Pergamon 0161~5890(95)00123-9 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Molecular Immunolop, Vol. 33, No. 1, pp. 41-56. 1996 Copyright :cf 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890196 $15.00+0.00 zyxwvutsrqp LIGHT CHAIN SHUFFLING OF A HIGH AFFINITY ANTIBODY RESULTS IN A DRIFT IN EPITOPE RECOGNITION MATS OHLIN,*$ HENRIK OWMAN.* MICHAEL MACH? and CARL A. K. BORREBAECK* *Department of Immunotechnology, Lund University, P.O. Box 703 1, S-220 07 Lund, Sweden: TInstitut fiir Klinische und Molekulare Virologie, der Friedrich-Alexander-Universitst Erlangen- Niirnberg, Erlangen, Germany (First received 25 May 1995; accepted in recised,fhm 29 August 1995) Abstract-Human polyclonal and monoclonal antibodies against pathogens and toxins are poten- tially useful in the treatment of various diseases. A number of human monoclonal antibodies with protective capacity in vitro have been established by conventional hybridoma technology. However, with the development of phage-display technology, the possibility of specifically tailoring antigen- binding properties has improved substantially. We show here that the reactivity of a high affinity, virus-neutralizing human antibody against the AD-2 epitope of cytomegalovirus gB can be modified by introducing other VK sequences together with the original VH sequence. The fine specificity, as determined by the requirement of particular amino acid residues in the epitope, is shifted in these new antibody fragments. It was also evident that the VH/VK pairing was not promiscuous, since antibody fragments selected by phage display retained light chain sequences very similar to the original hybridoma-derived light chain, proving that a high affinity interaction was very dependent on a co-operativity between both variable domains. These findings show that phage display technology might modify the binding properties of pre-existing, high affinity antibodies. Kej, words: cytomegalovirus, glycoprotein B (gB), human monoclonal antibody. phage-display tech- nology, antibody specificity. INTRODUCTION Establishing human monoclonal antibody specificities has proven very labour intensive using conventional hybridoma technology. Recently, however, the intro- duction of antibody libraries (Huse et al., 1989) and the use of antibody-display on filamentous phage (McCafferty et al., 1990) have opened new possibilities. Thus, it has been possible to obtain specific antibodies against a variety of antigens from both pre-immunized (see e.g. Burton et al., 1991 and Williamson et al., 1993) and also from apparently non-immune donors (Gram et a/., 1992; Hoogenboom and Winter, 1992; Marks ef al.. 1991). Synthetic (Barbas et al., 1992; Griffiths et al., 1994) repertoires have also recently been used for the same purpose. Furthermore, the phage technique has been used to select for antibody fragments exhibiting specific characteristics with respect to kinetic parameters (Haw- $Author to whom correspondence should be addressed. Abbreaiatiow CDR, complementarity-determining region. CMV. cytomegalovirus, ELISA, enzyme-linked immu- nosorbent assay. FR, framework region, gB, CMV gly- coprotein B, kdlss, dissociation rate constant, PBMC, peripheral blood mononuclear cells, scFv, single chain vari- able region fragment (VH-linker-VK). SPR, surface plasmon resonance. kins et al., 1992; Duefias et al., submitted) and to evaluate repertoires developed in uivo (Rapoport c,t al., 1995). In this report, we demonstrate the use of phage-display technology to modify the reactivity of high-affinity human antibodies. Specifically, a monoclonal antibody recognizing the AD-2 epitope expressed by human cyto- megalovirus (CMV) glycoprotein B (gB) (Ohlin et al., 1993) was used for this purpose. The AD-2 epitope, which is conserved among different isolates of CMV (Lehner et al., 1991; Meyer et al., 1992), is an important target for complement-independent CMV-neutralizing antibodies (Meyer et al., 1990; Ohlin et al., 1993) and might conse- quently be a useful target for passive immunotherapy. Clinical trials have already been initiated with one human antibody specific for CMV AD-2 (Azuma et al., 1991). Although the two human monoclonal antibodies which have been well characterized for their binding to the AD- 2 epitope show essentially identical reactivity patterns (Meyer et al., 1992; Ohlin et al., 1993), it appears that a polyclonal response can recognize the AD-2 epitope in different ways (Silvestri et al., 1993). Since the gB antigen is also a leading CMV vaccine candidate (Spaete et ul.. 1994), an understanding of antibody recognition of the most important neutralization epitopes is crucial to evaluate the benefits of an antibody response in vivo. Furthermore, development of antibodies with improved affinity (in particular reduced dissociation rate constant) 47