Synthesis and Evaluation of the Aldolase Antibody-Derived Chemical-Antibodies Targeting α5β1 Integrin

Abstract

Integrin α5β1 is an important therapeutic target that can be inhibited using an aldolase antibody (Ab)-derived chemical-Ab (chem-Ab) for the treatment of multiple human diseases, including cancers. A fairly optimized anti-integrin α5β1 chem-Ab 38C2-<b>4e</b> was obtained using an <i>in situ</i> convergent chemical programming (CP) approach, which minimized the time and effort needed to develop a chem-Ab. Multiple Ab-programming agents (PAs) <b>4a</b>–<b>e</b> could be prepared rapidly using the Cu-catalyzed alkyne–azide coupling (Cu-AAC) reaction of an α5β1 inhibitor <b>2</b> with multiple linkers <b>3a</b>–<b>e</b>, either before or after conjugating the linkers into Ab 38C2 binding sites. In these two-steps processes, the products after step 1 can be used in the next step without performing an extensive purification or analysis of the Ab-PAs or Ab-linker conjugates affording chem-Abs 38C2-(<b>4a</b>–<b>e</b>). Flow cytometry assay was used to determine the binding of the chem-Abs to U87 human glioblastoma cells expressing α5β1 integrin and identify 38C2-<b>3e</b> as the strongest binder. Further studies revealed that 38C2-<b>3e</b> strongly inhibited proliferation of U87 cells and tube formation of HUVEC in the matrigel assay, as well as tumor growth and metastasis of 4T1 cells <i>in vivo</i>