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-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

Tumor-treatment schedule.

<p>Tumor-treatment schedule.</p

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

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

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

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

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.

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. 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. 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. LTX-315 has an acceptable safety profile, is clinically active, induces changes in the tumor microenvironment and contributes to immune-mediated anticancer activity