Exploiting the plasticity of the single domain VHH antibody scaffold: Unique hapten recognition and engineered metalloregulation

Antibodies are invaluable affinity reagents in many diagnostic and therapeutic applications. Camelid heavy chain-only single domain (VHH) antibodies are of particular interest because despite lacking a variable light chain, they possess affinities and specificities rivaling full length IgG antibodies. In addition, their relative simplicity and ideal biophysical properties make the VHH antibody an excellent scaffold for protein engineering.;The first study reveals the structural and biophysical details for an anti-Methotrexate (MTX) VHH antibody. Initial grafting of the CDRs 1-3 from a previously published anti-MTX VHH antibody onto an anti-RNase A VHH scaffold produced the anti-MTX VHH CDR1-3 Graft. This graft displayed a significantly weaker binding affinity than the published value (KD, observed = 6.7 microM versus a KD, published = 29 nM). Surprisingly, the anti-MTX VHH CDR1-3 Graft-MTX complex structure revealed a novel cryptic binding site whereby the MTX tunneled underneath CDR1 and formed contacts with the non-hypervariable CDR4. As the CDR4 loop was not initially included in generating the anti-MTX VHH CDR1-3 Graft, the CDR4 loop was grafted onto the CDR1-3 scaffold, generating the anti-MTX VHH CDR1-4 Graft. ITC revealed a 1,000-fold increased affinity (KD = 4 nM) with the anti-MTX CDR1-4 graft VHH. Crystal structure determination of both the unbound and complex anti-MTX CDR1-4 graft VHH revealed that the CDR4 loop forms different contacts with MTX in the two VHH grafts. Additionally, both anti-MTX VHH Grafts were specific for MTX versus the analogues aminopterin and folate as judged by binding dissociation constants.;The second half of the dissertation focuses on developing novel synthetic combinatorial library techniques to engineer multi-specificity into an anti-RNase A VHH antibody. A combinatorial histidine scanning library was designed to introduce a competitive metal binding site into the anti-RNase A VHH antigen binding interface. When combined with a novel co-selection strategy, the histidine scanning library produced a VHH variant termed the Metal VHH which incorporated three histidines on the periphery of the antigen binding interface. Biophysical investigations of Metal VHH revealed that it was specific for Ni2+ versus other transition metal ions and its RNase A affinity could be tuned based on the amount of metal ion present. Further structural and biophysical investigations demonstrated that metalloregulation of VHH/RNase A binding was facilitated by a dual-function histidine and a flexible CDR1.;Despite the large number of possible variants within the combinatorial histidine scanning library, only one dual-specific VHH emerged. To determine whether sampling an enriched pool of metal coordinating amino acids could produce additional metal sensitive anti-RNase A VHH variants, a combinatorial library, which sampled His/Glu/Asp/Gln/WT (HEDQ) across 18 positions of the anti-RNase A VHH antigen binding interface, was explored. After three rounds of co-selection, four anti-RNase A VHH variants termed HEDQs 1-4 emerged. It was discovered that an error was made in generating the HEDQ3 for biophysical and structural studies as it contained remnants of the original Metal VHH, this variant was subsequently termed HEDQ3*. Biophysical characterizations reveal that both HEDQs 3* and 4 display negligible energetic penalties towards intrinsic RNase A affinity. Further, HEDQ3* appears to possess multiple copper (II) binding sites and HEDQ4 possesses one enriched affinity Cu2+ binding site compared to the Metal VHH-Ni2+ binding affinity.;This dissertation demonstrates the malleable nature of the single domain VHH antibody. The anti-MTX VHH revealed a novel antibody-hapten binding site while the Metal and HEDQ VHHs demonstrated that a VHH binding interface can accommodate multiple specificities with minimal impact to the original intrinsic binding affinity. In each case, the VHH binding interface underwent significant transformations to recognize its antigens.