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Biophysical and Mechanistic Dissection of Cytosol-Penetrating IgGs Reveals Their Potential for Targeted Intracellular Delivery

  • 김승은 (SEUNG-EUN KIM, 아주대학교 일반대학원)

초록/요약

Enabling immunoglobulin G (IgG) antibodies to autonomously internalize and localize in the cytosol of targeted cells—referred to as cytosol-penetrating antibodies (cytotransmabs, CTs)—is challenging yet highly promising. Our group has developed a second-generation (2G) CT, named in2C41, featuring an acidic pH-responsive endosomal escape motif (R-W/E motif) with Arg-Trp pairs and a Glu patch in the CH3 and CL domains of IgG1/κ antibody. Herein, I first examined the intracellular trafficking of in2C41 following receptor-mediated endocytosis, and subsequently elucidated its endosomal escape mechanism. in2C41 follows the endosomal maturation pathway and traffics to late endosomes, where the reduced pH environment specifically induces its membrane association and subsequent destabilization to enable cytosolic entry, whereas it remains inactive at neutral pH. Importantly, this motif does not compromise essential IgG properties, as in2C41 retains thermal stability, FcRn-binding affinity, and pharmacokinetic profile comparable to that of conventional IgG1/κ antibodies. To validate the receptor-targeting modularity of the 2G CT platform, a chimeric IgG was constructed by pairing the variable regions of the clinically approved anti-HER2 antibody pertuzumab with the engineered IgG1/κ constant domains of in2C41. The resulting construct, pertuzumab-2C41, retained HER2-binding affinity and acquired cytosol-penetrating ability. These findings establish 2G CT as a versatile antibody platform that enables cytosolic localization via diverse receptor-mediated endocytosis pathways.

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초록/요약

표적 세포의 세포질로 자율적으로 내부화 되어 도달할 수 있는 면역글로불린 G (IgG) 형식의 항체 (세포질 침투 항체, cytotransmabs 또는 CTs)를 개발하는 것은 여전히 도전적인 과제이지만, 매우 유망한 전략으로 간주된다. 본 연구진의 연구실에서는 IgG1/κ 항체의 CH3 및 CL 도메인에 Arg-Trp 쌍과 Glu 패치를 포함한 산성 pH 반응성 엔도좀 탈출 구조 모티프 (RW/E 모티프)를 도입하여, 2 세대 (2G) 세포질 침투 항체인 in2C41 을 개발하였다. 본 연구에서는 먼저, in2C41 이 수용체 매개 내재화(receptor-mediated endocytosis) 이후 세포 내에서 어떻게 이동하는지를 규명하였고, 이어서 endosomal escape 메커니즘을 밝혔다. in2C41 은 endosomal maturation 경로를 따라 이동하며 late endosome 에 도달하고, 이곳의 낮은 pH 환경에서 선택적으로 세포막에 결합하고 불안정화를 유도하여 세포질로의 진입을 가능하게 한다. 반면, 중성 pH 에서는 활성을 나타내지 않는다. 주목할 점은, 이러한 모티프의 도입이 IgG 항체의 본질적인 특성을 손상시키지 않는다는 것이다. in2C41 은 기존 IgG1/κ 항체와 유사한 열적 안정성, FcRn 결합능, 그리고 약리학적 특성(pharmacokinetic profile)을 유지하였다. 2G CT 플랫폼의 수용체 특이적 모듈성을 검증하기 위해, 승인된 항-HER2 항체인 퍼투주맙(pertuzumab)의 variable domain(VH 및 VL)을 in2C41 의 엔지니어드된 IgG1/κ constant domain 에 결합하여 키메라 IgG 인 pertuzumab-2C41 을 제작하였다. 이 항체는 HER2 에 대한 결합 능력을 유지하면서 세포질 침투 능력을 획득하였다. 이러한 결과는 2G CT 플랫폼이 다양한 수용체 매개 내재화 경로를 통해 세포질로 전달될 수 있는 범용적인 항체 플랫폼임을 입증한다.

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목차

1. Introduction 1
1.1. Structure and Function of IgG Antibodies 1
1.2. Limitations of Conventional IgG Therapeutics 3
1.3. Cytosol-Penetrating Antibodies (Cytotransmabs) 4
2. Materials and Methods 7
2.1. Cell lines 7
2.2. Construction of antibody expression plasmids. 7
2.3. Expression and purification of antibodies 8
2.4. Pulse-chase experiments to monitor intracellular trafficking of in2C41. 8
2.5. Cytosolic calcein release assay with pharmacological inhibitors 9
2.6. Trypan blue uptake assay and membrane pore formation recovery assay 9
2.7. pH-dependent plasma membrane binding assay 10
2.8. pH-dependent plasma membrane translocation assay 10
2.9. Differential scanning calorimetry 11
2.10. pH-dependent FcRn binding kinetics 11
2.11. PK studies in mice. 12
2.12. HER2 binding kinetics. 13
2.13. Split-GFP complementation assay on live cells 13
2.14. Statistical analysis 14
3. Results 15
3.1. Internalized in2C41 predominantly localizes to early and late endosomes prior to cytosolic release. 15
3.2. Cytosolic release of in2C41 occurs mainly from acidified late endosomes, not from early endosomes or the Golgi/ER 15
3.3. in2C41 binds selectively to integrin αvβ3/αvβ5-deficient membranes at acidic pH. 17
3.4. in2C41 induces pore formation in membranes exclusively at acidic pH without causing lysis 18
3.5. in2C41-induced membrane pores are transient, reversible, and enable IgG translocation at acidic pH 20
3.6. in2C41 maintains overall thermal stability despite modest CH 3 destabilization caused by endosomal escape motif insertion. 22
3.7. in2C41 retains pH-dependent FcRn binding comparable to conventional IgG 24
3.8. in2C41 demonstrates a serum half-life comparable to wild-type IgG1 in mice. 24
3.9. The 2G CT confers cytosol-penetrating ability to receptor-targeting antibody via constant region replacement 26
4. Discussion 29
REFERENCE 32
PUBLICATIONS AND PATENTS 34
ABSTRACT IN KOREAN 35

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