Isolation and optimization for affinity and biophysical characteristics of anti-CCL17 antibodies from the VH1-69 germline gene.

CCL17 is a homeostatic chemokine associated with several human inflammatory pathologies. This makes CCL17 a potential point of intervention in inflammatory diseases. Using a Fab-pIX phage display system we were able to select antibodies that specifically bind to CCL17 and neutralize CCL17-mediated signaling. Many of the selected antibodies belong to the VH1-69 germline gene family. The VH1-69 germline gene is represented at a high frequency in the human antibody repertoire and is seen in the early immune response to a variety of pathogens. The heavy chain CDR2 of this germline gene is notably hydrophobic and can insert into hydrophobic pockets of antigens, providing much of the binding energy for these antibodies. Affinity maturation of our primary binders by light chain mutagenesis produced antibodies with sub-nanomolar affinities, with affinity improvements up to 100-fold. These were screened for non-specific protein-protein interactions as a filter for solubility. All of our high affinity antibodies were found to have high levels of non-specific protein-protein interactions. We speculated that this was due to the hydrophobicity within the germline heavy chain CDR1 and CDR2. To ameliorate this problem, we generated a phage display library for one of the clones, where the surface-exposed residues within H-CDR1 and H-CDR2 were randomized. High stringency panning of this library against human CCL17 resulted in further affinity improvement, along with reduction in protein-protein interaction in some new variants. In addition, we improved the cross-reactivity to cynomolgus CCL17. We demonstrate that affinity maturation through targeted libraries in the VH1-69 germline gene can improve both affinity and biophysical characteristics of antibodies derived from this gene scaffold.

Selection and maturation of antibodies by phage display through fusion to pIX.

Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM.

Proteomic Approach to Evaluate Mechanisms That Contribute to Food Allergenicity: Comparative 2D-DIGE Analysis of Radioallergosorbent Test Positive and Negative Patients.

Proteomic profiles of RAST(+) subjects with severe food allergies and RAST(-) subjects were compared using 2D-DIGE analysis to obtain candidate biomarkers specific to food allergies. Our analysis highlighted 52 proteins that were differentially expressed between the RAST(+) and RAST(-) groups of which 37 were successfully identified that include chondroitin sulfates, zinc finger proteins, C-type lectins, retinoic acid binding proteins, heat shock proteins, myosin, cytokines, mast cell expressed proteins, and MAP kinases. Biological network analysis tool Metacore revealed that most of these regulated proteins play a role in immune tolerance, hypersensitivity and modulate cytokine patterns inducing a Th2 response that typically results in IgE-mediated allergic response which has a direct or indirect biological link to food allergy. Identifying unique biomarkers associated with certain allergic phenotypes and potentially cross-reactive proteins through bioinformatics analyses will provide enormous insight into the mechanisms that underlie allergic response in patients with food allergies.

De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins.

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V(L) and three V(H) domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V(H) by keeping V(L) in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V(H) diversity was sufficient for isolating Fabs to most antigens, whereas variability in V(L) was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V(L) diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.

A DNA-binding-independent pathway of repression by the Drosophila Runt protein.

DNA-binding proteins are important for regulating gene expression during development. It is widely assumed that this regulation involves sequence-specific DNA binding by these transcription factors to cognate cis-regulatory sequences of their downstream target genes. However, studies in both the Drosophila and the mouse model systems have provided examples in which the DNA-binding activity of a transcription factor is not essential for in vivo function. Using a system that allows for quantitative analysis of gene function in the Drosophila embryo, we have discovered a DNA-binding-independent activity of Runt, the founding member of the RUNX family of transcriptional regulators. Examination of the in vivo potency of a DNA-binding-defective form of Runt reveals differential requirements for DNA binding in the regulation of different downstream target genes. DNA binding is not required for establishing repression of the odd-numbered stripes of the segment polarity gene engrailed, but does contribute to Runt's role as a regulator of sloppy-paired, another downstream target gene in the pathway of segmentation. We investigate this DNA-binding-independent pathway using a genetic screen for dose-dependent modifiers of runt activity. These studies reveal that DNA-binding proteins encoded by the tramtrack locus cooperate with Runt to repress engrailed. These results provide new insights into the context-dependent regulatory functions of Runt domain proteins and provide a paradigm for understanding DNA-binding-independent regulation by developmentally important transcription factors.

Distinct in vivo requirements for establishment versus maintenance of transcriptional repression.

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. Here we show, by using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos, which indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression.