May 25, 2016

Checkpoint Inhibition Differentiation or Death: Proprietary Combinations with Protectable Agents

From this project's Twitter feed: Dr. Sally Church, PhD, ‏@MaverickNY
Click to enlarge. Tweet image source
How would Big Pharma distinguish each of its immune checkpoint inhibitors (CIs) from another? Efficacy? Tolerability? Cancer indication? Cost? Within groups (e.g., PD-1s, PD-L1s, etc.), efficacy and safety should equivalent. Different antibodies — for example, with the PD-1s, like nivolumab/Opdivo and pembrolizumab/Keytruda — function in almost identical fashion and differ only by means of manufacture and corporate decisions made with regards to clinical development (e.g., dose schedule, target indication). Magically, there is parity in pricing.

Based on drug sales to date and sales projections thus far, captured in part by @grhyasen's tweet graphic above, Bristol-Myers (nivolumab/Opdivo) and Merck & Co. (pembrolizumab/Keytruda) may be in no serious rush to differentiate their CIs beyond their current, respective corporate strategies. But Roche (atezolizumab/Tecentriq), AstraZeneca (durvalumab) and Merck KGaA/Pfizer (avelumab), all with "late to market" CIs, surely must be thinking about ways to differentiate their respective compounds.

Does meaningful, sustainable and profitable differentiation come in the form of combination, and thus in the form of a partner compound for a CI? In other words, could combination create branded differentiation — ultimately based on efficacy, tolerability, indication and cost of the combination. Proprietary combinations with protectable agents.

Novartis, one Big Pharma without a "lead CI" but having PD-1, TIM-3, LAG-3 and PD-L1 CIs within its pipeline, seems to have begun its brand differentiation by expanding its strategic thinking to include or focus on first-in-class combinations. See Pharmaceuticals and Oncology Business Units, Meet Novartis Management, May 24-25, 2016:
Click to enlarge. Fuzzy orange rectangle is mine
Click to enlarge. Fuzzy orange rectangle is mine
PV-10's, and thus Provectus', value proposition to the likes of Roche, AstraZeneca, Merck KGaA, Novartis, etc. might be to (a) combine their CI with PV-10 and (b) use Provectus' combo patent* to defend the unique combination thus formed — a proprietary combination with a protected agent.

* United States Patent No. 9,107,887, Eagle et al., August 18, 2015, Combination of local and systemic immunomodulative therapies for enhanced treatment of cancer

May 21, 2016

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

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

N.B. There is no reference to Rose Bengal (generic name) or PV-10 (proprietary name) in the February 2015 Garg et al. article.
Article Abstract: Phagocytosis of dying cells is a major homeostatic process that represents the final stage of cell death in a tissue context. Under basal conditions, in a diseased tissue (such as cancer) or after treatment with cytotoxic therapies (such as anticancer therapies), phagocytosis has a major role in avoiding toxic accumulation of cellular corpses. Recognition and phagocytosis of dying cancer cells dictate the eventual immunological consequences (i.e., tolerogenic, inflammatory or immunogenic) depending on a series of factors, including the type of ‘eat me’ signals. Homeostatic clearance of dying cancer cells (i.e., tolerogenic phagocytosis) tends to facilitate pro-tumorigenic processes and actively suppress antitumour immunity. Conversely, cancer cells killed by immunogenic anticancer therapies may stimulate non-homeostatic clearance by antigen-presenting cells and drive cancer antigen-directed immunity. On the other hand, (a general) inflammatory clearance of dying cancer cells could have pro-tumorigenic or antitumorigenic consequences depending on the context. Interestingly, the immunosuppressive consequences that accompany tolerogenic phagocytosis can be reversed through immune-checkpoint therapies. In the present review, we discuss the pivotal role of phagocytosis in regulating responses to anticancer therapy. We give particular attention to the role of phagocytosis following treatment with immunogenic or immune-checkpoint therapies, the clinical prognostic and predictive significance of phagocytic signals for cancer patients and the therapeutic strategies that can be employed for direct targeting of phagocytic determinants.
Provectus says PV-10/Rose Bengal:
  • Does not rely on a single pathway to work [I assume 'signalling pathway'],
  • Does not focus on a single receptor to work [I assume 'cell receptor'], and
  • Has no known resistance [I assume little no cancer drug resistance].
A highly specific compound in its targeting of only diseased (cancerous) tumors/lesions, tissue and cells, sparing healthy ones in the process, might the veracity of PV-10/Rose Bengal's "multiplicity" be based in its lack of "specificity" in regards to cell death?

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

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

The table of major cell death pathways includes (i) apoptosis, (ii) autophagy (autophagic cell death) and (iii) immunogenic cell death.
Click to enlarge. Image source
The illustration appears to draw three paths to anti-tumor immunity; one that is direct (e.g., DAMPs like HMGB1), and two that additionally employ co-stimulatory signals such as TLR agonists or co-inhibitory signals such as immune checkpoint therapy.
Click to enlarge. Image source
A sampling of Provectus and independent medical researcher work implicates PV-10 in apoptosis, autophagy, necrosis, and immunogenic cell death:
Garg et al. observe "The mechanisms of cancer cell death elicited by anticancer therapy and the type of phagocytes (e.g., tumour-resident versus therapy-recruited) interacting with dying cells are decisive factors in making a difference between anti-inflammatory or pro-inflammatory responses."

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

May 18, 2016

Rose Bengal (PV-10) at ASCO 2016

[Colored emphasis below is mine.]

A phase 2 study of intralesional PV-10 followed by radiotherapy for localized in transit or recurrent metastatic melanoma.

Author(s): Matthew C Foote, Bryan H Burmeister, Janine Thomas, Tavis Read, Bernard Mark Smithers; Princess Alexandra Hospital and University of Queensland, Brisbane, Australia; Princess Alexandra Hospital, Brisbane, Australia

Background: Intralesional rose bengal (IL PV-10) can elicit ablation of injected tumors and a T-cell mediated abscopal effect in untreated lesions. Phase 2 testing in patients with Stage III-IV melanoma yielded a 51% objective response rate (ORR) with 50% complete response (CR) when all disease was injected. Three patients who progressed received external beam radiotherapy (XRT) to their recurrent lesions with an impressive response without an increased radiation reaction. Methods: An open-label, single-arm phase 2 study was performed to assess efficacy and safety of IL PV-10 followed by XRT. Eligibility included recurrent localized dermal, subcutaneous, in-transit or metastatic malignant melanoma (stage IIIb / IIIc) suitable for intralesional therapy and XRT. Patients received a single course of PV-10 into lesions treatable within a localized radiotherapy field. If CR was not achieved patients received 30 Gy (6 fractions of 5 Gy twice weekly over 3 weeks) 3D conformal radiotherapy (photons or electrons) commencing 6-10 weeks after PV-10. Outcome assessments included ORR and clinical benefit (CR+PR+SD) of in-field target lesions by RECIST criteria, toxicity using CTCAE V3.0, and progression free survival (PFS). Results: There were 15 patients enrolled with 13 completing the radiotherapy component. Two patients had rapidly progressive distant disease following PV-10 injection. The mean age of patients was 69 years. With a median follow up duration of 19.3 months the overall response rate was 87% (CR 33%, PR 53%) with 93% clinical benefit on an intent-to-treat basis. The mean time to best response was 3.8 months, mean duration of complete response (PFS) 12.2 months, overall loco regional progression rate 80% and melanoma specific survival 65.5 months. Size of metastases ( < 10mm) predicted potential for lesion complete response. Treatments were well tolerated with no treatment associated grade 4 or 5 adverse events. Conclusions: The combination of IL PV-10 and radiotherapy resulted in lesion specific, normal tissue sparing, ablation of melanoma tumors with minimal local or systemic adverse effects. The study results justify expanded evaluation in a randomized trial.

Intralesional rose bengal for treatment of melanoma.

Author(s): Sanjiv S. Agarwala, Robert Hans Ingemar Andtbacka, Kristen N. Rice, Merrick I. Ross, Charles Raben Scoggins, Bernard Mark Smithers, Eric D. Whitman, Eric Andrew Wachter; St. Luke's Hospital and Health Network and Temple University, Bethlehem, PA; Huntsman Cancer Institute, University of Utah, Salt Lake City, UT; Medcl Onc Assoc of San Diego, San Diego, CA; The University of Texas MD Anderson Cancer Center, Houston, TX; University of Louisville, Louisville, KY; Princess Alexandra Hospital and University of Queensland, Brisbane, Australia; Atlantic Melanoma Ctr, Morristown, NJ; Provectus Biopharmaceuticals, Inc, Knoxville, TN

Background: Intralesional rose bengal (PV-10) is an investigational small molecule ablative immunotherapy that can elicit primary ablation of injected tumors and secondary T-cell activation. Phase 2 testing in Stage III-IV melanoma yielded a 51% objective response rate (ORR) with 50% complete response (CR) when all disease was injected. PV-10 is currently undergoing phase 3 testing as a single agent in patients with locally advanced cutaneous melanoma and phase 1b testing in combination with immune checkpoint inhibition for more advanced disease. Methods: Study PV-10-MM-31 (NCT02288897) is an international multicenter, open-label, randomized controlled trial of PV-10 versus investigator’s choice of chemotherapy (dacarbazine or temozolomide) or oncolytic viral therapy (talimogene laherparepvec). A total of 225 subjects with locally advanced cutaneous melanoma (Stage IIIB or Stage IIIC recurrent, satellite or in-transit melanoma) randomized 2:1 will be assessed for progression free survival (PFS) by RECIST 1.1 (using blinded Independent Review Committee assessment of study photography and radiology data). Comprehensive disease assessments, including review of photography and radiology data, are performed at 12 week intervals; clinical assessments of progression status are performed at 28-day intervals. Study PV-10-MM-1201 (NCT02557321) is an international multicenter, open-label, sequential phase study of PV-10 in combination with pembrolizumab. Stage IV metastatic melanoma patients with at least one injectable cutaneous or subcutaneous lesion who are candidates for pembrolizumab are eligible. In the current phase 1b portion of the study, up to 24 subjects will receive the combination of PV-10 and pembrolizumab (PV-10 + standard of care). In phase 2 an estimated 120 participants will be randomized 1:1 to receive either PV-10 and pembrolizumab or pembrolizumab alone. The primary endpoint for phase 1b is safety and tolerability with PFS a key secondary endpoint; PFS is the primary endpoint for phase 2. Clinical trial information: NCT02288897

May 12, 2016

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

Updated below, again.

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

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

Click to enlarge.

These data/results:
  • " the role of IL [intralesional] RB to activate dendritic cells at the site of tumor necrosis for the induction of a systemic anti-tumor immune response,"
  • "...suggest that IL PV-10 can induce tumor-specific T cells with memory characteristics in M05 melanoma-bearing mice,"
  • " that CD8+ T cells are crucial for the tumor-specific immune response induced by IL injection of PV-10,"
  • " that IL injection of PV-10 can boost T cell infiltration in tumors,"
  • " a role for IL PV-10 to induce DCs [dendritic cells] to take up antigens at the tumor site, infiltrate into the DLN [draining lymph node], and become functionally mature,"
  • "...suggest that PV-10-treated tumors may release factors that activate DCs,"
  • "...suggest that PV-10 can kill tumor cells at a dose that is not toxic to non-tumor cells,"
  • " the role of IL PV-10 treatment to induce a systemic anti-tumor immune response in patients with metastatic melanoma," and
  • " the design of additional clinical studies to measure anti-tumor immune responses after IL injection of PV-10 in patients with melanoma."
The Cancer Immunity Cycle & PV-10
Click to enlarge.

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

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

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

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

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

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

MATERIALS AND METHODS, Incl. Human subjects

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


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

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

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

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

Mouse-to-man-to-mouse: I'd venture, in my limited experience as a biotechnology or pharmaceutical industry investor, that Moffitt's work might be the epitome of a translational study, going from mouse to human, and back to mice before returning to human, as the cancer center team confirmed and/or discovered new things in their work. As Eric said in the PR {underlined emphasis below is mine}:
"The Moffitt researchers have systematically documented each of the key steps in the immuno-oncology cycle described by Chen and Mellman in their landmark review article (Oncology Meets Immunology: the Cancer-Immunity Cycle. Immunity 2013; 39: 1-10). In an exemplary demonstration of translational medicine, this team identified important immunologic markers in model systems and verified key facets of these in clinical trial participants, and similarly identified other markers in clinical trial participants and substantiated these in mouse models. While a number of their main observations were previously reported at scientific meetings, these are presented here in detailed, integrated fashion for the first time."
Moffitt team leader Dr. Shari Pilon-Thomas also broached this mouse-to-man-to mouse approach:
"Concordance of tumor-specific T cells in peripheral blood of clinical trial participants and mice led us to look for triggers of T cell activation. Working back from these observations, we found that HMGB1 release was common in mouse and man after tumor ablation with PV-10. These results support PV-10 ablation and the resulting tumor necrosis as the upstream trigger for systemic anti-tumor response." {concordance = agreement}
PV-10 is an immunotherapy: With Moffitt's work, presentation as Eric noted in a "detailed, integrated fashion for the first time," I'm hard pressed to understand anyone saying, in an intellectually honest fashion of course, that PV-10 is anything but an immunotherapy. As Eric further noted:
"This paper is a watershed event in the development of PV-10, walking the reader through all the steps of immune activation after PV-10 injection, from immunogenic cell death and signaling via release of HMGB1, dendritic cell recruitment and infiltration into draining lymph nodes, activation of tumor-specific T cells, and killing of uninjected tumors upon infiltration by these T cells."
Additionally, Eric underscored the immunotherapeutic role PV-10 plays as a single agent or monotherapy, and in combination with other therapies and therapeutics {bolded emphasis is mine, too}:
"This mechanism of action informed the design of the two active PV-10 clinical trials: NCT02288897 in patients with locally advanced cutaneous melanoma (melanoma limited to the skin) to test the hypothesis that PV-10 alone can produce a systemic immune response that translates to longer progression free survival (PFS); and NCT02557321 in patients with later stage melanoma to test whether combination of PV-10 with the recently approved systemic immunotherapy, pembrolizumab, can 'induce and boost' an immune response against melanoma."
Updated (5/13/16): I discussed choice of medical journal with Eric. He said New England Journal of Medicine (NEJM), Journal of Clinical Oncology (JCO) and the like focus on relatively large clinical trials. Moffitt's topic and study were not a good match for NEJM, JCO, etc. Oncotarget is a high-impact journal specializing in oncology mechanism and therapeutics (i.e., translational medicine), having a 2014 impact factor of 6.4. For comparison*, for example:
  • NEJM's 2014 impact factor was 55.9,
  • Lancet, 45.2,
  • JCO, 18.4,
  • Cancer Research 9.3,
  • Clinical Cancer Research, 8.7,
  • Oncotarget, 6.4,
  • Cancer, 4.9,
  • Journal of Immunotherapy, 4.0
  • Immunology 3.8, and
  • Melanoma Research, 2.2.

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

* The information above can be downloaded from this file.

May 5, 2016

Advancing from “occasional cures” to “routine cures”

On the blog's Current News today I compared an article in The Australian from last month, April 2016, "Melanoma drug Keytruda denied full listing on PBS," to one in The ABC from June 2015, about a year later, "'Revolutionary' melanoma drug worth $150,000 a year listed on PBS, saving Australian patients thousands."

Last year, Australian patients with advanced melanoma gained access to Merck & Co.'s anti-PD-1 or immune checkpoint inhibitor drug pembrolizumab (Keytruda) following its initial, "partial" listing on the country's Pharmaceutical Benefits Scheme (PBS). This year Keytruda was denied full listing on the PBS because there was insufficient evidence of clinical benefit to justify its cost — with The Australian calling Keytruda "a much-hyped melanoma drug."

The above dialog from Australia is one part of an ongoing, maturing, global discussion with respect to this topic: whether current “revolutionary” drugs are truly revolutionary, and ultimately worth the requested price. A paragraph from The Australian's article goes to the very heart of this topic:
"The knock-back comes less than a year after the PBAC flagged concerns about a “substantial mismatch” between the public’s expectations for the so-called “breakthrough drug” and the supporting scientific data." [PBAC is Australia's Pharmaceutical Benefits Advisory Committee]
Keytruda (aka lambrolizumab) received the FDA's breakthrough therapy designation (BTD) for advanced melanoma in 2013.

Since the beginning of 2016 there has been an increasing realization that declaring “mission accomplished” in the fight against cancer might be premature. At the Vatican's recent 2016 healthcare conference, Cellular Horizons: How Science, Technology, Information and Communication will Impact Society, it was noted that the current immuno-oncology (I-O) drugs lead to “occasional cures,” but that this outcome is not good enough. Part-and-parcel with this view is the realization that combinations — combinations of different cancer treatments, whether therapies or therapeutics — likely will be paramount to advancing from occasional cures to routine cures.

The global oncology community is actually now talking about cures, realizes the immune system is crucial for this (as was first noted by the Society for Immunotherapy of Cancer in 2013), and is looking at cancer with a markedly increased sophistication in the search for necessary treatment algorithms for various tumors and tumor subtypes.

As with the rest of the oncology community, Provectus has challenges ahead, but PV-10 appears to be congruent with the mainstream more and more daily.
Click to enlarge. Image source

May 3, 2016

Catching On

Updated below.

Takeaways and a Question:
  • Getting T cells into cancer tumors is important to more successfully fighting the disease.
  • Traditional cancer treatments do not kill enough cancer cells. Immunotherapies like immune checkpoint inhibitors kill more cells than traditional treatments, but they still do not kill enough.
  • Ways of getting T cells into tumors include chemotherapy, radiotherapy, targeted therapies and intralesional (IL) or intratumoral (IT) agents like Amgen's talimogene laherparepvec (T-Vec) (Imylgic®) [approved as a single agent], Provectus' PV-10 [in a pivotal Phase 3 trial as a single agent], Viralytics' Cavatak (another oncolytic virus) [having done Phase 2 trial work as a single agent], etc.
  • The more (quantity) and better (quality) T cells that get into cancer tumors, the greater the immunologic signalling of the drug or drug compound (immunotherapy) that led to said infiltration.
  • If a drug or drug compound's immunologic signalling is so powerful and broad as to enable the Cancer Immunity Cycle to cycle more sustainably and durably, what would have come first, the chicken or the egg (i.e., the left-hand side, or the right-hand side, the antigen cascader or the checkpoint inhibitor)?
Big Pharma-oriented blogger and publisher, and industry consultant, Dr. Sally Church, MD recently wrote a post (on her blog Biotech Strategy Blog; subscription required) asking if chemotherapy was immunotherapy: "Controversy: Is Chemotherapy Immunotherapy?" Her thoughts derived, in part or in whole, from her visit to AACR 2016. She noted in this post (alluding to a prior one) that one of the rate limiting steps in the Cancer Immunity Cycle was "getting more T cells into the tumours so that subsequent immunotherapy can be even more effective." One way to get more T cells into tumors, according to Dr. Church, was chemotherapy.
Click to enlarge. Tweet image source
Getting T cells into cancer tumors, from the perspective of a traditional treatment like chemotherapy, begins with generating antigens caused by killing cancer cells.
Click to enlarge. Figure 3, Chen and Mellman. Immunity 2013; 39: 1-10.
Utilizing therapeutics or therapies (immunotherapies) from the left-hand side of the Cycle to facilitate the infiltration of presumably educated and trained T cells into cancer tumors, therapeutics (immunotherapies) from the right-hand side can be, to use Dr. Church's words "even more effective."

Following Dr. Church's writing, primarily her tweets (and related or associated material) on Twitter (@maverickNY), is necessary for this investment project. She was kind enough to answer some basic questions of mine early on in my due diligence (basic for her, not so basic for me at the time). With 20 years of experience in the pharmaceutical industry that included the development and launch of Gleevec by Novartis (bios here and here), Dr. Church is smart, intelligent, thoughtful, and (I believe) a weather vane of Big Pharma's market/marketing (and, possibly, R&D) interest and direction. For example, she was and presumably remains a fan of checkpoint inhibitors, but has evolved her views (as I believe them to have developed) in regards to treating late-stage disease from such agents strictly as stand-alone, single agent therapies to their greater relevance and effectiveness in combination with other therapeutics and therapies (presumably stimulatory ones complementary to these inhibitory agents).

AACR 2016 not only discussed chemotherapy as a "potential immunotherapy" (viewing this through Dr. Church's prism), the conference also considered and contemplated radiation therapy or radiotherapy — but fractionated treatment, where radiation treatment is given in several, small doses over a period of time. This is about killing cancer cells to begin the antigen cascade (immunogenic cell death). This is about harnessing the cancer patient's tumor(s) to become a vaccine, so to speak (in situ vaccination).

PV-10 + Chemotherapy. Ironically, as an aside, at SITC 2012 (October), three-and-a-half years before AACR 2016 (April), Provectus presented the results of murine model work combining PV-10 and chemotherapy that generated the best response in non-injected (untreated) tumors {underlined emphasis below is mine}:
"The mechanism by which PV-10's bystander effect is produced was investigated using hepatocellular carcinoma (HCC) and melanoma tumors in immunocompetent and immunodeficient mice. In one set of experiments using immunocompetent mice with bilateral HCC tumors (i.e., two HCC tumors in opposite flanks), intralesional injection of PV-10 into one of the two tumors led not only to eradication of the injected tumor, but also to regression of the uninjected tumor. Controls treated with saline exhibited no effect in either tumor. Treatment of mice with systemic chemotherapy (i.e., 5-fluorouracil, "5-FU") had minimal effect on either tumor, while combination of intralesional PV-10 with systemic 5-FU elicited maximal response in uninjected tumors. Data were analyzed by tumor (i.e., injected tumor, uninjected tumor and total tumor burden) for time to progression and for tumor growth. PV-10 alone was favorable to saline control in all categories, while PV-10 combination therapy produced highly significant advantage vs. control for time to progression of treated tumors (p = 0.010), untreated tumors (p = 0.011) and total tumor burden (p = 0.004)." (Source: Provectus press release, October 2012)
Click to enlarge. Image source
Click to enlarge. Image source
PV-10 + Radiation. Even more ironic, and as a further aside, Provectus shareholders await the presentation or publication of an investigator-initiated study in Australia combining PV-10 and radiotherapy in patients with advanced melanoma. You know...what the company's CTO Dr. Eric Wachter, PhD said more than a year ago in response to a question of mine.

As background, Radiation therapy has been combined with PV-10 in two situations. First, in 2010, Foote et al. reported on their combination work on 3 patients in A novel treatment for metastatic melanoma with intralesional rose bengal and radiotherapy: a case seriesMelanoma Research, 20:48, Jan 2010. All of the patients originally were enrolled in Provectus' metastatic melanoma Phase 2 trial (I believe). All patients achieved complete response (CR) (note detailed descriptions in the article's text); however, no discussion was provided regarding survival (that is, keep in the mind the difference or differentiation between [objective] response rate and overall survival). Note: PV-10 was administered firstfollowed by radiation.
Click to enlarge. Fuzzy purple underlined emphasis is mine.
Later, in 2013, Tan and Neuhaus reported on their combination work on 2 patients in Novel use of Rose Bengal (PV-10) in two cases of refractory scalp sarcomaANZ Journal of Surgery, 83:1-2:93, Jan 2013. Both patients were treated in the company's compassionate use/expanded access program (I believe). One patient achieved a complete response, while the other enjoyed "good" clinical effect before progressing again. Also, no discussion was provided regarding survival of the patient achieving CR. Note: Here, radiation was administered firstfollowed by PV-10.

During the 4Q15/CY 2014 business update conference call on March 11, 2015, I asked about the timing, and possibly the potential venue(s) (medical conference, journal), of Foote et al.'s clinical follow-up work combining PV-10 and radiotherapy? Eric said: "But regarding the radiation therapy publication by Foote, that is of record as having led to an investigator-initiated study that you mentioned, 25 patients. That study has been enrolling slowly. It had very specific eligibility criteria at a single center. We have had discussions over the last several months with the investigator about ways to get data from that study available publicly and we anticipate that sometime in the coming months, that is sometime this year, that there will be some presentation of interim data from that study" {my underlined emphasis; this year = 2015}.

Step #5, Infiltration of T cells into Tumors. PV-10 has been potentially implicated in each and every step of the Cancer Immunity Cycle, with data on Step #5 to come.
Click to enlarge. Image source
Click to enlarge. Image source
If PV-10/Rose Bengal can (has) overcome one of the Cancer Immunity Cycle's rate limiting steps (of getting T cells into cancer tumors) in greater quantity and better quality (than other treatments), would Dr. Church then refer to PV-10 as immunotherapy?

Updated (5/20/16): Dr. Church updated or extended her thinking with one of her Blue Ice Publishing episodes at Novel Targets entitled Episode 12: Of Mice and Men. In it she further expands on chemotherapy as immunotherapy, and potentially a partner for other immunomodulatory agents like checkpoint inhibitors.
Click to enlarge. Tweet image source

Inception to date: PV-10 + [something else]
Click to enlarge.

April 21, 2016

Burning Down The House

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

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

Autologous (below left) and genetically engineered (below right) ACT are illustrated below from W. Joost Lesterhuis and Cornelis J. A. Punt, “Harnessing the immune system to combat cancer,” 2012, Nature Reviews/Drug Discovery, supplement to Nature Publishing Group Journals.
Click to enlarge.
Harnessing the immune system to combat cancer, in the context of intralesional (IL) or intratumoral (IT) compounds like Bacillus Calmette–GuĂ©rin (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.”