Targeted Radioimmunotherapy Using Anti-CD45 Antibody-Mediated Delivery Of An In Vivo Nanoparticle-Based Alpha Generator In a Xenograft Model Of Human Leukemia
Authors: Kenoyer Aimee L. , Hernandez Alexandra H. , Shadman Mazyar , McLaughlin Mark , Robertson David , Fang Chen , Press Oliver W. , Pagel John M. 
Published: October 2019

Abstract

Introduction
Acute Myeloid Leukemia (AML) relapse rates remain high despite treatment with combination chemotherapy and hematopoietic cell transplantation. New targeted treatment modalities including radioimmunotherapy (RIT) have been developed to reduce relapse. However, minimally toxic means of targeting AML cells for delivery of radionuclides may not be optimal for all therapeutically favorable isotopes. Alpha (α) particles have a higher linear energy transfer and shorter path length in contrast to beta (β) particles, thus leading to the promising exploration of α-particle RIT for AML. Treatment efficacies may be further improved by the use of in vivo α-generators such as 225Ac, which emits a total of 4 α-particles upon decay. However, targeted therapy utilizing traditional 225Ac chelates poses a challenge since the α-emitting daughter radionuclides while short-lived, are difficult to retain near the targeted malignant cells and may be released into circulation, leading to unwanted non-specific toxicities such as to the renal or hepato-biliary systems.

Methods
To improve the therapeutic potential of 225Ac-based RIT for AML and reduce toxicities we have developed an anti-CD45 antibody (Ab)-conjugated gold-coated lanthanide phosphate nanoparticle that contains multiple gadolinium (Gd) shells designed to sequester 225Ac in the core and retain the α-daughters within the nanoparticle. Previous work demonstrated the in vivo retention of the 221Fr daughter radionuclide of over 90% for at least 3 weeks past the core nanoparticle synthesis. In this work the nanoparticle core was first synthesized using LaCl3 and GdCl3 using 177Lu as a radioactive surrogate for 225Ac. Four consecutive GdPO4 layers were added to the lanthanide core, followed by NaAuCl4 to form a metallic gold coat. Anti-human CD45 Ab (BC8) was separately labeled with 125I and then conjugated to the gold coat of the nanoparticle via a polyethylene glycol linker. This dual-labeled approach allowed for verification of stability of the Ab-nanoparticle. A competitive binding flow cytometry assay was used to measure the efficiency of the Ab conjugation to the nanoparticle and determine the concentration of Ab in solution. In vivo targeting of the Ab-nanoparticle was initially tested in athymic nude mice bearing human AML xenografts by injection with 50 µCi 177Lu-anti-CD45 Ab-nanoparticle at 100 µg per dose of Ab injected.

Results
Nanoparticle-BC8 dual-labeled with 177Lu and 125I bound effectively in vitro to human AML cells (HEL) with an increase in mean fluorescence index (MFI) of 9.2-fold compared to non-binding isotype control. Labeled nanoparticle-BC8 also effectively bound to human Burkitt’s lymphoma Ramos and Raji cells with an increased MFI of 86- and 36-fold compared to control, respectively. A 96-well plate-based assay for cell binding to test radioactive conjugates was also employed to verify that anti-CD45 Ab-nanoparticle remained stable in vitro. Non-conjugated BC8 effectively blocked binding of 125I-BC8-conjugated 177Lu-nanoparticle to Ramos cells in comparison with control with a reduction in binding by 21.3 % with respect to 125I-BC8 (p = 0.016) and 20.2 % with respect to the 177Lu-NP core (p = 0.026). 125I-labeled nanoparticle-BC8 conjugate bound effectively in vivo to human leukemia xenografts. Favorable targeting to sites of disease was seen by 4 hours post-injection, with 18.0 ±2.9 % injected dose per gram of targeting Ab in the tumor.

Conclusion
Combined nanoparticle-antibody therapy is a promising, novel approach to target malignant cells. Antibody-mediated delivery of α-particle generators represents a potential solution for the difficulties of safe and effective targeting using 225Ac, largely nonspecific toxicities due to dispersal of α-particle daughters. We have shown that 177Lu is a useful surrogate for 225Ac for preliminary characterization assays, and that 177Lu is retained over time in the nanoparticle core. We have shown in vitro targeting of leukemia and lymphoma cells and have made strides towards obtaining a favorable biodistribution in a model of human AML. However, challenges remain as liver uptake by nanoparticles cleared through the reticuloendothelial system is unfavorable and may cause dose-limiting toxicities. Future work will further characterize nanoparticle-Ab conjugates and progress toward studies involving 225Ac for AML therapy.

Disclosures:
Press: Genentech, inc.: Consultancy, Research Funding.