microRNA-2110 (miR-2110) was previously identified as inducing neurite outgrowth in a neuroblastoma cell lines BE(2)-C, suggesting its differentiation-inducing and oncosuppressive function in neuroblastoma. In this study, we demonstrated that synthetic miR-2110 mimic had a generic effect on reducing cell survival in neuroblastoma cell lines with distinct genetic backgrounds, although the induction of cell differentiation traits varied between cell lines. In investigating the mechanisms underlying such functions of miR-2110, we identified that among its predicted target genes down-regulated by miR-2110, knockdown of Tsukushi (TSKU) expression showed the most potent effect in inducing cell differentiation and reducing cell survival, suggesting that TSKU protein plays a key role in mediating the functions of miR-2110. In investigating the clinical relevance of miR-2110 and TSKU expression in neuroblastoma patients, we found that low tumor miR-2110 levels were significantly correlated with high tumor TSKU mRNA levels, and that both low miR-2110 and high TSKU mRNA levels were significantly correlated with poor patient survival. These findings altogether support the oncosuppressive function of miR-2110 and suggest an important role for miR-2110 and its target TSKU in neuroblastoma tumorigenesis and in determining patient prognosis.

Neuroblastoma is one of the most common and aggressive types of pediatric cancers, making up about 7% of all childhood cancers. Neuroblastoma arises from the failure of neural crest cell precursors to differentiate, and inducing cell differentiation is one of the most important treatment approaches for neuroblastoma. MicroRNAs regulate gene expression by performing post-transcriptional gene modification by mainly translational suppression and mRNA degradation. The dysregulation of these molecules has been shown to be related to tumor development, tumor metastasis and drug resistance, and the promise of developing microRNA-based therapeutics for cancers has been demonstrated. Many recent studies have also provided evidence for the involvement of microRNAs in differentiation of neuroblastoma cells, suggesting the potential of developing microRNA-based differentiation therapies for neuroblastoma. Here we review the recent findings on the role of microRNAs in regulating cell differentiation, with a main focus on neuroblastoma cells. The investigations on the therapeutic potential of microRNAs in neuroblastoma therapy and differentiation therapy are also reviewed.

CTLA-4-targeted engineered toxin bodies (ETBs) are designed to deplete immune-suppressive regulatory T cells (Tregs) in the tumor microenvironment (TME) directly and in a manner independent of Fc-mediated effector functions, offering a unique approach to CTLA-4 targeted therapy that may show efficacy where blocking antibodies have failed. ETBs are large molecule proteins consisting of an antibody fragment genetically fused to a proprietary de-immunized (DI) form of the Shiga-like toxin-1 subunit A (SLTA). Once engaged to the specific cell surface target of interest, ETBs internalize, route to the cytosol, and permanently inactivate ribosomes through an irreversible enzymatic process. This results in cell death via apoptotic mechanisms. Here we describe the preclinical characterization of a candidate CTLA-4-targeted ETB. The candidate ETB kills gain-of-function model cell lines that express CTLA-4 at levels observed on primary human tumor Tregs. Notably, the potency of the candidate ETB is limited when CTLA-4 is expressed at low levels, thereby allowing a mechanism to spare CD8+ cytotoxic T lymphocytes (CTLs) and effector CD4+ T cells from targeted depletion. The candidate ETB binds CTLA-4 with high affinity utilizing a unique biparatopic binding domain. Using in vitro systems, we show that the candidate ETB can block CTLA-4:B7 interactions and repress Treg-mediated T-cell suppression. In a syngeneic mouse pharmacodynamic model, the candidate ETB depletes tumor-resident Tregs and increases the CD8:Treg ratio in the TME. A toxicological study in naive NHP demonstrate that the candidate ETB, which is cross-reactive to NHP, is well-tolerated at 450 mcg/kg (highest dose tested) administered intravenously once weekly for 4-weeks and does not significantly alter the CTLA-4 low/null peripheral T cell populations. Overall, the candidate ETB is a unique CTLA-4 targeting modality that is designed to deplete Tregs selectively and directly in the tumor, spare CD8+ CTLs, and represents an alternative treatment option to traditional blocking antibodies. Development activities are underway to support the upcoming IND application. Citation Format: Asis Sarkar, Rebecca Martin, Lauren R. Byrne, Caleigh Howard, Swati Khanna, Lilia A. Rabia, Diana Adhikari, Michaela M. Sousares, Alvaro Aldana, Garrett L. Robinson, Jay Zhao, Chris B. Moore, Aimee Iberg. A CTLA-4 targeted ETB for Treg depletion shows favorable preclinical efficacy and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3538.

Targeting PD-L1 has shown clinical efficacy in multiple solid tumor indications. Currently approved PD-L1 targeted approaches rely on monoclonal antibodies which sterically inhibit PD-L1 and prevent PD-1 mediated checkpoint activity. While these molecules have shown great activity in the clinic, the need for pre-existing tumor specific immunity and immune infiltration precludes responses in some patients and leads to resistance in others. Therefore, there remains a need for new modalities and treatment paradigms in these indications. Molecular Templates has developed MT-6402, an engineered toxin body (ETB) targeting PD-L1, as a single agent immunotoxin designed to overcome the challenges of current PD-L1 targeting approaches by 1) directly depleting PD-L1 positive tumor cells or immunosuppressive immune cells displaying PD-L1 in the tumor microenvironment and 2) delivery of an HLA: A*02 restricted viral peptide to alter the tumor immunophenotype for recruitment of CMV-restricted CTLs to target the tumor for depletion (antigen seeding). MT-6402 is slated for clinical development in 2021. Here we describe the preclinical characterization of several ETB candidates derived from MT-6402 delivering antigenic peptides restricted to the most prevalent MHC haplotypes in the U.S. population to broaden the patient population suitable for antigen seeding. ETBs were engineered with the ability to deliver viral peptides across a range of HLA restriction, including HLA: A*01, HLA: A*03, and HLA: A*24. ETBs were screened and benchmarked against MT-6402 and candidates were identified that retain comparable specificity, selectivity, and potency. Alteration of peptide antigen did not change the specificity or selectivity of ETBs which retained similar PD-L1 binding profiles to MT-6402. Binding profiles correlated to targeted potency and ETBs with varied HLA restricted peptides were found to target tumor and immune cells for depletion with similar potency to MT-6402. ETBs delivered an antigen seeding response in a PD-L1 dependent manner and only in conditions in which tumor cell and CTLs shared a matched HLA to the delivered antigenic peptide specificity. Preclinical assessment of the in vivo efficacy and safety profile of candidates is ongoing and further development is slated for 2021. Citation Format: Joseph D. Dekker, Swati Khanna, Elizabeth Saputra, Wenzhao Dong, Lindsey Aschenbach, Lilia A. Rabia, Garrett L. Cornelison, Michaela Sousares, Jay Zhao, Garrett L. Robinson, Betty Chang, Hilario J. Ramos. Engineered toxin bodies targeting PD-L1 to alter tumor immunophenotypes and deliver broad antigenic diversity and patient coverage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1628.

Engineered Toxin Bodies (ETBs) are comprised of a deimmunized Shiga-like toxin subunit A (SLTA) genetically fused to an antibody-like targeting domain. The antibody targeting domain allows for specific targeting of cancer cells while the SLTA component promotes self-internalization of ETBs, an activity that allows for the delivery of an enzymatic and permanent ribosomal destruction against targeted cells even in the context of non-or-poorly internalizing receptor targets. Molecular Templates has developed PD-L1-targeting ETBs as an approach to directly target tumor cells and overcome resistance mechanisms against PD-1 and PD-L1 antibodies. The cytotoxicity delivered by PD-L1-specific ETBs is engineered to be independent of a requirement for tumor infiltrating lymphocytes (TILs), high tumor mutational burden, or modulatory effects of the tumor microenvironment. Further, the activity is not dependent on blockade of the PD-1/PD-L1 checkpoint axis. Thus, PD-L1 targeting ETBs represent a distinct class of therapeutics with direct cell-kill mechanism of action and ability for activity in patients who have progressed on current standard of care or checkpoint therapy. In this study, we highlight the efficacy and safety profile of MT-6020, a human and cynomolgus cross-reactive, PD-L1 targeted, ETB. MT-6020 retains potent catalytic activity and mediates enzymatic destruction of ribosomes at comparable levels to wild-type SLTA in a cell free model. In addition, MT-6020 binds to human NSCLC, Melanoma, and TNBC tumor cell lines with nM affinity and mediates cellular cytotoxicity via ribosomal destruction at low nM to sub-nM potency. MT-6020 binds to cell lines expressing non-human primate (NHP)-PD-L1 and elicits cytotoxic responses comparable to those observed on human tumor target cells. MT-6020 demonstrated pharmacodynamic and pharmacokinetic effects and displayed a favorable tolerability profile in a repeat dose NHP study at doses that are above the presumed therapeutically active concentration. Further our lead PD-L1 ETB, MT-6035, is built upon the MT-6020 scaffold and can deliver a viral peptide for cell surface presentation to and targeting by a specific antiviral CTL population (antigen seeding technology (AST)) for a second and complementary mechanism for tumor cell destruction. MT-6020 and MT-6035 represent a novel approach to targeting and destroying tumors expressing PD-L1 that is unlikely to be inhibited by resistance mechanisms to current checkpoint inhibitors, is well tolerated in relevant toxicity models, and has the capacity for activity in indications where standard of care has failed. Molecular Templates is poised to initiate clinical development of the PD-L1 targeted-ETB (AST), MT-6035, in 2H - 2019. Citation Format: Hilario J. Ramos, Asis K. Sarkar, Sara Le Mar, Brigitte Brieschke, Joseph D. Dekker, Veronica R. Partridge, Pablo A. Maceda, Michaela M. Sousares, Garrett L. Robinson, Aimee Iberg, Shaoyou Chu, Jensing Liu, Jack P. Higgins, Erin K. Willert. The Safety and efficacy profile of a PD-L1 directed, Engineered Toxin Body, as a novel targeted direct-cell kill approach for the treatment of PD-L1 expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3900.