Near-Infrared Dye-Labeled Anti-Prostate Stem Cell Antigen Minibody Enables Real-Time Fluorescence Imaging and Targeted Surgery in Translational Mouse Models

Abstract

Purpose: The inability to intraoperatively distinguish the primary tumor, as well as lymphatic spread, increases the probability of positive surgical margins, tumor recurrence, and surgical toxicity. The goal of this study was to develop a tumor-specific optical probe for real-time fluorescence-guided surgery. Experimental design: A humanized antibody fragment against PSCA (A11 minibody, A11 Mb) was conjugated with a near-infrared fluorophore, IRDye800CW. The integrity and binding of the probe to PSCA were confirmed by gel electrophoresis, size exclusion chromatography, and flow cytometry, respectively. The ability of the probe to detect tumor infiltrated lymph nodes and metastatic lesions was evaluated in two xenograft models, and in transgenic mice expressing human PSCA (hPSCA). An invasive intramuscular model was utilized to evaluate the efficacy of the A11 Mb-IRDye800CW-guided surgery. Results: A11 Mb was successfully conjugated with IRDye800CW and retained specific binding to PSCA. In vivo imaging showed maximal signal to background ratios at 48 hours. The A11 Mb-IRDye800CW specifically detected PSCA positive primary tumors, tumor infiltrated lymph nodes, and distant metastases with high contrast. Fluorescence guidance facilitated more complete tumor resection, reduced tumor recurrence, and improved overall survival, compared with conventional white light surgery. The probe successfully identified primary orthotopic tumors and metastatic lesions in hPSCA transgenic mice. Conclusions: Real-time fluorescence image-guided surgery with A11 Mb-IRDye800CW enabled detection of lymph node metastases and positive surgical margins, facilitated more complete tumor removal, and improved survival, compared to white light surgery. These results may be translatable into clinical practice to improve surgical and patient outcomes.

Emerging Fluorescent Molecular Tracers to Guide Intra-Operative Surgical Decision-Making

“…To this end, a free cysteine residue is incorporated in the targeting moiety's structure at the location of predilection, such as the C- or N-terminus of protein scaffolds, peptides and small antibody fragments (Sun et al, 2005; Massa et al, 2014). Antibodies and larger fragments such as minibodies and diabodies can furthermore be partially reduced, freeing the sulfhydryl residues of more exposed disulfide bridges for conjugation (Olafsen et al, 2004; Sonn et al, 2016; Tsai et al, 2018; Zettlitz et al, 2018; Zhang et al, 2018). Many other site-specific methods have gained popularity over the last years, such as the azide-alkyne cycloaddition-click chemistry (Li et al, 2014), enzymatic modification of peptide tags (e.g., transglutaminase-, sortase-, or intein-mediated conjugation) (Massa et al, 2016a), incorporation of unnatural amino acids for biorthogonal conjugation or the use of linking peptides such as the SPY/SPYcatcher method (Massa et al, 2016b; Alam et al, 2018).…”

“…A recent study demonstrated the use of an anti-(human) PSCA minibody, called A11, conjugated with IRDye800CW in different mouse models. Subcutaneous, orthotopic and intramuscular implanted tumors and tumor positive lymph nodes could be clearly visualized, even in knock-in models expressing the human homolog of PSCA (Zhang et al, 2018). Furthermore, fluorescence-guided resection of intramuscular tumors, revealed superior overall survival compared to conventional white light surgery (Zhang et al, 2018).…”

A promising cancer diagnosis and treatment strategy: targeted cancer therapy and imaging based on antibody fragment

“…Unlike mAbs, antibody fragments with short half-life and strong permeability are more suitable for tumour imaging [45,46]. Antibody fragment-based tumour imaging is widely used for (1) non-invasive detection of tumours; (2) real-time guidance of surgery; (3) monitoring of tumourigenic receptor expression status, tumour development status, and tumour treatment prognosis [47,48]. At present, imaging diagnosis based on antibody fragments is rapidly developing and various methods have emerged.…”

“…These antibody fragments not only retain important ability to specifically bind antigen but also have more ideal half-life and penetration functions than mAbs. Immunoimaging agents, which are composed of antibody fragments coupled with radionuclides, fluorescent dyes, fluorophores, and nanoparticles, can achieve high-resolution and clearly visible tumour imaging in a variety of ways [17,18]. Moreover, antibody fragments can also be used for targeted-tumour treatments for a variety of methods.…”

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

“…Coupled with radionuclides, fluorescent dyes, or nanoparticles, antibody fragments serve as imaging modalities for noninvasive detection and staging of tumors, and the evaluation of the surface molecule portfolio of cancer cells. Moreover, fragment-based imaging provides additional information beneficial for treatment including real-time fluorescence-guided surgery [11][12][13][14].…”