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. 2016 May 5;5(6):e1168557.
doi: 10.1080/2162402X.2016.1168557. eCollection 2016 Jun.

Successful Engineering of a Highly Potent Single-Chain Variable-Fragment (scFv) Bispecific Antibody to Target Disialoganglioside (GD2) Positive Tumors

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Free PMC article

Successful Engineering of a Highly Potent Single-Chain Variable-Fragment (scFv) Bispecific Antibody to Target Disialoganglioside (GD2) Positive Tumors

Ming Cheng et al. Oncoimmunology. .
Free PMC article

Abstract

Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca(2+) flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity.

Keywords: Affinity; bispecific; disialoganglioside GD2; immunotherapy; scFv; stability.

Figures

Figure 1.
Figure 1.
Design and characterization of hu3F8-scBA. (A) Structural model showing a top down view of the antigen-biding site of hu3F8 scFv in the VL–VH orientation. CDR loops are colored in blue. A homology model was generated on Discovery Studio 4.1 (Dassault Systemes, San Diego, CA) using the crystal structure of murine 3F8 as a template (PDB 3VFG). The model was rendered in PyMol (Schrödinger LLC, New York, NY). (B) Diagram of hu3F8 scBA, with anti-GD2 scFv in the VL–VH format, and the anti-CD3 scFv in the VH–VL format. (C) Reduced SDS-PAGE analysis of hu3F8-scBA (D) HPLC profile of purified hu3F8-scBA. The peak with a retention time of 21 minutes (*) is hu3F8-scBA, while the peak with a retention time of 25 min is from absorbance of salt in the buffer (sodium citrate).
Figure 2.
Figure 2.
GD2 binding properties of hu3F8-scBA and 5F11-scBA. (A) Comparison of hu3F8-scBA and 5F11-scBA GD2 binding by ELISA. (B) Comparison of hu3F8-scBA and 5F11-scBA GD2 binding kinetics by SPR. Sensorgram depicts 1,000 nM run from each scBA binding to GD2, normalized to 100 RU.
Figure 3.
Figure 3.
T-cell activation by hu3F8-scBA and 5F11-scBA. Human T cells were preincubated with three different concentrations of scBA (1–10 nM) and imaged on artificial lipid bilayers containing ICAM-1 and GD2. Ca2+ responses were measured using Fura-2AM. The Fura ratio was calculated during the plateau phase of the calcium response (15–30 min), from six to eight imaging fields per condition (∼100 cells/field). Each dot represents the mean Fura ratio of one field acquired over 15 min.
Figure 4.
Figure 4.
Hu3F8-scBA and 5F11-scBA redirected T-cell cytotoxicity. (A) Neuroblastoma IMR-32. (B) Melanoma M14. (C) Melanoma SKMEL-28. (D) Ovarian Carcinoma SKOV3. T cells were incubated with 51Cr-labeled target cancer cells (10:1 E:T ratio) in the presence of a dilution series of scBA for 4 h. Cytotoxicity was measured by the amount of 51Cr released in the supernatant, counted by a γ-counter. Killing curves were analyzed with Graphpad Prism 6 using a nonlinear fit (log(agonist) vs response-Variable slope (four parameters)).
Figure 5.
Figure 5.
Efficacy of 5F11-scBA and hu3F8-scBA against human neuroblastoma/melanoma xenografts in DKO mice. (A) Neuroblastoma (IMR-32) or (B) melanoma (M14) and PBMCs were mixed (1:1) and coinjected subcutaneously on day 0. Treatment with scBA initiated on day 5, with 180 picomoles daily for 12 d (2 weeks). (C). Neuroblastoma (IMR32) cells and PBMCs were injected intravenously. 0.5 million neuroblastoma cells were injected on day 0, PBMCs were injected on days 6 and 12, and scBA treatment was initiated on day 6 at 180 picomoles daily for 13 d. n = 5 for each group. Bioluminescence is quantified as emitted photos per whole mouse per second.

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