Pre-Targeted Radioimmunotherapy Employing a Recombinant Bispecific Antibody Using a Murine Xenograft Model of Human Leukemia
Authors: Kenoyer Aimee L. , Lin Yukang , Orozco Johnnie J. , Balkin Ethan , Hamlin Don , Hernandez Alexandra H. , Fang Chen , Laird Kelly , Frost Sofia H.L. , Hylarides Mark , Orcutt Kelly D. , Wilbur D Scott , Wittrup K. Dane , Press Oliver W. , Pagel John 
Published: October 2019

Abstract
Introduction: Acute Myeloid Leukemia (AML) has a high rate of relapse despite hematopoietic cell transplantation (HCT). Pre-targeted radioimmunotherapy (PRIT) prior to HCT offers a promising approach to reduce relapse for high-risk patients. PRIT studies with antibody-streptavidin (Ab-SAV) conjugates have shown promise, but may be limited by SAV immunogenicity. We have developed a recombinant anti-human CD45 bispecific Ab with picomolar affinity to radiolabeled DOTA chelates for targeting AML xenografts in a preclinical model.
Methods: We engineered a bispecific Ab recognizing CD45 and DOTA-metal chelates. The light chain sequence from anti-human CD45 Ab (BC8) was cloned into a pFUSE vector along with a single-chain variable region developed to bind to DOTA chelates of multiple radionuclides. Ab was co-transfected into HEK 293 F cells with a pFUSE vector containing heavy chain sequence from Rituximab. Cultures were grown to extinction, and Ab purified via Protein A ion-exchange chromatography. Ab was characterized by SDS-PAGE, HPLC, and flow cytometry to determine specificity and integrity of material. Ab was tested for in vivo targeting in a xenograft model of human AML. Athymic mice bearing flank HEL tumors (30 to 100 µg) were injected with 280 µg bispecific Ab or 300 µg Ab-SAV. Twenty two hours later mice were treated with or without clearing agent (5 µg DOTAY-dextran for bispecific Ab and 50 µg NAGB for Ab-SAV groups). Two hours later mice were given Yttrium-90 (90Y)-DOTA-biotin. Blood was taken at time points over 22 hours post-injection for pharmacokinetic study; xenografts and normal tissues were taken 4, 24, and 48 hours after DOTA-biotin injection for gamma counts.
Results: Bispecific Ab targeting CD45 bound to human AML cells in vitro with an increase in mean fluorescence intensity >2 logs compared to control. SDS-PAGE and HPLC demonstrated a construct of expected size. Blood clearance of 90Y-DOTA-biotin was assessed following injection of bispecific Ab with or without clearing agent and compared to mice that received BC8-SAV as the first-step reagent. Even without clearing agent, mice that had received bispecific Ab showed rapid blood clearance of 90Y-DOTA-biotin similar to that of Ab-SAV-injected mice that had received clearing agent, with 8.15 ± 0.56 % ID/g in the blood 5 minutes post-DOTA-biotin injection falling to 1.02 ± 0.62% ID/g within 4 hours and 0.40 ± 0.16 % ID/g at 20 hours. Mice that received BC8-SAV followed by clearing agent had 7.32 ± 2.0 % ID/g in the blood 5 minutes post-injection, falling to 1.13 ± 0.77 % within 4 hours and 0.73 ± 0.46 % at 20 hours. Mice receiving bispecific Ab followed by DOTAY-dextran clearing agent and 90Y-DOTA-biotin showed a rapid blood clearance with a radioactivity concentration of 8.07 ± 3.0% ID/g at 5 minutes and falling to 0.14 ± 0.03 % within 4 hours post-injection. In vivo targeting of the bispecific Ab was similar to Ab-SAV groups; within 4 hours 3.62 ± 0.91 % ID/g was detected in the tumor compared to 0.12 ± 0.05 % in the blood for mice receiving bispecific Ab, while Ab-SAV mice had a tumor value of 3.85 ± 2.5 % and blood was 1.17 ± 0.63 %. Uptake in non-target tissues was low at 4 hours post-injection compared to tumor, with liver and kidney values of 0.05 ± 0.014 % ID/g and 0.518 ± 0.09 % ID/g, respectively. Tumor retention remained steady at 24 and 48 hours with values of 4.13 ± 1.0% ID/g and 4.14 ± 2.47% ID/g respectively. Groups that received bispecific Ab as the first-step agent followed by clearing agent and then 90Y-DOTA-biotin showed slightly higher uptake in normal organs early on, but displayed higher uptake in target tissue. Tumor values at 4, 24, and 48 hours post-injection were 4.65 ± 2.4, 5.89 ± 4.2, and 3.95 ± 2.3 % ID/g, respectively.
Conclusion: Recombinant bispecific Ab targeting both human CD45 and radiometal-labeled DOTA is promising as a novel therapeutic approach for AML prior to HCT. Bispecific Ab targets human AML cells in vitro and in vivo with favorable biodistribution showing specific uptake in tumors while normal organs displayed minimal uptake. Blood clearance of the bispecific Ab is rapid and comparable to that of Ab-SAV, even without the addition of a clearing agent. Future studies will assess therapeutic potential of this bispecific Ab PRIT approach in a murine model of AML.
Funding Source Acknowledgment: Research reported in this publication was supported by the National Cancer Institute under award numbers R01CA109663, R01CA136639, and R01CA138720.

Disclosures
No relevant conflicts of interest to declare.