Ketil André Camilio

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.

Discovery of a 9-mer Cationic Peptide (LTX-315) as a Potential First in Class Oncolytic Peptide

Oncolytic immunotherapies represent a new promising strategy in the treatment of cancer. In our efforts to develop oncolytic peptides, we identified a series of chemically modified 9-mer cationic peptides that were highly effective against both drug-resistant and drug-sensitive cancer cells and with lower toxicity toward normal cells. Among these peptides, LTX-315 displayed superior anticancer activity and was selected as a lead candidate. This peptide showed relative high plasma protein binding abilities and a human plasma half-life of 160 min, resulting in formation of nontoxic metabolites. In addition, the lead candidate demonstrated relatively low ability to inhibit CYP450 enzymes. Collectively these data indicated that this peptide has potential to be developed as a new anticancer agent for intratumoral administration and is currently being evaluated in a phase I/IIa study.

LTX-315 (Oncopore™): A short synthetic anticancer peptide and novel immunotherapeutic agent

Several cationic antimicrobial peptides demonstrate promising anticancer effects. We have recently described the anticancer properties of LTX-315, a novel synthetic anticancer peptide, against syngeneic B16 melanomas. LTX-315 induced a complete regression of B16 melanomas and systemic protective immune responses following intralesional administration of the peptide.

Cancer-associated fibroblasts from lung tumors maintain their immunosuppressive abilities after high-dose irradiation.

Accumulating evidence supports the notion that high-dose (>5 Gy) radiotherapy (RT) regimens are triggering stronger pro-immunogenic effects than standard low-dose (2 Gy) regimens. However, the effects of RT on certain immunoregulatory elements in tumors remain unexplored. In this study, we have investigated the effects of high-dose radiotherapy (HD-RT) on the immunomodulating functions of cancer-associated fibroblasts (CAFs). Primary CAF cultures were established from lung cancer specimens derived from patients diagnosed for non-small cell lung cancer. Irradiated and non-irradiated CAFs were examined for immunomodulation in experiments with peripheral blood mononuclear cells from random, healthy donors. Regulation of lymphocytes behavior was checked by lymphocyte proliferation assays, lymphocyte migration assays, and T-cell cytokine production. Additionally, CAF-secreted immunoregulatory factors were studied by multiplex protein arrays, ELISAs, and by LC-MS/MS proteomics. In all functional assays, we observed a powerful immunosuppressive effect exerted by CAF-conditioned medium on activated T-cells (p > 0.001), and this effect was sustained after a single radiation dose of 18 Gy. Relevant immunosuppressive molecules such as prostaglandin E2, interleukin-6, and -10, or transforming growth factor-β were found in CAF-conditioned medium, but their secretion was unchanged after irradiation. Finally, immunogenic cell death responses in CAFs were studied by exploring the release of high motility group box-1 and ATP. Both alarmins remained undetectable before and after irradiation. In conclusion, CAFs play a powerful immunosuppressive effect over activated T-cells, and this effect remains unchanged after HD-RT. Importantly, CAFs do not switch on immunogenic cell death responses after exposure to HD-RT.

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.

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.

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.

Abstract 11: The oncolytic peptide LTX-315 enhances T cell clonality and induces synergy with chemotherapy

LTX-315, a novel oncolytic peptide is effective against both drug-resistant and drug-sensitive cancer cells with lower toxicity towards normal cells. Intratumoral treatment with LTX-315 results in growth inhibition, complete regression and long lasting tumor-specific immune responses. The oncolytic effect of LTX-315 involves perturbation of the plasma membrane and distortion of intracellular organelles including the mitochondria with subsequent release of Damage-Associated Molecular Pattern molecules (DAMPs) such as ATP, cytochrome c and HMGB1. LTX-315 effectively induces necrosis within the tumor followed by the release of tumor antigens as demonstrated by a greater increase in tumor infiltrating CD8+ T cells, expansion of T cell clonality, and number of clones within the tumor microenvironment. LTX-315`s ability to modify the tumor microenvironment makes it ideal as a combination partner for other cancer therapies, including chemotherapy and immune checkpoint inhibitors. In preclinical tumor models, combination of LTX-315 and chemotherapeutic agents such as cyclophosphamide and doxorubicin demonstrates significant synergy. Citation Format: Ketil Andre Camilio, Meng Yu Wang, Janne Nestvold, Gunhild Maelandsmo, Baldur Sveinbjornsson, Oystein Rekdal. The oncolytic peptide LTX-315 enhances T cell clonality and induces synergy with chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 11. doi:10.1158/1538-7445.AM2017-11

Abstract 328: Antitumor activity of the oncolytic peptide LTX-315 in syngeneic tumor models

Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in a number of species as a defense mechanism for eukaryotic cells against pathogens and are an integral part of the innate immune system. CAPs have a broad spectrum of activities including antimicrobial and anticancer while being less cytotoxic toward non-malignant cells. The potential therapeutic application against cancer has spawned an interest in developing oncolytic agents that display a new mode of action to overcome the potential drug resistance associated with other current therapeutics. The anticancer effects of CAPs are still under investigation, but several peptides have already exhibited a promising potential with cytotoxic activities against a broad spectrum of tumor cells. Oncolytic peptides exert their activity through either a membranolytic mode of action or an interaction with intracellular targets, or a combination of both. LTX-315 (K-K-W-W-K-K-W-Dip-K-NH2), a novel oncolytic peptide developed by Lytix Biopharma AS has the potential to adopt an amphipathic helical coil structure. In vitro studies have demonstrated that it was highly effective against both drug-resistant and drug-sensitive cancer cells from several organ origins, with lower toxicity toward normal cells. LTX-315 was designed for intratumoral treatment of transdermal lesions. Previously, LTX-315 has been shown to induce complete regression of B16 melanomas and long lasting antitumor immune responses. Histological analyses of treated tumors revealed extensive hemorrhagic necrosis and infiltration of CD3+ T cells. Moreover, mRNA levels of inflammatory cytokines such as IL1β, IL6 and IL18 were found to be increased in the tumor tissue after LTX-315 treatment. The treatment did also prevent lung metastasis in mice re-challenged with B16F1 cells intravenously. Due to its oncolytic mode of action, LTX-315 induces immunogenic cell death through the release of danger-associated molecular pattern molecules and tumor antigens. Recently, we have demonstrated that when subcutaneously established EMT-6 tumors (inoculated into both flanks of the animal) were treated intratumorally with LTX-315, an antitumor response was observed with a T/C ratio of 17% 19 days post start of treatment. Furthermore, an abscopal effect of LTX-315 on the untreated tumor was also reported but only when it was combined with anti-PD-L1 antibody. At the end of study, 50% of mice that had received the combination therapy were still alive vs 30% and 40% in the groups treated with LTX-315 or anti-PD-L1 antibody alone, respectively. In conclusion, LTX-315 seems to be an ideal combinations partner for immune checkpoint inhibitors. Citation Format: Ketil Andre Camilio, Baldur Sveinbjornsson, Sylvie Maubant, Guillaume Serin, Jean-Francois Mirjolet, Francis Bichat, Oystein Rekdal. Antitumor activity of the oncolytic peptide LTX-315 in syngeneic tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 328.