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

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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
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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.

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