Molecular Templates (United States)

Abstract Immunotoxins (the fusion of a bacterial cytotoxic protein with a targeting moiety) have been explored since the 1970s but have been limited in their therapeutic utility because of the propensity to drive a host innate immune response resulting in potentially fatal cytokine elevations and capillary leak syndrome (CLS). Engineered Toxin Bodies (ETBs) represent an important evolution in immunotoxins, overcoming the issues with innate immunity and adding biologic utility not previously seen. ETBs use an antibody-based targeting domain genetically fused to a de-immunized form of the Shiga-like toxin A subunit (SLTA). Like previous immunotoxins, ETBs bind and induce cell-death in a target-specific manner but have additional biology not inherent to other immunotoxins. ETBs can induce their own internalization, allowing for the targeting of poorly/non internalizing receptors. ETBs can deliver other payloads like viral class I antigen or small molecules in addition to SLTA, allowing for multiple MOAs. And, most importantly, we have de-immunized SLTA through genetic engineering to create ETBs devoid of innate immune effects/CLS seen with previous immunotoxins. Three ETBs (MT-0169, MT-5111, and MT-6402) are currently in clinical studies across different targets (CD38, HER2, PD-L1) and across hematologic malignancies, solid tumor, and immuno-oncology indications. To date, no clinical manifestations of CLS have been observed in 80+ patients; in contrast, the rate of CLS for approved immunotoxins ranges for 33% to >50%. In general, all three ETBs have shown good tolerability with little off-target toxicity and all three ETBs have shown monotherapy clinical benefit in heavily pre-treated patients. MT-6402, an ETB targeting PD-L1, is the first ETB to carry the additional mechanism of antigen seeding. Along with its ability to destroy ribosome in a PD-L1-targeted fashion, MT-6402 can deliver a class I antigen derived from human cytomegalovirus (CMV) to the ER, allowing for presentation of the antigen in complex with MHC class I on the cell surface of a tumor cell. Early clinical data demonstrate antigen seeding with MT-6402 can result in T-cells specific to the seeded CMV antigen mounting an immune response to the tumor. ETBs represent a unique and wholly distinct scaffold for drug development in oncology. ETBs allow for the targeting of non-internalizing receptors that are not amenable to ADCs. ETBs have a mechanism of cell-kill (enzymatic and irreversible ribosomal destruction) that is distinct from any approved agent in oncology. ETBs can also deliver additional payloads to drive unique biology like the alteration of tumor immunophenotype. Here we describe three active clinical stage programs with encouraging safety and efficacy data that represent a transformation of the immunotoxin landscape into a more viable therapeutic approach to target validated as well as typically intractable clinical cancer targets. Citation Format: Chris Moore, Lee Robinson, Garrett Cornelison, Joseph Dekker, Roger Waltzman, John Majercak, Joseph Phillips, Jay Zhao, Jason Kim, Eric Poma. Engineered Toxin Bodies (ETBs): Clinical stage immunotoxins with a safer and differentiated profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2661.

BackgroundTIGIT (T cell immunoreceptor with Ig and ITIM domains) is an immune inhibitory receptor, which is known to be over-expressed on Tregs in the tumor microenvironment (TME) of multiple solid tumors and functions as an immunological checkpoint. TIGIT is often co-expressed with PD-1 on Tregs and CD4+ and CD8+ T cells in the TME.1-3 Monoclonal antibodies (mAb) blocking TIGIT have shown little efficacy as a monotherapy or in combination with αPD-L1 mAb in clinical studies.4-5 We have shown previously that TIGIT targeting Engineered Toxin Bodies (ETBs) show cytotoxicity in vitro on TIGIT over-expressing cell lines and TIGIT expressing immune cells, including Tregs.6 Here, we demonstrate the efficacy and pharmacodynamic (PD) effects of TIGIT ETB in a hTIGIT/PD-1 expressing humanized mouse model. Contrary to mAbs, which function by steric hinderance of the TIGIT-CD155 axis, TIGIT ETBs function by direct cell kill of TIGIT expressing cells and represent a novel approach to targeting TIGIT expressing cells in cancers.MethodsMC38 tumor bearing humanized mice (hTIGIT/PD-1) were treated with TIGIT ETB as a monotherapy and in combination with αPD-1 mAb. The effect of TIGIT ETB was compared with αPD-1 and αTIGIT mAbs used alone or in combination. Tumor volumes were measured during the study and all tumors were harvested at study conclusion on day 18 for evaluation of test article effects on tumor immunophenotype.ResultsTIGIT ETB monotherapy resulted in the best overall reduction in tumor burden and was comparable with the αPD-1 mAb group. Strikingly, TIGIT ETB monotherapy worked better than the αTIGIT+ αPD-1 mAb combination. TIGIT ETB+ αPD-1 mAb showed similar effects on tumor growth as the αTIGIT+ αPD-1 mAb combination. These effects on tumor growth coincided with significant reduction in Tregs and increased CD8:CD4 ratio in the TME across all TIGIT ETB treatment groups. This TME Treg depletion was not observed with αTIGIT+αPD-1 mAb combination.ConclusionsThese data demonstrate that targeting TIGIT using our Engineered Toxin Body platform promotes tumor regression through elimination of TIGIT/PD-1 co-expressing immune cells within the TME. Our data supports using ETB as a monotherapy to target TIGIT and represents a wholly novel approach for modulating TIGIT within 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: 1–13Johnston 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.Roche News Release. Roche reports interim results for the phase III SKYSCRAPER-01 study in PD-L1-high metastatic non–small cell lung cancer. News Release. May 11, 2022. https://bit.ly/39e8QY4.Saputra E, Cornelison G, Mitchell J, et al. 884 Engineered toxin body mediated depletion of TIGIT expressing immune cells for cancer immunotherapy. Journal for ImmunoTherapy of Cancer 2021;9: doi: 10.1136/jitc-2021-SITC2021.884.Ethics ApprovalBiocytogen Inc. IACUC #2018N001

Abstract Engineered toxin bodies (ETBs) are next-generation immunotoxins that harbor an antibody-derived targeting domain and a cytotoxic payload derived from Shiga-like toxin-1 catalytic subunit A (SLTA). SLTA has been engineered to reduce innate immunogenicity and therefore increase the safety of ETBs whilst retaining potent cytotoxic properties. When targeted via a binding domain, SLTA is internalized and routes to the cytosol leading to irreversible ribosome inactivation and ultimately cell death. ETBs have been developed to treat a wide variety of cancers, including breast, lymphoma, multiple myeloma and PD-L1 positive solid tumors. This technology holds promise for non-oncology indications as well, particularly as a targeted and non-genotoxic conditioning regimen for hematopoietic stem cell (HSC) ablation to prepare patients for autologous stem cell transplant. c-KIT (CD117) is a well-known marker of hematopoietic stem and progenitor cells and is overexpressed in a high percentage of certain cancers including GIST, SCLC and AML. While tyrosine kinase inhibitors such as imatinib are effective therapies for c-KIT mutant GIST, resistance often occurs by the development of secondary mutations in the intracellular signaling domains. Thus, c-KIT represents a potential ETB target for both oncological and HSC transplant conditioning indications. Here we present data highlighting the in vitro potency and efficacy of CD117-targeting ETBs on cancer cell lines as well as on primary human CD34+ HSCs. CD117-targeted ETBs demonstrate exquisite specificity in vitro by killing only target positive CD34+ cells at picomolar potency while sparing the progenitors that lack CD117 expression. However, relatively low CD117 receptor levels on CD34+ HSCs prevent complete killing in vitro, limiting the observed efficacy. To overcome this challenge, an additional cytotoxic payload was conjugated to the ETB molecule to capitalize on ETBs’ unique internalization and routing properties to deliver a secondary mechanism of action. ETB-drug conjugates (ETB-DCs) exhibited improved cytotoxicity in vitro, especially in CD117-low target cells. Moving forward, we plan to explore both the ETB and the ETB-DC therapeutic index in vivo with a series of CD117+ tumor efficacy and HSC depletion models. Citation Format: Caleigh Howard, Shu Wiley, Wenzhao Dong, Andrea Mendiola, Veronica Partridge, Sara LeMar, Paul Amador, Amit K. Chaudhary, Joseph D. Dekker, Jay Zhao, Ross Durland, Aimee Iberg. c-KIT targeted ETBs for cancer therapy and HSC transplant conditioning [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 335.

Abstract This is the first-in-human study of MT-6402, a unique, first-in-class potent PD-L1-targeted engineered toxin body (ETB) capable of direct killing of PD-L1 expressing cells via rapid PD-L1-mediated internalization of a fused Shiga-like toxin A subunit (SLTA) resulting in permanent ribosomal inactivation. MT-6402 also delivers an HLA-A*02 restricted pp65 cytomegalovirus (CMV) antigen into PD-L1 expressing tumor cells leading to MHC-I presentation to existing CMV-specific cytotoxic T cells (antigen seeding). MT-6402 functions by targeting tumor and inhibitory immune cells directly and altering tumor immunophenotype to re-direct antiviral cytotoxic T cells into the tumor microenvironment. MT-6402 shows picomolar cytotoxic activity across several PD-L1 expressing cancer cell lines and treatment of human PBMCs results in selective depletion of PD-L1 positive cells. MT-6402 was well tolerated in non-human primates at doses above those which induce pharmacodynamic (PD) effect (reduction of CD14+ monocytes) in humans. This first-in-human study in patients with PD-L1-expressing advanced solid tumors will employ a modified toxicity probability interval design to determine MTD and will then enroll additional subjects at the MTD, to further explore safety and efficacy and determine RP2D. Results of the first dose cohort (16 micrograms/kg) are presented. Six patients received MT-6402. A significant and sustained reduction in CD14+ monocytes starting in cycle 2 was observed in patients on therapy beyond one cycle and was maintained with each MT-6402 administration, indicating HLA-independent PD effect consistent with preclinical observations. One HLA-A*02 and CMV+ patient with osseous metastases from NSCLC showed marked CMV-specific T-cell extravasation at day 8 and serum cytokine signatures consistent with antigen dependent responses and T cell mobilizations, suggesting engagement of MT-6402 antigen seeding. This patient has reduced intensity of metastatic bone lesions with 3/4 lesions resolving; the remaining lesion showing reduced uptake. Two patients had cytokine elevations at day 15, manifested by transient (1-12h), grade 2 infusion-related reactions and grade 2 cytokine release syndrome, which were subsequently prevented by steroid premedication. These results describe a novel approach to checkpoint modulation, leveraging direct PD-L1 cell kill and antigen seeding technology by the ETB. The results support further dose escalation and hold promise for development of MT-6402 for solid tumors, including in the R/R setting. Citation Format: David R. Spigel, Eugene Ahn, Herbert L. Duvivier, Drew Rasco, Agnes Rethy, Chris Moore, Amy Yuet, Sandra Hankins, Swati Khanna, Joseph Dekker, Brian Van Tine. Phase I study of MT-6402, a novel engineered toxin body (ETB) targeting PD-L1, in patients with PD-L1 expressing advanced solid tumors [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 CT152.

Abstract 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.

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.

TPS8074 Background: 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 forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. MT-3724 represents a novel ETB modality comprised of an anti-CD20 single-chain variable fragment genetically fused to SLT-A. It is capable of efficient internalization once bound to CD20 and can induce potent direct cell-kill via enzymatic ribosome inactivation. MT-3724 is currently being studied in three ongoing Phase 2 studies for relapsed or refractory diffuse large B-cell lymphoma (r/rDLBCL). Methods: The primary objective of this single-arm, Phase 2 study (NCT02361346) is to determine the efficacy of MT-3724 monotherapy in r/rDLBCL based on overall response rate (ORR), defined as the proportion of subjects with a complete/partial response according to the Lugano criteria, as assessed by independent, central review. Key secondary objectives include safety, progression-free survival, investigator‐assessed ORR, duration of response, overall survival, and pharmacodynamics. Adverse events will be assessed and documented according to Common Terminology Criteria for Adverse Events version 5.0. Key eligibility criteria include adult subjects with histologically confirmed, r/rDLBCL, with ≥2 prior standard of care systemic NHL treatment regimens, and ≥1 measurable lesion. As rituximab and other CD20-targeting antibodies compete with MT-3724 for the same CD20 domain, minimum washout periods from these agents must be observed. Subjects remain eligible post stem cell transplant or chimeric antigen receptor T-cell therapy. Subjects will receive 50 µg/kg MT-3724 IV over 1 hour on Days 1, 3, 5, 8, 10 and 12 of a 21-day treatment cycle. The anticipated sample size is N = 100. Interim analyses will be performed to confirm minimum efficacy thresholds based on the encouraging data observed in the completed phase 1 portion of the study [Hamlin et al. Blood 2019;134(Suppl 1):4098]. Multiple global sites are enrolling subjects. Clinical trial information: NCT02361346 .

Abstract Molecular Templates is developing engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit (SLTA). ETBs can induce their own internalization, route through the cell in a predictable manner, enzymatically and irreversibly destroy ribosomes to shutdown protein synthesis and induce apoptosis of tumor cells. This mechanism of action is distinct from that of other therapeutics, making ETBs an attractive treatment for patients who have become resistant to chemotherapy and other treatments. Safety and efficacy in refractory non-Hodgkin’s lymphoma patients has been observed in a phase I study with Molecular Template’s first-generation CD20-targeting compound, MT-3724. CD38 is a surface receptor that is highly expressed on malignant plasma cells. CD38 is a clinically validated target of monoclonal antibodies for treatment of multiple myeloma and is known to persist after failure of antibody treatment. MT-4019 is a CD38-targeted next-generation ETB, utilizing a modified SLTA. The SLTA subunit of the next-generation ETB scaffold has been modified through proprietary genetic engineering to systematically and comprehensively reduce B cell and CD4+T cell epitopes, as well as to dampen the innate response by decreasing binding to TLR-4. This de-immunized next-generation scaffold retains the potency and specificity of an ETB containing an unmodified SLTA. Pre-clinical studies in rodents and non-human primate models have demonstrated the tolerability of MT-4019, along with a decreased anti-drug antibody response. Additionally, in the non-human primate model, MT-4019 showed reduced neutrophil and monocyte activation as compared to an ETB with an unmodified SLTA subunit, indicating that SLTA modification also exhibits a diminished innate immune response. Molecular Template’s ETB technology has resulted in potent, targeted therapeutic agents that have a unique mechanism of action in the field of oncology. MT-3724, a first-generation CD20-targeted ETB, has shown promising clinical results in the refractory setting for non-Hodgkin’s lymphoma. The next-generation scaffold, as exemplified in the CD38-targeted MT-4019, retains potent and specific direct cell kill activities, and additionally reduces the innate and adaptive immune response to the therapeutic. MT-4019 is a promising lead and is under further development to enable clinical studies in multiple myeloma. Citation Format: Garrett L. Robinson, Sangeetha Rajagopalan, Brigitte Brieschke, Jane Neill, Jennifer Erdman, Rodney Flores, Jensing Liu, Jack P. Higgins, Erin K. Willert. MT-4019: a de-immunized engineered toxin body targeting CD38 for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2659. doi:10.1158/1538-7445.AM2017-2659