SPLIT PRIT: A Novel Clearing Agent-Independent Approach for Pretargeted Alpha Therapy with 212Pb

Abstract

Introduction

Pretargeted radioimmunotherapy (PRIT) aims to improve the therapeutic index of systemic radiotherapy, typically involving a tumour-targeting bispecific antibody (BsAb) followed by a rapidly clearing small radiolabelled molecule. This strategy minimises healthy tissue exposure while increasing the absorbed dose to tumours. Rapid radioligand pharmacokinetics are particularly critical for efficacy and tolerability when using short-lived radionuclides like 212Pb (t1/2 = 10.6 h). While earlier PRIT regimens required an intermediate clearing agent to neutralise circulating BsAb, efforts are now focusing on developing clearing agent-independent approaches to decrease logistical complexity and mitigate safety risks.

Objectives

We developed a novel two-step, clearing agent-independent PRIT regimen for carcinoembryonic antigen (CEA)-positive tumours. This regimen involves a complementary SeParated v-domains LInkage Technology (SPLIT) antibody pair and a 212Pb radioligand. We asse

Materials and Methods

Two human SPLIT antibodies were developed that bind specifically and bivalently to human CEA, each fused to one half of a split high-affinity sub-pM 1,4,7,10-Tetrakis(carbamoylmethyl)1,4,7,10-tetraazacyclododecane (DOTAM) antibody variable region fragment: one antibody with the variable heavy (VH) and the other with the variable light (VL) domain. Bound to CEA on the cell surface, these split VH/VL domains assemble to form the active binding site for the subsequently administered therapeutic radioligand, 212Pb-DOTAM, capturing it significantly more efficiently than circulating SPLIT antibodies. To assess the functionality of this approach, we treated mice bearing subcutaneous CEA-expressing BxPC3 xenografts with the two pretargeting SPLIT antibodies, followed 7 days later by 212Pb-DOTAM. Another group of mice received a CEA-DOTAM BsAb, followed 7 days later by a dextran-based clearing agent to neutralise circulating BsAb, and then 212Pb-DOTAM after 24 hours. The two- and three-step CEA-PRIT regimens were compared for 212Pb biodistribution, tumour growth inhibition, and tolerability after three treatment cycles of 0.74 MBq (20 μCi).

Results

Both CEA-PRIT regimens achieved significant and comparable tumour growth delay. Only mild transient body weight loss was observed in both groups, confirming comparable tolerability. Excellent 212Pb tumour specificity was confirmed in both regimens. Tumour uptake for the two-step PRIT was 25–30% IA/g at 24 h p.i., compared to 36–45% IA/g for the three-step PRIT. Importantly, blood and kidney retention remained low for both regimens (<0.5% IA/g and <2 %IA/g, respectively).

Conclusion

The novel two-step CEA-targeted SPLIT PRIT approach demonstrated therapeutic efficacy and tolerability comparable to the three-step regimen. Biodistribution data confirmed the preferential retention of 212Pb-DOTAM in tumours and successful rapid excretion of the non-tumour bound radioligand. This optimised SPLIT PRIT approach offers potential improvements in clinical PRIT implementation, and its translation to a Phase I trial for patients with metastatic colorectal cancer is currently underway.

Funding Acknowledgements

This research was funded by and performed as a collaboration between F. Hoffman-La Roche and Orano Med.