Preclinical Evaluation of a Novel Clearing Agent-Independent Approach for Pretargeted Alpha Therapy with ²¹²Pb

Aim

Over the years, various pretargeted radioimmunotherapy (PRIT) modalities have been proposed to enhance the therapeutic window of systemic radiotherapy. These approaches typically involve a tumour-targeting bispecific antibody (BsAb) that distributes within target tissue, followed by a small radiolabelled molecule that efficiently binds pretargeted sites or is quickly eliminated from the body. A common challenge has been ensuring rapid excretion of the radioactive payload while maintaining a high affinity for the slower-clearing pretargeting molecule. An intermediate step to clear or neutralise circulating pretargeting antibodies has often been employed to prevent off-tumour capture of the radioligand, but this adds complexity and safety risks, posing challenges to the clinical implementation of PRIT. Consequently, the field has shifted towards clearing agent-independent strategies, such as complementary oligomeric pretargeting and engineered bispecific antibodies. 

Matching the biological half-life of the radioligand with the physical half-life of the radionuclide is particularly impactful using short-lived radionuclides such as ²¹²Pb (t_1/2 = 10.6 h), to minimise healthy tissue exposure while increasing the tumour absorbed dose. In this context, we developed a novel two-step, clearing agent-independent PRIT regimen for carcinoembryonic antigen (CEA)-positive tumours, involving a complementary bispecific antibody pair and a ²¹²Pb radioligand. The efficacy and tolerability of this two-step PRIT approach were compared with a corresponding three-step PRIT scheme.

Materials and Methods

A technique was developed which relies on complementary "SPLIT" (SeParated v-domains LInkage Technology) pretargeting antibodies with specificity for CEA and a split high-affinity sub-pM 1,4,7,10 Tetrakis(carbamoylmethyl)1,4,7,10-tetraazacyclododecane (DOTAM) binder: one antibody carrying the anti-DOTAM VH and the other the anti-DOTAM VL domain. Independently, these antibodies are unable to bind
²¹²Pb-DOTAM, as an active anti-DOTAM binding site is only generated in the presence of the complementary variable domain. This assembly is less likely to occur without target binding, but when tumour-bound, the split VH and VL domains form the complete binder in a concentration-dependent manner. Therefore, no clearing agent is required to prevent capturing of ²¹²Pb-DOTAM in circulation.

Severe combined immunodeficiency (SCID) mice bearing subcutaneous (s.c.) CEA-expressing BxPC3 xenografts were administered the two pretargeting antibodies via intraperitoneal injection, followed by ²¹²Pb-DOTAM (0.74 MBq) administered intravenously 7 days later. Another group of SCID mice received a pretargeting CEA-DOTAM BsAb with a complete ²¹²Pb-DOTAM binder. After 7 days, circulating BsAb was neutralised using a dextran-based clearing agent, followed by ²¹²Pb-DOTAM (0.74 MBq) after 24 hours. Two-and three-step CEA-PRIT were compared in terms of ²¹²Pb biodistribution, tumour growth inhibition, and tolerability after three treatment cycles as described above.

Conclusion

In vivo proof of mechanism and therapeutic efficacy was successfully demonstrated for two-step PRIT using the novel SPLIT pretargeting antibodies, rendering the logistically complex yet efficacious three-step PRIT regimen obsolete. Translation of this optimised SPLIT PRIT approach to a phase I trial is currently underway, offering potential improvements in clinical PRIT implementation.