Preclinical Activity Support DLL3-TOPAbody in DLL3-Expressing SCLC and Other Tumors

DLL3-TOPAbody

The tri-specific T-cell engager DLL3-TOPAbody demonstrated both in vitro and in vivo antitumor efficacy, enhanced cytokine release, and T-cell proliferation, as well as preliminary safety in DLL3-expressing tumor cells, according to data from a preclinical study presented at the 2024 SITC Annual Meeting.¹

DLL3-TOPAbody was found to induce DLL3-dependent cytokine release and proliferation of CD8– and CD4-positive T cells. Moreover, DLL3-TOPAbody inducted DLL3-dependent tumor cell lysis in DLL3-positive tumor cells. Potent and specific activity against human DLL3, CD3 and 4-IBB was observed with the agent, and the binding activity of DLL3-TOPAbody to different target proteins was also well-balanced. Initial assessment of safety through a cytokine release assay showed that treatment with DLL3-TOPAbody induced the release of interleukin-6. In the absence of the target, minimal cytokine release occurred following treatment.

In vivo assessment of the efficacy of DLL3-TOPAbody in mice models showed that the agent significantly inhibited tumor growth in a dose-dependent manner. Notably, DLL3-TOPAbody showed evidence of turning immunologically “cold” tumors into “hot’ tumors, as represented by a significant increase in CD8-positive infiltration in both the invasive margin (P < .05) and central tumor (P < .001).

“This approach has the potential to improve clinical outcomes for [patients with] SCLC, particularly those with poorly infiltrated tumors,” presenting author Lei Fang, PhD, and colleagues wrote in a poster presentation of the data. “Preliminary chemistry, manufacturing, and control developability assessment suggested a good stability of the molecule, which [will support] an investigational new drug submission [for DLL3-TOPAbody] in the first half of 2025.”

In patients with neuroendocrine tumors, particularly small cell lung cancer (SCLC), DLL3 is abnormally expressed on the surface of tumor cells but is minimally expressed in normal adult tissue. Other DLL3-targeted agents have demonstrated efficacy in this tumor type, such as the bispecific antibody tarlatamab-dlle (Imdelltra). On May 16, 2024, the FDA granted accelerated approval to tarlatamab for the treatment of patients with extensive-stage SCLC who experienced disease progression on or after platinum-based chemotherapy based on data from the open-label, multicenter, multi-cohort phase 2 DeLLphi-301 trial (NCT05060016).² However, the clinical activity of CD3 inhibitors may be hindered by poor T cell infiltration in the tumor microenvironment.¹

Accordingly, DLL3-TOPAbody was developed to address this unmet need. DLL3-TOPAbody comprises a membrane-proximal epitope of DLL3, a fine-tuned 4-1BB antibody with no systemic toxicity, an anti-CD3 antibody, and disabled Fc effector function. The agent was designed to have high affinity and specificity against tumor targets, target-dependent T-cell activation, balanced CD3 and 4-1BB signaling to maintain long-term activity; limited cytokine release without the target being present; a molecular weight close to that of a monoclonal antibody; and to be optimized for drug development.

The compound binds to DLL3 on cancer cells and stimulates both CD3 and 4-1BB on T cells. This mechanism could redirect bolstered T cell responses to DLL3-expressing tumor cells while maintaining a manageable safety profile.

Study Methodology and Assays

The binding affinity and potency of DLL3-TOPAbody to human DLL3, CD3ɛδ, CD3ɛ and 4-1BB was assessed using an enzyme linked immunosorbent assay.

For the in vitro portion of the study, tumor cells (SHP77) and DLL3-negative cells (HCT116) were incubated with peripheral blood mononuclear cells (PBMC) and antibodies for 48 hours. To assess cytokine release, TNF-alpha (TNFα) levels in the supernatant were assessed using the LANCE Ultra Human TNFα Detection Kit. Evaluation of T-cell proliferation involved co-culturing of carboxyfluorescein succinimidyl ester–labeled PBMCs with SHP77 and antibodies for 5 days.

Tumor killing in SHP77 and SCT116 cells were evaluated by lactate dehydrogenase assay, which was used to detect dead cells. Both SHP77 and HCT116 cells were incubated with PBMC and antibodies for 72 hours, using AMG757 as a benchmark.

IL-6 levels in supernatant were examined using the human Th1/TH2 Cytokine Cytometric Bead Array Kit. Human PBMCs were stimulated with various antibodies for 48 hours prior to assessment.

For in vivo efficacy assessment, B16F10-hDLL3 cells were implanted in humanized CD3ɛ/4-1BB mice. Upon tumor volume reaching approximately 100 mm³, 5 doses of DLL3-TOPAbody and AMG757, a half-life extended, DLL3-targeted bispecific T-cell engager, were administered twice weekly to the mice (n = 6 per group). DLL3-TOPAbody could be administered at doses of 1 mg/kg, 3 mg/kg or 6 mg/kg; AMG757 was administered at doses of either 0.7 mg/kg, 2.1 mg/kg, or 7 mg/kg. Mean tumor volume and survival was assessed.

Representative immunohistochemistry staining of CD8-positive cells in B16F10-hDLL3 tumor tissues among different groups was conducted, and the presence of infiltrated cells in the central tumor vs invasive margin was quantified to determine whether tumors remained “cold” or “hot”.

References

1. Yang L, Gao S, Cai Z, et al. DLL3-TOPAbody: A next-generation trispecific T cell engager with 4-1BB co-stimulation for the treatment of SCLC. Presented at: 2024 SITC Annual Meeting; November 8-19, 2023; Houston, TX. Abstract 1292.
2. FDA grants accelerated approval to tarlatamab-dlle for extensive stage small cell lung cancer. FDA. May 16, 2024. Accessed November 8 2024. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-tarlatamab-dlle-extensive-stage-small-cell-lung-cancer