RESUMO
Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure-function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure-function relationship of terminal galactose to complement activation in mAb therapeutics.
Assuntos
Anticorpos Monoclonais/farmacologia , Ativação do Complemento/efeitos dos fármacos , Complemento C1q/agonistas , Citotoxicidade Imunológica/efeitos dos fármacos , Galactose/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/farmacologia , Processamento de Proteína Pós-Traducional , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Células CHO , Complemento C1q/metabolismo , Cricetulus , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Cinética , Modelos Biológicos , Multimerização Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: A 48-year-old woman with chronic urinary tract infections presented with uremia. Imaging studies revealed bilateral hydronephrosis and segmental ureteral thickening. INVESTIGATIONS: Physical examination, urine and blood analysis, radiography of the chest, chest CT, abdominal and pelvis CT, renal ultrasonography, cystoscopy, anterograde pyelogram, bladder biopsy. DIAGNOSIS: Malacoplakia involving bilatateral ureters and bladder; cavitary pneumonia. MANAGEMENT: Hemodialysis, placement of bilateral percutaneous nephrostomy tubes, administration of ciprofloxacin, ascorbic acid and bethanechol.