Development of Nanobodies (VHH) against human Zinc-alpha-2-glycoprotein (ZAG) and synaptosome-associated protein of 23kDa (SNAP-23)

Abstract

All camelid species (i.e. camel, dromedary, and llama) belong to the family of the Camelidae and possess apart from conventional antibodies, also an exclusive class of function antibodies that lack L-chains, denoted as heavy-chain only antibodies (HCAB). Since antigen recognition by HCABs occurs solely by the variable domain of heavy-chain antibodies (VHH), it is desirable to produce Ag specific VHHs, which can be otained after immunization of camelids. VHHs, are succesfully expressed and produced in vitro as independent single-domain proteins, with full retention of Ag-recognition. These VHHs are denoted as Nanobodies (Nbs), which are the smallest functional Ag-binding proteins at present (15kDa, single digit nm-dimensions).
Nanobodies offer many therapeutical opportunities that are currently being optimized : competitive enzyme inhibition, inhibition of amyloidosis, oral immunotherapy, intracellular application as intrabodies, molecular building units in fusion constructs. They can also be developed as a potent diagnostic probe and used as a non-invasive imaging tool and novel biosensor probes where small size, high affinity and specificity are important.
We used two markers to generate VHHs : zinck-alpha-2-glycoprotein and synaptosome-associated protein of 23 kDa Zinc-alpha-2-glycoprotein (ZAG) is a member of the nonconventional major histocompatibility complex class I molecules, and is the sole soluble member implicated in cachexia, a poorly understood yet life-threatening, sever wasting syndrome. Cachexia is defined by massive depletion of both adipose and skeletal muscle tissues and occurs an a number of patients suffering from cancer, AIDS and other chronic illnesses.
Synaptosome-associated protein of 23kDa (SNAP-23) was identified in human B lymphocytes and plays an important role in the regulation of vesicle trafficking in mammalian cells. SNAP-23 is an esential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion in all mammalian cells.
In this work, a dromedary and llamas were immunized with SNAP-23 and ZAG antigens respectively, and a strong immune response was observed in the HCAb isotypes against SNAP23 and ZAG antigen. Peripheral blood lymphocytes were purified from the immune blood and used to construct two independent libraries of their VHHs. Since these VHH libraries were generated from immune blood, it is expected that they contain several antigen-specific VHHs. Such antigen-specific Nbs were retrieved after several rounds of panning of these phage-displayed VHH libraries. Remarkably, many of the antigen-specific binders had a striking homologous amino acid sequence motif in their CDR3 region. These sequences are indeed representative of true VHHs as they contain the key-amino acid substitutions (Muyldermans et al., 1994; Vu et al., 1997) in framework 2 at position 37, 44, 45 and 47 (Kabat numbering) occupied by Phe, Glu, Arg and Phe respectively.
The recombinant VHH selected from these libraries were well expressed, soluble and showed high epitope specificity for the target antigen. They exhibited a broad range of kinetic rate constants for their interaction with antigens, leading to equililbrium constant in the nanomolar range [from (9nM to 781 nM) and 11 nM to 110nM) for SNA-23 and ZAG respectively].
Because of their small size, robust nature and the above properties, the recombinant Nbs recognizing SNAP-23 and ZAG with affinity in the lower nM range might lead to the design of potent probes in new diagnostic detection tools.

Date of Award	1 Sep 2008
Original language	English
Supervisor	Serge Muyldermans (Promotor) & Dirk Saerens (Co-promotor)