Optimized Generation of High-Affinity, High-Specificity Single-Chain Fv Antibodies from Multi-Antigen Immunized Chickens.

High-affinity, highly specific binding proteins are a key class of molecules used in the development of new affinity chromatography methods. Traditionally, antibody-based methods have relied on the use of immunoglobulins purified from immune animal sera, from egg yolks, or from murine monoclonal hybridoma supernatants. To accelerate and refine the reagent antibody generation process, we have developed optimized methods that allow the rapid assembly of scFv libraries from chickens immunized with pools of immunogens. These methods allow the simplified generation of a single, moderately sized library of single chain Fv (scFv) and the subsequent isolation of diverse, high affinity, and high specificity monoclonals for each individual immunogen, via phage display. Using these methods, antibodies can be derived that exhibit the desired selectivity, including exquisite specificity or cross-reactivity to multiple orthologues of the same protein.

CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery.

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and>18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with<2% high molecular weight species present after 7 weeks at 4 °C and viscosity<15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.

Comprehensive interrogation of a minimalist synthetic CDR-H3 library and its ability to generate antibodies with therapeutic potential.

We have generated large libraries of single-chain Fv antibody fragments (>10(10) transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3. Both libraries provided diverse, specific antibodies, yielding a total of 243 unique hits against 7 different targets, but WySH2B produced fewer hits than WySH2A when selected in parallel. WySH2A also consistently produced hits of similar quality to WySH2B, demonstrating that the diversification of the CDR-L3 reduces library fitness. Despite the absence of deliberate bias in the library design, CDR length was strongly associated with the number of hits produced, leading to a functional loop length distribution profile that mimics the biases observed in the natural repertoire. A similar trend was also observed for the CDR-L3. After target selections, several key amino acids were enriched in the CDR-H3 (e.g., small and aromatic residues) while others were reduced (e.g., strongly charged residues) in a manner that was specific to position, preferentially occurred in CDR-H3 stem positions, and tended towards residues associated with loop stabilization. As proof of principle for the WySH2 libraries to produce viable lead candidate antibodies, 114 unique hits were produced against Delta-like ligand 4 (DLL4). Leads exhibited nanomolar binding affinities, highly specific staining of DLL4+ cells, and biochemical neutralization of DLL4-NOTCH1 interaction.

An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.

Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.

Fundamental characteristics of the immunoglobulin VH repertoire of chickens in comparison with those of humans, mice, and camelids.

Examination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R(2) = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the V(H) that exhibits 64 nM (K(D)) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.