Rebecca To

Aggregation-resistant VHs selected by in vitro evolution tend to have disulfide-bonded loops and acidic isoelectric points*

When panned with a transient heat denaturation approach against target enzymes, a human V(H) (antibody heavy chain variable domain) phage display library yielded V(H)s with composite characteristics of binding, non-aggregation and reversible thermal unfolding. Moreover, selection was characterized by enrichment for V(H)s with (i) an even number of disulfide forming Cys residues in complementarity-determining region (CDR) 1 and CDR3 and (ii) acidic isoelectric points. This parallels naturally occurring camelid and shark single-domain antibodies (sdAbs) which are also characterized by (i) solubility and reversible unfolding, (ii) a high occurrence of disulfide forming Cys in their CDRs, particularly, in CDR1 and CDR3 and (iii) acidic V(H)s as inferred here by a pI distribution analysis, reported here, of pools of human and camelid V(H) and V(H)H (camelid heavy chain antibody V(H)) sequences. Our results, reinforced by previous observations by others, suggest that protein acidification may yet be another mechanism nature has devised to create functional sdAbs and that this concept along with the inclusion of inter-CDR disulfide linkages may be applied to human V(H) domains/libraries for non-aggregation optimization. In addition, calculation of theoretical pIs of V(H)s selected by panning may be used for rapid and precise identification of non-aggregating V(H)s.

Transient expression and purification of chimeric heavy chain antibodies

Monoclonal antibodies have been successfully engineered as approved therapeutics. However, their large size is considered a major factor preventing them from having a more efficient tissue penetration. As the first step to establish a possibly more efficient antibody platform, we present here transient expression, purification and characterization of six chimeric heavy chain antibodies (cHCAbs), or fusion of camelid single domain antibodies (sdAbs) to human fragment crystallizable (Fc). All six HCAbs have a MW of approximately 80 kDa, expressed well in a HEK293 expression system and have G0, G1 and G2 types of glycosylation. The transient expression also provided a very fast way to generate high milligram to low gram amount of proteins for in vitro assays and preliminary animal studies.

The role of valency in the selection of anti-carbohydrate single-chain Fvs from phage display libraries

Several strategies were investigated for phage display of anti-carbohydrate single-chain Fvs (scFvs) using the anti-Salmonella Se155-4 scFv as a model system. All were based on the knowledge that panning V(H) CDR libraries displayed in a standard pIII phagemid/helper phage format against immobilized multivalent carbohydrate antigens selects almost solely for mutants with higher yields of soluble product or scFvs that form dimers or higher oligomers even when the linker length is chosen to give monomeric molecules. Construction of scFv libraries, in a phagemid vector, with mutations that already provide higher yields and/or short linkers to promote dimeric display greatly reduced these undesired selection pressures. However. the panning of an error-prone library of the entire scFv in a short linker format yielded two mutants that existed partially in higher oligomeric forms, indicating that dimeric display did not entirely eliminate the selection pressure problem. In one mutant a Ser75Gly mutation led to the formation of greater amounts of dimeric, trimeric and tetrameric scFv and surface plasmon resonance analysis of these different forms gave quantitative data for the functional affinity of these different scFv forms. Finally, a phage vector was constructed and the original V(H) CDR library was transferred to this vector. This display format, in which scFv is displayed on all three to five copies of pIII, seemed to be superior in terms of selection on the basis of binding site affinity and as a display mode for scFvs with low intrinsic affinity. While the use of the phage vector did not lead to the isolation of higher affinity binders from the library employed here, it did almost entirely remove the undesired selection pressures in that there was selection for the wild-type sequence. It is suggested that the multivalency of display provided by phage vectors is preferable to any phagemid vector strategy for the de novo isolation of anti-carbohydrate antibodies from phage libraries.

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy.

We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (Tm), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their Tms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin.

Preliminary crystal structure analysis of an Fab specific for a Salmonella O-polysaccharide antigen.

The Fab from an IgG1, lambda murine monoclonal antibody with specificity for the O-polysaccharide antigen of Salmonella typhimurium has been crystallized in the absence and presence of hapten. The conditions for crystal growth were vapor diffusion equilibration with 16 to 23% polyethylene glycol 8000 solutions. Data have been collected from crystals of the complex in space group P212121, a = 60.6 A, b = 111.3 A, c = 61.1 A, and refinement of a molecular replacement solution is underway.