Preclinical Characterization and Phase I Trial Results of INBRX-109, A Third-Generation, Recombinant, Humanized, Death Receptor 5 Agonist Antibody, in Chondrosarcoma

Abstract

Purpose: Patients with unresectable/metastatic chondrosarcoma have poor prognoses; conventional chondrosarcoma is associated with a median progression-free survival (PFS) of <4 months after first-line chemotherapy. No standard targeted therapies are available. We present the preclinical characterization of INBRX-109, a third-generation death receptor 5 (DR5) agonist, and clinical findings from a phase I trial of INBRX-109 in unresectable/metastatic chondrosarcoma (NCT03715933).

Patients and methods: INBRX-109 was first characterized preclinically as a DR5 agonist, with binding specificity and hepatotoxicity evaluated in vitro and antitumor activity evaluated both in vitro and in vivo. INBRX-109 (3 mg/kg every 3 weeks) was then evaluated in a phase I study of solid tumors, which included a cohort with any subtype of chondrosarcoma and a cohort with IDH1/IDH2-mutant conventional chondrosarcoma. The primary endpoint was safety. Efficacy was an exploratory endpoint, with measures including objective response, disease control rate, and PFS.

Results: In preclinical studies, INBRX-109 led to antitumor activity in vitro and in patient-derived xenograft models, with minimal hepatotoxicity. In the phase I study, INBRX-109 was well tolerated and demonstrated antitumor activity in unresectable/metastatic chondrosarcoma. INBRX-109 led to a disease control rate of 87.1% [27/31; durable clinical benefit, 40.7% (11/27)], including two partial responses, and median PFS of 7.6 months. Most treatment-related adverse events, including liver-related events, were low grade (grade ≥3 events in chondrosarcoma cohorts, 5.7%).

Conclusions: INBRX-109 demonstrated encouraging antitumor activity with a favorable safety profile in patients with unresectable/metastatic chondrosarcoma. A randomized, placebo-controlled, phase II trial (ChonDRAgon, NCT04950075) will further evaluate INBRX-109 in conventional chondrosarcoma.

Figure 1.

INBRX-109 characterization. A, Schematic representation of INBRX-109 structure. B, Representative binding curve of INBRX-109 on ExpiCHO-S transfected with full-length human DR5; untransfected ExpiCHO-S cells served as a negative control. Apparent affinity of the observed binding interaction was determined using a One Site total analysis. C, INBRX-109 competition with TRAIL for binding to DR5 expressed by ExpiCHO-S cells. Detection of a constant concentration of TRAIL (3.5 nmol/L) in the presence of increasing concentrations of INBRX-109 is shown. D, ADCC capability of INBRX-109 was evaluated in the Promega Jurkat CD16a (V158) ADCC reporter assay using DR5-transfected ExpiCHO-S cells as targets and untransfected cells as negative controls. Daratumumab, the target of which is expressed on Jurkat cells, serves as a positive control. E, The ability of INBRX-109 to bind the complement component C1q contained within normal human serum was measured by ELISA. Daratumumab is used as a positive control. Abbreviations: Ab, antibody; ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; CHO, Chinese hamster ovary cells; DR5, death receptor 5; Fc, crystallizable fragment; K_d, equilibrium dissociation constant; sdAb, single-domain antibody; UT, untransfected.

Figure 2.

In vitro and in vivo 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, Impact 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 four-parameter agonist concentration versus response curve, and calculated EC₅₀ values are reported. Activity of INBRX-109 was assessed in the presence of the pan-caspase inhibitor Z-VAD-FMK. C, Impact of valency on potency. The fold improvement in potency from bivalent to trivalent, trivalent to tetravalent, and tetravalent to hexavalent is 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 0, as indicated by arrows). Each symbol represents the mean tumor volume of 8 animals, with error bars to denote SEM. Abbreviations: DR5, death receptor 5; K_d, equilibrium dissociation constant.

Figure 3.

Hepatotoxicity analyses. A, The impact of valency and ADA on the risk of hepatoxicity was evaluated in HepaRG cells. Percent viability, as measured by CellTiter-Glo and using average relative light unit value for untreated cell samples for normalization, 41 hours after treatment with INBRX-109, hexavalent sdAb, TAS266 analogue, and TAS266 mut (a re-engineered version of the TAS266 analogue with no ADA recognition sites) in the presence or absence of IVIG as a source of preexisting ADA. B, The impact of valency on cell viability of treated 3D human liver microtissues as determined using intracellular ATP content after 7 days of treatment measured by CellTiter-Glo. C and D, The existence of preexisting anti-sdAb antibodies to INBRX-109 (C) or an analogue of TAS266 (D) was assessed in an ELISA-based competitive inhibition assay in sera from healthy individuals and patients with cancer. C and D show preexisting anti-sdAb antibodies that were at or above the cutpoint in an initial screen. Serum samples from healthy individuals and patients with cancer that had low screen signal to INBRX-109 (C) or TAS266 (D) were not analyzed in the confirmatory assay and assumed negative for ADA. TAS266 is an sdAb-based therapeutic with known preexisting ADA reactivity. Abbreviations: ADA, antidrug antibody; IVIG, intravenous immunoglobulin; mut, mutant.

Figure 4.

Clinical efficacy of INBRX-109 in chondrosarcoma. A, Best tumor response and time receiving treatment up to the first event of progression or death. B, Best response up to data cutoff date. C and D, Representative baseline and posttreatment contrast-enhanced CT scans of patients who experienced a PR. Orange arrows show the diameter of target lesions. E, Representative baseline and posttreatment contrast-enhanced CT scans of a patient who experienced SD. All patients in C–E had grade 3, metastatic, conventional chondrosarcoma. F, PFS by Kaplan–Meier analysis. Median follow-up was 11.6 months. Crosses indicate censored data. G, Mean modified growth modulation index (modified GMI), as determined by the ratio of PFS with INBRX-109 to prior treatment duration. Best response to treatment with INBRX-109 is indicated within each bar, and the ratio of PFS on INBRX-109 to prior treatment duration is indicated at the right of each bar. Data cutoff: May 26, 2022. Abbreviations: GMI, growth modulation index; IDHmt, isocitrate dehydrogenase 1/2 mutant; NA, no postbaseline scan available; PD, progressive disease. ^aA total of 31 patients were included in the analysis. Four patients from cohort B6 were excluded for taking prohibited medications (n = 1), not having conventional chondrosarcoma (n = 1), or not having first scan data (n = 2). ^bPatient has a mutation in IDH1 (R132) or IDH2 (R172). ^cOne patient is from dose-escalation cohort A4 and received INBRX-109 at a dose of 10 mg/kg; all other patients are from dose-expansion cohort B4 (INBRX-109 3 mg/kg). Patients with nonconventional chondrosarcoma are indicated with a stippled pattern. ^dDurable disease control is SD, PR, or CR for >6 months. ^ePatient's first tumor assessment during treatment showed PD. A second scan post PD showed tumor shrinkage, and the best change from baseline up to data cutoff is shown; patient was not receiving a subsequent therapy at the time of the second scan. ^fA total of 30 patients were evaluable. Five patients were excluded for taking prohibited medications (n = 1; cohort B6), not having conventional chondrosarcoma (n = 1; cohort B6), not having first scan data (n = 2; cohort B6), or due to death (n = 1; cohort B4). ^gTwo patients were excluded due to taking prohibited medication (n = 1) or having dedifferentiated chondrosarcoma (n = 1). ^hOverall, 23 of 35 patients were included in this analysis. One patient had a long prior treatment duration of 84 months (cohort B6) and, although included in the analysis, is not included in the figure (GMI, 0.03). Patients were excluded for taking prohibited medications (n = 1; cohort B6), not having conventional chondrosarcoma (n = 1; cohort B6), or having a treatment duration that could not be estimated. (n = 10). Partial start and stop dates were ignored.