Monoclonal Antibodies for Targeted Fluorescence-Guided Surgery

Monoclonal Antibodies for Targeted Fluorescence-Guided Surgery: History

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Subjects: Oncology | Surgery | Radiology, Nuclear Medicine & Medical Imaging

Contributor:

Myles Smith

Vinidh Paleri

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
HER2	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
GD2	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 [19,20].

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 [19,20].

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 [25]. 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 [26]. 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 [27].

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 [31].

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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