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Humanization of Nanobodies and Identification of Nanobodies for Alzheimer's Disease Immunotherapy

Tuesday, 30 June, 2009 - 17:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculty: Science and Bio-engineering Sciences
Cécile Vincke
phd defence

Almost a century ago, antibodies were envisioned as ‘magic bullets’ for the specific targeting of disease sites,
but until recently they struggled to meet their expectations. Clinical success with monoclonal antibodies has
now reinforced the role of these molecules as a major source of drugs for the treatments of human diseases.
Furthermore, protein engineering led to the design of smaller recombinant antibody fragments, such as Fabs
(~50 kDa) and single-chain Fv fragments (~25 kDa), which are emerging as credible alternatives. Even single
immunoglobulin variable domains have been engineered and shown to retain their antigen-binding capacity
and functionality. It is anticipated that these domain antibodies (dAbs) will expand the repertoire of antibodybased
reagents against a vast range of novel biomarkers. Unfortunately, dAbs are often associated with poor
stability and solubility and low levels of expression. Nanobodies, single-domain antigen-binding fragments of
camelid-specific heavy-chain only antibodies offer in this respect special advantages over classical antibody
fragments because of their smaller size, robustness, improved solubility and a high degree of specificity and
affinity for their antigen without requiring VL-domain pairing.

The beneficial properties of Nanobodies have been attributed to the unique presence of four hallmark amino
acids in the framework-2 region (positions 42, 49, 50, 52) and a longer third antigen-binding loop (H3) folding
over this area. Human dAbs have been engineered, i.e. camelized, by integrating in their sequences the
Nanobody hallmark residues, in an attempt to develop well-behaved entities. Additional studies suggested that
other mutations within a VH domain might contribute to the solubility of dAbs as well. We evaluated, in this
study the substitution of a residue at position 118, abutting the CDR3 region as an alternative approach to
generate an autonomous human dAb library.

However, in many aspects, Nanobodies remain superior to camelized VH domains. Obviously, for therapeutic
applications, Nanobodies require to be humanized, i.e. mutating camelid-specific amino acid sequences in the
framework to their human VH equivalent. We performed this humanization exercise on Nanobodies from
different VHH subfamilies and investigated the effects on their biochemical and biophysical properties. We
demonstrated that the humanization of Nanobody-specific residues outside framework-2, are neutral to the
Nanobody properties. Surprisingly, the Glu49Gly and Arg50Leu humanization of hallmark amino acids
generates a single domain that is more stable though probably less soluble. The other framework-2
substitutions, Tyr/Phe42Val and Gly/Ala52Trp, are detrimental for antigen affinity, due to a repositioning of
the H3 loop as shown by their crystal structures. In addition, imprinting the sequence signature of a human VH
in a VHH restores the VL binding capacity of a Nanobody with a short CDR3 loop that does not cover the
former VL binding site. These insights were employed to identify a soluble, stable, well expressed universal
humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies for transfer
of the antigen specificity and affinity.

Nanobodies are expected to provide new binding specificities and are creating possibilities for the development
of therapeutic compounds to treat amyloid disorders such as Alzheimer’s disease, a neurodegenerative
impairment evolving towards a major public health issue. The amyloid cascade hypothesis holds that
generation and deposition of the amyloid β-peptide (Aβ; 40-42 amino acids), obtained by cleavage of the
amyloid precursor protein (APP) by β- and γ-secretase, are key events driving neurodegeneration in AD. We
explored several approaches based on Nanobodies aiming at reducing or preventing Aβ production and
fibrillation to develop next-generation therapeutics. Successful immunization of a llama with Aβ resulted in the
isolation of several Aβ-specific Nanobodies, which recognize their antigen with affinities in the nanomolar
range. The targeted epitope was identified to be located between the residues 18 and 25 of the Aβ-peptide.
This region of the peptide plays a central role in the peptide-peptide interactions, which was confirmed by the
ability of the Aβ-specific Nanobodies to inhibit fibril formation in a concentration dependent manner.
Alternatively, the unique property of Nanobodies to preferentially target the catalytic site of enzymes provides a
unique opportunity to reduce Aβ accumulation by inhibiting β-secretase (BACE1). BACE1 inhibitors are
widely accepted as a promising therapeutic strategy since this enzyme represents the initiating and rate-limiting
step in the production of the Aβ-peptide. A myriad of Nanobodies were selected targeting different regions of
BACE1 and were characterized on their ability to inhibit the enzyme’s activity. Three different inhibitory
Nanobodies were identified and demonstrated to influence APP processing in primary neurons.