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1.
Mol Cancer Ther ; 11(3): 582-93, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22248472

RESUMO

The prevalence of ErbB2 amplification in breast cancer has resulted in the heavy pursuit of ErbB2 as a therapeutic target. Although both the ErbB2 monoclonal antibody trastuzumab and ErbB1/ErbB2 dual kinase inhibitor lapatinib have met with success in the clinic, many patients fail to benefit. In addition, the majority of patients who initially respond will unfortunately ultimately progress on these therapies. Activation of ErbB3, the preferred dimerization partner of ErbB2, plays a key role in driving ErbB2-amplified tumor growth, but we have found that current ErbB2-directed therapies are poor inhibitors of ligand-induced activation. By simulating ErbB3 inhibition in a computational model of ErbB2/ErbB3 receptor signaling, we predicted that a bispecific antibody that docks onto ErbB2 and subsequently binds to ErbB3 and blocks ligand-induced receptor activation would be highly effective in ErbB2-amplified tumors, with superior activity to a monospecific ErbB3 inhibitor. We have developed a bispecific antibody suitable for both large scale production and systemic therapy by generating a single polypeptide fusion protein of two human scFv antibodies linked to modified human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling and showing antitumor activity in preclinical models that is dependent on ErbB2 overexpression. MM-111 can be rationally combined with trastuzumab or lapatinib for increased antitumor activity and may in the future complement existing ErbB2-directed therapies to treat resistant tumors or deter relapse.


Assuntos
Anticorpos Biespecíficos/farmacologia , Neoplasias/tratamento farmacológico , Neuregulina-1/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/metabolismo , Anticorpos Biespecíficos/farmacocinética , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Desenho de Fármacos , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Complexos Multiproteicos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Int J Biol Macromol ; 39(1-3): 141-50, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16678252

RESUMO

Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His6-tag. These were separated by concanavalin A chromatography followed by HiTrap IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. The binding interaction of MFE-23 to N-A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9)M for the P. pastoris expressed, native N-A1, and 5.33 x 10(-9) M for the Endo H-treated N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-A1.


Assuntos
Biomarcadores Tumorais/química , Antígeno Carcinoembrionário/química , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/química , Pichia , Proteínas Recombinantes de Fusão/química , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/isolamento & purificação , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/isolamento & purificação , Cromatografia Líquida , Glicosilação , Humanos , Pichia/química , Pichia/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação
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