Preclinical and Nonclinical Characterization of HPN217: A Tri-Specific T Cell Activating Construct (TriTAC) Targeting B Cell Maturation Antigen (BCMA) for the Treatment of Multiple Myeloma
Authors: Law Che-Leung , Aaron Wade , Austin Rick , Barath Manasi , Callihan Evan , Evans Thomas , Gamez Guerrero Maria , Hemmati Golzar , Jones Adrie , Kwant Kathryn , Lao Llewelyn , Lemon Bryan , Moldt Katrine , Patnaik Purbasa , Sexton Kenneth , Wesche Holger , Xiao Yinghua , Yu Stephen , Yu Tim 
Published: October 2019

Abstract
About 31,000 new cases of multiple myeloma (MM) will be diagnosed in the US in 2018. In addition to chemotherapeutic agents, several targeted therapies utilizing distinct mechanisms of action, e.g., proteasome inhibitors (bortezomib, carfilzomib, ixazomib), HDAC inhibitors (panobinostat), Cullin-RING E3 ubiquitin ligase activators (thalidomide, lenalidomide, pomalidomide), and antibodies (daratumumab, elotuzumab) have become available for treating MM. However, MM remains an incurable disease. Patients who relapse after or are refractory to standard of care treatments generally have poor prognosis. In 2018, close to 13,000 patients will die of the disease in the US. Targeting the B cell maturation antigen (BCMA), a BAFF/BLyS and APRIL receptor, for treating MM patients can provide a new treatment approach complementary to existing therapies. CAR-T therapies and an antibody-drug conjugate targeting BCMA have demonstrated early clinical success in the treatment of relapsed refractory MM (RRMM).
HPN217 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: an N-terminal single domain antibody (sdAb) that binds to human BCMA, a middle sdAb that binds to human serum albumin (HSA), and a C-terminal single chain Fv (scFv) that binds to CD3ε of the T cell receptor (TCR) complex. HPN217 is a highly stable single polypeptide of ~ 53 kDa expressed by CHO cells. Simultaneous engagement of BCMA on a target MM cell and CD3 on a T cell results in T cell activation, functional differentiation and the eventual lysis of the target MM cell. Engineering of an HSA binding domain into HPN217 represents a unique strategy in extending serum half-life, giving the TriTAC molecule a small molecular size and flexibility. This approach is different from Fc-engineering applied in other CD3-based bispecific T cell engaging molecules.
The KD of HPN217 binding to recombinant human BCMA, HSA, and recombinant human CD3ε was determined to be 5.5 nM, 6 nM, and 17 nM, respectively, as measured by biolayer interferometry. Flow cytometric analysis on a panel of T cells from normal donors and BCMA positive and BCMA negative tumor cell lines confirmed binding of HPN217 to its native targets expressed on cell surface. The in vitro pharmacological activity of HPN217 was evaluated by T cell-dependent cellular cytotoxicity (TDCC) assays. In co-cultures of T cells from normal human or cynomolgus monkey donors, target tumor cells, and HSA, HPN217 mediated dose-dependent and BCMA-dependent cytotoxicity with EC50 values ranging from 0.05 to 0.7 nM. Killing was dependent on expression of BCMA on target tumor cells. Concomitant with target tumor cell killing, HPN217 also mediated dose-dependent upregulation of CD25 and CD69 on T cells in the TDCC co-cultures when BCMA positive tumor cells were presence. Consistent with the mechanism of action of CD3-based T cell engaging molecules, T cell derived cytokines, e.g., TNFα and IFNγ, were detected. Similar T cell activation could be observed using human or cynomolgus monkey whole blood as a source of T cells.
Nonclinical in vivo properties of HPN217 were evaluated in xenograft models and a single dose pharmacokinetic (PK) study in cynomolgus monkeys. HPN217 mediated dose-dependent growth suppression against the RPMI-8226 MM model and Jeko-1 mantle cell lymphoma model expressing relatively low levels of 5,600 and 2,200 copies of BCMA per cell, respectively. In the PK study, a single dose of HPN217 at 0.01, 0.1, or 1 mg/kg was given to cynomolgus monkeys. HPN217 exhibited linear PK behavior over this dose range. Serum half-life was in the range of 64 to 85 hours. Serum half-life, volume of distribution, and clearance appeared to be independent of dose. HPN217 was demonstrated to be stable and remained intact up to 3 weeks in vivo as demonstrated by a functional ligand binding assay using recombinant CD3ε and BCMA, respectively, to capture and detect HPN217. Importantly, serum samples collected one week after dosing were as potent as stock HPN217 in MM tumor cell killing in TDCC assays.
Collectively, preclinical and nonclinical characterization suggests that HPN217 is an efficacious novel therapeutic candidate that can provide a convenient dosing schedule for patients. A first-in-human phase 1 clinical trial is planned to evaluate HPN217 in RRMM.

Disclosures
Law: Harpoon Therapeutics: Employment. Aaron:Harpoon Therapeutics: Employment. Austin:Harpoon Therapeutics: Employment. Barath:Harpoon Therapeutics: Employment. Callihan:Harpoon Therapeutics: Employment. Evans:Harpoon Therapeutics: Employment. Gamez Guerrero:Harpoon Therapeutics: Employment. Hemmati:Harpoon Therapeutics: Employment. Jones:Harpoon Therapeutics: Employment. Kwant:Harpoon Therapeutics: Employment. Lao:Harpoon Therapeutics: Employment. Lemon:Harpoon Therapeutics: Employment. Patnaik:Harpoon Therapeutics: Employment. Sexton:Harpoon Therapeutics: Employment. Wesche:Harpoon Therapeutics: Employment. Xiao:Harpoon Therapeutics: Employment. Yu:Harpoon Therapeutics: Employment. Yu:Harpoon Therapeutics: Employment.