Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen-binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen-binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra-articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL-1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL-1 receptor. In vitro, IL-1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra-articular IL-1-induced IL-6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL-1Ra_M26 and native IL-1Ra inhibited IL-6 output when co-administered with the IL-1 challenge, only IL-1Ra_M26 inhibited when administered 1 week prior to IL-1 challenge. Collagen-binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238-1247, 2018.
BackgroundCD3-targeted T cell engagers are potent anti-tumor therapies, but their development often requires management of cytokine release syndrome (CRS). Subcutaneous dosing is a promising strategy to reduce CRS, but its application is limited by its increased immunogenicity risks. Subcutaneous dosing is hypothesized to mitigate CRS by reducing the maximum drug concentration (Cmax) and preserve efficacy by maintaining the same minimum drug concentration (Cmin) as intravenous dosing. A T cell engager designed to be dosed intravenously but engineered to mimic the PK properties of subcutaneous dosing could alleviate CRS without increasing immunogenicity.MethodsTriTAC-XR molecules are engineered T cell engager prodrugs that become slowly activated in systemic circulation. This extended-release mechanism results in a slow build-up of circulating active drug, similar to subcutaneous dosing, and extends drug exposure to enable longer dosing intervals. The prodrug was engineered by adding a peptide mask and protease-cleavable linker to the N-terminus of a TriTAC, a constitutively active and half-life extended T cell engager. The mask binds to the anti-CD3ε domain and prevents T cell binding. Upon cleavage by systemic proteases, active T cell engager is released. Binding was assessed using ELISA on recombinant CD3ε protein and using flow cytometry on primary T cells. T cell engager function was assessed using T cell-dependent cellular cytotoxicity (TDCC) assays with resting human T cells. Safety and efficacy were modeled in non-human primates.ResultsTriTAC-XR had markedly reduced binding to recombinant CD3ε protein and to primary T cells as well as reduced potency in functional TDCC assays compared to its unmasked active drug. In cynomolgus monkeys, TriTAC-XR had significantly attenuated cytokine production while maintaining comparable pharmacodynamic effects as a non-masked active drug. The ratio of Cmax to Cmin for the active TriTAC-XR was significantly smaller than a non-masked control.ConclusionsTriTAC-XR is activated in a time released manner by systemic proteases to minimize differences between the Cmax and Cmin of systemic active drug. This mechanism is different from other protease-activated T cell engager prodrugs that are only activated by tumor-associated proteases. Compared to canonical T cell engagers, TriTAC-XR is expected to improve safety by reducing CRS and to provide convenience by extending dosing intervals.
BackgroundEpithelial cell adhesion molecule (EpCAM) is highly expressed in many solid tumors. However, therapeutics targeting EpCAM have had limited clinical utility or failed in clinical development likely due to the expression of EpCAM in normal tissues. For example, clinical testing of solitomab, an EpCAM-targeting T cell engager, resulted in severe dose-limiting toxicities, including elevated liver transaminases, hyperbilirubinemia, and diarrhea. Designing an EpCAM-targeting T cell engager that is only active in the tumor would expand its therapeutic window and improve its safety profile.MethodsUsing a T cell engager prodrug platform named ProTriTAC that couples therapeutic half-life extension with functional masking, we have engineered HPN601, a protease-activated EpCAM-targeting T cell engager. HPN601 is a single polypeptide with three binding domains: anti-albumin for half-life extension, anti-CD3e for T cell engagement, and anti-EpCAM for tumor cell engagement. The anti-albumin domain contains a masking moiety and a protease-cleavable linker and keeps the molecule inert outside the tumor microenvironment. Activation by tumor-associated proteases removes the anti-albumin domain along with the masking moiety to reveal a potently active drug inside the tumor. This active drug has minimal activity outside of tumor because, without an albumin binding domain, it is rapidly cleared in circulation.ResultsA humanized rodent tumor model was used to simultaneously measure anti-tumor efficacy and clinically relevant toxicity endpoints. In this model, a surrogate molecule of HPN601 was safely administered at a dose ten-fold higher than the minimal efficacious dose required for durable tumor regression. Higher doses produced toxicities including elevated ALT/AST and bilirubin, body weight loss, and evidence of tissue damage by histopathology. In contrast, a constitutively active EpCAM-targeting T cell engager could only be dosed safely up to its minimal efficacious dose. The improved safety profile of HPN601 is further supported by a toxicokinetic study in non-human primates: compared to a constitutively active EpCAM-targeting T cell engager, HPN601 had significantly attenuated cytokine production, including IFN-g, IL-2, IL-6, and IL-10.ConclusionsHPN601 is a conditionally active EpCAM-targeting T cell engager with a ten-fold improved therapeutic window compared to a constitutively active EpCAM-targeting T cell engager. An EpCAM-specific T cell engager with an improved safety profile could address unmet needs in many solid tumors and demonstrate the feasibility of using conditionally active T cell engagers to target more solid tumor antigens.Ethics ApprovalThe study was reviewed and approved by Harpoon’s Institutional Animal Care and Use Committee.
CD3-targeted T cell engagers are potent anti-tumor therapies, but their development often requires management of cytokine release syndrome (CRS). One strategy to reduce CRS is subcutaneous dosing, which is hypothesized to mitigate CRS by reducing the maximum drug concentration (Cmax) and preserve efficacy by maintaining the same minimum drug concentration (Cmin) as intravenous dosing. Although promising for mitigating CRS, this approach is limited by its increased immunogenicity risks. A T cell engager designed to be dosed intravenously but engineered to mimic the PK properties of subcutaneous dosing could alleviate CRS without increasing immunogenicity. Here we describe TriTAC-XR, a platform of T cell engager prodrugs designed to become slowly activated in systemic circulation. This extended-release mechanism results in a slow build-up of circulating active drug and minimizes the Cmax/Cmin ratio, similar to subcutaneous dosing. TriTAC-XR prodrugs were engineered by adding a peptide mask and protease-cleavable linker to the N-terminus of a TriTAC, a constitutively active and half-life extended T cell engager. The mask binds to the anti-CD3ε domain and inhibits T cell binding. Upon cleavage by systemic proteases, active T cell engager is released. In vitro, TriTAC-XR has markedly reduced binding to recombinant CD3ε protein and to primary T cells as well as reduced potency in functional T cell-dependent cellular cytotoxicity (TDCC) assays compared to its unmasked active drug. In cynomolgus monkeys, TriTAC-XRs targeting multiple tumor antigens resulted in a gradual build-up of active drug during the first week post dose and significantly lower Cmax/Cmin ratios than comparable constitutively active TriTACs. Modeling based on these PK data predicts that TriTAC-XR dosed intravenously will result in a slower build-up of active drug and smaller Cmax/Cmin ratios than TriTAC dosed intravenously or subcutaneously. Cytokine release and target cell depletion in cynomolgus monkeys were used to compare the therapeutic index of TriTAC-XR to TriTAC. A single dose of FLT3-targeting TriTAC-XR resulted in 100-fold protection in cytokine release and similar FLT3 expressing cell depletion when compared to an equivalent FLT3-targeting TriTAC. Similarly, repeat dosing of a TriTAC-XR targeting B cells resulted in complete B cell depletion with substantially lower cytokines than a comparable TriTAC. TriTAC-XR is expected to improve the safety of T cell engagers by reducing CRS and may increase clinical dosing convenience by enabling higher doses that will extend dosing intervals. Citation Format: Kathryn Strobel Kwant, Sony S. Rocha, Timothy Yu, Katrina Stephenson, Raphaela Rose Banzon, Sydney Vollhardt, Golzar Hemmati, Evan Callihan, Wade H. Aaron, Subramanian Thothathri, Jessica O'Rear, Eric Bragg, Willis Kwong, Hubert Situ, Avneel Hundal, Stephen Yu, Taggra Jackson, Kevin Carlin, Yinghua Xiao, Maria Rosalyn Dayao, Linh To, Nick Bergo, Kevin Wright, Richard Austin, Holger Wesche, Bryan Lemon, S. Jack Lin. TriTAC-XR: An extended-release T cell engager platform designed to minimize cytokine release syndrome [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 2861.
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