Bispecific antibodies (BsAbs) have confirmed highly efficient T cell recruiters for cancer immunotherapy by virtue of one tumor antigen-reactive single chain variable fragment (scFv) and another that binds CD3. induce dimerization was assayed by dynamic light scattering and size exclusion chromatography (observe Table 1 and Fig. S1). GD2xCD3 BsAb (MW 54?kDa) had a diameter of 7.2 1.7?nm and was 95% monomeric, whereas GD2xCD3-HDD BsAb (MW 59?kDa for monomer, 118?kDa for dimer) had a diameter of 11.1 0.5?nm and was 97% dimeric. In addition to higher purity, the GD2xCD3-HDD BsAb experienced a 2-fold increase in yield when stably expressed in CHO cells. Table 1. Bispecific antibody (BsAb) biophysical characterization by Dynamic Light Scattering buy Lapatinib (free base) (DLS), Size Exclusion Chromatography (SEC), and Surface Plasmon Resonance (SPR). To test whether the HDD tag enhanced the functional affinity to tumor buy Lapatinib (free base) antigen GD2, Surface Plasmon Resonance (SPR) and ELISA experiments were carried out using purified GD2 (Table 1, Fig. 2 and Table 2). SPR analysis showed that the Kon for GD2xCD3 BsAb and GD2xCD3-HDD BsAb were comparable (9.07 104 1/Ms and 8.83 104 1/Ms, respectively), but the Koff were very different buy Lapatinib (free base) (2.27 10?2 1/s and 3.45 10?3 1/s, respectively). This resulted in a 6-7 fold difference in the overall KD (250?nM for GD2xCD3 BsAb and 39?nM for GD2xCD3-HDD BsAb). The kinetic analysis confirmed that the HDD tag enhanced the ability of the BsAb to retain its distal target. ELISA binding assays showed an 8-fold enhancement of GD2 binding for GD2xCD3-HDD BsAb when compared with GD2xCD3 BsAb. We also compared the HDD PLD1 tag to the synthetic dHLX and the human Fc domain name. To this end, GD2xCD3 BsAb was produced with either the HDD, dHLX domain name, or the Fc domain name at their respective C-termini and assayed for GD2 antigen binding (Table 2 and Fig. 2C). The GD2 binding showed that covalent dimers created buy Lapatinib (free base) by GD2xCD3-Fc BsAb (0.3?nM EC50, 99% dimeric by HPLC-SEC) had a two-fold enhancement in avidity to GD2, when compared with the non-covalent dimers formed by GD2xCD3-HDD BsAb (0.6?nM EC50). ELISA results showed that binding of both of these constructs were several fold higher than monomeric GD2xCD3 BsAb (5.0?nM EC50) or the GD2xCD3-dHLX (2.9?nM EC50). GD2xCD3-dHLX BsAb did not form homogeneous dimers, and contained >50% aggregates by HPLC-SEC analysis. Physique 2. Comparison of bispecific antibody GD2 tumor antigen-binding kinetics. Bispecific antibody GD2 binding kinetics by surface plasmon resonance of (A) GD2xCD3 BsAb and (W) GD2xCD3-HDD. Remnants are shown at the following BsAb concentrations: 62.5, 125, 250, … Table 2. Analysis of binding of the indicated bispecific antibody (BsAb) to GD2 tumor antigen by ELISA (mean SE). CD3 binding, cytokine release, and T-cell proliferation We also compared T cell CD3 binding by fluorescence cytometry for all 4 constructs (GD2xCD3, GD2xCD3-HDD, GD2xCD3-dHLX, GD2xCD3-Fc BsAbs) (Fig. 3A and Table 3) and found no significant difference between the binding GD2xCD3 and GD2xCD3-HDD BsAbs (= 0.6963), indicating functional monovalency to CD3. GD2xCD3-Fc BsAb showed buy Lapatinib (free base) significantly higher CD3 binding (= 0.0043), with a nearly 2-fold decrease in EC50. This result indicates that the orientation of the anti-CD3 scFv fragments (HDD tagged versus Fc-tagged) can directly influence CD3 binding avidity. The GD2xCD3-dHLX BsAb showed the least expensive CD3 binding, likely due to the observation that the sample did not form homogeneous dimers and contained aggregates. Physique 3. Bispecific antibody binding to human T cell CD3 and cytokine release. T cells purified from human peripheral blood mononuclear cells were incubated with the indicated bispecific antibody (BsAb) and characterized for CD3 binding (cytofluorimetric analysis) … Table 3. Analysis of binding of the indicated bispecific antibody (BsAb) to CD3 on T cells by cytofluorimetric analysis (mean SE). We also characterized the ability of GD2xCD3 and GD2xCD3-HDD BsAb to induce T-cell mediated cytokine release in the presence or absence of GD2-conveying tumor cells (Fig. 3C and 3D, Table H1 and Table H2). The BsAb stimulated release of the cytokines tumor necrosis (TNF), interferon (IFN) and the interleukins.