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. 2015 Jan 15;136(2):476-86.
doi: 10.1002/ijc.29007. Epub 2014 Jun 19.

Structural Design of Disialoganglioside GD2 and CD3-bispecific Antibodies to Redirect T Cells for Tumor Therapy

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Free PMC article

Structural Design of Disialoganglioside GD2 and CD3-bispecific Antibodies to Redirect T Cells for Tumor Therapy

Ming Cheng et al. Int J Cancer. .
Free PMC article

Abstract

Antibody-based immunotherapy has proven efficacy for patients with high-risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single-chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T-cell-mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2-positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell-mediated therapy.

Keywords: bispecificity; disialoganglioside; immunotherapy; neuroblastoma; structure.

Figures

Figure 1
Figure 1. Molecular Modeling of anti-GD2 5F11-scFv in the 5HLDS(15)BA and 5LHDS(15)BA constructs
Molecular Modeling of anti-GD2 5F11-scFv in the 5HLDS(15)BA and 5LHDS(15)BA constructs. A) Ribbon diagram of 5F11-scFv in 5HLDS(15)BA (5F11 VH - linker - VL - disulfide stabilized), where VH-VL disulfide stabilizing residues are shown in stick rendering, and CDR main chain residues are shown in Blue. B) Ribbon diagram of 5F11-scFv in 5LHDS(15)BA (5F11 VL - linker - VH - disulfide stabilized), where VH-VL disulfide stabilizing residues are shown in stick rendering, and CDR main chain residues are shown in Green. C) Overlay of CDR loops of 5HLDS (Blue) and 5LHDS (Green). D) Overlay of CDR residues with docked model of GD2 for 5HLDS(15)BA:GD2 (Blue) and 5LHDS15BA:GD2 (Green). E) Interaction diagram of 5HLDS(15)BA:GD2 and F) Interaction diagram of 5LHDS(15)BA:GD2. Interacting residues are displayed as colored discs. Residues with H-bonding, charged or polar interactions are colored in magenta. Residues having van der Waals interactions are colored in green. H-bonds, and charge-charge interactions are displayed as dashed lines. The solvent accessible surface is shown as a diffuse background circle with the radius proportional to the exposure. The per residue interaction energies are shown in Supplemental Table III. The residues with the largest contributions to the interaction with GD2 are L: Arg90 and L: Tyr93, which are significantly reduced in 5LHDS(15)BA:GD2 (noted with *).
Figure 2
Figure 2. T cell cytotoxicity of LAN-1 cells in the presence of 5F11-BsAbs
T cell cytotoxicity of LAN-1 cells labelled with 51Cr was assayed in the presence of increasing concentrations of 5F11-BsAbs. Specific lysis was measured by 51Cr release in supernatant counted by a γ-counter.
Figure 3
Figure 3. In vivo tumor therapy using 5HLDS(15)BA(Y)
BALB-Rag2-/-IL-2R-rC-KO (DKO) mice were implanted with (A) 3×106 M14 (melanoma) only or mixed with 3×106 human PBMC (B) 5×106 IMR-32 (neuroblastoma) only or mixed with 5×106 human PBMC on day 0 and then treated with 5HLDS(15)BA(Y) at 20 µg daily intravenously starting on day 3 for a total of 20 days. Tumor size was measured twice a week and calculated with the formula: volume = (width) × length/2. *P < 0.05, unpaired t test. (C and D) M14 cells (1.5 million) were injected into DKO mice intravenously, treatment with intravenous BsAb was initiated on day 6 daily for a total of two weeks. PBMC were injected intravenously on day 6 and 13. Tumor growth was assessed by luminescence once a week starting on day 2. ***P < 0.001, unpaired t test.

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