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Engineered Toxin Bodies Specific for TROP2 Positive Cancers Authors: Garrett L. Cornelison, Ileana Pedraza, Kendra Garrison, Elizabeth M. Kapeel, Channing Pletka, Abdul Khan, Jessica Momb, Rebecca Martin, Adam Bartos, Joseph D. Dekker, Jay Zhao, John Majercak, Garrett L. Robinson Molecular Templates, Austin, TX Lanier Biotherapeutics, Bogart, GA Molecular Templates produces next generation immunotoxins called Engineered toxin bodies (ETBs). ETBs are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forced internalization, undergoing retrograde translocation to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes resulting in the inhibition of protein synthesis and induction of apoptosis. In addition, Molecular Templates has expanded the ETB platform to include Antigen Seeding Technology (AST) to generate ETBs with the ability to deliver foreign protein antigen to targeted populations of tumor cells. This mechanism of action allows for the intracellular processing of antigen and subsequent surface MHC-I presentation required for activation of a re-directed T lymphocyte response and the capacity to restore a functional immune clearance program against the tumor. Three ETBs are in clinical development (MT-5111 targeting HER2, MT-0169 targeting CD38, and AST enabled MT-6402 targeting PD-L1). The novel mechanisms of action have potential benefit in different indications including in the relapsed setting, when disease has progressed after chemotherapies and other targeted therapies, and additionally may be able to combine with standard of care. ETBs are being developed that target other cell surface receptors expressed on solid tumors including tumor-associated calcium signal transducer 2 (TROP2). TROP2 is a clinically validated target in metastatic triple-negative breast cancer (mTNBC) and other cancers such as metastatic urothelial carcinoma (mUC) using antibody drug conjugate (ADC) therapies such as sacituzumab govitecan (Trodelvy®). In vitro, ETBs targeting TROP2 specifically and directly kill tumor cells expressing TROP2 with picomolar activity. ETBs can bind to TROP2 in the presence of the Trodelvy parent monoclonal antibody, sacituzumab, and ETBs retain potency on TROP2 positive cell lines in the presence of clinically relevant concentrations of sacituzumab. AST enabled Trop2 targeted ETBs retain direct cell killing activity and can deliver multiple viral antigens to induce cytokine secretion and T-cell mediated killing in a co-culture assay of TROP2 target cells with antigen matched HLA type and antigen specific T-cells. In vivo, TROP2 targeted ETBs demonstrate good tolerability in a murine HCC1806 triple-negative breast cancer xenograft model and significantly reduce tumor burden relative to vehicle control. These pre-clinical in vitro and in vivo data suggest AST enabled Trop2 targeted ETBs have the potential to deplete Trop2 positive malignancies through multiple unique mechanisms of action. Citation Format: Garrett L. Cornelison, Ileana Pedraza, Kendra Garrison, Elizabeth M. Kapeel, Channing Pletka, Abdul Khan, Jessica Momb, Rebecca Martin, Adam Bartos, Joseph D. Dekker, Jay Zhao, John Majercak, Garrett L. Robinson. Engineered Toxin Bodies Specific for TROP2 Positive Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-18.

Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forced internalization, undergoing retrograde translocation to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes resulting in the inhibition of protein synthesis and induction of apoptosis through ribotoxic stress mechanisms. Additionally, Molecular Templates has developed the Antigen Seeding Technology (AST) platform to generate ETBs with the unique ability to deliver foreign protein antigen to targeted populations of tumor cells. This MOA allows for the intracellular processing of antigen and subsequent surface MHC-I presentation required for activation of a re-directed T lymphocyte response and the capacity to restore a functional immune clearance program against the tumor. Three ETBs are in clinical development (MT-5111 targeting HER2, MT-0169 targeting CD38, and AST enabled MT-6402 targeting PD-L1). The novel mechanisms of action have potential benefit in different indications including in the relapsed setting, when disease has progressed after chemotherapies and other targeted therapies, and additionally may be able to combine with standard of care. ETBs are being developed that target other cell surface receptors expressed on solid tumors including tumor-associated calcium signal transducer 2 (Trop2). Trop2 is a clinically validated target of antibody drug conjugate (ADC) therapy in metastatic triple-negative breast cancer (mTNBC) and other cancers such as metastatic urothelial carcinoma (mUC). In vitro, tumor cells expressing Trop2 are effectively, specifically, and directly killed with picomolar activity by targeted ETBs. AST enabled Trop2 targeted ETBs are capable of delivering viral antigens for multiple HLA types and inducing cytokine secretion and T-cell mediated killing in a co-culture assay of Trop2 target cells with antigen matched HLA type and antigen specific T-cells. These pre-clinical in vitro data suggest AST enabled Trop2 targeted ETBs have the potential to deplete Trop2 positive malignancies through multiple unique mechanisms of action. Citation Format: Garrett L. Cornelison, Adam Bartos, Brigitte Brieschke, Jessica Momb, Ileana Pedraza, Elizabeth M. Kapeel, Rebecca Martin, Channing Pletka, Adrian Gonzalez, Joseph D. Dekker, Jay Zhao, John Majercak, Garrett L. Robinson. Engineered toxin bodies (ETBs) targeting Trop2 [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 326.

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.

Engineered Toxin Bodies (ETBs) represent a unique therapeutic strategy for fighting cancer by targeting and selectively destroying cancer cells or cancer-promoting immune cells. Featuring an antibody-based targeting domain fused with the cytotoxic, ribosome inactivating Shiga-like toxin A subunit (SLTA), ETBs enable killing of target expressing cells. Although immunotoxins have been explored for decades as promising cancer therapeutics, activation of innate immunity limited this approach. MTEM has developed 1st, 2nd, and 3rd generation ETBs with iterative improvements to reduce immunogenicity and add mechanisms of action. Here, we describe the development of an ex vivo cytokine release assay used to enhance the safety, tolerance, and therapeutic index (TI) of ETBs as cancer therapeutics. MT-3724, a 1st gen CD20 targeting ETB comprising wild type SLTA (WT SLTA) genetically fused to a CD20 targeting scFv, displayed efficacy as monotherapy in relapsed/refractory non-Hodgkin lymphoma. However, a subset of MT-3724-treated patients experienced capillary leak syndrome (CLS), likely driven by innate immune recognition of specific components of WT SLTA. Adverse events were also associated with a particular lot of MT-3724 that contained a higher proportion of aggregated species. Therefore, next gen ETBs were de-immunized by replacing surface exposed residues of WT SLTA while retaining cytotoxicity and were designed to have reduced propensity for aggregation. An ex vivo cytokine release assay, developed to triage candidate ETBs that generate innate immune activation, was validated by probing WT-SLTA and aggregated MT-3724. Cancer free human peripheral blood mononuclear cells (PBMCs) or PBMCs depleted of B cells were treated with positive and negative controls, non-targeted SLTAs, and ETBs. Cytokine concentrations were quantified by Luminex. The positive control TLR4 agonist lipopolysaccharide (LPS) activated robust release of proinflammatory cytokines including IL-1β, TNFα, and IL-6, as well as CCL3, CCL4, GM-CSF, IL-10, IFNγ, and Granzyme B. Non-targeted WT SLTA displayed a similar, but not identical, pattern of cytokine release. Conversely, the de-immunized (DI) SLTA did not activate cytokine or chemokine release, remaining at levels equal to PBS, indicating that scrubbing WT SLTA of immunogenic components can improve the safety profile and TI of our 2nd gen ETBs. Aggregated MT-3724 induced moderate increases in TNFα, IL-6, CCL3, and CCL4 relative to a non-aggregated MT-3724 lot. Removal of B cells from PBMCs did not alter cytokine release patterns of LPS, WT SLTA, or aggregated MT-3724. These data suggest that cytokines were released in response to WT SLTA and protein aggregates in an off-target manner. This assay will be used to evaluate ETB safety by testing candidate ETBs for likelihood of cytokine release and/or innate immune activation prior to selection as candidates for clinical trials. Citation Format: Rachael M. Orlandella, Elizabeth M. Kapeel, Brigitte Brieschke, Garrett L. Robinson, Joseph D. Dekker, Chris B. Moore. Improving immunotoxin-based therapeutics for cancer with de-immunized 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 2579.