Background
EVOLVE104 is the first CD3 T-cell engager (TcE) being developed for solid tumors which utilizes integrated CD2 T-cell co-stimulation to enhance durable T-cell effector function. EVOLVE104 is a tri-specific molecule which targets ULBP2/5/6 on tumor cells and utilizes an affinity-tuned CD3 binder together with a CD2-selected fusion protein to deliver coordinated co-stimulatory signal integration to the T-cell. ULBP2/5/6 are closely related proteins belonging to the UL16 binding protein family which are recognized by the NKG2D receptor and are induced on tumor tissues while maintaining restricted expression on normal human tissues. In this study, we evaluated the expression of ULBP2/5/6 in cynomolgus monkeys, assessed the cross-reactivity of EVOLVE104 for ULBP orthologs and T-cells in cynomolgus monkeys, and investigated the preclinical safety and tolerability of EVOLVE104.
Methods
ULBP2/5/6 expression on normal tissues was evaluated by gene expression and immunohistochemistry analysis. EVOLVE104 cross-species binding affinity to recombinant ULBP2/5/6 orthologs CD3 and CD2 was assessed by SPR analysis. In vitro target cell occupancy and functional readouts were assessed using recombinant CHO-K1 cells expressing the above recombinant cynomolgus molecules. Safety assessment, pharmacokinetics, and pharmacodynamics for EVOLVE104 were evaluated in cynomolgus monkeys in single-dose and repeat-dose studies. Immunophenotyping was evaluated by flow cytometry, and Luminex was used for cytokine/chemokine measurements.
Results
ULBP2/5/6 expression is restricted to normal mid-differentiated esophageal and ectocervical tissues in human and gastrointestinal tissues in cynomolgus EVOLVE104 binding affinities between human and cynomolgus T-cells was within 2-fold. Evaluation of EVOLVE104 T-cell killing of CHO cells expressing human and cynomolgus ULBP orthologs showed antigen-dependent T-cell-redirected killing, and increased T-cell proliferation and activation. Doses up to 24 mg/kg of EVOLVE104 were well tolerated, with no adverse findings or organ-specific toxicities at supra-pharmacologic levels for human efficacious exposures. In vivo EVOLVE104 showed expected IgG-like pharmacokinetics in vivo. EVOLVE104 demonstrated > 95% positive peripheral T-cell binding for at least 168 hours post dose. Transient T-cell margination was evident within 24 hours post dose. No evidence for cytokine release was observed.
Conclusions
EVOLVE104 demonstrates target-dependent cynomolgus T-cell activation in vitro, and a favorable in vivo safety profile including no evidence for cytokine release in cynomolgus monkeys at supra-pharmacologic exposures. These data support the clinical development of EVOLVE104 for the treatment of solid tumors where significant unmet medical needs remain. These favorable safety and tolerability features highlight EVOLVE104’s differentiated profile as a promising option for solid tumor treatments.