Background Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide expressed in various tissues including the pancreas, intestines and central nervous system.1, 2 The overexpression of VIP and its receptors is associated with increased growth and metastasis of breast, prostate, and lung malignancies.3 In addition, the interaction of VIP with its receptors on activated T cells results in immune suppression which further supports tumor growth.4, 5 Furthermore, tumor-supporting regulatory T cells have been found to be promoted by VIP-dependent mechanisms.6 Altogether, prior literature implies that blockade of VIP signaling may inhibit tumor-mediated immune suppression and augment antitumor immune responses. Recent preclinical studies in acute myeloid leukemia and T lymphoblastic leukemia demonstrated that VIP receptor antagonists increase T cell-dependent anti-tumor responses.2. Unfortunately, the short-half lives of peptide antagonists limit their clinical utility. A more translatable approach is the development of long circulating antibodies that bind VIP and inhibit its immunosuppressive activities.
Methods In this study, we utilized a yeast display of a non-immune human single-chain variable fragment (scFv) library to identify VIP-binding scFvs.7-9 VIP binders were screened by several rounds of selection using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The enriched binder population was cloned into single colonies of yeast cells by limited dilution. The binding affinities of VIP-binding clones were evaluated via flow cytometry by titrating fluorescence-labeled VIP. Clones with high binding affinity (Kd < 500 nM) were selected for sequencing (figures 1-5).
Results Sequences of the isolated scFv revealed that a unique section of complementarity-determining region 3 (CDR3) of the heavy chain played an important role in VIP binding. Multiple clones with similar but distinct CDR3 sequences produced a useful range of binding affinities for further development (figure 6).
Conclusions Yeast display is an effective technology for identifying human scFvs that bind to the immunosuppressive neuropeptide, VIP. CDR3 of scFv heavy chains were influential in VIP recognition. Ongoing studies are focused on the production, purification, and validation of novel anti-VIP human antibodies.
Acknowledgements We thank Dr. K. Dane Wittrup (Massachusetts Institute of Technology) for kindly providing yeast libraries, Drs. Edmund K. Waller and Jens Wrammert (Emory University) for helpful discussions and comments, and Dr. Ryan W. Paerl (North Carolina State University) for technical advice on cell sorting.
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