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J. Control. Release, Available online 17 June 2026, 115106 | https://doi.org/10.1016/j.jconrel.2026.115106 Normalized internalization predicts potency of carcinoembryonic antigen-related cell adhesion molecule 6-targeting antibody–drug conjugates

Joo-Young Lim^a,1, Jisu Hong ^b,1, Su Hyeon Lee ^a,1, Seul-Gi Lee ^a, Byeong-Ho Chae ^a, Ga-Hyun Son ^a, Minho Park ^b, Tae Hyeon Yoo ^a,c,2, Yong-Sung Kim ^a,c,b,2, Chang-Han Lee^{b,d,e,f,g,b,2}

^aDepartment of Molecular Science and Technology, College of Engineering, Ajou University, Suwon 16499, Republic of Korea
^bDepartment of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
^cCollege of Advanced Bio-Convergence Engineering, Ajou University, Suwon 16499, Republic of Korea
^dCancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea
^eDepartment of Pharmacology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
^fBK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
^gWide River Institute of Immunology, Seoul National University, Hongcheon 25159, Republic of Korea

¹These authors contributed equally to this work as co-first authors.
²These senior authors contributed equally to this work as co-corresponding authors.
Corresponding authors: Tae Hyeon Yoo, Yong-Sung Kim, Chang-Han Lee

Abstract

Antibody–drug conjugate (ADC) efficacy in solid tumors frequently fails to track with target abundance or binding affinity, reflecting delivery bottlenecks that remain difficult to quantify during lead selection. Here, we develop a discovery-to-function workflow for carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a glycosylphosphatidylinositol (GPI)-anchored antigen with context-dependent trafficking and show that intracellular delivery-related parameters can better organize ADC potency than antigen abundance or binding affinity alone in the CEACAM6 model system. Using multiplexed single-cell screening, we generated a mechanistically diverse anti-CEACAM6 antibody panel spanning CEACAM-family specificity, domain-level epitope usage, kinetic interaction modes, and uptake behavior. Three leads were humanized while preserving specificity and epitope identity and were conjugated to MMAE using a cleavable MC–VC–PAB linker with comparable drug loading across constructs. Despite target-dependent cytotoxicity, ADC potency and selectivity varied markedly by clone and cellular background and could not be consistently predicted by antigen expression or binding affinity alone. We defined a unitless normalized internalization degree (NID), a composite ranking metric derived from conventional surface binding and internalization assays, to integrate surface engagement with internalization efficiency. NID was associated with potency across CEACAM-expressing contexts and provided a delivery-integrated descriptor distinct from conventional internalized-fluorescence amount metrics. This delivery-related interpretation was supported by pHrodo-based tracking of accumulation in acidic compartments relevant to linker processing. The CEACAM6-selective lead h9738, prioritized based on NID profiling, exhibited robust, dose-dependent antitumor activity in a Capan-1 pancreatic cancer xenograft model, achieving complete responses (CRs) at higher doses. Collectively, these findings support a delivery-aware framework for CEACAM6 ADC optimization and identify NID as a practical, context-sensitive ranking metric for candidate prioritization for delivery-limited, GPI-anchored targets such as CEACAM6.

논문소개전체 설명

항체-약물 접합체(ADC)는 암세포 표면 항원을 인식해 강력한 세포독성 약물을 세포 안으로 전달하는 치료 플랫폼이지만, 고형암에서는 항원 발현량이나 항체 결합친화도만으로 약효를 예측하기 어렵다. 본 연구는 췌장암 등에서 발현되는 GPI-anchored 항원 CEACAM6를 표적으로, ADC 후보항체를 선별하는 새로운 평가체계를 제시했다. 연구진은 단일세포 optofluidic 스크리닝을 통해 CEACAM6 결합항체 패널을 확보하고, 특이성, 에피토프, 결합동역학, internalization 특성이 다른 3개 항체를 선별·인간화한 뒤 MMAE payload를 접합해 ADC로 평가했다. 그 결과 ADC의 세포독성은 CEACAM6 발현량이나 결합친화도와 항상 일치하지 않았고, 항체별·세포주별로 큰 차이를 보였다. 이를 설명하기 위해 표면 결합량과 internalization 효율을 통합한 normalized internalization degree(NID)를 정의했으며, NID가 ADC potency와 더 잘 연관됨을 보였다. pHrodo 분석과 uptake 저해 실험도 NID가 실제 세포내 전달과 관련된 지표임을 뒷받침했다. 특히 NID 기반으로 우선순위가 매겨진 CEACAM6 선택적 h9738-vc-MMAE는 Capan-1 췌장암 xenograft 모델에서 용량의존적 종양억제와 고용량 완전관해를 유도했다. 이 연구는 CEACAM6와 같은 internalization 제한 표적에서 ADC 개발 시 단순 결합력 중심 평가를 넘어, 세포내 전달능을 반영한 후보선별 전략이 중요함을 제시한다.

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