Rapid induction of apoptosis in B‐cell lymphoma by functionally isolated human antibodies

Abstract

Novel panning and screening methodology was devised to isolate high affinity human recombinant scFv antibody fragments with functionally associated properties in B lymphoma cells. The approach was used to generate a panel of apoptosis-inducing antibodies specific for antigens differentially expressed in B lymphoma vs. T leukaemia cells. The selections resulted in an antibody pool with near perfect selectivity (>99%) for the B lymphoma target cells. Randomly picked clones (72) revealed 7 unique antibody genotypes. Six of these rapidly induced apoptosis in target cells. Following the conversion to full IgGs, the antibodies were shown to be specific for HLA-DR/DP, the B-cell receptor l chain and for CD54/ ICAM-1. The latter receptor was not previously associated with apoptotic properties in B-cell lymphomas. Anti-ICAM-1 IgG induced apoptosis in a broad range of B lymphoma cell lines and were shown by immunohistochemistry to bind strongly to B lymphoma tissue obtained from 5 different B lymphoma patients. The recombinant IgG antibodies had affinities in the subnanomolar (0.3 nM) to nanomolar (3 nM) range. The described technology is generally applicable for the rapid isolation of high affinity human antibodies with specificity for differentially expressed cell surface receptors with intrinsic negative or positive signalling properties from na€ ıve phage libraries. ' 2006 Wiley-Liss, Inc.

The nuclear DNA repair protein Ku70/80 is a tumor‐associated antigen displaying rapid receptor mediated endocytosis

“…The antigen recognized by the INCA‐X antibody was immunoprecipitated, as described earlier 10. Briefly, the membrane fraction was pre‐cleared with Protein A Sepharose 4 Fast Flow (Amersham Biosciences, Uppsala, Sweden) for 2 hr on rotation and immunoprecipitated overnight by 20 μg INCA‐X antibody, or a human IgG control.…”

Phenotypic screening: the future of antibody discovery

“…As early as 1995, de Kruif et al [11] screened for target cell specificity and more recent examples have followed [12][13][14]. Functional screens have also been performed that have identified antibodies that induce apoptosis [15], inhibit cell proliferation [16], or internalise [17,18].…”

“…To date, most antibodies identified by phenotypic screens have been found using a two-step process. First, antibody populations have been enriched against a target cell or tissue type before being screened for function [11][12][13][14][15][16][17][18]. A two-step process is dependent on the efficiency of both steps; however, the functional screen poses the greatest challenge and is often the bottleneck in the process.…”

“…However, they have also been successfully used in phenotypic screens: Sanchez-Martin et al [9] recently reviewed target-agnostic selection strategies for isolating anticancer antibodies. In 2006, Fransson et al [15] described the isolation of BI-505 using the FIRST™ platform. Using this platform, three rounds of affinity selection were performed with a naïve antibody phage library against intact Ramos B lymphoma cells in the presence of membrane vesicles derived from Jurkat T leukaemia cells for deselection.…”

Function-first antibody discovery

“…We have embarked in this daunting journey by developing a “function-first” approach. Such a platform relies on a differential cell-based screening methodology, which in a first step allows for the isolation of antibodies based on the targeting of a specific cell type over others, for instance a malignant cell over its normal counterpart 8 . Antibodies against tumor-associated targets are then screened in clinically and mechanistically relevant, high-throughput, functional in vitro assays.…”