Delivery Of A Cd20 Transferrin Receptor Vnar Bispecific Antibody To The Brain For Cns Lymphoma

Penetration of the blood brain barrier (BBB) remains a significant hurdle in the development of biologic therapeutics for CNS-related cancers. Antibody-based molecules typically do not cross the BBB in amounts required for therapeutic efficacy. Receptor-mediated transporters in the luminal membrane of brain capillary endothelium, which normally transport circulating endogenous peptides and proteins, offer a non-invasive approach for the delivery of therapeutic molecules to the brain.Using a combination of in vitro and in vivo phage display technology, we isolated a panel of cross species (rodent, primate and human) binders to the transferrin receptor 1 (TfR1) from synthetic libraries of shark variable antigen receptors (VNARs). Initially, the anti-TfR1 VNARs were fused to an immunoglobulin Fc backbone for testing in mice. At therapeutic (2 mg/kg) doses delivered by tail vein injection, high levels of the VNAR-Fc antibodies were found in the brain after 18 hours, which exceeded 5% of the plasma levels. Brain fractionation studies showed that the VNAR-Fc antibodies to TfR1 translocated to the brain parenchyma and were not retained in the capillaries. The lead anti-TfR1 VNAR was then used to target aberrant B cells in the brain for conditions such as cerebral lymphoma where existing CD20 therapies to not penetrate the CNS at therapeutic doses. A family of anti-CD20/TfR1 bispecific molecules were produced by linking the small VNAR to either the N- and C- terminus of heavy and light chains to create both monovalent and bivalent fusions . We used rituxan as the CD20 agent. The bispecifics were assessed for binding to the two cognate antigens and then tested for brain penetration after tail vein injection. At least 4 different formats gave excellent brain penetration 18 hours after dosing at 3.75mg/kg. Unmodified rituxan had a brain plasma ratio of 0.25 in line with published data while the best anti-TfR1-rituximab bispecific formats were between 2 to 5%. The bispecific formats also retain antibody-dependent cell-mediated cytotoxicity and will be further tested for brain penetration in non-human primates and in rodent models of CNS lymphoma. If these results successfully translate from rodents to larger animals, the peripheral delivery of a brain penetrant CD20/TfR1 bispecific antibody would provide a safer and more effective alternative to CD20 antibody delivery by the intrathecal route and could have broad utility in B cell driven brain cancer. Citation Format: Frank S. Walsh, Krzysztof Wicher, Jaroslaw Szary, Pawel Stocki, Michael Demydchuk, Lynn Rutkowski. Delivery of a CD20 transferrin receptor VNAR bispecific antibody to the brain for CNS lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3631. doi:10.1158/1538-7445.AM2017-3631