Data cutoff: May 26, 2022. these models was consistent with standard chondrosarcoma. Study mice were treated weekly for 3 weeks, with intravenous administration of vehicle control or 1 mg/kg INBRX-109. Effects on tumor growth were evaluated by twice-weekly tumor volume measurements. Tolerability was assessed by body weight loss (measured twice weekly) or animal morbidity/mortality. The study endpoint was reached when the mean tumor volume of the vehicle control group (uncensored) reached 1,500 mm3. All animal studies were approved by local regulatory government bodies for animal welfare. All mice were kept in accordance with federal and state policies on animal research. Data visualization and analysis Data were graphed and analyzed using GraphPad Prism 9. Clinical Study design The INBRX-109 phase I trial is an ongoing, open-label, three-part study evaluating INBRX-109 in individuals with advanced/metastatic solid tumors that was initiated in November 2018. Part 1 (3+3 single-agent dose escalation) was completed in 2019. Individuals were treated with dose levels of 0.3, 1, 3, 10, and 30 mg/kg. Intrapatient dose escalation was not permitted. INBRX-109 was given as an intravenous infusion over 60 moments every 21 days. INBRX-109 was well tolerated, with no significant toxicities observed; Saccharin 1-methylimidazole an MTD was not reached, and a dose of 3 mg/kg was chosen for further investigation. In part 2 (single-agent dose development), INBRX-109 3 mg/kg i.v. every 3 weeks is being evaluated in several tumor types, including chondrosarcoma (cohort B4) and and Given its high DR5 manifestation, the human being extraskeletal myxoid chondrosarcoma cell collection H-EMC-SS was used to characterize the antitumor activity of INBRX-109 (Fig. 2). The cytotoxicity of tetravalent INBRX-109 was compared with hexavalent and bivalent antibodies using Saccharin 1-methylimidazole the same sdAb within INBRX-109 as well as with recombinant, soluble TRAIL, a naturally occurring trimer. Although molecules with valencies 3 showed related (within four-fold) high binding affinity to H-EMC-SS cells (Fig. 2A), their cytotoxic activities diverged substantially, demonstrating the effect of valency (Fig. 2B). Considerable raises in the potency Saccharin 1-methylimidazole of INBRX-109 and the hexavalent agent over trimeric TRAIL and the bivalent DR5 agonist were observed, indicating that increasing valency increases potency of signaling (Figs. 2B and ?andC).C). Of notice, the hexavalent comparator resulted in only a moderate increase in potency over INBRX-109 (Fig. 2C). Open in a separate window Number 2. and antitumor activity. A, Representative dose-dependent binding of bivalent, trivalent (TRAIL), tetravalent (INBRX-109), and hexavalent anti-DR5 molecules on H-EMC-SS chondrosarcoma cells. B, Effect of valency on H-EMC-SS cell death. Death of H-EMC-SS cells 16 hours after treatment with the indicated concentrations of molecules of increasing valency was measured by CellTiter-Glo. Data were fit with a nonlinear Saccharin 1-methylimidazole four-parameter agonist concentration versus response curve, and determined EC50 ideals are reported. Activity of INBRX-109 was assessed in the presence of the pan-caspase inhibitor Z-VAD-FMK. C, Effect of valency on potency. The fold improvement in potency from bivalent to trivalent, trivalent to tetravalent, and tetravalent to hexavalent is definitely shown. D, Activation of caspase-3 and -7 in H-EMC-SS cells treated with bivalent, trivalent (TRAIL), tetravalent (INBRX-109), and hexavalent anti-DR5 molecules for the indicated durations of time was measured by real-time imaging on an Incucyte live cell imaging system. Activity of INBRX-109 was assessed in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK. E, Tumor volume over time in animals harboring patient-derived chondrosarcoma tumors and treated with vehicle or INBRX-109 (1 mg/kg, i.v. once every week 3 weeks starting on study day time 0, as indicated by arrows). Each sign represents the mean tumor volume of 8 animals, with error bars to denote SEM. Abbreviations: DR5, death receptor 5; in TSPAN33 chondrosarcoma PDX models (Fig. 2E). In evaluating a wide range of doses (0.1C10 mg/kg weekly) in mouse tumor models, INBRX-109 at a dose of 1 1 mg/kg weekly was generally the most efficacious and led to sustained tumor inhibition in two chondrosarcoma PDX models, with near-complete tumor regressions observed in one model. Hepatotoxicity analyses In Good Laboratory Practice toxicology studies in cynomolgus monkeys, INBRX-109 was well tolerated and the no-observed-adverse-effect levels were the highest doses.