Selection and characterization of cell binding and internalizing phage antibodies.

Many therapeutic targets are cell surface receptors, which can be challenging antigens for antibody generation. For many therapeutic applications, one needs antibodies that not only bind the cell surface receptor but also are internalized into the cell. This allows use of the antibody to deliver various payloads into the cell to achieve a therapeutic effect. Phage antibody technology has proven a powerful tool for the generation and optimization of human antibodies to any antigen. While applied to the generation of antibodies to purified proteins, it is possible to directly select cell binding and internalizing antibodies on cells. Potential advantages of this approach include: cell surface receptors are in native conformation on intact cells while this might not be so for recombinant proteins; antibodies can be selected for both cell binding and internalization properties; the antibodies can be used to identify their tumor associated antigens; and such antibodies can be used for human treatment directly since they are human in sequence. This review will discuss the factors that impact the successful selection of cell binding and internalizing antibodies. These factors include the cell types used for selection, the impact of different phage antibody library formats, and the specific selection protocols used.

Multicolor fluorescence activated cell sorting to generate humanized monoclonal antibody binding seven subtypes of BoNT/F.

Generating specific monoclonal antibodies (mAbs) that neutralize multiple antigen variants is challenging. Here, we present a strategy to generate mAbs that bind seven subtypes of botulinum neurotoxin serotype F (BoNT/F) that differ from each other in amino acid sequence by up to 36%. Previously, we identified 28H4, a mouse mAb with poor cross-reactivity to BoNT/F1, F3, F4, and F6 and with no detectable binding to BoNT/F2, F5, or F7. Using multicolor labeling of the different BoNT/F subtypes and fluorescence-activated cell sorting (FACS) of yeast displayed single-chain Fv (scFv) mutant libraries, 28H4 was evolved to a humanized mAb hu6F15.4 that bound each of seven BoNT/F subtypes with high affinity (KD 5.81 pM to 659.78 pM). In contrast, using single antigen FACS sorting, affinity was increased to the subtype used for sorting but with a decrease in affinity for other subtypes. None of the mAb variants showed any binding to other BoNT serotypes or to HEK293 or CHO cell lysates by flow cytometry, thus demonstrating stringent BoNT/F specificity. Multicolor FACS-mediated antibody library screening is thus proposed as a general method to generate multi-specific antibodies to protein subtypes such as toxins or species variants.

A regulating role of the JAK2 FERM domain in hyperactivation of JAK2(V617F).

JAK2 (Janus tyrosine kinase 2) is important for signalling through many cytokine receptors, and a gain-of-function JAK2 mutation in its pseudokinase domain, V617F, has been implicated in Philadelphia chromosome-negative myeloproliferative neoplasms. How this mutation hyperactivates JAK2 is poorly understood. In the present paper we report our findings that the V617F mutation has little effect on the Vmax of JAK2 kinase activity, but lowers the Km value for substrates. Therefore under physiological conditions where the concentration level of substrates is presumably below saturation, JAK2(V617F) exhibits hyperactivation compared with wild-type JAK2. This lower Km of JAK2(V617F) towards substrates requires the JAK2 FERM (4.1/ezrin/radixin/moesin) domain, as deletion of the FERM domain abolished this effect. We also show that, in contrast with its positive role in JAK2(V617F) hyperactivation, the FERM domain in wild-type JAK2 is inhibitory. Deletion or mutations of the FERM domain resulted in increased basal JAK2 kinase activity. The results of the present study provide the biochemical basis for how V617F hyperactivates JAK2, and identifies novel regulating roles of the JAK2 FERM domain to control kinase activity at different activation states.

A JAK2 interdomain linker relays Epo receptor engagement signals to kinase activation.

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu(543)-Asp(544) in this linker or Leu(611), Arg(683), or Phe(694) in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.

Discovery of internalizing antibodies to basal breast cancer cells.

We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies. The unique mAbs were further screened on nine basal and seven luminal cell lines to identify those with the greatest affinity, specificity, and internalizing capability for basal-like breast cancer cells. Among the internalizing basal-specific mAbs were those recognizing four transmembrane receptors (EphA2, CD44, CD73 and EGFR), identified by immunoprecipitation-mass spectrometry and yeast-displayed antigen screening. Basal-like breast cancer expression of these four receptors was confirmed using a bioinformatic approach, and expression microarray data on 683 intrinsically subtyped primary breast tumors. This overall approach, which sequentially employs phage display antibody library selection, antigen identification and bioinformatic confirmation of antigen expression by cancer subtypes, offers efficient production of high-affinity mAbs with diagnostic and therapeutic utility against specific cancer subtypes.

High throughput identification of monoclonal antibodies to membrane bound and secreted proteins using yeast and phage display.

Antibodies are ubiquitous and essential reagents for biomedical research. Uses of antibodies include quantifying proteins, identifying the temporal and spatial pattern of expression in cells and tissue, and determining how proteins function under normal or pathological conditions. Specific antibodies are only available for a small portion of the proteome, limiting study of those proteins for which antibodies do not exist. The technologies to generate target-specific antibodies need to be improved to obtain high quality antibodies to the proteome at reasonable cost. Here we show that renewable, validated, and standardized monoclonal antibodies can be generated at high throughput, without the need for antigen production or animal immunizations. In this study, 60 protein domains from 24 selected secreted proteins were expressed on the surface of yeast and used for selection of phage antibodies, over 400 monoclonal antibodies were identified within 3 weeks. A subset of these antibodies was validated for binding to cancer cells that overexpress the target protein by flow cytometry or immunohistochemistry. This approach will be applicable to many of the membrane-bound and the secreted proteins, 20-40% of the proteome, accelerating the timeline for Ab generation while reducing the cost.