High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

Abstract

Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.

Bispecific Antibody-Functionalized Upconversion Nanoprobe

“…previous work, the BsAb shows high binding affinity to both mPEG (K D 10 nM) and EphA2 (K D 1 nM) 20 , which is comparable to the normal EphA2 IgG antibody (K D 0.1 nM) and binders (K D 10~100 nM) 21 .…”

Engineered Autonomous Human Variable Domains

“…Autonomous constant (C H 2) domains derived from human IgG have also been engineered as antigen-binding scaffolds [14]. An attractive feature of engineered C H 2 domains is their potential for both antigen and FcRn binding, the later of which prolongs serum half-life [15, 16]. Soluble autonomous C H 3 domains have been described [17] and loops on C H 3 have been recruited for antigen binding in so-called Fcabs (Fc antigen binding) [18–20].…”

Component‐resolved microarray analysis of IgE sensitization profiles to Culicoides recombinant allergens in horses with insect bite hypersensitivity

“…A total of 347 horses were included, of which 235 horses belonged to the Icelandic breed. The other 112 horses belonged to various breeds pastoris, VALIDOGEN GMBH (formerly VTU Technology), Grambach, AT),42 resulting in a total of 44 Culicoides r-proteins included on the array (Table2and TableS1).Additionally, a Cul n thorax extract (CN-TE43 ) and a whole-body extract from Cul o group midges (CO-WBE)16 as well as a black fly extract (Simulium vittatum [SV-WBE]44 were used. As negative control antigens, the recombinant mould allergen Alternaria alternate 1 (Alt a 1, Biomay, www.biomay.com) and a house dust mite extract (Dermatophagoides farinae [Der f], Stallergenes Greer, www.stall ergen esgre er.com) were also assessed (Table2).…”