A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization

Abstract

Various formats of bispecific antibodies exist, among them Two-in-One antibodies in which each Fab arm can bind to two different antigens. Their IgG-like architecture accounts for low immunogenicity and also circumvents laborious engineering and purification steps to facilitate correct chain pairing. Here we report for the first time the identification of a Two‐in‐One antibody by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibody simultaneously targets the epidermal growth factor receptor (EGFR) and programmed death‐ligand 1 (PD-L1) at the same Fv fragment with two non-overlapping paratopes. The dual action Fab is capable of inhibiting EGFR signaling by binding to dimerization domain II as well as blocking the PD-1/PD-L1 interaction. Furthermore, the Two-in-One antibody demonstrates specific cellular binding properties on EGFR/PD-L1 double positive tumor cells. The presented strategy relies solely on screening of combinational immune-libraries and obviates the need for any additional CDR engineering as described in previous reports. Therefore, this study paves the way for further development of therapeutic antibodies derived from avian immunization with novel and tailor-made binding properties.

TriTECM: A tetrafunctional T-cell engaging antibody with built-in risk mitigation of cytokine release syndrome

“…Antibodies were loaded onto AHC biosensors and varying concentrations of antigen were associated to the mAb. The determined affinities were in similar ranges to those cited in literature from previous studies ( 65 , 74 ), with only minor changes where the fused scFvs could presumably interfere with binding ( Table 2 ; Supplementary Figure 2 ). Nevertheless, the affinity to neither EGFR nor PD-L1 was greatly influenced by N- or C-terminal scFv fusions.…”

“…In this proof-of-concept study, we combined existing and well-characterised binding modules in different antibody formats to generate a trispecific T-cell engager with TME and cytokine release modulation (TriTECM). Due to its ability to bind and block both EGFR and PD-L1 pathways and having two binding sites for each target, a recently isolated two-in-one antibody ( 65 ) was chosen as the backbone, carrying an effector-silenced Fc to minimise cytotoxicity and extend the half-life. This molecule carries dual targeting abilities in a generic IgG1 antibody format, where the light chain mainly contributes to EGFR-binding and the heavy chain to PD-L1-binding.…”

“…(A) Molecular design of two TriTECM constructs, (αCD3ϵ)K(αIL-6R) + H(αIL-6R) as scFv(VH)-IgG1(H)-scFv 2 (purple) and K(αCD3ϵ) + H(αIL-6R) as an IgG1(H)-scFv 2 (orange). The blue/green variable regions represent the anti-CD3 NI0401-derived scFv, the red/blue variable regions represent the anti-IL-6R sarilumab-derived scFv (αIL-6R), the Fabs originate from HCP-LCE ( 65 ) and an effector-silenced IgG1 backbone was used. The light blue circle between the CH3 domains represent the Knob-into-Hole technology for heterodimerisation.…”

“…The HCP-LCE IgG backbone was used as a starting block ( 65 ). For the different scFvs, gene fragments were ordered from Twist Biosciences based on their published protein sequences ( 66 ) with respective Sap I overhangs.…”

“…Competition assays for PD-L1/PD-1 and IL-6/IL-6R were performed as previously described ( 65 , 69 , 70 ).…”