Abstract
Background: In the context of precision diagnosis for various subtypes of melanoma, identifying biomarkers with clinical translational potential from a molecular standpoint is crucial for a more comprehensive characterization of the disease. MUC18 is highly expressed in both tumor cells and tumor vasculature in major melanoma subtypes and is restricted to normal tissues.
Methods: A noninvasive imaging approach for MUC18 in melanoma utilizing an immune Positron Emission Tomography (PET) radionuclide-conjugated drug (RDC) with an 89Zr-labeled humanized anti-MUC18 monoclonal antibody (mAb) was developed. A375, Sk-Mel-28, HMVII, and A549 cells and tumor model mice were conducted. Immuno-PET was employed to assess the specificity and targeting of three distinct melanoma cell line-derived xenografts (CDXs) and patient-derived tumor xenografts (PDXs) in immunodeficient mice.
Results: The developed RDC, named 89Zr-IP150, demonstrated robust in vitro stability and high binding affinity, ensuring reliable and specific PET imaging of small, medium, and large subcutaneous tumors in human melanoma mouse xenotransplantation models. Notably, for the first time, the clinical translational potential of89Zr-IP150 was successfully validated using a PDX model.
Conclusions: These findings present a noninvasive, real-time method for the early screening of MUC18 (+) melanoma patients and are important for studying the early-stage biological distribution of MUC18-targeted antibody‒drug conjugates (ADCs).
Keywords: Immuno-PET imaging; Humanized mAb; Melanoma; PDX model
Introduction:
Melanoma stands out as the most lethal form of skin cancer and originates from the malignant transformation of melanocytes[1]. These melanocytes, which arise from the neuroectoderm, migrate extensively throughout the body, including through the skin, uvea, mucous membranes, inner ear, and rectum. These cells are highly dendritic cells that produce melanin to shield against light damage[2]. According to statistics, an estimated 100,640 individuals will receive a new diagnosis of this invasive disease, and 8,290 individuals will die from melanoma of the skin in America by 2024[3]. Benefiting from advancements in early clinical detection and systemic treatment, the 5-year relative survival rate for skin melanoma patients increased to 93% between 2011 and 2017, a notable improvement from 82% in the mid-1970s[4]. Approximately 71% of melanoma patients receive a diagnosis at stage 1, with a remarkable 5-year relative survival rate nearing 100%. In addition, although the incidence of mucosal melanoma is low, due to the lack of early identification, its mortality rate is much greater than that of skin melanoma patients. Consequently, the early screening and detection of melanoma holds profound significance in guiding subsequent treatment choices and enhancing overall survival rates.
MUC18, also known as melanoma cell adhesion molecule (MCAM), is a transmembrane glycoprotein identified by Johnson et al. using the anti-human melanoma monoclonal antibody MUC18, which exhibits distinct expression differences between malignant melanoma cells and normal cells[5]. The resulting amino acid sequences revealed that MUC18 belongs to the immunoglobulin superfamily and shares significant similarity with the sequences of a group of nerve cell adhesion molecules expressed during organogenesis[6, 7]. As research progressed, MUC18 acquired various names, including CD146, melanoma adhesion molecule, melanoma-associated antigen A32, melanoma-associated antigen Mel-CAM, MET-CAM, and HEMCAM. Through bidirectional interactions with multiple specific ligands, such as laminin 411 and 421, galectin-1 and -3, S100A8/A9, and matriptase, MUC18 actively participates in numerous physiological and pathological cellular processes. Overexpression of MUC18 is commonly observed in most malignancies and is implicated in nearly every stage of cancer development and progression, particularly in vascular and lymphatic metastasis[8]. In recent decades, immunotherapy, including immune checkpoint blockade, has revolutionized cancer treatment[9, 10]. However, T-cell exhaustion is associated with decreased efficacy of immune checkpoint inhibitors and adoptive T-cell therapies[11], and a significant number of patients still do not benefit from current forms of immunotherapy. Given the unusually high expression of MUC18 in a variety of tumors and its various roles in reshaping the tumor microenvironment, specific diagnosis and targeted therapies targeting MUC18 may overcome this barrier[12].
Routine clinical methods, such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), for analyzing biomarker expression levels in tumors are invasive and lack real-time access to biomarker expression throughout the body. Recent advancements in nuclear medicine devices, such as whole-body positron emission tomography/computed tomography (PET/CT), now enable the acquisition of higher quality images while reducing patient and staff doses and acquisition times [13-15].
Imaging targeting MUC18 currently relies predominantly on PET probes, with radiolabeled precursors encompassing MUC18-targeted monoclonal antibodies (mAbs), F(ab’)2 fragments, and scFv[16]. To align with the precursors’ half-life, nuclides such as 52Mn, 64Cu,68Ga, 89Zr, and99mTc are utilized for labeling. These probes have been investigated in various cancer models, including models of malignant melanoma[17, 18], brain tumors[19, 20], lung cancer[21, 22], hepatocellular carcinoma[23], and breast cancer[24, 25]. Despite the significance of these studies, the antibodies YY146 and Fab’2 TsCD146 employed in the present study are nonhumanized antibodies. In clinical applications, the human body recognizes these monoclonal antibodies as alloproteins, leading to immune rejection and faster clearance rates. Consequently, there is a compelling need to develop humanized mAb-based radiopharmaceuticals targeting MUC18 to address these challenges. We previously employed a Zr-89-labeled humanized antibody for CLDN18.2-targeted imaging[26].
Here, we developed an MUC18-specific probe through the radiolabeling of89Zr with a humanized monoclonal antibody, IP150. The resulting radiolabeled probe, denoted RDC (89Zr-IP150), demonstrated robust binding specificity for MUC18 in vitro and exhibited reliable imaging capabilities for both skin and mucosal CDX models in vivo. To enhance the clinical relevance of this study, probe specificity was also assessed in melanoma PDX models. These findings offer a promising approach for the early diagnosis of melanoma patients, signifying the importance of exploring the biological distribution of ADCs and guiding therapy.
Materials and Methods
General: 89Zr is produced using a medical cyclotron via the nuclear reaction of 89Y (p, n)89Zr (Peking University Cancer Hospital & Institute). MUC18 mAb (IP150, a humanized antibody) was generated in a CHO expression system and purified by MabSelectTM Sure Resin (GE Healthcare Life Sciences).