Gerd Berge

The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo.

Antimicrobial peptides have been shown to exert cytotoxic activity towards cancer cells through their ability to interact with negatively charged cell membranes. In this study the cytotoxic effect of the antimicrobial peptide, LfcinB was tested in a panel of human neuroblastoma cell lines. LfcinB displayed a selective cytotoxic activity against both MYCN‐amplified and non‐MYCN‐amplified cell lines. Non‐transformed fibroblasts were not substantially affected by LfcinB. Treatment of neuroblastoma cells with LfcinB induced rapid destabilization of the cytoplasmic membrane and formation of membrane blebs. Depolarization of the mitochondria membranes and irreversible changes in the mitochondria morphology was also evident. Immuno‐ and fluorescence‐labeled LfcinB revealed that the peptide co‐localized with mitochondria. Furthermore, treatment of neuroblastoma cells with LfcinB induced cleavage of caspase‐6, ‐7 and ‐9 followed by cell death. However, neither addition of the pan‐caspase inhibitor, zVAD‐fmk, or specific caspase inhibitors could reverse the cytotoxic effect induced by LfcinB. Treatment of established SH‐SY‐5Y neuroblastoma xenografts with repeated injections of LfcinB resulted in significant tumor growth inhibition. These results revealed a selective destabilizing effect of LfcinB on two important targets in the neuroblastoma cells, the cytoplasmic‐ and the mitochondria membrane.

Therapeutic vaccination against a murine lymphoma by intratumoral injection of a cationic anticancer peptide

Cationic antimicrobial peptides (CAPs) exhibit promising anticancer activities. In the present study, we have examined the in vivo antitumoral effects of a 9-mer peptide, LTX-302, which is derived from the CAP bovine lactoferricin (LfcinB). A20 B cell lymphomas of BALB/c origin were established by subcutaneous inoculation in syngeneic mice. Intratumoral LTX-302 injection resulted in tumor necrosis and infiltration of inflammatory cells followed by complete regression of the tumors in the majority of the animals. This effect was T cell dependent, since the intervention was inefficient in nude mice. Successfully treated mice were protected against rechallenge with A20 cells, but not against Meth A sarcoma cells. Tumor resistance could be adoptively transferred with spleen cells from LTX-302-treated mice. Resistance was abrogated by depletion of T lymphocytes, or either the CD4+ or CD8+ T cell subsets. Taken together, these data suggest that LTX-302 treatment induced long-term, specific cellular immunity against the A20 lymphoma and that both CD4+ and CD8+ T cells were required. Thus, intratumoral administration of lytic peptide might, in addition to providing local tumor control, confer a novel strategy for therapeutic vaccination against cancer.

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

Malignant melanoma is the most aggressive and deadliest form of skin cancer due to its highly metastatic potential, which calls for new and improved therapies. Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line of defense against pathogens, and several CAPs have shown promising potential as novel anticancer agents. Structure–activity relationship studies on the CAP bovine lactoferricin allowed us to de novo design short chemically modified lytic anticancer peptides. In the present study, we investigated the in vivo antitumor effects of LTX-315 against intradermally established B16 melanomas in syngeneic mice. Intratumoral administration of LTX-315 resulted in tumor necrosis and the infiltration of immune cells into the tumor parenchyma followed by complete regression of the tumor in the majority of the animals. LTX-315 induced the release of danger-associated molecular pattern molecules such as the high mobility group box-1 protein in vitro and the subsequent upregulation of proinflammatory cytokines such as interleukin (IL) 1β, IL6 and IL18 in vivo. Animals cured by LTX-315 treatment were protected against a re-challenge with live B16 tumor cells both intradermally and intravenously. Together, our data indicate that intratumoral treatment with LTX-315 can provide local tumor control followed by protective immune responses and has potential as a new immunotherapeutic agent.

Inhibition of tumour growth

THE retardation of the growth-rate of tumours in experimental animals by chemical compounds and by extracts of tissue has been the subject of numerous investigations, and the fact that inhibition can be produced by so large a number of compounds suggests some general effect on metabolic processes. Samuel and Kugelmass1 have shown that an acid-forming diet fed to young rats produces a much slower rate of growth than does an alkali-forming diet.

Abstract 2584: Complete regression and protective immune responses obtained in B16 melanomas after treatment with LTX-315 (Oncopore®)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Malignant melanoma is the most aggressive and deadliest form of skin cancer, with a high mortality rate among late-stage melanoma patients. This calls for new and improved therapies within the field. Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line of defense against pathogens, and several CAPs have shown promising anticancer activity. Structure-activity relationship studies on the CAP bovine lactoferricin has resulted in the design of a short chemically modified lytic anticancer peptide, LTX-315, which has potential as a new anticancer drug. Previous animal studies conducted with LTX-315 have demonstrated that intratumoral (i.t.) treatment of syngeneic murine A20 B-cell lymphomas and CT26WT colon carcinomas resulted in complete tumor regression. In the present study, we investigated the antitumor effects of LTX-315 against highly aggressive B16 melanomas in syngeneic mice. Methods: The cytotoxicity of LTX-315 was tested against a series of cell lines in vitro using the MTT assay. B16F1 melanoma cells (5 x 104) were intradermally inoculated into the abdomen of syngeneic C57BL/6N mice and established tumors treated i.t. with LTX-315. Tumor tissue and blood samples were taken for analysis to elaborate the mechanism of action of LTX-315 and for potential immune modulatory effects following i.t. treatment. Blood plasma was analyzed for IL 1β and IL 6 content and tumor tissue was analyzed for infiltrating immune cells and expression of pro-inflammatory cytokines using immunohistochemistry and real time qPCR, respectively. Results: LTX-315 rapidly kills B16 melanoma cells and induces the release of Danger-Associated Molecular Pattern molecules such as the High Mobility Group Box-1 protein in vitro. Intratumoral (i.t.) administration of LTX-315 resulted in tumor necrosis and the infiltration of immune cells into the tumor parenchyma followed by a complete regression of the tumor in the majority of the animals. Animals cured by LTX-315 treatment were protected against a re-challenge with live B16 tumor cells. Conclusions: Our results demonstrate that LTX-315 i.t. treatments with LTX-315 induces complete regression of solid B16F1 melanoma tumors, thus leading to local tumor control, followed by protective immune responses. Thus, LTX-315 has potential as a novel immunotherapeutic agent. Citation Format: Ketil Andre Camilio, Gerd Berge, Chandra Sekhar Ravuri, Oystein Rekdal, Baldur Sveinbjornsson. Complete regression and protective immune responses obtained in B16 melanomas after treatment with LTX-315 (Oncopore®). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2584. doi:10.1158/1538-7445.AM2014-2584

Abstract 474: Complete regression and long-term specific protective immune responses obtained in rodent tumor models after intratumoral treatment with LTX-315 .

LTX-315 is a de novo designed peptide derived from a naturally occurring host defence peptide. LTX-315 has the potential to induce long-term specific protective immune responses by stimulating immune cells, inducing tumor cell lysis with subsequent release of danger signals (e.g. HMGB1) and tumor associated antigens (TAA`s). A complete tumor regression has been obtained in several syngenic rodent tumor models by intratumoral (i.t.) injection with LTX-315. The effect was T- cell- dependent since the intervention was inefficient in immune-deficient animals. Studies on treated tumor tissue confirmed infiltration of immune cells and a switch in the cytokine profile towards a Th1 response. Successfully treated animals were protected against re-challenge with the tumor cell type treated, but not against other types of tumor cells. Moreover, tumor resistance could be adoptively transferred by spleen cells from LTX-315-treated animals. The resistance was abrogated by depletion of T- lymphocytes. Additional studies also indicate that LTX-315`s potential to locally activate the innate immune system by the immunogenic stressing of cells, in addition to the subsequent release of endogenous adjuvants and natural danger signals, provides a strong rationale for using LTX-315 as an adjuvant for vaccines based on tumor-associated antigens (TAA) and for combination with other types of immune–modulatory therapies. LTX-315 is currently being tested in a Phase I dose escalation clinical study and may represents a novel strategy for personalized in situ vaccination against cancer. Citation Format: Oystein Rekdal, Gunnar Kvalheim, Pal-Dag Line, Bent Rolstad, Ketil Camilio, Gerd Berge, Janne Nestvold, Mengyu Wang, Jihua Shi, Ali Areffard, Baldur Sveinbjornsson. Complete regression and long-term specific protective immune responses obtained in rodent tumor models after intratumoral treatment with LTX-315 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 474. doi:10.1158/1538-7445.AM2013-474

Abstract 477: Long-term protection against B16F1 melanoma upon vaccination with tumor cell lysate combined with LTX-315 as a novel adjuvant.

Here, we demonstrate the potency of LTX-315 as a novel adjuvant in combination with B16F1 tumor cell lysate vaccine. LTX-315 is a chemically modified cationic peptide derived from a family of molecules called host defense peptides. Through its cytolytic activity, the main mechanism of LTX-315 is the induction of immunogenic cell death which leads to local inflammation by recruitment and activation of the immune system at the injection site. This is important to obtain strong vaccine-specific immune responses. We have selected the B16F1 melanoma model for this study since this tumor model is poorly immunogenic and have been described for being notoriously difficult to treat. Mice were vaccinated once a week for 4 weeks and challenged with viable B16F1 cells s.c. two weeks after the last vaccination. Tumor protected mice were re-challenged 10 weeks post primary challenge. In contrast to other studies where tumor growth inhibition is observed for this model, our results not only demonstrate complete tumor regression, but also long-term protection against the B16F1 melanoma. In total, 14/18 animals had established long-term protection which indicates induction of immunological memory against B16F1 melanoma. In conclusion, LTX-315 is a novel adjuvant with substantial potency to induce strong and durable immune responses against the poorly immunogenic B16F1 in vivo. The efficacy of vaccinations to further document the potency of LTX-315 as an adjuvant are currently studied in therapeutic and metastatic B16 models and other orthotopic models. The present results indicate the potential of LTX-315 to be a next generation adjuvant for therapeutic vaccines based on tumor-associated antigens (TAA). Citation Format: Gerd Berge, Ketil Andre Camilio, Oystein Rekdal, Ali Areffard. Long-term protection against B16F1 melanoma upon vaccination with tumor cell lysate combined with LTX-315 as a novel adjuvant. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 477. doi:10.1158/1538-7445.AM2013-477