development is that the initial demonstrations that genetically modified autologous T cells could be reinfused into patients to yield substantial clinical benefit, at least in certain B cell malignancies (Grupp et al., 2013; reviewed in Kalos and June, 2013). The most well developed of these is the use of “CARs,” or chimeric antigen receptors, in which a patient’s T cells are transfected with a construct encoding an antibody against a tumor surface antigen (typically CD19) fused to T cell signaling domains (Kochenderfer and Rosenberg, 2013). Similar approaches are under investigation with recombinant T cell receptors (reviewed in Kalos and June, 2013). The procedure avoids the need for immunization and may even overcome mechanisms of immune suppression by overwhelming the system through infusion of large quantities of the modified T cells. This can force the revolution of the Cancer-Immunity Cycle, enhancing the accumulation of stimulatory immune factors, and potentially promotes eventual self-propagation of the cycle. The potential limitations here, which are yet to be fully determined, include whether the approach can be extended to cancers beyond hematologic malignancies, whether the delivery of large numbers of monospecific T cells will cause resistance due to antigenic drift, and whether the toxicity issues already identified can be safely managed. {Underlined emphasis is mine}

See Chen & Mellman's section entitled T Cell Priming and Activation.

Whether tumor antigens are delivered exogenously or are captured and presented by DCs endogenously, another strategy for intervening in the Cancer-Immunity Cycle involves the control of T cell activation. This is the presumed primary mechanism of action of anti-CTLA4 antibodies, such as ipilimumab, which blocks the interaction of the major negative regulator of T cells (CTLA4) with its ligands B7.1 and B7.2 (CD80 and CD86; Qureshi et al., 2011). Thus, during antigen presentation in lymphoid organs (or in the periphery), the expansion of T cell responses is disinhibited, thereby promoting the production of autoreactive T cells, including tumor-specific T cells. The lack of selectivity in T cell expansion combined with the fundamental importance of CTLA4 as a checkpoint may underlie the significant immune-related toxicities seen in patients treated with ipilimumab (Hodi et al., 2010). {Underlined emphasis is mine}

This second illustration below builds on (is revised by me of) Chen & Mellman's Figure 2:

The numerous factors that come into play in the Cancer-Immunity Cycle provide a wide range of potential therapeutic targets. This figure 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. Abbreviations are as follows: GM-CSF, granulocyte macrophage colony-stimulating factor; CARs, chimeric antigen receptors. {Underlined emphasis is mine}

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In the cancer immunity cycle, where certain drugs (based on known and yet to be known factors, notably stimulatory and inhibitory) promote a step (or potentially more than one step), who owns what "real estate" (i.e., who owns what drugs) should be key to understanding the competitive landscape, and where it eventually leads. As the researchers, industry and the FDA combine therapies to address the still unmet needs of late-stage cancer patients, ownership of cancer assets used in whatever combinations may and do work (and thus eventually are approved) translates into sales, profit and return on investment (in R&D).

Step #1: Chemotherapies and radiation therapies likely produce more antigen fragments than whole antigens, but antigens nevertheless. Chemotherapies are commoditized (read: inexpensive, not so profitable, and growing obsolete). Radiation therapy is owned and delivered by physicians. There are numerous targeted therapies owned by various pharmaceutical companies. They may or may not generate whole and/or fragmented antigens, but it seems their owners are worried about the dismantlement of their franchises, as some rush to combine and partner with PD-1 and PD-L1 agents.

Step #2: These agents presumably facilitate the presentation of the antigens released in Step #1, and include failed (or as yet ineffectively utilized) vaccines, CD40 agent owners (like Genentech-Roche, among others), and not-so-valuable-properties-because-of-widespread-use (read: un-patentable, like INF alpha).

Step #3: There potentially is more real estate here. Already owned includes, notably, Bristol-Myers' approved CTLA-4 agent (ipilimumab), and CD137, OX40 and CD27 agents by Pfizer and Roche, among others.

Step #7: The PD-1 owners include Bristol-Myers (Opdivo) and Merck (Keytruda). The PD-L1 owners include Roche and AstraZeneca. IDO owners include, among others, Incyte (non-exclusive combination study relationships/agreements with AstraZeneca, Bristol-Myers, Merck, and Roche) and NewLink (exclusive, now, to Roche)

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Celgene's expansion of its license relationship with Sutro today (building on 2012's initial arrangement), provides Celgene with real estate, and potentially special ones at that. In terms of real estate, while the Big Biotech can access CTLA4, PD-1 and PD-L1 via Sutro, it notably also gets LAG-3 and TIM-3 (the next generation of immune checkpoint inhibitors?).

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The illustration below builds on (is revised by me of) Chen & Mellman's Figure 3 (note LAG-3 and TIM-3 inhibitors, alongside PD-1 and PD-L-1):

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. Immune checkpoint proteins, such as CTLA4, can inhibit the development of an active immune response by acting primarily at the level of T cell development and proliferation (step 3). We distinguish these from immune rheostat (“immunostat”) factors, such as PD-L1, can have an inhibitory function that primarily acts to modulate active immune responses in the tumor bed (step 7). Examples of such factors and the primary steps at which they can act are shown. Abbreviations are as follows: IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; CDN, cyclic dinucleotide; ATP, adenosine triphosphate; HMGB1, high-mobility group protein B1; TLR, Toll-like receptor; HVEM, herpes virus entry mediator; GITR, glucocorticoid-induced TNFR family-related gene; CTLA4, cytotoxic T-lympocyte antigen-4; PD-L1, programmed death-ligand 1; CXCL/CCL, chemokine motif ligands; LFA1, lymphocyte function-associated antigen-1; ICAM1, intracellular adhesion molecule 1; VEGF, vascular endothelial growth factor; IDO, indoleamine 2,3-dioxygenase; TGF, transforming growth factor; BTLA, B- and T-lymphocyte attenuator; VISTA, V-domain Ig suppressor of T cell activation; LAG-3, lymphocyte-activation gene 3 protein; MIC, MHC class I polypeptide-related sequence protein; TIM-3, T cell immunoglobulin domain and mucin domain-3. Although not illustrated, it is important to note that intratumoral T regulatory cells, macrophages, and myeloid-derived suppressor cells are key sources of many of these inhibitory factors.

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Additionally, according to FierceBiotech, the real estate is special:

"Sutro's team believes it has devised a much better way to build ADCs--those precise cancer cell-killing constructs made up of a targeting antibody, linker and payload--and bispecifics, teeing up potentially best-in-class products that promise to be more efficiently and consistently manufactured. Using biochemical synthesis, they've hatched a technology that can bypass the current approach to biologics by genetically engineering drugs that are much simpler to make, more akin to small molecules." {Underlined and bold emphasis is mine}

PV-10's effectiveness is attributed to its physical chemistry and small molecule nature.

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Moffitt presents pre-clinical (murine model) data about PV-10 in combination with co-inhibitory blockade at SITC 2014 (Provectus' press release on the topic is here). Chen and Mellman note "inhibitors shown in red [in their Figure 3] help keep the process in check and reduce immune activity and/or prevent autoimmunity distinguish between checkpoint," but distinguish between CTLA-4, and PD-1 and PD-L-1:

"Immune checkpoint proteins, such as CTLA4, can inhibit the development of an active immune response by acting primarily at the level of T cell development and proliferation (step 3). We distinguish these from immune rheostat (“immunostat”) factors, such as PD-L1, can have an inhibitory function that primarily acts to modulate active immune responses in the tumor bed (step 7). "

The presentation could explain Moffitt's Dr. Jeff Weber, M.D., Ph.D.'s contention PV-10 may be the perfect immune system primer. What does being the perfect primer mean, and entail?

Provectus recently revised its Fact Sheet to note more work by Moffitt, this time on biomarkers:

What happened to the next cohort of the Phase 1 feasibility study? Of the total enrollment of 15 patients, 8 were reported on at AACR/ASCO 2014. April 2014 article PV-10 decreases melanoma cells in tumours, which followed AACR 2014, noted:

Studies are now underway in an additional seven patients to take biopsies and blood samples at more frequent time intervals after PV-10 injection to elucidate the pathways more clearly.

Moffitt's presentation could discuss whether or not PV-10 promotes steps #4, #5 and/or #6 of the cancer immunity cycle. If so, why and how?

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Moffitt

H. Lee Moffitt Cancer Center & Research Institute has issued strongly worded press releases related to recent pre-clinical and clinical cancer trial work and research successes.

Single Injection May Revolutionize Melanoma Treatment, Moffitt Study Shows

August 22, 2013, Tampa, Fla. – A new study at Moffitt Cancer Center could offer hope to people with melanoma, the deadliest form of skin cancer. Researchers are investigating whether an injectable known as PV-10 can shrink tumors and reduce the spread of cancer. PV-10 is a solution developed from Rose Bengal, a water-soluble dye commonly used to stain damaged cells in the eye. Early clinical trials show PV-10 can boost immune response in melanoma tumors, as well as the blood stream.

Moffitt Cancer Center Instrumental in FDA Approval of Revolutionary Two-Drug Combo to Treat Advanced Melanoma

January 23, 2014, Tampa, Fla. – Moffitt Cancer Center researchers have laid the groundwork for a revolutionary new combination therapy for the treatment of advanced melanoma – melanoma that cannot be removed surgically or has spread to other areas of the body. The newly FDA-approved therapy, Mekinist (trametinib) in combination with Tafinlar (dabrafenib), is one of the biggest advancements in melanoma treatment in the past 30 years.

Moffitt Cancer Center Plays Pivotal Role in FDA Approval of New Anti-PD-1 Inhibitor Keytruda for Metastatic Melanoma

September 4, 2014, Tampa Fla. – The U.S. Food and Drug Administration (FDA) announced the approval of a new cancer immunotherapy today to treat patients with metastatic melanoma, Keytruda (pembrolizumab) by Merck & Co.

Quotes and statements from Moffitt about PV-10

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

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

At the 2013 AACR annual meeting last April, Moffitt's then abstract related to their murine model work on PV-10 concluded: "In total, these studies support the induction of tumor-specific T cell-mediated immunity after single treatment with IL-PV-10 in multiple histologic subtypes." The upshot of this appeared to me to be:

• One injection of PV-10 ("a single treatment," later heralded in Moffitt's August 2013 press release Single Injection May Revolutionize Melanoma Treatment, Moffitt Study Shows),
• Could bring about a natural, potentially permanent or very long-lasting immune response ("tumor-specific T cell-mediated immunity"),
• To the disease ("multiple histologic subtypes").

Multiple histologic subtypes. I don't think melanoma is one disease but rather has multiple histologic subtypes. Courtesy of a fellow shareholder and internist: "Interestingly, Rosenberg’s comments in a recent editorial makes the case for nurturing in vivo adaptive T-cell immunity such as PV-10 triggers. Adaptive as in dynamically adapt to the continuous mutations that occur, not try to identify, adopt and try to clone T-cells responding to some random tumor mutation." Rosenberg is Dr. Steven Rosenberg of the National Cancer Institute.

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

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

This year (2014), Moffitt concluded: "In sum, these clinical and preclinical results increase our understanding of the cytotoxic and immunological mechanisms that may play a role in systemic immunity induced by PV-10 tumor ablation." The upshot of this appears to me to be:

• Ablation of tumors following PV-10 injection (mechanism #1 [cytotoxic mechanism] of action #1 [destroy injected tumors]),
• Leads to immunity around the body (mechanism #2 [immunological mechanism] of action #2 [destroy non-injected tumors]).

My further thoughts related to Moffitt's 2014 abstract:

Immunotherapeutic strategies incorporating intralesional (IL) ablative therapy to elicit a tumor specific immune response are under investigation as a non-surgical option to induce tumor regression of cutaneous neoplasms.

[My] takeaway: "Non-surgical option" is a translational outcome from Moffitt's work. By injecting PV-10 into tumors, turn un- or non-resectable melanoma into resectable. Make larger amounts of resectable disease much less before resecting it. Eliminate lesser amounts of disease without the need for resection or excision (surgery) at all.

Rose Bengal (RB) is a water-soluble xanthene dye that was originally used as an intravenous liver diagnostic and is in use by ophthalmologists to stain damaged cells in the eye. In murine models of breast cancer and melanoma, we have shown that IL injection of PV-10 (10% RB in saline solution) leads to ablation of injected tumors and regression of non-injected bystander tumors. In these models, increased anti-tumor T cell responses were measured, supporting the induction of systemic anti-tumor immunity after tumor ablation with PV-10.

Takeaway: Confirmation of Moffitt's murine model work (for both melanoma and breast cancer) by the cancer center's human study results of local and systemic efficacy and results (for melanoma). This repeatability, from mice to humans, is consistent with Craig, Tim and Eric's ("Provectus'") repeatability experience in cell lines to mice to higher-level animals (like horses and dogs) to human clinical trials and the compassionate use program. It would appear Moffitt fully reproduced Provectus' work. Repeatability.

In our ongoing phase I clinical trial exploring melanoma regression in patients, IL PV-10 has led to a significant decrease of melA positive melanoma cells in the biopsies of both PV10-injected and non-injected lesions.

Takeaway: I think more specific confirmation of local and systemic efficacy by virtue of said cell decreases in injected and non-injected lesions, respectively.

This regression correlated with increased circulating CD3+T cells (p=0.03) in peripheral blood mononuclear cells (PBMC).

Takeaway: I think this confirms a primary immune response derived from PV-10 by virtue of more circulating CD3+ T-cells (naïve T-cells, I believe). I think a high or higher levels of this metric corresponds with a potent (or more potent) immune response. Update 3/6/14: As I later was informed, the CD3+ protein group is associated with the T-cell receptor. "The T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells) that is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules." It's a very specific marker for T-cells.

T cells purified from PBMC from a melanoma patient produced increased IFN-gamma in response to autologous tumor after treatment with PV-10.

Takeaway: I believe this means feasibility study patients had tumor tissue banked from a resection or excision prior to PV-10 treatment. The patient then was treated with PV-10. Blood subsequently taken from him or her showed reactivity to previously resected tumor tissue; that is, Moffitt could specifically target the previously resected tumor tissue by using immune cells isolated from post-PV-10 treatment-drawn blood. I imagine if the human work was consistent with murine model work, blood drawn pre-PV-10 treatment would not have the same effect (i.e., it would not react).

Ex vivo models implemented to investigate this phenomenon indicate that the cytotoxicity induced by PV-10 is not apoptosis-dependent as evidenced by Annexin staining of melanoma cells following PV-10 treatment.

Takeaway: I think this is confirmation that while PV-10's ablation is apoptosis-like, it is not apoptosis-dependent. Management has previously described this cytotoxic mechanism as eliciting cell destruction naturally (a form of cell death that mimics both features of necrosis and apoptosis).

PV-10 directly induced necrosis of melanoma cells at 50 uM, but was not toxic to healthy fibroblasts at the same dose.

Takeaway: I think this is confirmation PV-10 targets diseased tissue only, and spares healthy tissue. Again, aspects of the drug's value proposition management has described for quite a while.

Further preclinical translational testing has shown that treatment of murine B16 cells with PV-10 leads to release of HMGB1, a soluble Damage Associated Molecule Pattern (DAMP) that is important for activation of dendritic cells (DCs). In the murine B16 melanoma model, there is a significant increase in the number of DCs infiltrating the tumor-draining lymph nodes after IL injection of PV-10. These findings suggest that PV-10 treatment leads to the release of DC activating factors and DC recruitment. Further studies to determine the role of PV-10 on T cell activation are ongoing.

Takeaway: Returning to their murine model work not previously presented or detailed, I believe, the potential for DC activation through HMBG1 release. In Moffitt's July 2013 PLoS paper, they hypothesized large amounts of tumor debris created by the rapid ablation that followed PV-10's injection into tumors, debris containing antigens (thus, large amounts of debris should mean large numbers of antigens), are taken up by DCs (see blog post, red underlined portion 4 in paragraph 3). DCs, antigen-presenting cells, present (display) antigens they've taken up to the body's immune system's T-cells. It's all about the T-cells. For example, "Cytotoxic T cells ("CTLs") specific for tumor antigens play a major role in the immunity against cancer." Will Moffitt show PV-10 specifically activates these CTLs?

I would presume this aspect of their human work, the confirmation of DC activation in patients in the feasibility study, will make-up their next presentation somewhere sometime (ASCO 2014?, a journal article?, another medical conference?).

In sum, these clinical and preclinical results increase our understanding of the cytotoxic and immunological mechanisms that may play a role in systemic immunity induced by PV-10 tumor ablation.

Takeaway: Moffitt now knows more (way more?) about PV-10's dual MOAs. PV-10's rapid tumor ablation leads to body-wide (systemic) immunity.

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

In the year 2013

In Provectus' annual CEO letter published May 2013, management wrote under Regulatory Progress:

"Provectus is finalizing details for submission of a pivotal Phase 3 randomized controlled trial ("RCT") of PV-10 for metastatic melanoma, suitable for Special Protocol Assessment ("SPA"), to the Food and Drug Administration ("FDA"). While preparation for submission of our SPA has taken longer than expected, it is crucial to remember that oncology presents a moving playing field. Fine tuning of the study design is expected to mitigate clinical efficacy risk, optimize patient accrual, and increase FDA's confidence that the study design and protocol will ensure the best possible outcome for our pivotal trial. We have every reason to believe this key milestone will be achieved in 2013."

"Provectus is also considering applying for the new Breakthrough Therapy Designation for PV-10 to treat melanoma. This new regulatory pathway was announced with the passage of The Food and Drug Administration Safety and Innovation Act (FDASIA) in July 2012. Breakthrough Therapy Designation is intended to expedite the development and review of drugs for serious or life-threatening conditions. The criteria for breakthrough therapy designation require preliminary clinical evidence that demonstrates the drug may have substantial improvement on at least one clinically significant endpoint over available therapy. A breakthrough therapy designation conveys all of the fast track program features as well as more intensive FDA guidance on an efficient drug development program. However, because this program is relatively new, the potential impact of receiving such designation is still unclear, but could be pivotal in achieving an accelerated path for approval of PV-10."

It now appears Provectus has made its [final] submission of a pivotal Phase 3 RCT of PV-10 for metastatic melanoma ("MM"), suitable for SPA, to the FDA, and also submitted its application for BTD for PV-10 to treat melanoma.

Based on connecting some dots, the submission date very likely was after October 1st. Below is a table of CDER BTD requests from October 1-25, where I think Provectus' request is one of the eight the Agency received during this period:

The recent partial federal government shutdown ran from October 1-16, with operations resuming October 17th. Assuming Provectus hears within 60 days of submission of its BTD request, and adjusting for a delay of about half-a-month related to the shutdown (there might be an adjustment for when the FDA actually received the application itself), we should learn about this outcome around or before mid-December.

Updated 11/1/13: The next six weeks, maybe sooner or potentially later, may provide a fuller picture of the regulatory clarity path management has hoed the last several years and the final step(s) or finish line. I'm still foggy about how and the process by which management is requesting exactly whatever they're requesting.

The SPA is a step on the way to approval, albeit no guarantee of such upon completion. Accelerated approval ("AA") and outright approval ("OA") are steps too (but closer to being actual destinations). Fast Track, BTD and Priority Review, while designations, are processes that speed access to new important therapies by leading to subsequent steps. A good, recent article on BTD is FDA Speeds Things Up: Breakthrough Therapy Designation Is Changing How the Agency Operates (October 1, 2013, Genetic Engineering & Biotechnology News). Some of the article's quotes are striking:

• "...the designation’s greatest value was that it prompted an “all-hands-on-deck” mentality at CDER."
• "...the breakthrough designation can rely on preliminary clinical evidence demonstrating substantial improvement on a significant clinical endpoint, while the fast-track could be based on nonclinical data such as the drug’s mechanism of action."
• "Under breakthrough designation, he said, “everything is on the table” for discussion in order to move the process along as quickly as possible. Communications that might typically take weeks and months take minutes under the breakthrough pathway."

BTD and the SPA are two independent regulatory pathways, so it makes sense management has been cultivating multiple options (including pursuit of approval via AA). I'm not clear, however, if AA is another independent pathway or an outcome of BTD in this case. The Agency notes "...a drug that has received a breakthrough therapy designation or a fast track designation can be eligible for the accelerated approval pathway, if the relevant criteria are met."

Setting aside the parallel pursuit of the SPA, it would seem several outcomes are possible if BTD is awarded: the SPA (and a "full" Phase 3 trial), a modified version of the Phase 3 trial under the SPA, AA or OA.

What's the ask? Whether directly or unrelated to the BTD application, the first ask very likely is AA, or potentially OA, for Stage IIIb-IIIc melanoma patients refractory to treatment on the basis and strength of Provectus' multi-thousand page final MM Phase 2 clinical study report and Moffitt's PLoS paper Intralesional Injection of Rose Bengal Induces a Systemic Tumor-Specific Immune Response in Murine Models of Melanoma and Breast Cancer: compelling clinical data showing PV-10 can forestall the onset of metastatic disease, and the elucidation of PV-10's mechanism of action (and systemic benefit). The second ask, or perhaps the "other side of the coin" of the first ask, should be, of course, BTD.

$PVCT's PV-10 Verified, Translated, Elucidated and Optimized by Moffitt

When Provectus issued a PR in January 2013 about Moffitt's Phase 1 feasibility study to elucidate PV-10's bystander effect, Moffitt's Dr. Amod Sarnaik, MD was quoted as "We look forward to verifying the promising pre-clinical data from our ongoing work in this translational study. These results should help elucidate the immunologic basis of the 'bystander effect' noted in previous clinical studies of PV-10 and help optimize PV-10 treatment, particularly in combination with other therapies. As Moffitt pursues its mission of contributing to the prevention and cure of cancer, we are pleased to spearhead this important clinical work."

It appears this work has been completed, preliminary results or interim analysis are available, the bystander effect has been elucidated, and Moffitt already has moved on to combining PV-10 with other notable categories of treatments, such as immunotherapies (i.e., anti-CTLA-4, anti-PD-1 and anti-PDL-1 agents).

Moffitt and, more specifically, Dr. Weber, are critical and key to PV-10 and Provectus, having been notably involved in translational studies and other work contributing greatly to the approvals of, among other treatments and therapies, ipilimumab (Yervoy) and vemurafenib (Zelboraf).

Moffitt's Phase 1 Feasibility Trial for $PVCT's PV-10 (follow-up)

A blog reader reminder me about the February article "Back to Phase 1: Understanding Systemic Effects of PV-10" in CancerWatch (Vol. 22 Feb. 2013) that discussed the background and motivation for Moffitt's Phase 1 study of the systemic antitumor effects of intralesional PV-10 treatment. It's worth re-reading the article again in the context of Moffitt purportedly completing the bulk of its feasibility study work.

“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.”

“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.

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.

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

Moffitt's Phase 1 Feasibility Trial for $PVCT's PV-10 Complete?

Gathering and connecting different informational tidbits from multiple sources, it is quite possible the bulk of Moffitt's Phase 1 feasibility study to explore PV-10's mechanism of action may already be complete (i.e., enrollment, treatment, preliminary analysis).

By detecting immune cell infiltration into melanomas treated by PV-10, Moffitt researchers (led by Dr. Amod Sarnaik MD) want to find out more about how PV-10 works in melanoma tumors, including if there are changes in the body's immune cells after PV-10 is given, both inside the melanoma tumors and circulating in the blood.

These results build upon Moffitt's prior work presented at SSO in March 2012 (Toomey et al.)* and AACR in April 2013 (Pilon-Thomas et al.)**. They could (should) be very relevant to Provectus' pursuit of breakthrough therapy designation from the FDA, as well as to Big Pharma.

* PV-10 induces anti-tumor immunity (murine model work)
** PV-10 induces systemic immune response in multiple tumor types (murine model work)