Characterization of a novel bispecific antibody that mediates

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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.
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