Comparative biodistributions of pretargeted radioimmunoconjugates targeting CD20, CD22, and DR molecules on human B-cell lymphomas

Abstract

Pretargeted radioimmunotherapy (PRIT) using streptavidin (SA)-conjugated antibodies (Abs), followed by clearing agent and radiolabeled biotin is a promising method that can increase the effectiveness of RIT, while decreasing the toxicities associated with directly labeled Abs. Although CD20 has been the traditional target antigen for RIT of non-Hodgkin lymphoma (NHL), studies targeting HLA DR and CD22 have yielded promising results. Targeting all 3 antigens at once may further augment the effect of PRIT. This study compares the targeting of Ramos, Raji, and FL-18 lymphoma xenografts with either anti-CD20 Ab/SA (1F5/SA), anti-HLA DR Ab/SA (Lym-1/SA), anti-CD22 Ab/SA (HD39/SA), or all 3 conjugates in combination, followed 24 hours later by a biotin-N-acetyl-galactosamine clearing agent, and 3 hours after that by (111)In-DOTA-biotin. The Ab/SA conjugate yielding the best tumor uptake and tumor-to-normal organ ratios of radioactivity varied depending on the target antigen expression on the cell line used, with 1F5/SA and Lym-1/SA yielding the most promising results overall. Also, the best tumor-to-normal organ ratios of absorbed radioactivity were obtained using single conjugates optimized for target tumor antigen expression rather than the combination therapy. This study highlights the importance of screening the antigenic expression on lymphomas to select the optimal reagent for PRIT.

Figure 1

Cell-binding studies. Flow cytometric analysis of the cell-binding capabilities of 1F5/SA, Lym-1/SA, and HD39/SA to 3 different B lymphoma cell lines: Ramos (A), Raji (B), and FL-18 (C). For all studies, 2 controls were included, one omitting the primary conjugate and one using a nonbinding control conjugate HB8181/SA.

Figure 2

Pharmacokinetics and blood clearance of Ab/SA conjugates. (A) Whole blood clearance of 1.4 nmol of either ¹²⁵I-labeled 1F5/SA (●), Lym-1/SA (○) or HD39/SA (◇) injected intravenously into athymic BALB/c mice (n = 4/group). (B-D) The effect of the NAGB CA on circulating levels of each of the Ab/SA conjugates: 1F5/SA (B), Lym-1/SA (C), and HD39/SA (D). Athymic BALB/c mice (n = 4/group) were injected intravenously at t = 0 with 1.4 nmol ¹²⁵I-labeled conjugate followed 24 hours later with an intravenous injection of 5.8 nmol CA. In each study, serial blood samples were obtained from the retro-orbital venous plexus and radiation levels present determined by γ-counting and percent injected dose/gram (%ID/g) calculated.

Figure 3

Biodistributions of ¹¹¹In-DOTA-biotin in tumor xenografts and normal organs after pretargeting with each Ab/SA conjugate. Athymic BALB/c mice bearing Ramos, Raji, or FL-18 tumor xenografts were injected intravenously with 1.4 nmol of a single conjugate. After 24 hours, mice were injected with 5.8 nmol CA followed 3 hours later with 1.2 nmol ¹¹¹In-labeled DOTA-biotin. Mice were killed 24 and 48 hours later and blood, tumors, and normal organs were harvested, weighed, and analyzed for levels of radioactivity by γ-counting to determine %ID/g. Results are shown for PRIT with 1F5/SA (A), Lym-1 (B), HD39/SA (C), and the nonbinding control conjugate HB8181/SA (D). For each organ, the first bar represents results 24 hours after ¹¹¹In-DOTA-biotin injection and the second bar represents results after 48 hours.

Figure 4

Comparative biodistributions using all 3 conjugates in combination or with each conjugate singly at 24 hours. For the combination studies, mice bearing Ramos (A), Raji (B), or FL-18 (C) tumor xenografts were injected intravenously with a cocktail of either 0.47 or 1.4 nmol each of 1F5/SA, Lym-1/SA, and HD39/SA followed 24 hours later with 5.8 or 17.4 nmol, respectively, of CA and 3 hours after that with 1.2 nmol ¹¹¹In-DOTA-biotin. Mice were killed 24 and 48 hours after injection of ¹¹¹In-DOTA-biotin and tumor and organs were harvested and analyzed as for the studies in Figure 3. Each graph shows %ID/g 24 hours after injecting 1.4 nmol 1F5/SA alone (□), Lym-1/SA alone (▩), HD39 alone (■), and all 3 used in combination at 0.47 nmol/conjugate (▤) and at 1.4 nmol/conjugate (▧).

Figure 5

Comparative biodistributions using all 3 conjugates in combination or with each conjugate singly at 48 hours. Combination studies in Ramos (A), Raji (B), and FL-18 (C) as described for Figure 4. Each graph shows results 48 hours after injecting ¹¹¹In-DOTA-biotin pretargeted with either 1.4 nmol 1F5/SA alone (□), Lym-1/SA alone (▩), HD39 alone (■), and all 3 used in combination at 0.47 nmol/conjugate (▤) and at 1.4 nmol/conjugate (▧).

Figure 6

Tumor-to–normal organ ratios of absorbed radioactivity using each Ab/SA conjugate individually or all 3 in combination. Mice were treated as described in Figure 3. Tumor-to–normal organ ratios of radioactivity are shown 48 hours after injection of ¹¹¹In-DOTA-biotin. Animals were pretargeted with 1.4 nmol of either 1F5/SA (□), Lym-1/SA (▩), or HD39/SA (■) or a combination of either 0.47 nmol (▤) or 1.4 nmol of each conjugate (▧). Results are shown for mice bearing Ramos (A), Raji (B), and FL-18 (C) xenografts.