Lytix Biopharma (Norway)

BackgroundOncolytic peptides are emerging as attractive candidates for the development of novel anticancer regimens1, reflecting broad cytolytic activities against a variety of malignant (but not normal) cells and a pronounced potential for immunostimulation.2 LTX-315 is a synthetic nonameric cationic peptide derived from bovine lactotransferrin3 that has been associated with a pronounced capacity to elicit tumor-targeting immune responses in various preclinical models of cancer.2 Specifically, LTX-315 has been shown to (1) cause immunogenic cell death (ICD) 4 coupled to the release of immunostimulatory cytokines and damage-associated molecular patterns (DAMPs)5,6, (2) deplete the tumor microenvironment (TME) of immunosuppressive cells such as CD4+CD25+FOXP3+ regulatory T (TREG) cells and myeloid-derived suppressor cells (MDSCs)7, and (3) synergize with immunogenic chemotherapy8 or immune checkpoint inhibitors (ICIs)7 in the control of syngeneic mouse tumor models. However, the effects of tumor cells responding to LTX-315 on dendritic cells (DCs) remain to be precisely elucidated.MethodsHuman A375 and mouse B16F10 cells were used as models of melanoma, in vitro (A375, B16F10) and in vivo (B16F10) upon subcutaneous injection in immunocompetent C57BL/6 mice. Flow cytometry, ELISA, RT-PCR and immunoblotting were used to assess DC maturation in vitro and in vivo, immunoblotting was employed to investigate the molecular mechanisms underlying DC activation, knockout and knockdown systems were harnessed to demonstrate mechanistic involvement. Therapeutic assays with B16F10 melanoma cells growing in immunocompetent vs Myd88-/- mice coupled to tumor rechallenge were employed to confirm activation of tumor-targeting immunity coupled to the establishment of immunological memory.ResultsLTX-315 mediated the release of immunostimulatory DAMPs and nucleic acids (NAs) from melanoma cells that drove robust DC activation as monitored by surface immunophenotyping for CD80, CD83, CD86 and MHC class II and cytokine (TNF, IL-1β) secretion. This immunostimulatory effect mechanistically depended on TLR9 (detecting LTX-315-DNA complexes), TLR8 (detecting LTX315-RNA complexes) and TLR7 (which was activated by LTX-315 per se), as well as on the common TLR signal transducer MYD88. Accordingly, the in vivo anticancer activity of LTX-315 against B16F10 melanoma cells was significantly reduced in Myd88-/- mice, and Myd88-/- mice eradicating B16F10 tumors upon LTX-315 treatment remained susceptible to a rechallenge with B16F10 cells.ConclusionsIn addition to induce immunogenic cell death, LTX-315 also activates DC via MYD88-dependent pathways that mediate optimal immunostimulatory effects coupled to the establishment of cancer-specific immunological memory in preclinical melanoma models.ReferencesCamilio KA, Berge G, Ravuri CS, Rekdal O, Sveinbjørnsson B. Complete regression and systemic protective immune responses obtained in B16 melanomas after treatment with LTX-315. Cancer Immunol Immunother 2014;63(6):601–613.Camilio KA, Wang MY, Mauseth B, et al. Combining the oncolytic peptide LTX-315 with doxorubicin demonstrates therapeutic potential in a triple-negative breast cancer model. Breast Cancer Res. 2019;21(1):9.Eike LM, Yang N, Rekdal Ø, Sveinbjørnsson B. The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells. Oncotarget 2015;6(33):34910–34923.Kepp O, Marabelle A, Zitvogel L, Kroemer G. Oncolysis without viruses – inducing systemic anticancer immune responses with local therapies. Nat Rev Clin Oncol 2020;17(1):49–64.Kroemer G, Galassi C, Zitvogel L, Galluzzi L. Immunogenic cell stress and death. Nat Immunol 2022;23(4):487–500.Vitale I, Yamazaki T, Wennerberg E, et al. Targeting cancer heterogeneity with immune responses driven by oncolytic peptides. Trends Cancer 2021;7(6):557–572.Yamazaki T, Pitt JM, Vétizou M, et al. The oncolytic peptide LTX-315 overcomes resistance of cancers to immunotherapy with CTLA4 checkpoint blockade. Cell Death Differ. 2016;23(6):1004–1015.Zhou H, Forveille S, Sauvat A, et al. The oncolytic peptide LTX-315 triggers immunogenic cell death. Cell Death Dis 2016;7(3):e2134.Ethics ApprovalThis study was approved by Weill Cornell Medicine IACUC.

11567 Background: Adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TILs) is a potent treatment that can induce complete and durable tumor regression as documented in patients with metastatic melanoma. To our knowledge, ACT has not been utilized for patients with metastatic soft-tissue sarcoma (STS). LTX-315 is an oncolytic peptide that has been shown to increase TILs in malignant tumors after intratumoral injection. In this trial, patients with metastatic sarcoma were treated with the combination of LTX-315 and TIL - based ACT. Methods: Patients with progressive metastatic STS after a minimum of one systemic treatment were eligible for inclusion. In step 1, patients received up to five treatments over 2-3 weeks with LTX-315 injections. Afterwards, the injected tumor was surgically removed, and TILs were expanded in vitro. In step 2, patients received preparative chemotherapy followed by infusion of the expanded TILs, and follow-up treatment with subcutaneous IL-2. The impact of LTX-315 on tumor microenvironment was assessed by immunohistochemistry (IHC) on biopsies collected before and after injections. Clinical effect of the treatment was assessed by follow-up CT-scans using RECIST 1.1. Exploratory analyses aimed at identifying tumor reactive T cells in the expanded TILs and in peripheral blood (PBMC) in the patients before and after treatment. Expanded TILs or PBMC were cocultured with autologous tumor or selected neo-peptides and reactivity was assessed by measurement by Elispot or flow cytometry. Results: Six patients received intratumoral injections with LTX-315. In 3 out of 5 samples available for IHC, increased infiltration of CD4+ cells following LTX-315 injections was observed. Four patients received the full treatment with both LTX-315 injections and ACT. Total number of infused cells was 44-63x109, comprising 0,4-52% CD8+ T cells. Treatment was tolerated with manageable toxicity. After completed treatment, all four patients had stable disease as best overall response. Two patients with leiomyosarcoma and solitary fibrous tumor had stable disease for 25 and 20 weeks, respectively. In these two patients, and one additional patient, Elispot analyses showed presence of tumor-reactive cells in the PBMC following treatment. Conclusions: This trial demonstrates that the combination of LTX-315 and ACT is feasible and tolerable with manageable toxicity. CD4+ and CD8+ TILs can be expanded in vitro from sarcomas that have been pretreated with the oncolytic peptide LTX-315. Furthermore, the data suggest that LTX-315 can modulate the tumor microenvironment in sarcomas, thus potentially affect the expanded TILs that can be used for ACT. Further optimization of the treatment schedule will position LTX-315 as technology to invoke tumor specific T cells that can be cultured and infused as part of an adoptive transfer regimen for several subtypes of soft tissue sarcoma. Clinical trial information: NCT03725605.

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.

Novel antifungals are in high demand due to the challenges associated with resistant, persistent, and systemic fungal infections. Synthetic mimics of antimicrobial peptides are emerging as a promising class of compounds for antifungal treatment. In the current study, five synthetic cationic antimicrobial tripeptides were evaluated as antifungal therapeutics against 24 pathogenic strains of fungi. Three of the peptides displayed strong general antifungal properties at low micromolar inhibitory concentrations. The most promising peptide, compound 5, was selected and evaluated as an antifungal remedy for Candida albicans candidiasis in a human skin model and for the treatment of Trichophyton rubrum induced onychomycosis in an infected human nail model. Compound 5 was shown to display antifungal properties and a rapid mode of action superior to those of both the commercial comparators Loceryl and Lamisil. Compound 5 was also active against a clinical isolate of Candida albicans with acquired fluconazole resistance.

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.