Enhanced Fluorescence of Near‑Infrared Anti‑CEA Antibodies for Visualizing Colorectal Cancers Using Modified Heptamethine Cyanines

Sep 22, 2026

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Fluorescence-guided surgery depends on tumor-specific fluorescent probes that achieve high tumor-to-background contrast. Conventional near-infrared fluorophores can disrupt pharmacokinetics of the parental molecule, leading to aggregation, altered biodistribution, and reduced tumor targeting. This study compares two charge-balanced heptamethine cyanine dyes, FNIR-Tag-766 and FNIR-Tag-804, conjugated to a humanized anti-CEA antibody (M5A), versus a standard M5A-IR800CW conjugate in mouse colorectal cancer xenograft models. Results show improved mean fluorescence intensity and tumor-to-background ratio with reduced hepatic signal.

LanguageEnglish

CategoryResearch & Report

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Pages13

Reading Time33 min

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Abstract

Background

Methods

Results

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Introduction

Molecular Imaging and Biology (2026) 28:369–381 [https://doi.org/10.1007/s1](https://doi.org/10.1007/s1) 1307-025-02076-3 Enhanced Fluorescence of Near‑Infrared Anti‑CEA Antibodies for Visualizing Colorectal Cancers Using Modified Heptamethine Cyanines K. Cox1,2 · J. Jitender3 · S. Mehta3 · Dong‑Hao Li4 · S. Amirfakhri1,2 · R. M. Hoffman1,2 · J. Shively3 · P. Yazaki3 · M. J. Schnermann4 · M. Bouvet1,2 · T. M. Lwin3,5 Received: 30 August 2025 / Revised: 6 November 2025 / Accepted: 11 December 2025 / Published online: 10 February 2026 This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2026 Abstract Background The clinical success of fluorescence-guided surgery is dependent on tumor-specific probes that can achieve high tumor-to-background contrast. Conventional near-infrared (NIR) fluorophores often alter the pharmacokinetic properties of the parental molecule that result in aggregation, altered biodistribution, and impaired tumor targeting. Methods This present study evaluates two charge-balanced heptamethine cyanine dyes, FNIR-Tag-766 and FNIR-Tag-804, conjugated to a humanized anti-carcinoembryonic antigen antibody (M5A). Their performance was compared to the standard M5A-IR800CW conjugate in both subcutaneous and orthotopic colorectal cancer xenograft models in mice. Mean fluorescence intensity (MFI), tumor-to-background ratio (TBR), and ex vivo biodistribution were compared. Results The M5A-FNIR-766 conjugate produced a greater MFI in tumors at all timepoints compared to M5A-IR800CW, resulting in a significantly improved TBR. Ex vivo analysis at 96 h confirmed higher tumor accumulation for M5A-FNIR-766 and revealed a reduction in hepatic signal for both M5A-FNIR-Tag conjugates. The observations were concordant in the clinically relevant orthotopic model. Conclusion Antibody-fluorophore conjugates linked to the charge-modified FNIR-Tag dyes provide improved tumor-specific signals and a more favorable biodistribution profile than the same antibody conjugated to a conventional dye. By achieving superior performance through intrinsic fluorophore design rather than complex bioconjugation strategies, the approach provides a clinically translatable advancement for tumor-specific FGS. Keywords Fluorescence-guided surgery · Molecular imaging · Near-infrared fluorescent dyes · Anti-carcinoembryonic antigen antibody · Heptamethine cyanine dyes K. Cox and J. Jitender contributed equally as first authors * T. M. Lwin [tlwin@coh.org](tlwin@coh.org) 1 Department of Surgery, University of California San Diego, San Diego, CA, USA 2 VA San Diego Healthcare System, San Diego, CA, USA 3 Department of Immunology & Theranostics, Beckman Research Institute, Duarte, CA, USA 4 Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 5 Department of Surgery, City of Hope National Medical Center, Duarte, CA, USA Introduction Advances in molecular biology and protein engineering have facilitated the development of probes that can be linked to fluorophores for fluorescent intra-operative tumor visualization and fluorescence-guided surgery (FGS) [1] . Among these, the use of monoclonal antibodies (mAb’s) as fluorescent tracers have demonstrated promise since mAb’shave been well established in the clinic for their specificity, pharmacokinetics parameters, and engineering ability [2] . The selection of the fluorophore that is linked to the antibody is critical as the dye molecule itself changes the invivo behavior of the entire conjugate and the final pharmacokinetic properties of the antibody-dye conjugate [3] . The biochemical behavior of the fluorophore molecule affects the overall stability, fluorescence intensity, target avidity, and biodistribution of the probe which impacts the clinical applicability and success of the molecule [4] . Near-infrared (NIR) heptamethine cyanine fluorophores are preferre

FAQs

Why are tumor-specific probes critical for fluorescence-guided surgery?

Fluorescence-guided surgery requires probes that provide high tumor-to-background contrast. The study emphasizes that probe performance depends on maintaining favorable pharmacokinetics and strong tumor targeting.

What fluorophores and antibody construct were compared in the study?

Two charge-balanced heptamethine cyanine dyes, FNIR-Tag-766 and FNIR-Tag-804, were conjugated to a humanized anti-CEA antibody (M5A). Their performance was compared with the standard M5A-IR800CW conjugate.

What were the main findings about tumor signal and biodistribution?

The M5A-FNIR-766 conjugate produced higher mean fluorescence intensity in tumors across timepoints and improved tumor-to-background ratio. Ex vivo analysis also indicated reduced hepatic signal for the FNIR-Tag conjugates, consistent with the orthotopic model.

Sep 22, 2026

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