Single chain antibodies speci¢c for fatty acids derived from a
semi-synthetic phage display library
Ari Gargir
a
, Itzhak Ofek
aY
*, Shiri Meron-Sudai
a
, Meital Gal Tanamy
c
,
Panagiotis S. Kabouridis
b
, Ahuva Nissim
bYc
a
The Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
b
Bone and Joint Research Unit, Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK
c
Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel Aviv University, Rabin Medical Center, Belinson Campus, Petach Tikva 49100, Israel
Received 2 August 2001; received in revised form 15 November 2001; accepted 20 November 2001
Abstract
The biological activities of many acylated molecules are lipid dependent. Lipids, however, are poorly immunogenic or non-immunogenic.
We employed a phage display semi-synthetic human antibody library to isolate anti-lipid antibodies. Selection was done against methyl
palmitate, a 16 carbon aliphatic chain, and a major component of bacterial glycolipids and lipoproteins in animal cells. The selected single
chain variable fragment (scFv) bound specifically to a 16 carbon aliphatic chain and to a lesser extent to a 14 or 18 carbon aliphatic chain
and poorly to either 12, 22 or 8 carbon aliphatic chains. Furthermore, the scFv prevented micelle formation of lipoteichoic acid from Gram-
positive bacteria; inhibited lipopolysaccharide-induced tumor necrosis factor K release in mononuclear cells; bound to hydrophobic
bacterial surfaces, especially those of Gram-positive bacteria, and bound to Lck, a mammalian palmitated lipoprotein. Our data suggest
that the phage antibody library can be successfully employed to obtain human anti-aliphatic scFv human antibody fragment with potential
therapeutic applications in neutralizing the deleterious effects of bacterial toxins as well as in structure^function analysis of lipoproteins in
animal cells. ß 2002 Elsevier Science B.V. All rights reserved.
Keywords: Phage display; Single chain variable fragment ; Methyl palmitate ; Lipoteichoic acid
1. Introduction
Acylation plays important roles in prokaryotes and eu-
karyotes. For example, the active portion of lipopolysac-
charide (LPS) from Gram-negative bacteria, as well as of
lipoteichoic acid (LTA), from Gram-positive bacteria
binds and activates monocytes and macrophages in a lipid
dependent manner [1^3]. In eukaryotes, posttranslational
acylation of proteins plays a major role in their function,
in particular proteins involved in signalling pathways via
cell surface receptors [4]. Anti-lipid antibodies and in par-
ticular antibodies against the aliphatic portion are, there-
fore, required to study the structure^function relationships
as well as to neutralize the deleterious e¡ects of fatty acid
containing agents. Lipids, however, are considered to be
very poor immunogens [5]. Attempts to develop immuno-
therapy of LPS by neutralizing anti-lipid A antibodies
have been fraught with pitfalls, since such antibodies did
not react speci¢cally with the fatty acids of the molecule
[6,7]. One of these antibodies, the human^mouse chimeric
HA-1A antibody, has been shown to react with hydro-
phobic patches on lipid A but also reacted against bovine
serum albumin (BSA)-conjugated to 12 carbon aliphatic
chain or to single-stranded DNA [8]. Recently, however,
rabbits immunized with di¡erent subclasses of mycolic
acid, a cell wall constituent and virulence factor of Myco-
bacteria, produced antibodies that were highly speci¢c for
methoxy derivatives of this fatty acid [9]. Antibodies that
bind BSA-conjugated aliphatic chains were found in sera
of multiple sclerosis patients and patients su¡ering from
other autoimmune diseases, suggesting a possible connec-
tion between these antibodies and these diseases [5].
An alternative approach for development of antibodies
or peptides against weak immunogens is the phage display
technology. The phage display of antibody repertoires of-
0304-4165 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved.
PII: S0304-4165(01)00245-8
Abbreviations: LTA, lipoteichoic acid; LPS, lipopolysaccharide ; scFv,
single chain variable fragment ; MP, methyl palmitate; PBS, phosphate
bu¡ered saline ; MPBS, skimmed milk in phosphate bu¡ered saline ;
TPBS, phosphate bu¡ered saline containing 0.05% Tween 20
* Corresponding author. Fax: +972-3-640-9160.
E-mail address : aofek@ccsg.tau.ac.il (I. Ofek).
BBAGEN 25279 24-1-02
Biochimica et Biophysica Acta 1569 (2002) 167^173
www.bba-direct.com