Oncolytic Activity and Mechanisms of Action by a small β2,2-amino acid derivative LTX-401

Antimicrobial peptides are part of the innate immune defence of many organisms and represent a novel class of therapeutics due to their broad-spectrum activities, including cytotoxic activity against cancer cells. It has recently been reported that small antimicrobial β2,2-amino acid derivatives (Mw<500), such as LTX-401, possess potent anticancer activity against Ramos human Burkitt’s lymphoma cells. In the present thesis, the anticancer properties displayed by LTX-401 became subject for a continued investigation. In vitro cytotoxicity studies revealed that LTX-401 was highly active against a panel of malignant cells including murine and human cancer cell lines. Moreover, LTX-401 was found to induce known features of immunogenic cell death as shown by the release of Damage-Associated Molecular Pattern molecules such as High-Mobility Group Box-1 protein, adenosine triphosphate and cytochrome c in vitro. Intralesional administration of LTX-401 into intradermally established B16 melanomas in syngeneic mice resulted in complete regression of the tumor. Moreover, cured animals that were given a second tumor challenge with live B16F1 cells did not develop tumor and displayed immune protection against the cancer. Altogether, these results underscore the therapeutic potential of LTX-401 in conveying local tumor control and generation of protective immunity, presumably by the engagement of immunogenic cell death

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

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 induces the release of danger signals.

<p>(A) Release of HMGB1 into the supernatant of LTX-401-treated cells as determined with Western blot. Translocation of the nuclear protein HMGB1 from the cell lysate (L) to the culture supernatant (S) was evident after 30 minutes of treatment with LTX-401 (108 μM), and the translocation was absolute after 90 minutes of incubation. Control cells showed no translocation after 240 minutes. Experiment was conducted thrice, and this figure is a digital image of a representative blot; (B) B16F1 cells were treated with LTX-401 (108 μM) for different time points (30, 60, 90, 120 and 240 minutes) before determining the amount of cytochrome <i>c</i> in supernatants using an ELISA assay. The results are presented as mean +/- SEM; (C) B16F1 cells were treated with LTX-401 (54 μM) for designated time points (10, 30, 60, 90 and 120 min). The quantification of ATP level was performed by luciferase bioluminescence, and the results are presented as mean +/- SEM.</p

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

LTX-401 treatment induced loss of lysosomal integrity.

<p>(A) B16F1 cells were treated with LTX-401 (27 μM) for 60 minutes before determining the signal strength from LysoTracker (DND-26) using flow cytometry. The figure is representative for one of three conducted experiments; (B) B16F1 cells were treated with LTX-401 (27 μM) for 60 minutes, and the signal from LysoTracker was imaged using confocal microscopy (z-stack, maximum intensity projection).</p

Combining the oncolytic peptide LTX-315 with doxorubicin demonstrates therapeutic potential in a triple-negative breast cancer model

Background Immunochemotherapy, the combined use of immunotherapy and chemotherapy, has demonstrated great promise in several cancers. LTX-315 is an oncolytic peptide with potent immunomodulatory properties designed for the local treatment of solid tumors. By inducing rapid immunogenic cell death through the release of danger-associated molecular pattern molecules (DAMPs), LTX-315 is capable of reshaping the tumor microenvironment, turning “cold” tumors “hot” through a significant increase in tumor-infiltrating lymphocytes. Methods We investigated the potential of LTX-315 to be used in combination with standard-of-care chemotherapy (doxorubicin, brand name CAELYX®) against triple-negative breast cancer in an orthotopic 4 T1 mammary fat pad model. Tumor growth curves were compared using one-way ANOVA analysis of variance and Tukey’s multiple comparisons test, and animal survival curves were compared using the log-rank (Mantel-Cox) test. We considered p values ≤0.05 to indicate statistical significance. Results We found that LTX-315 displayed a strong additive antitumoral effect when used in combination with CAELYX®, and induced immune-mediated changes in the tumor microenvironment, followed by complete regression in the majority of animals treated. Furthermore, imaging techniques and histological examination showed that the combination induced strong local necrosis, followed by an increase in the infiltration of CD4+ and CD8+ immune cells into the tumor parenchymal tissue. Conclusions Our data demonstrate that LTX-315 is a promising combination partner with CAELYX® for the treatment of triple-negative breast cancer

Tumor-treatment schedule.

<p>Tumor-treatment schedule.</p

Intratumoral treatment induced complete regression in B16F1 melanomas.

<p>(A) The intratumoral treatment of murine melanoma induced a rapid and complete regression in 9/11 animals, and offered long-term protection against a rechallenge in the majority of the cured mice. Palpable B16F1 tumors were injected with either a sterile 0.9% NaCl (vehicle controls) or 5 mg/ml LTX-401 once a day for three consecutive days. P<0.0001 (Mantel-Cox test); (B) Infiltrating CD3+ lymphocytes were detected in tumors following one single injection of LTX-401 (7 days post-injection) in a murine experimental animal model.</p

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

Abstract 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