Improved therapeutic results by pretargeted radioimmunotherapy of non-Hodgkin's lymphoma with a new recombinant, trivalent, anti-CD20, bispecific antibody

Abstract

We examined whether a pretargeting method using a new recombinant anti-CD20 bispecific antibody (bsMAb) followed by (90)Y-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid ((90)Y-DOTA)-peptide could reduce hematologic toxicity yet improve therapeutic responses compared with conventional (90)Y-anti-CD20 IgG and a chemically conjugated bsMAb. TF4, a humanized, tri-Fab bsMAb with two Fabs binding CD20 and one Fab binding histamine-succinyl-glycine (HSG), developed by the dock and lock (DNL) method, was tested in nude mice with Ramos B-cell lymphomas. Optimal pretargeting required a 29-h interval between TF4 and (90)Y-DOTA-HSG, and 20-fold more moles of TF4. TF4 cleared more rapidly from the blood than anti-CD20 IgG, with early processing in the liver, spleen, and kidney. At 24 h, TF4 improved tumor uptake of (111)In-HSG-peptide 2.6-fold [13% versus 5% injected dose per gram (ID/g)] and enhanced tumor to blood ratios >45-fold (770 versus 17), compared with an anti-CD20 Fab x anti-HSG Fab chemical conjugate, and by 1.6-fold (9.0% versus 5.6% ID/g) and 1,600-fold (522 versus 0.32), respectively, compared with radiolabeled anti-CD20 IgG. A severe (>or=90%) and prolonged reduction of WBCs was observed at the maximum dose of (90)Y-anti-CD20 IgG, whereas pretargeting resulted in a <or=60% transient drop. TF4 pretargeting resulted in highly significant improvement in survival, curing 33% to 90% of the animals, even at relatively low doses, whereas most tumors progressed quickly without cures with (90)Y-anti-CD20 IgG. These results indicate an improved therapeutic index with pretargeted radioimmunotherapy (RAIT) using a DNL-constructed tri-Fab, bsMAb, compared with conventional therapy with directly radiolabeled antibody or with a chemically conjugated bsMAb. These encouraging results prompt testing these constructs for pretargeting RAIT in patients.

Figure 1

Diagram depicts the structure of tri-Fab made by DNL method, showing DDD and AD domains bearing the anti-CD20 Fabs and the anti-HSG Fab, respectively. IMP-288's structure shows the single DOTA moiety (red), 2 HSG moieties (blue) and the D-amino acids (black) that compose this hapten-peptide.

Figure 2

Evaluation of optimal pretargeting conditions. Tumor-bearing nude mice (N = 5 per determination) were administered increasing amounts of TF4 (0.125 nmoles to 1.0 nmoles). At 29 or 39 h later, 0.025 nmoles (15 μCi) of ¹¹¹In-IMP-288 was given. Doses are expressed as the TF4 to IMP-288 mole ratio, ranging from 5:1 to 40:1. Values represent means ± SD. Tumor weights averaged 1.0 g for the animals studied at the 39-h interval and ∼2.0 g for the animals examined at the 29-h interval.

Figure 3

Comparison of direct and pretargeting methods. The left side-panels show the average biological data (% ID/g; mean ± SD, n = 5) for tumor and normal tissue uptake, and the right-side panels show the effective data (μCi/g; error bars not given because they were contained with the symbol). The effective data assume that 0.7 and 0.15 mCi of the pretargeting ⁹⁰Y-IMP-288 and ⁹⁰Y-IgG, respectively, were given. (see Supplementary Table S1 in for listing of means ± SD for % ID/g and tumor/nontumor ratios for the pretargeting and IgG data.)

Figure 4

Kaplan-Meier survival curves for six separate studies summarized in Table 2. Survival was based on the time required for the tumor to reach the size of ≥3.0 cm³. Wilcoxon two-sample analysis of tumor quadrupling time can be found in Supplementary Data Table S2.