Pretargeted alpha therapy in MUC16-positive high-grade serous ovarian cancer

Abstract

Background: Peritoneal metastasis with micrometastatic cell clusters is a common feature of advanced ovarian cancer. Targeted alpha therapy (TAT) is an attractive approach for treating micrometastatic diseases as alpha particles release enormous amounts of energy within a short distance. A pretargeting approach - leveraging the inverse-electron-demand Diels-Alder reaction between tetrazines (Tz) and trans-cyclooctene (TCO) - can minimize off-target toxicity related to TAT, often associated with full-length antibodies. We hypothesized that a pretargeting strategy could effectively treat high-grade serous (HGS) ovarian tumors while minimizing toxicity.

Methods: We utilized the humanized antibody, AR9.6, labeled with actinium-225 (²²⁵Ac). AR9.6 targets fully glycosylated and hypoglycosylated isoforms of MUC16. For biodistribution and radioimmunotherapy studies, AR9.6-TCO was injected into OVCAR3-bearing mice 72 h before administering [²²⁵Ac]Ac-mcp-PEG₈-Tz, e.g. using a 1,2,4,5-tetrazine conjugated to the macropa chelator via a polyethylene glycol (PEG) linker.

Results: Biodistribution data revealed that the pretargeting approach achieved substantial tumor uptake. Cerenkov luminescence imaging confirmed successful in vivo pretargeting during TAT studies. Compared to the control groups, TAT with AR9.6-TCO and [²²⁵Ac]Ac-mcp-PEG₈-Tz significantly suppressed tumor growth and improved overall survival in OVCAR3 tumor-bearing mice. Renal and ovarian pathology compatible with toxicity was observed in mice in addition to transient hematologic toxicity.

Conclusion: We confirmed that pretargeting with AR9.6-TCO and [²²⁵Ac]Ac-mcp-PEG₈-Tz has durable antitumor effects in high MUC16-expressing tumors. These findings demonstrate great potential for using pretargeting in combination with TAT for the treatment of ovarian cancer.

Classification: Biological Sciences; Applied Biological Sciences.

Keywords: AR9.6; MUC16; Ovarian cancer; Pretargeting; Targeted alpha therapy.

Figure 1.. Biodistribution studies confirm tumor uptake in MUC16-positive tumors.

(A) Biodistribution data for the RIT approach with [²²⁵Ac]Ac-mcp-PEG₈-AR9.6. Click reaction was conducted prior to injecting mice. (B) Biodistribution data for PRIT approach with [²²⁵Ac]Ac-mcp-PEG₈-AR9.6. Mice were injected with AR9.6-TCO on day 0 followed by an injection of [²²⁵Ac]Ac-mcp-PEG₈-Tz on day 3. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001; Mantel-Cox test. RO = reproductive organs.

Figure 2.. Pretargeted alpha radioimmunotherapy achieves complete responses in OVCAR3 xenografts.

(A) Tumor volume (mm³) growth curves and overall survival percentage (B) of athymic nude OVCAR3-bearing mice after treatment. Significant tumor growth inhibition was observed in both treatment groups compared to saline and IgG isotype controls. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001; Mantel-Cox test.

Figure 3.. Representative histopathology of the kidneys from mice in control and treatment groups.

(A) Histology of a normal kidney from the saline control group (n = 3). (B) and (C) Histology of the kidneys from the IgG RIT control group (n = 3) (B) and the AR9.6 RIT treatment group (n = 4) (C) shows multifocal subacute to chronic tubular injury areas in the renal corticomedullar junctions and cortices. (D) and (E) Histology of the kidneys from the IgG PRIT control group (n = 4) (D) and the AR9.6 PRIT treatment group (n = 4) (E) revealed multifocal areas of subacute to chronic tubular injury in the renal corticomedullar junctions and cortices. The top and middle panels represent low and high magnifications –10× and 40× respectively. (A–E) (bottom panels). Immunohistochemistry for γ-H2AX in the kidney from a mouse in the saline group (A) showed negative γ-H2AX renal tubular immunolabeling. In contrast, those from the other groups (B–E) showed occasional γ-H2AX-postive immunolabeling of cells (brown) in areas of tubular injury.