Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display

In antibody-ribosome-mRNA complex (ARM) ribosome display, stable complexes of nascent protein, mRNA and ribosomes are produced in a eukaryotic in vitro expression system, through coupled transcription and translation of DNA lacking a 3' stop codon. Selection of the protein simultaneously captures the relevant mRNA, which is recovered as DNA by coupled reverse transcription-polymerase chain reaction (RT-PCR) performed on the intact complexes. Here, we describe the use of ARM display to select a specific human antibody fragment from a transgenic mouse library. The mice carry unrearranged gene segments of the human heavy (H) and kappa light (L) chain loci, while the endogenous murine H and kappa loci are functionally silenced; they respond to immunisation by production of fully human IgM antibodies. A library encoding human single-chain (sc) antibody (V-H/K) fragments, in which V-H domains and kappa light chains were combined at random by PCR, was prepared from spleen cells of transgenic mice immunised with progesterone-bovine serum albumin (BSA). Library diversity was demonstrated by sequencing. Progesterone-binding fragments were selected over five cycles of ARM display and the selected DNA cloned and expressed in Escherichia coli. Soluble V-H/K fragments obtained in periplasmic extracts had the same specificity as ribosome-bound V-H/K, supporting the view that folding and specificity of the displayed and soluble proteins are equivalent. The affinity of the expressed V-H/K was similar to 10(-8) M. Sequencing showed that ARM display selected a single V-H/V-L combination (V(H)1-2, V kappa 4-1) and rearrangement, with a few mutational differences between clones. Monoclonal antibodies against progesterone-BSA obtained from hybridomas were encoded by the same V-H and V-L segments and had similar properties to the fragments obtained in vitro. The combination of ribosome display and transgenic mouse technologies is a rapid means of generating fully human antibody fragments in vitro for expression and further manipulation. (C) 1999 Elsevier Science B.V. All rights reserved.