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 approximately 10(-8) M. Sequencing showed that ARM display selected a single V(H)/V(L) combination (V(H)1-2, Vkappa4-1) 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.