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1.
Front Immunol ; 15: 1378813, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38720892

RESUMO

Background: Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. Methods: We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). Results: NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. Conclusion: NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.


Assuntos
Anticorpos Biespecíficos , Antígeno CD47 , Antígeno Carcinoembrionário , Animais , Feminino , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Antígeno CD47/imunologia , Antígeno CD47/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas Ligadas por GPI , Macaca fascicularis , Neoplasias/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Genome Res ; 14(7): 1324-32, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15231748

RESUMO

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Biologia Computacional/métodos , Bases de Dados de Proteínas , Biblioteca Gênica , Proteínas de Homeodomínio/fisiologia , Humanos , Rim/química , Rim/citologia , Rim/embriologia , Rim/metabolismo , Proteínas com Domínio LIM , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas de Membrana/fisiologia , Placenta/química , Placenta/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Técnicas do Sistema de Duplo-Híbrido
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