Pretargeted radioimmunotherapy using anti-CD45 monoclonal antibodies to deliver radiation to murine hematolymphoid tissues and human myeloid leukemia

Abstract

Radioimmunotherapy (RIT) for treatment of hematologic malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted (P)RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by a biotinylated clearing agent and radiolabeled 1,4,7,10-tetraazacylodode cane N,N',N",N'''-tetraacetic (DOTA)-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the PRIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a large amount of labeled Ab for a prolonged time. Therapy experiments showed that (90)Y-DOTA-biotin significantly prolonged survival of mice treated with pretargeted anti-hCD45 Ab-SA compared with mice treated with conventional RIT using (90)Y-labeled anti-hCD45 Ab at 200 muCi. Because human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and PRIT using an anti-murine (m)CD45 Ab where the target antigen is present on normal hematopoietic tissues. After 24 h, 27.3% +/- 2.8% of the injected dose of activity was delivered per gram (% ID/g) of lymph node using (131)I-A20-Ab compared with 40.0 +/- 5.4% ID/g for pretargeted (111)In-DOTA-biotin. These data suggest that pretargeted methods for delivering RIT may be superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.

Fig. 1. Fluorescent images of HEL xenograft-bearing athymic mice treated with either conventional or PRIT

Mice were injected with either (a) 1.4 nmol anti-hCD45 Ab directly labeled with fluorophore or (b) pretargeted anti-hCD45 Ab-SA conjugate (1.4 nmol) followed 22 hours later by CA and then by 100 μg R-Phycoerythrin-biotin. Images are shown at same camera intensity settings. Images of mice are shown at time = 12 hours after injection of fluorophore. Arrows indicate fluorophore in tumor (T) and blood pool (B). (c) Tumor-to-total body fluorescence ratios 8 hours after-injection in conventional and PRIT mice.

Fig. 1. Fluorescent images of HEL xenograft-bearing athymic mice treated with either conventional or PRIT

Mice were injected with either (a) 1.4 nmol anti-hCD45 Ab directly labeled with fluorophore or (b) pretargeted anti-hCD45 Ab-SA conjugate (1.4 nmol) followed 22 hours later by CA and then by 100 μg R-Phycoerythrin-biotin. Images are shown at same camera intensity settings. Images of mice are shown at time = 12 hours after injection of fluorophore. Arrows indicate fluorophore in tumor (T) and blood pool (B). (c) Tumor-to-total body fluorescence ratios 8 hours after-injection in conventional and PRIT mice.

Fig. 2. Biodistributions of radioactivity in HEL-xenograft bearing athymic mice injected with ¹¹¹In-anti-hCD45 (BC8) DOTA-Ab conjugate for (a) conventional RIT or with ¹¹¹In-DOTA-biotin after anti-hCD45 Ab-SA conjugate for (b) PRIT

HEL xenograft bearing mice were injected i.v. via the tail vein with 1.4 nmol of either: (a) conventional trace-labeled ¹¹¹In-anti-hCD45 DOTA-Ab or (b) anti-hCD45 Ab-SA conjugate followed 22 hours later with 5.8 nmol of CA and 2 hours subsequent with 1.2 nmol of ¹¹¹In-DOTA-biotin. Groups of 5 mice were euthanized 24 (open bars) and 48 (solid bars) hours after injection of ¹¹¹In. The radioactivity in blood, tumor, and normal organs was quantified by gamma counting, corrected for decay and expressed as % ID/g of tissue. (c) Tumor-to-normal organ ratios of radioactivity using either directly-labeled ¹¹¹In-anti-hCD45 DOTA-Ab or pretargeted ¹¹¹In-DOTA-biotin delivered after CA are shown 24 and 48 hrs after injection of radioactivity. In panel c, the first two bars represent results at 24 hours after administration of pretargeted ¹¹¹In-DOTA-biotin (■) and directly-radiolabeled anti-hCD45 DOTA-Ab conjugate (▨), whereas the third and fourth bars represent tumor-to-normal organ ratios at 48 hours after pretargeted ¹¹¹In-DOTA-biotin injection (□) and directly-radiolabeled anti-hCD45 DOTA-Ab conjugate ( ).

Fig. 3. Regression of HEL leukemia xenografts and survival of mice after conventional and PRIT

Athymic BALB/c mice bearing HEL leukemia xenografts were injected i.v. with either 1.4 nmol (a, c) BC8 Ab directly labeled with 200–400 μCi of ⁹⁰Y, or (b, d) unlabeled BC8 Ab-SA conjugate followed 20 hours later by 5.8 nmol of CA, and 2 hours after that with 400–1600 μCi ⁹⁰Y-DOTA-biotin. Control mice bearing xenograft tumors were either left untreated or treated in a similar manner with directly labeled BHV-1 Ab or pretargeted BHV-1-SA, respectively. Tumor volume curves (panel a represents conventional RIT; panel b represents PRIT) are truncated at the time of euthanasia of the first mouse in each group. These mice bearing HEL leukemia xenografts were also analyzed for survival as a function of time (panel c represents conventional RIT; panel d represents PRIT).

Fig. 4. Biodistributions of radioactivity in blood, bone marrow, spleen, lymph node, and lung of non-tumor bearing B6 Ly5a syngeneic mice injected with directly-radiolabeled anti-mCD45 DOTA-Ab or ¹¹¹In-DOTA-biotin after pretargeted anti-mCD45 Ab-SA conjugate

Non-tumor bearing B6 Ly5a mice were injected with 1.4 nmol of (a) conventional trace-labeled ¹³¹I-anti-mCD45 (A20) DOTA-Ab or (b) pretargeted anti-mCD45 Ab-SA conjugate followed 20 hours later by 5.8 nmol of CA and 2 hours after that by 1.2 nmol ¹¹¹In-DOTA-biotin. Groups of 5 mice were euthanized at either 2, 20, 24, 48, 72, and 96 hours after injection of the anti-murine Ab conjugates. The radioactivity concentrations in blood, bone marrow, spleen, lymph node, and lung were quantified by gamma counting and expressed as %ID/g. The shaded area-under-the-curve in panel a represents the initial 20 hours of non-specific radiation dose from blood-borne radiolabeled Ab that can potentially be eliminated with the use of PRIT.