Design and Characterization of Bispecific Chemically Self-assembled Antibody Nanorings (CSANs) T-cell Engagers that Target CD22+ B-Leukemias Jingjing Shen, Adrian Fegan, Qing Li, Daniel A. Vallera, and Carston R. Wagner* Medicinal Chemistry Department, College of Pharmacy, University of Minnesota Minneapolis, MN 55455 Recently, we have developed a protocol for the preparation of self-assembling protein macrocyclic oligomers, or nanorings of dihydrofolate reductase fusion proteins (DHFR2) by chemical induction with bismethotrexate (bis-MTX)1. Inspection of the X-ray crystal structure of the DHFR dimer formed with bis-MTX suggested that proteins, such as scFvs, could be appended to DHFR2 fusion proteins without loss of their ability to undergo chemically induced macrocyclic oligomerization. We have also been able to construct multivalent chemically selfassembled antibody nanorings (CSANs) from a fusion protein DHFR2-antiCD3 scFv containing a single glycine linker between the two DHFR scaffolding proteins. The antiCD3 CSANs were found to mimic the binding and internalization behavior of the parental mAb2,3. Our success in forming multivalent antiCD3 CSANs encouraged us to further develop bispecific antibody nanorings by self-assembling DHFR2 antiCD3 with DHFR2 antiCD22 fusion protein nanorings formed by bis-MTX. Potentially bispecific anti CD3×22 CSANs against the CD3ε subunit of the T-cell-receptor/CD3 complex and the CD22 antigen on malignant B cells can redirect cytotoxic T cells (CTLs) to B lymphoma Daudi or Raji cells for the treatment of acute lymphoblastic leukemia (ALL) and malignant lymphomas. In this study, human peripheral blood mononuclear cells (PBMC) were isolated from a donor and then incubated with bispecfic antiCD3×22 CSANs. Upon engagement with the tumor associated antigen, the T cell activation, cytokines release, and lysis of target cells in vitro were investigated. (1) Carlson, J. C. T.; Jena, S. S.; Flenniken, M.; Chou, T.; Siegel, R. A.; Wagner, C. R. Chemically Controlled Self-Assembly of Protein Nanorings. J. Am. Chem. Soc. 2006, 128, 7630–7638. (2) Li, Q.; So, C. R.; Fegan, A.; Cody, V.; Sarikaya, M.; Vallera, D. A.; Wagner, C. R. Chemically Self-Assembled Antibody Nanorings (CSANs): Design and Characterization of an Anti-CD3 IgM Biomimetic. J. Am. Chem. Soc. 2010, 132, 17247–17257. (3) Li, Q.; Hapka, D.; Chen, H.; Vallera, D. A.; Wagner, C. R. Self-Assembly of Antibodies by Chemical Induction. Angew. Chem. Int. Ed. 2008, 47, 10179–10182.