LTX-315 triggers anticancer immunity by inducing MyD88-dependent maturation of dendritic cells.

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer activity but limited toxicity for non-malignant cells. LTX-315 induces both immunogenic tumor cell death and generation of tumor-specific immune responses in multiple experimental tumor models. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immunity, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. We found that LTX-315 treatment induces the maturation of DCs, both indirectly through the release of cancer cell-derived damage-associated molecular patterns (DAMPs)/alarmins and nucleic acids (DNA and RNA) capable of triggering distinct Toll-like receptor (TLR) signaling, and, directly by activating TLR7. The latter results in the ignition of multiple intracellular signaling pathways that promotes DC maturation, including NF-κB, mitogen activated protein kinases (MAPKs), and inflammasome signaling, as well as increased type 1 interferon production. Critically, the effects of LTX-315 on DCs the consequent promotion of anti-melanoma immunity depend on the cytosolic signal transducer myeloid differentiation response gene 88 (MyD88). These results cast light on the mechanisms by which LTX-315 induces DC maturation and hence elicits anticancer immunity, with important implications for the use of LTX-315 as an anticancer immunotherapeutic.

LTX-315 and adoptive cell therapy using tumor-infiltrating lymphocytes generate tumor specific T cells in patients with metastatic soft tissue sarcoma.

LTX-315 is an oncolytic peptide that elicits both local and systemic immune responses upon intratumoral injection. In the present pilot trial, we treated patients with metastatic soft tissue sarcoma with the combination of LTX-315 and adoptive T-cell therapy using in vitro expanded tumor-infiltrating lymphocytes. Six heavily pretreated patients were included in the trial and treated with LTX-315 of which four patients proceeded to adoptive T-cell therapy. Overall, the treatment was considered safe with only expected and manageable toxicity. The best overall clinical response was stable disease for 208 days, and in this patient, we detected tumor-reactive T cells in the blood that lasted until disease progression. In three patients T-cell reactivity against in silico predicted neoantigens was demonstrated. Additionally, de novo T-cell clones were generated and expanded in the blood following LTX-315 injections. In conclusion, this pilot study provides proof that it is feasible to combine LTX-315 and adoptive T-cell therapy, and that this treatment can induce systemic immune responses that resulted in stabilization of the disease in sarcoma patients with otherwise progressive disease. Further optimization of the treatment protocol is warranted to increase clinical activity. ClinicalTrials.gov Identifier: NCT03725605.

LTX-315-enabled, radiotherapy-boosted immunotherapeutic control of breast cancer by NK cells.

LTX-315 is a nonameric oncolytic peptide in early clinical development for the treatment of solid malignancies. Preclinical and clinical evidence indicates that the anticancer properties of LTX-315 originate not only from its ability to selectively kill cancer cells, but also from its capacity to promote tumor-targeting immune responses. Here, we investigated the therapeutic activity and immunological correlates of intratumoral LTX-315 administration in three syngeneic mouse models of breast carcinoma, with a focus on the identification of possible combinatorial partners. We found that breast cancer control by LTX-315 is accompanied by a reconfiguration of the immunological tumor microenvironment that supports the activation of anticancer immunity and can be boosted by radiation therapy. Mechanistically, depletion of natural killer (NK) cells compromised the capacity of LTX-315 to limit local and systemic disease progression in a mouse model of triple-negative breast cancer, and to extend the survival of mice bearing hormone-accelerated, carcinogen-driven endogenous mammary carcinomas. Altogether, our data suggest that LTX-315 controls breast cancer progression by engaging NK cell-dependent immunity.

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.

Targeting Cancer Heterogeneity with Immune Responses Driven by Oncolytic Peptides.

Accumulating preclinical and clinical evidence indicates that high degrees of heterogeneity among malignant cells constitute a considerable obstacle to the success of cancer therapy. This calls for the development of approaches that operate - or enable established treatments to operate - despite such intratumoral heterogeneity (ITH). In this context, oncolytic peptides stand out as promising therapeutic tools based on their ability to drive immunogenic cell death associated with robust anticancer immune responses independently of ITH. We review the main molecular and immunological pathways engaged by oncolytic peptides, and discuss potential approaches to combine these agents with modern immunotherapeutics in support of superior tumor-targeting immunity and efficacy in patients with cancer.

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation.

Selective Intracellular Delivery of Thiolated Cargo to Tumor and Neovasculature Cells Using Histidine-Rich Peptides as Vectors.

Short histidine-rich peptides could serve as novel activatable vectors for delivering cytotoxic payloads to tumor and neovasculature cells. This explorative study reports preliminary results showing that zinc ions, which are found in elevated levels at neovasculature sites, can trigger the intracellular delivery of a short antimicrobial peptide when conjugated to a histidine-rich peptide through a disulfide bond. The importance of exofacial thiols in the mode of action of these disulfide-linked conjugates is also shown.

LTX-315 sequentially promotes lymphocyte-independent and lymphocyte-dependent antitumor effects.

LTX-315 is an oncolytic peptide that has antitumor efficacy in mice grafted with various tumor cell lines and is currently being tested in phase II clinical trials. Here we aimed to further evaluate LTX-315 in conditional genetic mouse models of cancer that typically resist current treatment options and to better understand the drug's mode of action in vivo. We report LTX-315 mediates profound antitumor effects against Braf- and Pten-driven melanoma and delays the progression of Kras- and P53-driven soft tissue sarcoma in mice. Additionally, we show in melanoma that LTX-315 triggers two sequential phases of antitumor response. The first phase of response, which begins within minutes of drug delivery into tumors, is defined by disrupted tumor vasculature and decreased tumor burden and occurs independently of lymphocytes. The second phase of response, which continues over weeks, is defined by long-term alteration of the tumor microenvironment; the changes induced by LTX-315 are most notably characterized by CD8+ T cell infiltration. We further show that these CD8+ T cells are involved in suppressing melanoma outgrowth in mice and report similar CD8+ T cell infiltration following LTX-315 treatment in melanoma and sarcoma patients. Taken together, these findings reveal LTX-315's multiple antitumor effects, including disrupting the tumor vasculature and promoting the conversion of poorly immunogenic tumors into ones that display antitumor T cell immunity.

Enhanced T-lymphocyte infiltration in a desmoid tumor of the thoracic wall in a young woman treated with intratumoral injections of the oncolytic peptide LTX-315: a case report.

BACKGROUND: Desmoid tumors are intermediary malignant, fibrous lesions occurring in various soft tissues. Surgical treatment is relentlessly challenging because of the propensity for local aggressive behavior and high risk of recurrence. Consequently, a wide range of oncological drugs and radiation therapy are being used; however, outcomes are unpredictable. We investigated whether local treatment with an oncolytic peptide could be beneficial in a patient with an unresectable desmoid tumor. CASE PRESENTATION: In a young 29-year-old Caucasian woman who was diagnosed with a retromammary desmoid tumor infiltrating deeply into the anterior thoracic wall, surgery was considered excessively mutilating, and observation was recommended. The lesion progressed, however, and caused debilitating pain, despite nonsteroidal anti-inflammatory medication. Subcutaneous injections of human interferon-α (Multiferon®) resulted in reduced growth kinetics but had to be terminated because of development of symptomatic pneumonitis. Frequently used oncological treatment was withheld because of the toxicity profile, and the patient was instead included in a phase I study investigating transdermal intratumoral injection of LTX-315, an oncolytic peptide that induces anticancer immune responses ( ClinicalTrials.gov , NCT01986426 ). A marked increase of CD8+ tumor-infiltrating T cells in the lesion was complemented by upregulation of immune gene signature (including effector T-cell, T-helper type 1 cell, chemokine, and cytokine genes). These changes were followed by gradual symptom relief and long-term disease stabilization, indicating clinical benefit. LTX-315 was well tolerated until termination in week 16 after a serious allergic reaction. CONCLUSIONS: Our patient was treated with repeated intratumoral injections of LTX-315, resulting in tumor regression accompanied by upregulation of immune genes and T-cell infiltration. Local application of immunotherapy, minimizing systemic side effects, represents a novel treatment modality in desmoid tumors that should be tested in further clinical trials.

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.

Tumor lysis with LTX-401 creates anticancer immunity.

Local immunotherapies such as the intratumoral injection of oncolytic compounds aim at reinstating and enhancing systemic anticancer immune responses. LTX-315 is a first-in-class, clinically evaluated oncolytic peptide-based local immunotherapy that meets these criteria. Here, we show that LTX-401, yet another oncolytic compound designed for local immunotherapy, depicts a similar safety profile and that sequential local inoculation of LTX-401 was able to cure immunocompetent host from subcutaneous MCA205 and TC-1 cancers. Cured animals exhibited long-term immune memory effects that rendered them resistant to rechallenge with syngeneic tumors. Nevertheless, the local treatment with LTX-401 alone had only limited abscopal effects on secondary contralateral lesions. Anticancer effects resulting from single as well as sequential injections of LTX-401 were boosted in combination with PD-1 and CTLA-4 immune checkpoint blockade (ICB), and sequential LTX-401 treatment combined with double ICB exhibited strong abscopal antineoplastic effects on contralateral tumors underlining the potency of this combination therapy.

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.

Recruitment of LC3 to damaged Golgi apparatus.

LC3 is a protein that can associate with autophagosomes, autolysosomes, and phagosomes. Here, we show that LC3 can also redistribute toward the damaged Golgi apparatus where it clusters with SQSTM1/p62 and lysosomes. This organelle-specific relocation, which did not involve the generation of double-membraned autophagosomes, could be observed after Golgi damage was induced by various strategies, namely (i) laser-induced localized cellular damage, (ii) local expression of peroxidase and exposure to peroxide and diaminobenzidine, (iii) treatment with the Golgi-tropic photosensitizer redaporfin and light, (iv) or exposure to the Golgi-tropic anticancer peptidomimetic LTX-401. Mechanistic exploration led to the conclusion that both reactive oxygen species-dependent and -independent Golgi damage induces a similar phenotype that depended on ATG5 yet did not depend on phosphatidylinositol-3-kinase catalytic subunit type 3 and Beclin-1. Interestingly, knockout of ATG5 sensitized cells to Golgi damage-induced cell death, suggesting that the pathway culminating in the relocation of LC3 to the damaged Golgi may have a cytoprotective function.

Oncolysis with DTT-205 and DTT-304 generates immunological memory in cured animals.

Oncolytic peptides and peptidomimetics are being optimized for the treatment of cancer by selecting agents with high cytotoxic potential to kill a maximum of tumor cells as well as the capacity to trigger anticancer immune responses and hence to achieve long-term effects beyond therapeutic discontinuation. Here, we report on the characterization of two novel oncolytic peptides, DTT-205 and DTT-304 that both selectively enrich in the lysosomal compartment of cancer cells yet differ to some extent in their cytotoxic mode of action. While DTT-304 can trigger the aggregation of RIP3 in ripoptosomes, coupled to the phosphorylation of MLKL by RIP3, DTT-205 fails to activate RIP3. Accordingly, knockout of either RIP3 or MLKL caused partial resistance against cell killing by DTT-304 but not DTT-205. In contrast, both agents shared common features in other aspects of pro-death signaling in the sense that their cytotoxic effects were strongly inhibited by both serum and antioxidants, partially reduced by lysosomal inhibition with bafilomycin A1 or double knockout of Bax and Bak, yet totally refractory to caspase inhibition. Both DTT-304 and DTT-205 caused the exposure of calreticulin at the cell surface, as well as the release of HMGB1 from the cells. Mice bearing established subcutaneous cancers could be cured by local injection of DTT-205 or DTT-304, and this effect depended on T lymphocytes, as it led to the establishment of a long-term memory response against tumor-associated antigens. Thus, mice that had been cured from cancer by the administration of DTT compounds were refractory against rechallenge with the same cancer type several months after the disappearance of the primary lesion. In summary, DTT-205 and DTT-304 both have the capacity to induce immunotherapeutic oncolysis.

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus.

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.

The oncolytic compound LTX-401 targets the Golgi apparatus.

This corrects the article DOI: 10.1038/cdd.2016.86.

Oncolytic peptide LTX-315 induces an immune-mediated abscopal effect in a rat sarcoma model.

LTX 315 is an oncolytic peptide with potent immunological properties. In the present study, we demonstrate that intratumoral treatment with LTX-315 resulted in a complete regression and systemic immune response in a rat fibrosarcoma model. The treatment was T-cell dependent, and also resulted in an abscopal effect as demonstrated by the regression of distal non-treated lesions. Significant infiltration of CD8+ T cells was observed in both treated and non-treated lesions, as shown by immunohistochemical and flow cytometric analysis. LTX-315 rapidly killed the cells in vitro with a lytic mode of action followed by the subsequent release of Danger-Associated Molecular Pattern (DAMP) molecules such as HMGB1, ATP and Cytochrome c. Together, our data demonstrate that LTX-315 represents a new approach to cancer immunotherapy, which has the potential as a novel immunotherapeutic agent.

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.