SINGLE DOMAIN CAMELID ANTIBODIES: STRUCTURES OF ANTIGEN COMPLEXES AND THEIR USE AS INHIBITORS AND SUBSTRATE MIMICS
Lode Wyns
Laboratorium voor Ultrastructuur, Vlaams Interuniversitair
Instituut voor Biotechnologie, Vrije Universiteit Brussel,
Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
In the context of many
biotechnological applications there have been many developments
aiming at the production of high affinity minimal size
antibodies: two domain variable fragments (Fv), or single chain
ones (scFv). Also the road towards production of single domain
VH's has been explored. Many obstacles with respect to large
scale production, low solubility, tendency to aggregate are
limiting and impairing progress. The discovery of naturally
occurring camelid antibodies devoid of light chains and of a CH1
domain has opened many new avenues of research both fundamental
and applied 1.
The crystal structures of two
camelid heavy-chain immunoglobin VHH domains have already been
published.(VHH :VH fragment of the heavy chain antibodies).
Desmyter et al. described the structure of a complex
between a cloned VHH from Camelus dromaderius and hen
eggwhite lysozyme (HEWL2) and Spinelli et al.
described the structure of an uncomplexed VHH fragment from Lama
lama raised against a subunit of the human chorionic
gonadotropin hormone (hCG)3 These two structures
reveal features of unique interest in structural immunoloy, and
paved the way for further studies aiming at deciphering the
recognition mode and the antigen-antibody interfaces of these new
antibodies. It appeared that the reduced interface offered to the
antigen by the single domain binding site can be compensated in
part by an extension of the CDR3 domain, especially in dromedary,
where in addition this loop is often stabilised by a disulphide
bridge towards CDR1. The structure of the VHH-lysozyme complex
also demonstrated that the extended CDR3-domain actually formed a
protruding loop, penetrating deeply into the active site of
lysozyme and thus acting as an inhibitor of the enzyme. A common
feature of the camelid-VHHs is that the first hypervariable loop
H1 adopted a unique structure, not recognised as one of the known
canonical structures in human or mouse VH.
The important unique feature of these antibodies to protrude into enzyme active sites and as such to provide a systematic approach towards the production of enzyme inhibitors and peptide leads has been further analyzed and documented.
In the specific case of the anti-lysozyme structure the mimicry of the carbohydrate substrate essentially by the CDR3 loop has been carefully analysed and documented. (4)
Meanwhile crystals of domains in complex with RNases, Amylases, Lactamases and Carbonic anhydrase have been obtained. (for the latter both free antibody and a complex). The structures of a number of these shed further light on the innovative solutions the camelid immune system has devised in order to bind at high affinity with a single domain VHH.
We are involved in a thorough
study of the organization of these genes : cloning, selection,
sequencing is performed. (Amongst others to study the ontogeny of
the response). This sequence data base which is accumulating also
allows us to foresee a number of original, noncanonical designs
of CDR's by the camelid immune system.
1. C. Hamers-Casterman, T. Atarhouch, S.
Muyldermans, G. Robinson, C. Hamers, E. Bajyana Songa, N.
Bendahman and 2. R. Hamers. Naturally occurring antibodies devoid
of light chains. Nature 363, 446-448,
1993.
3. A. Desmyter, T. R. Transue, M. Arbabi Ghahroudi, D. T.
Minh-Hoa, F. Poortmans, R. Hamers, S. Muyldermans, L. Wyns.
Crystal structure of a camel single-domain VH antibody fragment
in complex with lysozyme. Nature Structural Biology, 3,
nr. 9, 805-813, 1996
4. S. Spinelli et al. Nature Structural Biology, 3,
752-757, 1996
5. T. R. Transue, E. De Genst, M. Arbabi Ghahroudi, L. Wyns, S.
Muyldermans. Camel single-domain antibody inhibits enzyme by
mimicking carbohydrate substrate. Protein Structure, Functions,
and Genetics, In Press 1998