Oncolytic peptides DTT-205 and DTT-304 induce complete regression and protective immune response in experimental murine colorectal cancer.

Oncolytic peptides represent a novel, promising cancer treatment strategy with activity in a broad spectrum of cancer entities, including colorectal cancer (CRC). Cancer cells are killed by immunogenic cell death, causing long-lasting anticancer immune responses, a feature of particular interest in non-immunogenic CRC. Oncolytic peptides DTT-205 and DTT-304 were administered by intratumoral injection in subcutaneous tumors established from murine CRC cell lines CT26 and MC38, and complete regression was obtained in the majority of animals. When cured animals were rechallenged by splenic injection of tumor cells, 1/23 animals developed liver metastases, compared to 19/22 naïve animals. Treatment with both peptides was well tolerated, but monitoring post-injection hemodynamic parameters in rats, less extensive changes were observed with DTT-205 than DTT-304, favoring DTT-205 for future drug development. DTT-205 was subsequently shown to have strong in vitro activity in a panel of 33 cancer cell lines. In conclusion, both peptides exerted a strong inhibitory effect in two immunocompetent CRC models and induced a systemic effect preventing development of liver metastases upon splenic rechallenge. If a similar effect could be obtained in humans, these drugs would be of particular interest for combinatory treatment with immune checkpoint inhibitors in metastatic CRC.

Safety, Antitumor Activity, and T-cell Responses in a Dose-Ranging Phase I Trial of the Oncolytic Peptide LTX-315 in Patients with Solid Tumors.

PURPOSE: LTX-315 is a first-in-class, 9-mer membranolytic peptide that has shown potent immunomodulatory properties in preclinical models. We conducted a phase I dose-escalating study of intratumoral LTX-315 administration in patients with advanced solid tumors. PATIENTS AND METHODS: Thirty-nine patients were enrolled, receiving LTX-315 injections into accessible tumors. The primary objective was to assess the safety and tolerability of this approach, with antitumor and immunomodulatory activity as secondary objectives. Tumor biopsies were collected at baseline and posttreatment for analysis of immunologic parameters. RESULTS: The most common treatment-related grade 1-2 adverse events were vascular disorders including transient hypotension (18 patients, 46%), flushing (11 patients, 28%), and injection site reactions in 38% of patients. The most common grade 3 LTX-315-related toxicities were hypersensitivity or anaphylaxis (4 patients, 10%). Analysis of immune endpoints in serial biopsies indicated that LTX-315 induces necrosis and CD8+ T-cell infiltration into the tumor microenvironment. Sequencing of the T-cell receptor repertoire in peripheral blood identified significant expansion of T-cell clones after treatment, of which 49% were present in available tumor biopsies after treatment, suggesting that they were tumor associated. Substantial volume reduction (≥30%) of injected tumors occurred in 29% of the patients, and 86% (12/14 biopsies) had an increase in intralesional CD8+ T cells posttreatment. No partial responses by immune-related response criteria were seen, but evidence of abscopal effect was demonstrated following treatment with LTX-315. CONCLUSIONS: LTX-315 has an acceptable safety profile, is clinically active, induces changes in the tumor microenvironment and contributes to immune-mediated anticancer activity.

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-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.

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.

The Cytolytic Amphipathic ß(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.

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-ß 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.

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.

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) 1ß, 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.

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.