Monoclonal Antibodies for Targeted Fluorescence-Guided Surgery: History
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Contributor:
Stefano Giuliani
,
Irene Paraboschi
,
Angus McNair
,
Myles Smith
,
Kenneth S. Rankin
,
Daniel S. Elson
,
Vinidh Paleri
,
Daniel Leff
,
Graeme Stasiuk
,
John Anderson

Due to their specificity, monoclonal antibodies have significantly impacted cancer patients’ care, becoming one of the fastest-growing classes of new drugs approved for the treatment of solid tumors. Targeted fluorescence-guided surgery is a novel technology to better visualize tumor residuals intraoperatively. It consists of a fluorescent molecular probe, that, once injected, lights up the neoplastic cells during the surgical resection. In this regard, the development of an off-the-shelf large-scale production of clinically approved, fluorescently labeled monoclonal antibodies for targeted fluorescence-guided surgery is becoming an urgent need for oncological surgeons working in this field.

  • monoclonal antibodies
  • targeted fluorescence-guided surgery
  • solid malignancies
  • surgical oncology
  • fluorophores

1. Introduction

Due to their target specificity and immunomodulatory properties, monoclonal antibodies (mAbs) have significantly impacted cancer patients’ care, becoming one of the fastest growing groups of new drugs approved for the cure of solid tumors [1]. mAbs have been effectively used to elicit long-lasting effector responses against solid malignancies by exploiting their unique capability of directly killing cancer cells while simultaneously activating the host immune system [1]. As a form of radioimmunotherapy, mAbs have also been adopted to selectively deliver high doses of radiation to tumor cells while also reducing the exposure of the surrounding healthy tissues, thus enhancing the efficacy of standard radiotherapy while minimizing its side effects [2].
Recently, fluorescently labeled mAbs have been used in the operating theatre to help surgeons better visualize and remove solid tumors in real time. This has been a revolution in the field of targeted fluorescence-guided surgery (T-FGS). Even if the adoption of fluorescently labeled mAbs in surgical oncology is still in its infancy, its applications are growing fast, with real potential to revolutionize the surgical field [3][4][5][6][7][8][9][10]. In this regard, the development of an “off-the-shelf” large-scale production of clinically approved fluorescently labeled mAbs is becoming an urgent need for oncological surgeons working in this surgical field.

2. Fluorescence-Guided Surgery (FGS): Non-Specific and Monoclonal Antibody (mAb)-Targeted

FGS is an emerging imaging tool that permits surgeons to identify healthy and pathological tissues, in real time, by intravenously delivering non-specific fluorophores (e.g., indocyanine green (ICG), 5-aminolevulinic acid, or fluorescein sodium) or fluorescently labeled molecules with a particular tissue or tumor tropism (e.g., mAbs). Depending on their route of administration, systemically administered non-specific fluorescent dyes can be used to help surgeons identify blood, bile, and lymphatic vessels during surgery, bridging the gap existing between preoperative anatomical imaging and patient-specific surgical findings [11][12].
In the surgical oncology field, non-specific dyes could be employed to distinguish neoplastic lesions from the surrounding normal tissues, by exploiting the enhanced permeability and retention (EPR) effects caused by the leaky nature of tumor vessels and the compromised lymphatic drainage [3][4][5][6][7][8][9]. Unfortunately, non-specific dyes are often unable to cause tumors from a diverse range of cancer types to fluoresce effectively as the EPR effect is not homogenous or reliable.
Besides non-specific fluorescent dyes, fluorophores have been conjugated to mAbs to deliver T-FGS, thus enhancing the intraoperative visualization of cancer at the tumor margins, loco-regional lymph nodes, and occult disease with high resolution and deep tissue penetration. By binding to a specific site of the tumor antigen (i.e., epitope), fluorescently labeled mAbs can be administered a few days before surgery, and then visualized using new-generation near-infrared (NIR) camera systems for endoscopic and open-field procedures [3][4][5][6][7][8][9].
Building on the widespread use of non-specific FGS and ICG, biomedical device companies have developed several NIR fluorescence platforms, which can be used to detect mAb-based FGS [3][4][5][8]. Briefly, the fluorophore-conjugated mAb needs to be excited at a specific wavelength to produce an invisible light, which will be collected at a set wavelength range to be transformed into an image. For surgical indications, mAbs have been conjugated to NIR dyes (e.g., IRDye800CW; wavelengths: 650–900 nm), which provide low absorption in healthy tissue, high tumor-to-background-ratio (TBR) and tissue penetration, with low interference from intrinsic fluorescence, especially when compared to the visible light spectrum (wavelengths: 400–600 nm). By doing so, the non-specific tissue background can be minimized while the tumor cells can be illuminated in real-time helping surgeons with better visualization [3][4][5][6][7][8][9].

3. Monoclonal Antibodies (mAbs) Clinically Approved for the Treatment of Extra-Hematological Solid Malignancies under Evaluation in Clinical Trials for T-FGS Purposes

In this section, mAbs clinically approved for the treatment of extra-hematological solid malignancies are summarized in Table 1 and Table 2.
Table 1. Monoclonal antibodies (mAbs) clinically approved for the treatment of solid extra-hematological malignancies.
Antigen Drug Generic Name Drug Brand Name Format Clinical Indication
EGFR Cetuximab Erbitux Chimeric IgG1 Squamous-Cell Carcinoma of the Head and Neck, Colorectal Cancer
Panitumumab Vectibix Human IgG2 Colorectal Cancer
Necitumumab Portrazza Human IgG1 Squamous Non-Small-Cell Lung Cancer
HER2 Trastuzumab Herceptin Humanized IgG1 Breast Cancer
Pertuzumab Perjeta Humanized IgG1 Breast Cancer
VEGF Bevacizumab Avastin Humanized IgG1 Colorectal Cancer, Non-Squamous Non-Small Cell Lung Cancer, Glioblastoma, Renal-Cell Carcinoma, Cervical Cancer, Epithelial Ovarian, Fallopian Tube, Primary Peritoneal Cancer, Hepatocellular Carcinoma
VEGFR2 Ramucirumab Cyramza Human IgG1 Gastric Or Gastro-Esophageal Junction Adenocarcinoma, Non-Small-Cell Lung Cancer, Colorectal Cancer, Hepatocellular Carcinoma
PD-L1 Atezolizumab Tecentriq Humanized IgG1 Non-Small-Cell Lung Cancer, Small-Cell Lung Cancer, Hepatocellular Carcinoma, Melanoma, Alveolar Soft Part Sarcoma
Avelumab Bavencio Human IgG1 Merkel Cell Carcinoma
Durvalumab Imfinzi Human IgG1 Non-Small-Cell Lung Cancer, Small-Cell Lung Cancer, Biliary Tract Cancer, Hepatocellular Carcinoma
GD2 Dinutiximab Unituxin Chimeric IgG1 Neuroblastoma
Dinutiximab-beta Quarziba Chimeric IgG1 Neuroblastoma
PDGFRα Olaratumab Lartruvo Human IgG1 Soft-Tissue Sarcoma
Table 2. Monoclonal antibodies (mAbs) conjugated to the near-infrared I (NIR-I) fluorescent dye IRDye800CW for the intraoperative imaging of solid extra-hematological malignancies.
Drug Tumor Type Phase Recruitment Status Estimated Patient Enrollment Dose Timing ClinicalTrials.gov Identifier
Cetuximab-IRDye800CW Esophageal Cancer Phase I nd 40 1% of therapeutic dose (2.5 mg/m2); 10% of therapeutic dose (25 mg/m2); 25% of therapeutic dose (62.5 mg/m2) (iv) 2 days before surgery NCT04161560
Head and Neck Cancer Phase I; Phase II Recruiting 79 10 mg; 25 mg; 50 mg; 75mg unlabeled test/loading dose cetuximab + 15 mg Cetuximab-IRDye800CW; 75 mg unlabeled test/loading dose cetuximab + 25 mg Cetuximab-IRDye800CW (iv) 4 days before surgery NCT03134846
Head and Neck Cancer Phase I Terminated 12 10 mg; 100 mg unlabeled test/loading dose of cetuximab + 50 mg dose of cetuximab-IRDye800 (iv) Prior to surgery NCT01987375
Pancreatic Adenocarcinoma Phase II Terminated 10 100 mg unlabeled test/loading dose of cetuximab + 50 mg; 100 mg unlabeled test/loading dose of cetuximab + 100 mg (iv) 2–5 days before surgery NCT02736578
Brain Neoplasm Phase I; Phase II Terminated 10 unlabeled test/loading dose of cetuximab + 50 mg; unlabeled test/loading dose of cetuximab + 100 mg (iv) 2–5 days before surgery NCT02855086
Rectal Cancer Phase I nd 15 75 mg unlabeled test/loading dose of cetuximab + 15 mg (iv) Prior to surgery NCT04638036
Penile Cancer Phase I Recruiting 15 15 mg unlabeled test/loading dose of cetuximab + 15 mg (iv) 2 days before surgery NCT05376202
Head and Neck Cancer Phase II Not yet recruiting 20 75 mg unlabeled test/loading dose of cetuximab + 15 mg (iv) Prior to surgery NCT05499065
Panitumumab-IRDye800CW Head and Neck Cancer Phase II Recruiting 25 50 mg (iv) nd NCT04511078
Head and Neck Cancer Phase II Completed 20 50 mg (iv) 1–5 days before surgery NCT03405142
Head and Neck Cancer Phase I Completed 43 nd (iv) Prior to surgery NCT02415881
Head and Neck Cancer Phase I Completed 14 30 mg (iv) 2-5 days before surgery NCT03733210
Lung Cancer Phase I; Phase II Active, not recruiting 30 nd (iv) 1–2 days or 3–5 days prior to surgery NCT03582124
Brain Tumor Phase I; Phase II Not yet recruiting 12 0.006 mg/kg; 0.25 mg/kg; 0.5 mg/kg; 1.0 (with max cap dose 50 mg) 1–5 days before surgery NCT04085887
Brain Tumor Phase I; Phase II Recruiting 22 50 mg; 100 mg unlabeled test/loading dose of panitumumab + 50 mg; 100 mg; 100 mg unlabeled test/loading dose of panitumumab + 100 mg (iv) 1–5 days before surgery NCT03510208
Pancreatic Adenocarcinoma Phase I; Phase II Active, not recruiting 24 100 mg unlabeled test/loading dose of panitumumab + 25 mg; 100 mg unlabeled test/loading dose of panitumumab + 50 mg; 100 mg unlabeled test/loading dose of panitumumab + 75 mg; 50 mg (iv) 2–5 days before surgery NCT03384238
Bevacizumab-IRDye800CW Adenomatous Polyposis Coli Phase I Completed 15 4.5 mg; 10 mg; 25 mg (iv) 3 days before endoscopy NCT02113202
Rectal Cancer Phase I Completed 30 4.5 mg (iv) 2–3 days before endoscopy NCT01972373
Breast Cancer Phase I Completed 30 4.5 mg (iv) 4 h, 2 days before imaging and 3 days before surgery NCT01508572
Breast Cancer Phase I; Phase II Completed 26 4.5 mg; 10 mg; 25 mg; 50 mg (iv) 3 days before surgery NCT02583568
Soft-Tissue Sarcoma Phase I; Phase II Completed 23 10 mg; 25 mg; 50 mg (iv) Prior to surgery NCT03913806
Esophageal Cancer Phase I Completed 10 4.5 mg (iv) 2 days before endoscopy NCT02129933
Esophageal Cancer Phase I Terminated 25 4.5 mg; 10 mg; 25 mg (iv) Prior to endoscopy NCT03558724
Hilar Cholangiocarcinoma Phase I; Phase II Recruiting 12 10 mg; 25 mg; 50 mg (iv) 3 days before surgery NCT03620292
Barrett Esophagus Phase II nd 60 nd (topical) 5 min before endoscopy NCT03877601
Inverted Papilloma Phase I Active, not recruiting 6 10 mg; 25 mg (iv) 2–4 days before surgery NCT03925285
Pancreatic Cancer Phase I; Phase II Terminated 26 4.5 mg; 10 mg; 25 mg; 50 mg (iv) 3 days before surgery NCT02743975
Pituitary Tumor Phase I Recruiting 15 4.5 mg; 10 mg; 25 mg (iv) Prior to endoscopy NCT04212793
Girentuximab-IRDye800CW Renal Tumor Phase I Recruiting 22 nd (iv) 7 days before surgery NCT03558724
Labetuzumab-IRDye800CW Colorectal Cancer Phase I; Phase II Recruiting 29 nd (iv) 6–7 days before surgery NCT03699332
Nimotuzumab-IRDye800CW Lung Cancer Phase I; Phase II Recruiting 36 50 mg; 100 mg (iv) 4–6 days before surgery NCT04459065
  • mAbs targeting the epidermal growth factor receptor (EGFR)
The EGFR (c-ErbB1, HER1) is a 170 kDa transmembrane glycoprotein belonging to the epidermal growth factor receptor (ErbB) family [13][14].
The members of this family of type I receptor tyrosine kinases, which also include c-ErbB2 (HER2, neu in rodents), c-ErbB3 (HER3), and c-ErbB4 (HER4), share similarities in their structure (i.e., an extracellular domain, a lipophilic transmembrane region, an intracellular domain containing tyrosine kinase, and a carboxy-terminal region) and function [13][14].
Despite being a monomer in its inactive form, when it is bounded by its ligands (e.g., EGF, TNFα), EGFR forms homodimers or heterodimers with another member of its family of receptors and activates its intracellular tyrosine kinase region. This results in its autophosphorylation and in the initiation of a cascade of intracellular events, which regulate tumor cell differentiation, proliferation, angiogenesis, and migration [13][14].
Several alterations in EGFR are associated with the growth and spread of different solid tumors in humans. Hence, this surface antigen has been extensively studied over time together with the effects of anti-EGFR mAbs on EGFR-positive tumors [13][14].
Since anti-EGFR mAbs are bound to EGFR with a higher affinity compared with its ligands, they inhibit the subsequent activation of the tyrosine kinase-mediated cascade of intracellular events [13][14].
  • mAbs targeting the vascular endothelial growth factor (VEGF)
Angiogenesis is a complex phenomenon, involving several series of events, including the outgrowth of post-capillary vessels from pre-existing venules, vasodilation, increased permeability, and the migration of endothelial progenitor cells. Besides the tightly regulated process occurring during the normal growth of an organism and the tissue repair occurring during wound healing, solid malignancies also upregulate angiogenetic processes to induce nutrient delivery and tumor growth [15][16].
In this regard, the protein family of VEGF has been demonstrated to play major roles in physiological and pathological angiogenesis by providing signaling pathways and cascades of events crucial for endothelial cell division and migration. The VEGF family consists of six secreted proteins including VEGF-A (also known as VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placenta-induced growth factor (PDGF), characterized by eight conserved cysteines and functions as a homodimer structure. These ligands bind to three different, but structurally related, VEGF-receptor (VEGFR) tyrosine kinases, essential for hematopoietic, vascular endothelial, and lymphatic endothelial cell development [15][16].
  • mAbs targeting carbonic anhydrase IX (CAIX)
Carbonic anhydrase IX (CAIX) is a zinc metalloprotein enzyme. CAIX belongs to the carbonic anhydrase family. Under conditions associated with low pH, CAIX is triggered by the hypoxia-inducible factor 1-α (HIF1-α) and is highly upregulated in environments characterized by a low pressure of oxygen. By catalyzing the interconversion of CO2 and HCO3− to maintain intracellular pH homeostasis, the overexpression of CAIX contributes to cancer cell survival. Not only does CAIX modulate the microenvironment, but it also has a critical role in mediating tumor survival, spreading, and metastasis [17]. Hence, CAIX has become an interesting target not only for cancer diagnosis but also for treatment. Its expression is increased in a wide variety of solid tumors (e.g., renal-cell carcinoma, brain, bladder, cervical, head and neck, breast, lung, sarcoma, and kidney cancers). Conversely, its expression in normal healthy tissues is usually low [18]. It is worth noting that the Von Hippel–Lindau mutation in clear-cell renal-cell carcinomas causes HIF1-α overexpression on the cell surface in this group of tumors [19].
  • mAbs targeting carcinoembryonic antigen (CEA)
Carcinoembryonic antigen (CEA) is a non-specific serum biomarker that is elevated in various malignancies, and it is highly expressed on the surface of colorectal cancer cells in more than 90% of all cases with a significant antigenic density. Conversely, its expression is 60 times lower on average in healthy tissue [20].
Labetuzumab (CEA-CIDE) is a humanized IgG1 mAb that has a high specificity to CEA. It has been deeply studied as a therapeutic drug, radiotracer, and antibody–drug conjugate for the treatment of several tumors.

This entry is adapted from the peer-reviewed paper 10.3390/cancers16051045

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