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