Miniaturized Fab' Imaging Probe Derived from a Clinical Antibody: Characterization and Imaging in CRISPRi-attenuated Mammary Tumor Models

Overview

Journal iScience

Publisher Cell Press

Date 2024 Aug 26

PMID 39184438

Authors

Suresh Gupta

Rahul Pal

Eric J Schmidt

Murali Krishnamoorthy

Anita Leporati

Anand T N Kumar

Alexei Bogdanov Jr

Affiliations

Soon will be listed here.

Abstract

Clinical imaging-assisted oncosurgical navigation requires cancer-specific miniaturized optical imaging probes. We report a near-infrared (NIR) Fab'-based epidermal growth factor receptor (EGFR)-specific probe carrying 3 NIR fluorophores (Fab'-800CW), which retained high-affinity binding to EGFR ectodomain (equilibrium K = 1 nM). Fab'-800CW showed a robust 4-times gain of fluorescence intensity (FI) and a 20% lifetime (FLT) increase under the conditions mimicking intracellular degradation. The probe was tested by using triple-negative breast cancer (TNBC) cell lines obtained by applying CRISPR interference (CRISPRi) effect of -targeting sgRNA and dCas9-KRAB chimera coexpression in MDA-MB-231 cells (WT cells). FI imaging in cell culture proved a 50% EGFR expression attenuation by CRISPRi. FI imaging in animals harboring attenuated or WT TNBC tumors with corroboration identified differences between WT and CRISPRi tumors FI at 30 min post injection. Our results suggest the feasibility of EGFR expression imaging using a Fab'-based probe relevant for imaging-guided cancer surgery.

References

Kocaturk B, Versteeg H . Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth. J Vis Exp. 2015; (96). PMC: 4354624. DOI: 10.3791/51967. View

Kumar A, Rice W, Lopez J, Gupta S, Goergen C, Bogdanov Jr A . Substrate-based near-infrared imaging sensors enable fluorescence lifetime contrast via built-in dynamic fluorescence quenching elements. ACS Sens. 2017; 1(4):427-436. PMC: 5609830. DOI: 10.1021/acssensors.5b00252. View

Bauerle T, Gupta S, Zheng S, Seyler L, Leporati A, Marosfoi M . Multimodal Bone Metastasis-associated Epidermal Growth Factor Receptor Imaging in an Orthotopic Rat Model. Radiol Imaging Cancer. 2021; 3(4):e200069. PMC: 8344354. DOI: 10.1148/rycan.2021200069. View

Ding H, Carlton M, Povoski S, Milum K, Kumar K, Kothandaraman S . Site specific discrete PEGylation of (124)I-labeled mCC49 Fab' fragments improves tumor MicroPET/CT imaging in mice. Bioconjug Chem. 2013; 24(11):1945-54. PMC: 4240220. DOI: 10.1021/bc400375f. View

Ntziachristos V, Tung C, Bremer C, Weissleder R . Fluorescence molecular tomography resolves protease activity in vivo. Nat Med. 2002; 8(7):757-60. DOI: 10.1038/nm729. View

Ma H, Naseri A, Reyes-Gutierrez P, Wolfe S, Zhang S, Pederson T . Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci U S A. 2015; 112(10):3002-7. PMC: 4364232. DOI: 10.1073/pnas.1420024112. View

Kobayashi H, Choyke P . Target-cancer-cell-specific activatable fluorescence imaging probes: rational design and in vivo applications. Acc Chem Res. 2010; 44(2):83-90. PMC: 3040277. DOI: 10.1021/ar1000633. View

Bogdanov Jr A, Mazzanti M, Castillo G, Bolotin E . Protected Graft Copolymer (PGC) in Imaging and Therapy: A Platform for the Delivery of Covalently and Non-Covalently Bound Drugs. Theranostics. 2012; 2(6):553-76. PMC: 3381344. DOI: 10.7150/thno.4070. View

Ntziachristos V . Going deeper than microscopy: the optical imaging frontier in biology. Nat Methods. 2010; 7(8):603-14. DOI: 10.1038/nmeth.1483. View

10.

Shazeeb M, Gupta S, Bogdanov Jr A . MR signal amplification for imaging of the mutant EGF receptor in orthotopic human glioma model. Mol Imaging Biol. 2013; 15(6):675-84. PMC: 3800697. DOI: 10.1007/s11307-013-0653-8. View

11.

Ishikawa E, Yoshitake S . A general and mild procedure for the purification of rabbit Fab' antibodies. J Immunol Methods. 1980; 38(1-2):117-23. DOI: 10.1016/0022-1759(80)90336-1. View

12.

Lakowicz J . Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching. Scanning Microsc Suppl. 1996; 10:213-24. View

13.

Wilkins M, Gasteiger E, Bairoch A, Sanchez J, Williams K, Appel R . Protein identification and analysis tools in the ExPASy server. Methods Mol Biol. 1999; 112:531-52. DOI: 10.1385/1-59259-584-7:531. View

14.

Korb M, Hartman Y, Kovar J, Zinn K, Bland K, Rosenthal E . Use of monoclonal antibody-IRDye800CW bioconjugates in the resection of breast cancer. J Surg Res. 2013; 188(1):119-28. PMC: 4354808. DOI: 10.1016/j.jss.2013.11.1089. View

15.

Lawhorn I, Ferreira J, Wang C . Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53. PLoS One. 2014; 9(11):e113232. PMC: 4232525. DOI: 10.1371/journal.pone.0113232. View

16.

Berezin M, Achilefu S . Fluorescence lifetime measurements and biological imaging. Chem Rev. 2010; 110(5):2641-84. PMC: 2924670. DOI: 10.1021/cr900343z. View

17.

Yeo N, Chavez A, Lance-Byrne A, Chan Y, Menn D, Milanova D . An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat Methods. 2018; 15(8):611-616. PMC: 6129399. DOI: 10.1038/s41592-018-0048-5. View

18.

Gao R, Teraphongphom N, de Boer E, van den Berg N, Divi V, Kaplan M . Safety of panitumumab-IRDye800CW and cetuximab-IRDye800CW for fluorescence-guided surgical navigation in head and neck cancers. Theranostics. 2018; 8(9):2488-2495. PMC: 5928904. DOI: 10.7150/thno.24487. View

19.

Pal R, Hom M, van den Berg N, Lwin T, Lee Y, Prilutskiy A . First Clinical Results of Fluorescence Lifetime-enhanced Tumor Imaging Using Receptor-targeted Fluorescent Probes. Clin Cancer Res. 2022; 28(11):2373-2384. PMC: 9167767. DOI: 10.1158/1078-0432.CCR-21-3429. View

20.

Garron J, Moinereau M, Pasqualini R, Saccavini J . Direct 99mTc labeling of monoclonal antibodies: radiolabeling and in vitro stability. Int J Rad Appl Instrum B. 1991; 18(7):695-703. DOI: 10.1016/0883-2897(91)90007-8. View