Structural design of disialoganglioside GD2 and CD3-bispecific antibodies to redirect T cells for tumor therapy

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.

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

“…For example, diL2K scFv is constructed as V H -V L and 5-10xdiL2K BiTE as 5-10(V L -V H )xdiL2K(V H -V L ). However, other factors should also be taken into consideration during novel KIH design such as 1) the orientation within the scFv (V L -V H vs. V H -V L ), 59,60 2) the order of 2 scFvs on BiTE construct, 61,62 and 3) the length and composition of the linkers (between 2 scFv as well as between V L and V H ), 63,64 all of which likely contribute to KIH folding and assembly.…”

GD2-Targeted Immunotherapy and Radioimmunotherapy

“…131 Anti-GD2 BsAb can kill a wide spectrum of GD2(+) tumors with femtomolar EC50 in the presence of expanded human T-cells, a potency orders of magnitude stronger than the nanomolar or picomolar EC50 for IgG mediated ADCC or CMC. 132,133 GD2/CD3 BsAbs have demonstrated high efficiency in tumor ablation in xenograft models. 132–134 Both tandem scFv format (BiTE) 132 as well as IgG-scFv modular formats 133 have shown anti-tumor efficacy.…”

Human derived dimerization tag enhances tumor killing potency of a T-cell engaging bispecific antibody

“…More recently, several laboratories have reported the presence of GD2 on breast cancer stem cells, 14,15 neuroectodermal 16 and mesenchymal stem cells. 17,18 The anti-GD2£anti-CD3 tsc-BsAb (GD2xCD3) were composed of single polypeptide chains containing the scFv of anti-GD2 monoclonal antibody 5F11 [19][20][21] and the humanized scFv of the anti-CD3 antibody OKT3 22 without and with the HNF1a dimerization domain (HDD) (see Fig. 1A and 1B).…”

“…We previously described the potency of this 5F11 GD2xCD3 monomeric BsAb against GD2-positive tumor cell lines and tumor xenografts. 21 The HDD tag was placed at the carboxyl end of anti-CD3 scFv and distal to the anti-GD2 scFv. This was done to maximize the functional affinity for the tumor antigen (GD2) at the N-terminal end of the molecule, but not to CD3, since the anti-CD3 scFvs were sterically restricted due to the anti-parallel arrangement of the HDD helices, which would orient the anti-CD3 scFvs »180 apart.…”