A New Tri-Fab Bispecific Antibody for Pretargeting Trop-2–Expressing Epithelial Cancers

Abstract

RS7 is an internalizing anti-Trop-2 pancarcinoma antibody capable of targeting most epithelial cancers. Because pretargeting strategies could improve the tumor localization of radionuclides, a new anti-Trop-2 · antihapten bispecific antibody for pretargeting, based on humanized RS7, was prepared and evaluated with a radiolabeled hapten-peptide in vitro and in vivo to determine whether its internalization properties would interfere with pretargeting. Methods: The anti-Trop-2 · antihapten bispecific antibody, TF12, was prepared using the modular dock-and-lock method. TF12 and humanized RS7 binding was assessed by cell binding assays and fluorescence-activated cell sorting analysis in a variety of human carcinoma cell lines. The internalization of TF12 was evaluated in vitro using a fluorescent TF12 conjugate or hapten-peptide and 111 In-labeled TF12 and RS7. The biodistribution of TF12 and its use as a pretargeting agent with an 111 In-labeled hapten-peptide were assessed in several human epithelial cancer xenografts. Dose optimization was examined in 2 tumor models. Results: TF12 internalizes, but a substantial fraction remained accessible on the tumor surface. Fluorescence-activated cell sorting analysis showed only a minor change in fluorescent signal when the tumor was probed with a fluorescent hapten-peptide over 4 h, and microscopy showed substantial membrane staining when reassessed at 24 h after TF12 exposure. Only 40.1% of 111 In-TF12 was internalized after 24 h. In vivo, excellent tumor localization of the 111 In-labeled peptide was observed in several tumor models. Conclusion: TF12 was retained sufficiently on the cell surface in several epithelial cancers, thereby making it suitable for pretargeted imaging and therapy of various Trop-2-expressing carcinomas.

A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo

“…However, Trop2-related cancer treatments have been quickly developed in recent years and Trop2-targeted cancer therapy may offer novel and promising therapeutic strategies for cancer treatment. [31][32][33][34] In this study, we employed the human Fab phage library to isolate a human Fab fragment that recognizes EMD of Trop2. The biopanning strategy with the repeated panning with living cells and coated Trop2 EMD protein in microtiter plates guaranteed the enrichment of specific Trop2 EMDbinding phage.…”

A new class of bispecific antibodies to redirect T cells for cancer immunotherapy

“…Since its inception in 2005, the DNL method, by combining DDD2-and AD2 modules derived from assorted classes of molecules that encompass IgG, [38][39][40][41] Fab, 34,42,43 cytokines, [44][45][46] polyethylene glycols, 47 and enfuvirtide, 48 has been used to produce more than 100 different complexes with potential applications for targeted therapies of malignant, autoimmune, and infectious diseases. The generation of a functional Okt3-scFv-AD2 and the demonstration of the in vitro and in vivo activities of various (X)-3s to kill target tumor cells via T cells, as described in this study, expands the applications of the DNL repertoire to now include scFv as a viable building block for future exploration.…”

Redirected T-Cell Killing of Solid Cancers Targeted with an Anti-CD3/Trop-2–Bispecific Antibody Is Enhanced in Combination with Interferon-α

“…TF12 (Fig. 1B), which is a bispecific Tri-Fab generated by DNL to comprise two Trop-2 (hRS7)-binding Fabs fused to a third Fab that binds the hapten, histamine succinyl glycine, and not to any human cells/tissue, has been described previously (34). The 19-3 BiTE, which has the identical deduced amino acid sequence as blinatumomab (CD19XCD3 BiTE), was produced recombinantly from a stable SpESF transfectant cell line, and purified from culture supernatant fluids with Ni-Sepharose.…”