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1.
MAbs ; 13(1): 1850395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33459147

RESUMO

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.


Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Enterotoxina/imunologia , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Hibridomas , Macaca fascicularis/imunologia , Macaca fascicularis/metabolismo , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismo
2.
Antibodies (Basel) ; 5(1)2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-31557987

RESUMO

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

3.
J Biol Chem ; 289(15): 10865-10875, 2014 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-24567333

RESUMO

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.


Assuntos
Regulação Enzimológica da Expressão Gênica , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores Toll-Like/metabolismo , Sequência de Aminoácidos , Animais , Sistema Livre de Células , Clonagem Molecular , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Insetos , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases , Dados de Sequência Molecular , Monócitos/citologia , Mutação , NF-kappa B/metabolismo , Fases de Leitura Aberta , Fosforilação , Ligação Proteica , Conformação Proteica , Receptores de Interleucina-1/agonistas , Transdução de Sinais , Pele/metabolismo , Receptores Toll-Like/agonistas
4.
J Med Chem ; 53(3): 1238-49, 2010 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-20038108

RESUMO

To aid in the pursuit of selective kinase inhibitors, we have developed a unique ATP site binder tool for the detection of binders outside the ATP site by nuclear magnetic resonance (NMR). We report here the novel synthesis that led to this paramagnetic spin-labeled pyrazolopyrimidine probe (1), which exhibits nanomolar inhibitory activity against multiple kinases. We demonstrate the application of this probe by performing NMR binding experiments with Lck and Src kinases and utilize it to detect the binding of two compounds proximal to the ATP site. The complex structure of the probe with Lck is also presented, revealing how the probe fits in the ATP site and the specific interactions it has with the protein. We believe that this spin-labeled probe is a valuable tool that holds broad applicability in a screen for non-ATP site binders.


Assuntos
Trifosfato de Adenosina/metabolismo , Espectroscopia de Ressonância Magnética , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Marcadores de Spin/síntese química , Sítios de Ligação , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia
5.
OMICS ; 8(4): 267-88, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15703476

RESUMO

Multitiered quantitative analysis of biological systems is rapidly becoming the desired approach to study hierarchical functional interactions between proteins and metabolites. We describe here a novel systematic approach to analyze organisms with complex metabolic regulatory networks. By using precise analytical methods to measure biochemical constituents and their relative abundance in whole plasma of transgenic ApoE*3-Leiden mice and an isogenic wild-type control group, simultaneous snapshots of metabolic and protein states were obtained. Novel data processing and multivariate analysis tools such as Impurity Resolution Software (IMPRESS) and Windows-based linear fit program (WINLIN) were used to compare protein and metabolic profiles in parallel. Canonical correlations of the resulting data show quantitative relationships between heterogeneous components in the TG animals. These results, obtained solely from whole plasma analysis allowed us, in a rapid manner, to corroborate previous findings as well as find new events pertaining to dominant and peripheral events in lipoprotein metabolism of a genetically modified mammalian organism in relation to ApoE3, a key mediator of lipoprotein metabolism.


Assuntos
Apolipoproteínas E/sangue , Arteriosclerose/genética , Técnicas Genéticas , Hiperlipoproteinemias/genética , Camundongos Transgênicos , Animais , Apolipoproteína E3 , Apolipoproteínas E/química , Cromatografia Líquida , Cruzamentos Genéticos , Feminino , Genes Dominantes , Humanos , Metabolismo dos Lipídeos , Lipoproteínas/química , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Análise Multivariada , Mutação , Peptídeos/química , Análise de Componente Principal , Proteínas/química , Software , Fatores de Tempo , Tripsina/química
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