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Updated: Jul 4, 2025
Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity
Published on: May 1, 2018
Development of a [89Zr]Zr-labeled Human Antibody using a Novel Phage-displayed Human scFv Library
Abhay K Singh1, Calvin D Lewis1, Cristian A W V Boas1
1Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri.
Purpose:
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.
Experimental Design:
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).
Results:
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.
Conclusions:
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.
Insights
A novel human antibody, L111, targeting cancer antigen TIP1 was developed for PET imaging. Radiolabeled L111 demonstrated effective tumor targeting in preclinical models, showing promise for noninvasive cancer detection.
Area of Science:
- Oncology
- Immunology
- Radiochemistry
Background:
- Tax-interacting protein 1 (TIP1) is a cancer-specific antigen implicated in tumor progression and therapy resistance.
- Developing targeted imaging agents is crucial for noninvasive cancer diagnosis and monitoring.
Purpose of the Study:
- To develop a novel human antibody targeting TIP1 for noninvasive positron emission tomography (PET) imaging in cancer patients.
- To characterize the antibody and its radiolabeled form for potential clinical application.
Main Methods:
- Phage display was used to generate high-affinity single-chain variable fragments (scFvs) against TIP1, which were engineered into a human IgG1 antibody (L111).
- The antibody L111 was characterized using SEC-HPLC, native MS, ELISA, BIAcore, and flow cytometry.
- The antibody was radiolabeled with [89Zr]Zr using a deferoxamine (DFO) chelator, and its stability and immunoreactivity were assessed.
- Small animal PET studies were conducted in lung cancer models to evaluate tumor targeting efficacy.
Main Results:
- The human antibody L111 was successfully developed and characterized, showing high purity and specific binding.
- [89Zr]Zr-DFO-L111 demonstrated excellent radiochemical purity, stability in human serum, and high immunoreactivity.
- PET imaging in lung cancer models showed significant tumor uptake and enhanced tumor-to-muscle ratios with L111, particularly when administered with unlabeled antibody.
Conclusions:
- The developed human antibody L111 effectively targets TIP1-expressing lung cancer cells in vitro and in vivo.
- [89Zr]Zr-DFO-L111 shows promise as a PET imaging agent for cancer.
- This antibody will be advanced to a first-in-human study to assess safety and imaging capabilities.
