Figure 2

A. Schematic illustration of the functional activity of primary human T-cells engineered to express a chimeric bispecific antibody binding immune receptor (BsAb-IR) when redirected against tumor cells with a unique bispecific antibody (frBsAbs). The frBsAb, bound to tumor associated antigen (TAA) on tumor cell surface, facilitates crosslinking with BsAb-IRs on the T-cell surface and promotes BsAb-IR T-cell activation. Unlike traditional bispecific antibody (BsAbs) platforms that indiscriminately engage all T-cells via the CD3 molecule and lack costimulatory signaling capacity, incorporation of CD28 co-stimulation in a second generation BsAb-28z-IR augments antitumor activity of redirected human T-cells, in comparison to a first generation BsAb-z-IR lacking an engineered costimulatory domain. In addition, engineered expression of BsAb-IR in preselected human T cells ex vivo allows for a selective activation of the chosen T-cell population, not the open repertoire of all T cells. B. SDS-PAGE (4-15% gel) analysis of chemically heteroconjugated anti-CD3 [OKT3 (immunoglobulin (Ig)G2a); Orthobiotech], or MOV18 (anti-FR alpha) with anti-Her2/neu (Herceptin, Genentech, San Francisco, CA) or anti-CD20 (Rituxan, Genentech) and control IgG. The SDS-polyacrylamide gel electrophoresis shows anti-BsAb-IR monoclonal antibody, MOV18 (lane 2); anti-CD20 monoclonal antibody, (lane 4); and the heteroconjugated product containing monomers, dimers, and multimers (lane 3, 5 and 7 and 8). C. Graph showing the percentage of heterodimerized bispecific antibody in the mixture for each antibody format following chemical conjugation.