Abstract 3543: Altering tumor immunophenotypes with PD-L1 engineered toxin bodies

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.

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.

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 to specifically kill target expressing cells. Once delivered to appropriate cells, the Shiga toxin A subunit inhibits protein synthesis and promotes apoptosis of tumor cells. Bacterial or plant derived toxin moieties have the potential to induce an immune response, commonly limiting repeat dosing of immunotoxins. Our next-generation ETB scaffold has been modified to overcome this limitation through genetic engineering to systematically and comprehensively reduce B and CD4+T cell epitopes. Molecular Templates has developed a proprietary epitope class switching technology designed to both reduce the anti-drug response and promote the anti-tumor response by replacing naturally occurring CD4+ T cell epitopes with CD8+ T cell epitopes. We have found that a combination of surface remodeling and epitope class switching combined in one protein results in powerful reductions in the anti-drug response against ETBs after repeat administration. This de-immunized next-generation scaffold retains the potency and specificity of the unmodified ETB. Additionally, the engineering of CD8+ T cell epitopes on the ETB scaffold can allow for foreign antigen presentation in complex with MHC class I on the tumor cell surface. This antigen presentation may recruit activated cytotoxic T lymphocytes (CTLs) to the tumor microenvironment thereby adding an additional mechanism of action to the direct cell kill activity of the ETBs. The second generation ETB scaffold has been combined with multiple target binding domains, including scFvs that target CD38, PD-L1 and HER2. CD38 is a validated target for multiple myeloma, and our CD38 targeted ETB has shown potent activity in vitro and in vivo. Pre-clinical studies have shown effective combination of the CD38 targeted ETB with standard of care agents such as IMiDs. Additionally, we have developed a PD-L1 targeted ETB that can be used against various PD-L1 expressing malignancies including Hodgkin's lymphoma and melanoma. HER2 is a well characterized target in breast cancer that persists in many patients after HER2 targeted treatment relapse; a novel MOA to this target may impart additional clinical benefit. The potency, reduced immunogenicity, unique mechanism of action and immune modulating activities of ETBs in this second generation scaffold allows for these agents to fit in well in a refractory setting as well as in combination with other agents in the growing field of immuno-oncology. Citation Format: Sangeetha Rajagopalan, Garrett L. Robinson, Brigitte Brieschke, Jennifer Erdman, Jane Neill, Jack P. Higgins, Erin K. Willert. Next-generation engineered toxin bodies: CD38, PD-L1 and HER2 targeted ETBs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 595.

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

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.

CMV Infection after Transplant from Cord Blood Compared to Other Alternative Donors: The Importance of Donor-Negative CMV Serostatus

Detection of the potentiality before the actuality: Measurement of T-cell proliferation before cell division occurs.

Cytomegalovirus (CMV) infection is known to cause ulceration and mucosal hemorrhage in the gastrointestinal tract. Gastroduodenal and biliary complications were prospectively evaluated in 100 consecutive liver transplant patients in whom CMV was monitored during the first posttransplant year. Gastroduodenal biopsy specimens were taken from 36 patients by endoscopies and in 28 patients by endoscopic retrograde cholangiopancreatography, and bile duct specimens were taken from three patients who underwent surgical reconstruction because of biliary complication. CMV was demonstrated from blood by the pp65 antigenemia test and from frozen sections of tissue specimens by immunohistochemistry and in situ hybridization. Symptomatic CMV infection, treated with ganciclovir, developed in 49 recipients: 13 (100%) of CMV seropositive donor (D+) seronegative recipient (R-) cases, 29 (45%) D+/R+ cases, and 7 (32%) D-/R+ cases. Duodenal ulcer developed in three and hemorrhagic gastritis in three recipients. CMV antigens were found from the gastroduodenal mucosa in 37 (69%) of the 54 studied recipients. The biliary complication rate was 24%. Preceding or concomitant CMV antigenemia was demonstrated in 75% of patients with a biliary complication (68% in CMV D+/R+ or D-/R+ and 100% in D+/R- recipients). The biliary complication rate was higher among recipients with CMV antigenemia, compared with recipients without (P<0.05). CMV antigenemia, CMV infection, or both in the duodenal mucosa was found in 96% of patients with a biliary complication. In two patients who underwent surgical reconstruction, CMV antigens and DNA were demonstrated in the bile ducts. Liver transplant patients are at risk of developing biliary complications after CMV infection, especially those with primary CMV infection.

The Effect of Cytomegalovirus on Pediatric Acute Lymphoblastic Leukemia

Localization of cytomegalovirus antigens in liver allografts over time

Human cytomegalovirus (CMV) infection has been linked to chronic heart disease. The mechanism of CMV dissemination to the heart remains unknown. CMV antigens and nucleic acid sequences have been detected in endothelial cells (ECs) in vivo, and ECs are fully permissive hosts to CMV replication in vitro. This report examines the characteristics of CMV replication in primary cultures of human heart microvascular ECs (HHMECs). Capillary ECs were isolated from heart tissue biopsies of six patients at the time of heart surgery. HHMECs were infected with CMV and viral antigens were detected by immunofluorescence assay using monoclonal antibodies as specific reagents. Cytokine and chemokine release in the supernatant of sham- and CMV-infected cells was quantitated by ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyse expression of mRNA for adhesion molecules. CMV was found to productively infect HHMECs without cytolytic effects. Infected cultures released high levels of pro-inflammatory chemokines and enhanced their adhesion molecule expression. Our data provide new insights into the mechanism of CMV dissemination to the heart, signalling the need for further investigation of the pathogenetic role of this virus in cardiac disorders.

Cell-mediated immunity to murine cytomegalovirus (CMV) was tested by an in vitro lymphocyte proliferation assay. CMV antigen was prepared from infected mouse embryo fibroblasts by ultracentrifugation. Control antigen was prepared in a similar fashion from uninfected fibroblasts. Splenic lymphocytes from CMV-infected mice underwent significant proliferation when incubated with CMV antigen. Lymphocytes from uninfected mice did not proliferate when incubated in vitro with CMV antigen. The proliferation was shown to be specific, since lymphocytes from CMV-infected mice did not proliferate when incubated with control antigen or an indifferent (encephalomyocarditis) viral antigen. Lymphocyte proliferation was directly related to the number of lymphocytes in each culture and to the amount of viral antigen. However, at higher numbers of cells and with large amounts of viral antigen, the backgrounds increased, and the standard errors were larger. The optimal incubation time was eight days. Lymphocyte proliferation reached a maximum 15 days after CMV infection but was still significantly elevated above background 60 days later. This type of lymphocyte proliferation test with human lymphocytes and human CMV antigen can be used to study clinical questions regarding cell-mediated immunity to CMV in immunosuppressed patients, in children with congenital CMV, and in transplant patients after vaccination with attenuated CMV.

P802 Aims: The most common organ specific complication of cytomegalovirus (CMV) infection after liver transplantation is hepatitis. The purpose of the study was to describe retrospectively the detailed virological, histological and immunological observations associated with CMV infection of the liver transplant. Methods: The study included 253 liver transplantations performed on 229 adult recipients during 1991-2000. CMV infection was diagnosed by the frequent monitoring of CMVpp65 antigenemia. CMV antigens were demonstrated by immunohistochemistry and CMV-DNA by hybridization in situ from liver biopsy frozen sections. The histological findings were analyzed according to the Banff criteria. The expression of adhesion molecules (ICAM-1, VCAM-1, ELAM-1), their ligands (LFA-1, VLA-4, sLeX) and lymphoid activation markers (MHC Class II, IL-2-R) were demonstrated by immunohistochemistry. Results: CMV hepatitis was diagnosed in 26 patients (11%). The incidence of CMV hepatitis was higher among seronegative (26%) than in seropositive recipients (9%), but most cases 18/26 (70%) occurred during reactivation. Viral loads were usually high in primary infections (893±1069, range 50-3000, pp65+cells), but varied widely in reactivations (388±740, range 3-3000). CMV antigens were demonstrated in the liver biopsy specimens of all 26 patients and confirmed by a positive finding in CMV-DNA hybridization in situ. CMV antigens were mainly located in the inflammatory cells of the portal infiltrate, but also a few infected hepatocytes were seen. No CMV positive endothelial or bile duct epithelial cells were recorded. The histological Banff-score was slightly increased (2.3±0.9, range 0.5-4.0). Microabcesses, lymphocytic infiltration, ballooning of hepatocytes, slight cholangitis and Kupffer cell reaction were common, but viral inclusions rare. CMV significantly (p<0.05) increased ICAM-1 and VCAM-1 expression and the number of LFA-1, VLA-4 and Class II positive lymphocytes in the graft. CMV hepatitis had no influence on the long-term outcome, but biliary complications were common. Conclusions: CMV hepatitis was not necessarily associated with high viral loads or primary infections. Microabscesses and other histological alterations were common but viral inclusions rare. Increased adhesion molecule expression was associated with lymphocyte infiltration.

Cytomegalovirus (CMV) disease continues to be a major problem for lung transplant recipients. In CMV-seropositive individuals, we detected two types of CMV-specific responses: a self-restricted response stimulated by soluble CMV antigen (sCMV-Ag) and a non-self-restricted response induced by CMV-infected cells (cCMV-Ag). Lung transplant recipients who develop the CMV-specific self-restricted T helper response have a low risk of recurrent CMV disease. In contrast, during CMV disease, lung transplant recipients exhibit only the non-self-restricted T helper responses. We characterized the T cell activation and the kinetics of cytokine production of sorted CD4+ and CD8+ T cells from PBLs of CMV seropositive donors. The two types of CMV antigens induced cytokine production in both T cell subsets. We also performed competitive RT-PCR for Granzyme B (GB) in BAL cells of lung transplant recipients prior to, during and following CMV disease. CMV disease was associated with increase in GB gene expression when was accompanied by acute cellular rejection while it remained low in patients with CMV disease that did not have a complicated course. In summary, CMV-activated T cells within the allograft may produce inflammatory cytokines and effector molecules that may promote allograft rejection.

Cytomegalovirus Infection and Lymphopenia Are Associated with Increased Mortality Post Autologous Stem Cell Transplantation