Adam J. Rosenberg

Background: Epidermal Growth Factor Receptor (EGFR) overexpression in Head and Neck Squamous Cell Carcinoma (HNSCC) is well documented and strongly correlated with aggressive phenotypes. As such EGFR is an excellent choice for molecular imaging of HNSCC as the high overexpression levels allow for a specific marker of tumors. Currently the only established EGFR radiopharmaceuticals that have been translated into human subjects are labeled versions of antibodies and antibody fragments, which all have slow pharmacokinetics and have low specificity due to high background and blood pool. [ ¹⁸ F]NOTA-ABY-030 is a promising positron emission tomography (PET) radiotracer for in vivo study of the EGFR receptor that alleviates these issues; herein we report the cGMP automated radiosynthesis for use with human subjects. Results: To facilitate clinical trials a fully automated radiosynthesis procedure for [ ¹⁸ F]NOTA-ABY-030 was developed using commercially available materials on the GE HealthCare FASTlab cassette-based automated radiosynthesis system. The overall synthesis time was 20 min with a radiochemical yield of 47 ± 10% (n = 3). The radiochemical purity was greater than 90% for [ ¹⁸ F]NOTA-ABY-030 with a binding fraction of 90% ( n = 3). The protocol described herein reliably provides [ ¹⁸ F]NOTA-ABY-030 that meets all relevant release criteria for a GMP radiopharmaceutical. Conclusions: A fast, reliable and efficient automated radiosynthesis was developed using a commercial cassette-based module. This will enable clinical trials work as well as simple method transfer to alternate manufacturing sites; as well as being applicable to small biologic targeting vector radiolabeling.

Background: The clinical translation of molecularly targeted therapeutics and imaging agents represents a cornerstone of precision oncology, with the global theranostics market projected to exceed $25 billion by 2030. However, the development of theragnostic agents or diagnostic companions remains constrained by analytical bottlenecks in quality control, such as target-binding fraction, which are increasingly required by regulatory agencies as product release criteria during the translation process. Current methods, including enzyme-linked immunosorbent assay (ELISA), which require specialized resources or external CROs, or bead-based assays for radiolabeled compounds, which involve complex multi-step protocols; these limitations and others hamper their practical implementation in clinical manufacturing environments. Assay delays can postpone clinical trial initiation, increase development costs, and delay patient access to these agents. Results: We have developed and validated a rapid, size-exclusion high-performance liquid chromatography (SE-HPLC) method for the determination of target-binding fractions of labeled biologics. The method separates the unbound biologic from the larger antigen-bound complex, allowing for rapid quantification. We validated the method using a panel of fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030), assessing linearity, specificity, and concentration independence. The SE-HPLC method achieved excellent separation of bound and unbound species with a resolution (Rs) of 3.2. A strong linear relationship (R ² = 0.999) was observed between the antigen-to-antibody ratio and the measured binding fraction. The method demonstrated high specificity, with no binding detected with non-target antigens. The total assay and analysis time was less than 35 min, a significant improvement over traditional methods. Conclusions: SE-HPLC provides a rapid, specific, and cost-effective alternative to traditional binding fraction assessment methods, reducing quality control timelines from weeks/hours to minutes. The method’s compatibility with both fluorescent and radiolabeled biologics and integration with existing HPLC infrastructure represents a significant advancement in development workflows.

Background: Intraoperative ¹⁸ F‐fluorodeoxyglucose ( ¹⁸ F‐FDG) micro‐positron emission tomography/computed tomography (micro‐PET/CT) is an emerging modality for margin assessment. Prior to clinical use, micro‐PET/CT margin distances must be correlated with gold‐standard histopathology. Methods: Micro‐PET/CT uptake was co‐registered to 3D specimen models annotated with margin sampling sites using principal component analysis and iterative closest point surface registration. Margin distances were measured on the fused models using Gaussian Mixed Modeling and compared with histopathology. Results: Fused micro‐PET/CT and 3D specimen models were successfully generated for five patients with head and neck cancer. Among the 16 measured margins, the margin distance on the fused models was within 1 mm of histopathology in 15/16 cases. The Bland–Altman plot demonstrated excellent agreement (±1.25 mm). Conclusion: We report a novel technique to register micro‐PET/CT data to annotated 3D specimen models. Margin distance measured on the micro‐PET/CT scans correlated closely with histopathology. Intraoperative micro‐PET/CT warrants further investigation in head and neck margin assessment.

Background: Neoadjuvant chemoimmunotherapy is being explored in locally advanced head and neck squamous cell carcinoma (HNSCC), yet the spatial patterns of pathologic response and clinical implications remain poorly characterized. Methods: A 79‐year‐old female with recurrent left oral tongue SCC (PD‐L122C3 Combined Positive Score 90) who underwent prior resection and free flap reconstruction received neoadjuvant pembrolizumab, paclitaxel, and carboplatin followed by definitive surgical resection with intraoperative positron emission tomography/computed tomography (PET/CT) imaging. Results: Final histopathology revealed two discrete foci of residual poorly differentiated SCC, measuring 1.8 and 0.5 cm, separated by an 8 mm intervening bridge of inflammation without viable tumor. Intraoperative PET/CT with 3D specimen mapping confirmed the spatial relationship between residual foci and intervening treatment effect. Conclusions: Pathologic response to neoadjuvant chemoimmunotherapy in HNSCC can manifest as a multifocal regression pattern. Intraoperative real‐time approaches to assessing treatment response warrant further investigation. Trial Registration: ClinicalTrials.gov identifier: NCT06915454

Monoclonal antibodies are commonly used as targeted therapies for autoimmune, infectious and oncologic diseases. Here, we describe a simple protocol to optically label monoclonal antibodies for use as molecular imaging agents for clinical investigation. In preclinical settings, optical imaging has complemented the strengths of nuclear imaging while offering higher resolution and a safer method for antibody-ligand engagement. However, translation of optically labeled monoclonal antibodies to the clinic has been slow because antibodies require a manual process in current good manufacturing practices (cGMP)-compliant facilities; the cost barriers to establish the investigational new drug application by using the traditional contract research organization pathway (>US$1 million) exceed the resources of academic institutions and early-phase pharmaceutical companies. To address these challenges, we repurposed an existing radiolabeling cGMP method for optical labeling that uses an automated, self-contained synthesis module. This method depends on commercially available, single-use, cassette-based production, which simplifies the workflow and does not require a clean room facility. This automated production method reduces both the cost and time required to produce a clinical dose of near-IR fluorescently labeled monoclonal antibody-IRDye800CW, decreasing the development costs for pilot and initial batches by almost 90%, as well as the production time by 40% to 4 h plus quality control (~10 h total). Our cGMP manufacturing method can optically label any compatible monoclonal antibodies at any dedicated radiochemistry facility. We provide the detailed protocol for production of panitumumab-IRDye800CW and nivolumab-IRDye800CW under cGMP regulations, achieving excellent yield, optimal degree of labeling and high purity.

Background: The clinical translation of molecularly targeted therapeutics and imaging agents represents a cornerstone of precision oncology, with the global theranostics market projected to exceed $25 billion by 2030. However, the development of theragnostic agents or diagnostic companions remains constrained by analytical bottlenecks in quality control, such as target-binding specificity, which are increasingly required by regulatory agencies as product release criteria during the translation process. Current methods, including enzyme-linked immunosorbent assay (ELISA), which require specialized resources or external CROs, or bead-based assays for radiolabeled compounds, which involve complex multi-step protocols; these limitations and others hamper their practical implementation in clinical manufacturing environments. Assay delays can postpone clinical trial initiation, increase development costs, and delay patient access to these agents. Results: We have developed and validated a rapid, size-exclusion high-performance liquid chromatography (SE-HPLC) method for the determination of target-binding fractions of labeled biologics. The method separates the unbound biologic from the larger antigen-bound complex, allowing for rapid quantification. We validated the method using a panel of fluorescently labeled antibodies (panitumumab-IRDye800CW, nivolumab-IRDye800CW) and radiolabeled biologics ([18F]GEH200521, [18F]NOTA-ABY-030), assessing linearity, specificity, and concentration independence. The SE-HPLC method achieved excellent separation of bound and unbound species with a resolution (Rs) of 3.2. A strong linear relationship (R ² = 0.999) was observed between the antigen-to-antibody ratio and the measured binding fraction. The method demonstrated high specificity, with no binding detected with non-target antigens. The total assay and analysis time was less than 35 minutes, a significant improvement over traditional methods. Conclusions: SE-HPLC provides a rapid, specific, and cost-effective alternative to traditional binding fraction assessment methods, reducing quality control timelines from weeks/hours to minutes. The method’s compatibility with both fluorescent and radiolabeled biologics and integration with existing HPLC infrastructure represents a significant advancement in development workflows.

As the field of radiochemistry moves toward the rapid development and translation of radiopharmaceuticals, the radiosynthetic methodology and manufacturing are becoming more and more refined. As the use of radiopharmaceuticals in clinical research is one of the primary goals of radiochemistry research, compliance with cGMP regulations is a key factor in radiosynthesis developments. This review is primarily focused on the automation of the radiosynthesis on modules, with a special focus on the use of disposable cassettes for the reactors and flow‐paths. This review aims to cover the developments and current state‐of‐the‐art for cassette‐based radiosynthesis of carbon‐11 radiopharmaceuticals.

Background: Radiopharmaceuticals capable of targeting the fibroblast activation protein have become widely utilized in the research realm as well as show great promise to be commercialized; with [⁶⁸Ga]Ga-FAPI-46 being one of the most widely utilized. Until now the synthesis has relied on generator-produced gallium-68. Here we present a developed method to utilize liquid-target cyclotron-produced gallium-68 to prepare [⁶⁸Ga]Ga-FAPI-46. Results: A fully-automated manufacturing process for [⁶⁸Ga]Ga-FAPI-46 was developed starting with the ⁶⁸Zn[p,n]⁶⁸Ga cyclotron bombardment to provide [⁶⁸Ga]GaCl₃, automated purification of the [⁶⁸Ga]GaCl₃, chelation with the precursor, and final formulation/purification. The activity levels produced were sufficient for multiple clinical research doses, and the final product met all release criteria. Furthermore, the process consistently provides < 2% of Ga-66 and Ga-67 at the 4-h expiry, meeting the Ph. Eur. standards. Conclusions: The automated radiosynthesis on the GE FASTlab 2 module purifies the cyclotron output into [⁶⁸Ga]GaCl₃, performs the labeling, formulates the product, and sterilizes the product while transferring to the final vial. Production of > 40 mCi (> 1480 MBq) of [⁶⁸Ga]Ga-FAPI-46 in excellent radiochemical yield was achieved with all batches meeting release criteria.