journal contribution
posted on 2024-04-01, 07:22 authored by Abhay K. Singh, Calvin D. Lewis, Cristian A.W.V. Boas, Philipp Diebolder, Prashant N. Jethva, Aaron Rhee, Jong Hee Song, Young Ah Goo, Shunqian Li, Michael L. Nickels, Yongjian Liu, Buck E. Rogers, Vaishali Kapoor, Dennis E. Hallahan Supplementary Figure 1. Strategy for WashU II phage display library creation. The human antibody repertoires were amplified by PCR from the cDNA of mixed human peripheral blood mononuclear cells and cloned randomly into a phagemid vector. This vector encodes for a 16 aa linker (G4S)3T between the VH and VL domain of the scFv and adds an additional C-terminal 6xHis and Flag tag. First, the VH and VL repertoires were amplified in the first set of PCRs and completed by SfiI/XhoI (VH) or SalI/NotI (VL) restriction sites in the second set of PCRs. The VH repertories were cloned first by electroporation in E. coli TG1 (Lucigen, #60502), followed by cloning of the VLkappa or VL-lambda repertories.
Funding
National Institutes of Health (NIH)
Alvin J. Siteman Cancer Center (Siteman Cancer Center)
Elizabeth and James McDonnell endowment
History
ARTICLE ABSTRACT
Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer.
A phage-displayed single-chain variable fragment (scFv) library was created from healthy donors’ blood. High-affinity anti-TIP1 scFvs were selected from the library and engineered to human IgG1. Purified Abs were characterized by size exclusion chromatography high-performance liquid chromatography (SEC-HPLC), native mass spectrometry (native MS), ELISA, BIAcore, and flow cytometry. The labeling of positron emitter [89Zr]Zr to the lead Ab, L111, was optimized using deferoxamine (DFO) chelator. The stability of [89Zr]Zr-DFO-L111 was assessed in human serum. Small animal PET studies were performed in lung cancer tumor models (A549 and H460).
We obtained 95% pure L111 by SEC-HPLC. Native MS confirmed the intact mass and glycosylation pattern of L111. Conjugation of three molar equivalents of DFO led to the optimal DFO-to-L111 ratio of 1.05. Radiochemical purity of 99.9% and specific activity of 0.37 MBq/μg was obtained for [89Zr]Zr-DFO-L111. [89Zr]Zr-DFO-L111 was stable in human serum over 7 days. The immunoreactive fraction in cell surface binding studies was 96%. In PET, preinjection with 4 mg/kg cold L111 before [89Zr]Zr-DFO-L111 (7.4 MBq; 20 μg) significantly (P < 0.01) enhanced the tumor-to-muscle standard uptake values (SUVmax) ratios on day 5 compared with day 2 postinjection.
L111 Ab targets lung cancer cells in vitro and in vivo. [89Zr]Zr-DFO-L111 is a human antibody that will be evaluated in the first in-human study of safety and PET imaging.
