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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.

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BackgroundTIGIT (T cell immunoreceptor with Ig and ITIM domains) is an exciting novel target for immuno-oncology which functions as an immune checkpoint on multiple immune cell types including memory CD8+, CD4+ Treg, and memory CD4+ cells. TIGIT upregulation on tumor infiltrating lymphocytes (TILs) has been observed in multiple cancer types and contributes to an immunosuppressive tumor microenvironment (TME). Interestingly, TIGIT is commonly co-expressed with PD-1 on Tregs in the TME, tumor antigen specific CD8+ T cells and CD8+ TILs, leading to weakened anti-tumor immune responses.1–2 To date, TIGIT inhibiting monoclonal antibodies (mAb) have shown little activity as a monotherapy in clinical and preclinical studies. 3–4 Therefore, current clinical trials are now focused on combining TIGIT mAbs with known commercial PD-1 or PD-L1 mAbs. A TIGIT-specific engineered toxin body (ETB) represents a wholly new approach to targeting TIGIT expressing cells including those co-expressing TIGIT and PD-1.MethodsETBs targeting TIGIT were designed to deplete TIGIT-expressing TILs, including Tregs, directly in the TME. ETBs are proteins that consist of an antibody fragment genetically fused to a proprietary de-immunized (DI) form of the Shiga-like toxin A subunit (SLTA). These proteins are specific for a cell surface receptor, and function through triggering rapid internalization upon binding, followed by an enzymatic and irreversible termination of ribosomal protein synthesis resulting in cellular apoptosis. Here we provide proof of concept for ETBs as a novel modality for the depletion of TIGIT-expressing immune cells.ResultsTIGIT-targeting ETBs exhibit potent in vitro cytotoxicity of TIGIT over-expressing cell lines (IC50<1nM). These ETBs also lead to apoptotic depletion of ex vivo TIGIT-expressing regulatory T cells (Tregs) from healthy donors. In mixed culture assays, TIGIT ETBs increase the proliferation of TIGIT negative T cells by depleting TIGIT-expressing T cells.ConclusionsStudies to assess pharmacodynamics and efficacy of TIGIT targeting ETBs using a double knock-in (TIGIT and PD-1) mouse tumor model are ongoing, but these early proof of concept in vitro data support the hypothesis that ETBs can deplete TIGIT positive immune cell populations including those co-expressing PD-1. It is possible that targeted TIGIT inhibition through ETB-induced cell death could tip the balance towards tumor regression by eliminating this novel checkpoint (and TIGIT/PD-1 co-expression) at the level of the TME.ReferencesJinhua X, Ji W, Shouliang C, Liangfeng Z. Expression of immune checkpoints in T cells of esophageal cancer patients. Oncotarget 2016;7(39):1–10.Blessin NC, Simon R, Kluth M, Fischer K, et al. Patterns of TIGIT expression in lymphatic tissue, inflammation and cancer. Dis Markers 2019;2019:1–13.Johnston RJ, Comps-Agrar L, Hackney J, Yu X, et al. The immunoreceptor TIGIT regulates anti-tumor and antiviral CD8(+) T effector function. Cancer Cell 2014;26(6):923–927.Bendell JC, Bedrad P, Bang Y-J, LoRusso P, et al. Phase Ia/Ib dose-escalation study of the anti-TIGIT antibody Tiragolumab as a single agent and in combination with atezolizumab in patients with advanced solid tumors. Proceedings: AACR Annual Meeting 2020; April 27–28, 2020 and June 22–24, 2020; Philadelphia, PA.

Targeting of PD-L1 checkpoint has shown clinical efficacy in multiple solid tumor indications. Currently approved PD-L1 targeted approaches rely on the blocking activity of monoclonal antibodies (mAbs) which sterically inhibit PD-L1 thus preventing PD-1 mediated immune checkpoint activity. Although these mAbs have shown activity in the clinic, the need for pre-existing tumor specific immunity and tumor immune infiltration precludes responses in some patients and leads to resistance in others. Therefore, there remains a need for new modalities and treatment paradigms. Molecular Templates has developed MT-6402, an engineered toxin body (ETB) targeting PD-L1 designed to overcome the challenges of current PD-L1 targeting approaches by 1) directly depleting PD-L1 positive tumor or immunosuppressive immune cells displaying PD-L1 and 2) alteration of the tumor immunophenotype through the cytoplasmic delivery of an HLA:A*02 restricted cytomegalovirus (CMV) antigen. Delivery of this antigen is meant to leverage recruitment of existing CMV-restricted cytotoxic T lymphocytes (CTLs) for cell-mediated cytotoxic depletion (antigen seeding technology, AST) of CMV antigen presenting tumor cells. MT-6402 is currently in a phase I open-label, dose escalation and expansion study in subjects with advanced solid cancers that express PD-L1 (NCT04795713). Initial assessment of pharmacodynamic markers for PD-L1 and CMV-mediated ETB activity in a subject with confirmed PD-L1+ tumor cells, CMV positivity, and HLA:A*02 restriction has shown serum phenotypes associated with robust checkpoint inhibitor activity and full extravasation of circulating CMV-specific T cells. In addition to assessing early clinical data from the HLA: A*02 restricted MT-6402 trials, expansion of ETB AST to a broader patient population requires testing of ETBs engineered with the ability to deliver CMV antigens across a broad range of HLA restriction, including HLA:A*01, HLA:A*03, and HLA:A*24. To this end, ETBs were benchmarked against MT-6402 to identify candidates that retain comparable specificity, selectivity, and activity. Substitution of peptide antigens did not alter specificity or selectivity of ETBs compared to MT-6402. Candidate ETB binding profiles and potency were comparable to MT-6402. ETBs delivered an antigen seeding response in a PD-L1 dependent and HLA specific manner. In vivo efficacy of candidate ETBs was comparable in a murine efficacy xenograft model with MT-6402. An ex vivo cytokine release assay in a co-culture setting using HLA matched CTLs and PD-L1+ target cells, treatment with AST capable ETBs resulted in secretion of antigen specific T cell mediated immune cytokines compared to AST null controls. These cytokines overlap with cytokine signatures observed after dosing MT-6402 in HLA:A*02 patients. Preclinical assessment of the safety profile of candidates is ongoing and further development is slated for 2021. Citation Format: Swati Khanna, Elizabeth M. Kapeel, Lauren R. Byrne, Elizabeth Saputra, Steven Rivera, Lindsey Aschenbach, Lilia A. Rabia, Garrett L. Cornelison, Rachael M. Orlandella, Brigitte Brieschke, Michaela Sousares, Jay Zhao, Garrett L. Robinson, Chris Moore, Joseph D. Dekker. Altering tumor immunophenotypes with PD-L1 engineered toxin bodies [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 3543.