A panel of anti-influenza virus nucleoprotein antibodies selected from phage-displayed synthetic antibody libraries with rapid diagnostic capability to distinguish diverse influenza virus subtypes.

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10-15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90-94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.

Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models.

HER2-ECD (human epidermal growth factor receptor 2 - extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies' propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.

Predominant structural configuration of natural antibody repertoires enables potent antibody responses against protein antigens.

Humoral immunity against diverse pathogens is rapidly elicited from natural antibody repertoires of limited complexity. But the organizing principles underlying the antibody repertoires that facilitate this immunity are not well-understood. We used HER2 as a model immunogen and reverse-engineered murine antibody response through constructing an artificial antibody library encoded with rudimentary sequence and structural characteristics learned from high throughput sequencing of antibody variable domains. Antibodies selected in vitro from the phage-displayed synthetic antibody library bound to the model immunogen with high affinity and specificities, which reproduced the specificities of natural antibody responses. We conclude that natural antibody structural repertoires are shaped to allow functional antibodies to be encoded efficiently, within the complexity limit of an individual antibody repertoire, to bind to diverse protein antigens with high specificity and affinity. Phage-displayed synthetic antibody libraries, in conjunction with high-throughput sequencing, can thus be designed to replicate natural antibody responses and to generate novel antibodies against diverse antigens.

Antibody variable domain interface and framework sequence requirements for stability and function by high-throughput experiments.

Protein structural stability and biological functionality are dictated by the formation of intradomain cores and interdomain interfaces, but the intricate sequence-structure-function interrelationships in the packing of protein cores and interfaces remain difficult to elucidate due to the intractability of enumerating all packing possibilities and assessing the consequences of all the variations. In this work, groups of ß strand residues of model antibody variable domains were randomized with saturated mutagenesis and the functional variants were selected for high-throughput sequencing and high-throughput thermal stability measurements. The results show that the sequence preferences of the intradomain hydrophobic core residues are strikingly flexible among hydrophobic residues, implying that these residues are coupled indirectly with antigen binding through energetic stabilization of the protein structures. By contrast, the interdomain interface residues are directly coupled with antigen binding. The interdomain interface should be treated as an integral part of the antigen-binding site.

Rationalization and design of the complementarity determining region sequences in an antibody-antigen recognition interface.

Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes.

Effects of signal sequence on phage-displayed disulfide-stabilized single chain antibody variable fragment (sc-dsFv) libraries.

Phage-displayed single chain variable fragment (scFv) libraries are powerful tools in antibody engineering. Disulfide-stabilized scFv (sc-dsFv) with an interface disulfide bond is structure-wise more stable than the corresponding scFv. A set of recently discovered signal sequences replacing the wild type (pelB) signal peptidase cleavage site in the c-region has been shown to be effective in rescuing the expression of sc-dsFv libraries on the phage surface. However, the effects of the other regions of the signal sequence on the expression of the sc-dsFv libraries and on the formation of the interface disulfide bond in the phage-displayed sc-dsFv have not been clear. In this work, selected novel signal sequence variants in the h-region were shown to be equally effective in promoting sc-dsFv library expression on the phage surface; the expression level and complexity of the sc-dsFv libraries were comparable to the corresponding scFv libraries produced with the wild-type (pelB) signal sequence. The interface disulfide bond in the phage-displayed sc-dsFv was proven to form to a large extent in the library variant ensemble generated with signal sequence variants in both the h-region and the c-region. The sc-dsFv engineering platform established in this work can be applied to many of the known scFv molecules which are in need of a more stable version for the applications under harsh conditions or for longer shelf-life.

Engineering anti-vascular endothelial growth factor single chain disulfide-stabilized antibody variable fragments (sc-dsFv) with phage-displayed sc-dsFv libraries.

Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv.