Tetravalent Antibody–scTRAIL Fusion Proteins with Improved Properties

Abstract

We applied the immunoglobulin E (IgE) heavy-chain domain 2 (EHD2) as the covalently linked homodimerization module to generate antibody-scTRAIL fusion proteins. By fusing a humanized single-chain fragment variable (scFv) directed against EGFR to the N-terminus of the EHD2 and a single-chain derivative of TRAIL (scTRAIL) to the C-terminus of the EHD2, we produced a dimeric, tetravalent fusion protein. The fusion protein retained its binding activity for EGFR and TRAIL receptors. In vitro, the targeted antibody-scTRAIL fusion protein exhibited an approximately 8-to 18-fold increased cytotoxic activity compared with the untargeted EHD2-scTRAIL fusion protein. This resulted in increased antitumor activity in a subcutaneous Colo205 xenograft tumor murine model. In summary, the scFv-EHD2-scTRAIL fusion protein combines target cell selectivity with an increased TRAIL activity leading to improved antitumor activities.

An optimized antibody-single-chain TRAIL fusion protein for cancer therapy

“…18,20,21,26,27 In addition to a high specific activity, clinically useful recombinant protein therapeutics must also have suitable protein stability and pharmacokinetic properties. 24,28,29 Toward this end, we here describe singlechain variants of the apoptosis-inducing TRAIL with improved stability and superior product quality in the form of a tumortargeted fusion protein, resulting in high anti-tumor activity in a xenograft tumor model in vivo.…”

“…Aliquots were stored at ¡80 C. DR4-Fc, DR5-Fc and EGFR-Fc fusion proteins were produced and purified as previously described. 21 Biochemical and biophysical protein analysis Affinity-purified scTRAIL proteins were analyzed by SDS-PAGE under reducing conditions and stained with InstantBlue (Expedeon, ISB1L). For Western blotting, monoclonal anti-FLAG M2 antibody (1 mg/ml in PBS) was used, followed by anti-mouse IgG alkaline phosphatase-coupled secondary antibody (1:15,000 in PBS with 0.05% Tween 20, Sigma-Aldrich, A3562) for detection.…”

“…The thermal stability of the proteins was determined by measuring denaturation temperatures with dynamic light scattering using a ZetaSizer Nano ZS instrument (Malvern) as published. 21 ELISA ELISA plates were coated with TRAIL-R1-Fc, TRAIL-R2-Fc or EGFR-Fc 21 (300 ng/well in 0.1 M sodium carbonate buffer, pH 9.5) overnight at 4 C. The plates were blocked with 3% (w/v) skim milk powder in PBS (MPBS). The scTRAIL fusion proteins were diluted in MPBS, titrated 1:3 in duplicates starting from 500 nM and incubated for 2 h at room temperature on a shaker.…”

“…18,20 ScTRAIL molecules have been successfully used to generate antibody TRAIL fusion proteins to achieve specific targeting and to improve pharmacokinetic properties with the aim to enrich the therapeutic protein at the tumor site. 18,20,21 Moreover, depending on the antibody format used as fusion partner for scTRAIL, we recently showed that a controlled oligomerization of the scTRAIL module can be achieved, for example, through fusion of a diabody or a dimerization module such as the heavy chain domain 2 of IgE, resulting in substantially increased biological activity compared to sTRAIL, scTRAIL, and targeted scTRAIL in a monomeric scFv fusion protein. 20,21 Because we noted a rather low thermal stability of scTRAIL with a melting temperature of approximately 46 C, as determined by dynamic light scattering, we set out to develop derivatives of scTRAIL with increased biophysical and biochemical properties as a prerequisite for clinical development of tumor-targeted TRAIL-based therapeutics.…”

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

“…16 Interestingly, the homodimers of IgM or IgE CH2 domains have been used as homodimerization scaffolds for fusion proteins. 17 However, the use of Cɛ2 as a heterodimerization platform has not been reported. Here, we describe a novel solution called EFab domain substitution that is based on fusion of the variable domains of an antibody to the Cɛ2 domain of the IgE Fc.…”