Complete regression and systemic protective immune responses obtained in B16 melanomas after treatment with LTX-315

A manuscript version of this article is part of Ketil André Camilio's doctoral thesis, which is available in Munin at http://hdl.handle.net/10037/5489</a

Short Lytic Anticancer Peptides as a Novel Therapy against Cancer

Cancer is the leading cause of deaths worldwide, with a significant increase in the number of annual incidents. Concurrently, cancer-related therapy has been met with a number of challenges, such as toxic side effects and an increase in multi-drug resistant cancer cells, thereby spawning a need for new and improved therapies. Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in most species, often as an integral part of the first line of defense against pathogens. Several CAPs have shown promising potential as novel anticancer agents with an ability to selectively kill cancer cells. Structure-activity relationship studies on bovine lactoferricin allowed us to de novo design short chemically modified lytic anticancer peptides (ACPs) with an improved therapeutic potential compared to bovine lactoferricin. The intratumoral (i.t.) administration of LTX-302 induced a complete regression of- and a long-term and transferrable tumor-specific immune protection against syngeneic A20 B cell lymphomas. A more active nonapeptide, LTX-315, was able to induce complete regression and a long-term tumor immune protection against highly aggressive and low immunogenic syngeneic B16 melanomas. By inducing rapid necrosis and local inflammation due to the release of Danger-Associated Molecular Pattern molecules, i.t. administration of the ACPs stimulated the infiltration of immune cells into the tumor parenchyma, thus creating a synergistic relationship between the direct disruptive effects and the indirect immunomodulatory effects of the peptides. This thesis also demonstrates that ACPs with a high cell membrane disruptive potential such as LTX-315 can be used in an immune augmenting adjuvant setting due to its potential to stimulate immune responses. Intradermal administration of LTX-315 induced reversible tissue damage, leading to local inflammation and the infiltration of immune cells at the injection site. When used together with a tumor cell lysate, the combination was able to mount a long-term tumor immune protection against syngeneic B16 melanomas. Taken together, the data presented in this thesis demonstrates that i.t. treatment with short lytic LTX-ACPs can have potential as new immunotherapeutic agents by enlisting local tumor control, followed by protective immune responses. Moreover, LTX-315 has an immune augmenting adjuvant potential when used in combination with a whole cell cancer vaccine

LTX-315: a first-in-class oncolytic peptide that reprograms the tumor microenvironment

The oncolytic peptide LTX-315, which has been de novo designed based on structure– activity relationship studies of host defense peptides, has the ability to kill human cancer cells and induce specific anticancer immune response when injected locally into tumors established in immunocompetent mice. The oncolytic effect of LTX-315 involves perturbation of plasma membrane and the mitochondria with subsequent release of danger-associated molecular pattern molecules, which highlights the ability of LTX315 to induce complete regression and protective immune responses. Treatment with LTX-315 reprograms the tumor microenvironment by decreasing the local abundance of immunosuppressive cells and by increasing the frequency of effector T cells

LTX-315: a first-in-class oncolytic peptide that reprograms the tumor microenvironment

The oncolytic peptide LTX-315, which has been de novo designed based on structure–activity relationship studies of host defense peptides, has the ability to kill human cancer cells and induce specific anticancer immune response when injected locally into tumors established in immunocompetent mice. The oncolytic effect of LTX-315 involves perturbation of plasma membrane and the mitochondria with subsequent release of danger-associated molecular pattern molecules, which highlights the ability of LTX-315 to induce complete regression and protective immune responses. Treatment with LTX-315 reprograms the tumor microenvironment by decreasing the local abundance of immunosuppressive cells and by increasing the frequency of effector T cells

The cytolytic amphipathic β(2,2)-amino acid LTX-401 induces DAMP release in melanoma cells and causes complete regression of B16 melanoma

In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors

Ionizing radiation abrogates the pro-tumorigenic capacity of cancer-associated fibroblasts co-implanted in xenografts

Cancer-associated fibroblasts (CAFs) are abundantly present in solid tumors and affect tumorigenesis and therapeutic responses. In the context of clinical radiotherapy, the impact of irradiated CAFs to treatment outcomes is largely unexplored. Aiming at improving radiotherapy efficacy, we have here explored the effect of radiation on the inherent pro-tumorigenic capacity of CAFs in animals. Ionizing radiation was delivered to cultured CAFs as single-high or fractionated doses. Tumor development was compared in mice receiving A549 lung tumor cells admixed with irradiated or control CAFs. Biological mechanisms behind tumor growth regulation were investigated by quantitative histology and immunohistochemistry. Viability assessments confirmed that irradiated CAFs are fully functional prior to implantation. However, the enhanced tumorigenic effect observed in tumors co-implanted with control CAFs was abrogated in tumors established with irradiated CAFs. Experiments to ascertain fate of implanted fibroblasts showed that exogenously administered CAFs reside at the implantation site for few days, suggesting that tumor growth regulation from admixed CAFs take place during initial tumor formation. Our work demonstrate that irradiated CAFs lose their pro-tumorigenic potential in vivo, affecting angiogenesis and tumor engraftment. This finding propose a previously unknown advantageous effect induced by radiotherapy, adding to the direct cytotoxic effects on transformed epithelial cells

The Novel Oncolytic Compound LTX-401 Induces Antitumor Immune Responses in Experimental Hepatocellular Carcinoma

LTX-401 is a novel oncolytic compound designed for the local treatment of solid tumors. In the present study, we have examined the applicability and efficacy of LTX-401 in a rat model JM1 hepatocellular carcinoma, with particular interest in its ability to induce antitumor immunity. LTX-401 induces necrotic cell death followed by the release of immunogenic cell death mediators such as high-mobility group box 1 protein, ATP, and cytochrome c. When injected into subcutaneous and orthotopic JM1 tumors, LTX-401 treatment resulted in a strong antitumoral effect followed by complete tumor regression in the majority of animals. Additionally, LTX-401 could affect the growth of distal tumor deposits simulating metastases, hence indicating immune-mediated abscopal responses. Furthermore, LTX-401 treatment induced tumor-specific immune responses as seen by protection against tumor rechallenge and increased production of interferon-gamma (IFN-γ) by splenic cells in response to stimulation with tumor cells. Taken together, our data demonstrate that the oncolytic compound LTX-401 provides local tumor control followed by protective immune responses and may be exploited as a novel immunotherapeutic agent in hepatocellular carcinoma

LTX-401 treatment induced ultrastructural changes with vacuolization.

<p>Representative TEM micrographs of B16F1 cells treated with LTX-401 (108 μM). Untreated control cells (A and B) were kept in a serum-free RPMI 1640 only until the experimental endpoint (60 minutes), and compared with cells treated for both 5 min (C and D) and 60 min (E and F); scale bars = 10 μm for A, C, E, 5 μm for B, D, F.</p