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1.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35210365

RESUMO

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.


Assuntos
Anticorpos/metabolismo , Imunoconjugados/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Especificidade de Anticorpos , Humanos
2.
J Clin Invest ; 132(4)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35166238

RESUMO

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.


Assuntos
Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Epitopos/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Pancreáticas/imunologia , Proteólise , Animais , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Epitopos/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Pancreáticas/genética
3.
Clin Cancer Res ; 26(14): 3608-3615, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32341034

RESUMO

PURPOSE: The recent emergence of radioligand therapies for cancer treatment has increased enthusiasm for developing new theranostic strategies coupling both imaging and cytotoxicity in the same entity. In this study, we evaluated whether CUB domain containing protein 1 (CDCP1), a single-pass transmembrane protein highly overexpressed in diverse human cancers, might be a target for cancer theranostics. EXPERIMENTAL DESIGN: The ectodomain of CDCP1 was targeted using radiolabeled forms of 4A06, a potent and specific recombinant human antibody that we developed. Imaging and antitumor assessment studies were performed in animal models of pancreatic cancer, including two patient-derived xenograft models we developed for this study. For antitumor assessment studies, the endpoints were death due to tumor volume >3,000 mm3 or ≥20% loss in body weight. Specific tracer binding or antitumor effects were assessed with an unpaired, two-tailed Student t test and survival advantages were assessed with a log rank (Mantel-Cox) test. Differences at the 95% confidence level were interpreted to be significant. RESULTS: 89Zr-4A06 detected a broad dynamic range of full length or cleaved CDCP1 expression on seven human pancreatic cancer tumors (n = 4/tumor). Treating mice with single or fractionated doses of 177Lu-4A06 significantly reduced pancreatic cancer tumor volume compared with mice receiving vehicle or unlabeled 4A06 (n = 8; P < 0.01). A single dose of 225Ac-4A06 also inhibited tumor growth, although the effect was less profound compared with 177Lu-4A06 (n = 8; P < 0.01). A significant survival advantage was imparted by 225Ac-4A06 (HR = 2.56; P < 0.05). CONCLUSIONS: These data establish that CDCP1 can be exploited for theranostics, a finding with widespread implications given its breadth of overexpression in cancer.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Moléculas de Adesão Celular/antagonistas & inibidores , Pâncreas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Medicina de Precisão/métodos , Animais , Antígenos de Neoplasias/genética , Antineoplásicos Imunológicos/farmacocinética , Moléculas de Adesão Celular/genética , Humanos , Masculino , Camundongos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Proc Natl Acad Sci U S A ; 115(11): 2836-2841, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29476010

RESUMO

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.


Assuntos
Anticorpos/análise , Linfoma de Burkitt/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia/genética , Proteínas de Membrana/genética , Proteômica/métodos , Anticorpos/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Humanos , Leucemia/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo
5.
Elife ; 72018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29359686

RESUMO

While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRASG12V, and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell.


Assuntos
Anticorpos/metabolismo , Antineoplásicos/metabolismo , Portadores de Fármacos/metabolismo , Fatores Imunológicos/metabolismo , Proteínas de Membrana/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Anticorpos/imunologia , Linhagem Celular Tumoral , Humanos , Fatores Imunológicos/imunologia , Proteínas de Membrana/imunologia , Proteínas ras/metabolismo
6.
J Med Chem ; 54(13): 4659-69, 2011 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-21591694

RESUMO

Toll-like receptor 4 (TLR4) induced proinflammatory signaling has been directly implicated in severe sepsis and represents an attractive therapeutic target. Herein, we report our investigations into the structure-activity relationship and preliminary drug metabolism/pharmacokinetics study of ß-amino alcohol derivatives that inhibit the TLR4 signaling pathway. Lead compounds were identified from in vitro cellular examination with micromolar potency for their inhibitory effects on TLR4 signaling and subsequently assessed for their ability to suppress the TLR4-induced inflammatory response in an ex vivo whole blood model. In addition, the toxicology, specificity, solubility, brain-blood barrier permeability, and drug metabolism of several compounds were evaluated. Although further optimizations are needed, our findings lay the groundwork for the future drug development of this class of small molecule agents for the treatment of severe sepsis.


Assuntos
Amino Álcoois/síntese química , Anti-Inflamatórios/síntese química , Receptor 4 Toll-Like/antagonistas & inibidores , Amino Álcoois/farmacocinética , Amino Álcoois/farmacologia , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Modelos Moleculares , Óxido Nítrico/biossíntese , Permeabilidade , Sepse/tratamento farmacológico , Estereoisomerismo , Relação Estrutura-Atividade
7.
Curr Pharm Des ; 16(9): 1055-62, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20030619

RESUMO

Membrane proteins account for approximately one third of all proteins in eukaryotic and prokaryotic cells. These proteins are critical in a diverse array of cellular functions. Despite their obvious importance, the effectiveness of research tools to study the structure and function of integral membrane proteins lags behind that of water-soluble proteins. This is due in part to the lack of probing agents that can specifically and selectively recognize these targets. This review focuses on methods developed to overcome the obstacles of studying membrane proteins. We describe TM protein properties as well as biophysical properties of amino acids within the membrane bilayer. We also summarize the known characteristics of membrane regions in their distinctive environments and generate a summary of current research approaches that succeed in probing interactions of TM proteins within their native setting. This allows further insight into protein-protein interactions in a hydrophobic environment as it pertains to drug development.


Assuntos
Membrana Celular/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Proteínas de Membrana/metabolismo , Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Animais , Modelos Biológicos , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Mapeamento de Interação de Proteínas
8.
Chembiochem ; 10(4): 645-9, 2009 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-19184989

RESUMO

Toll-like receptors are an integral part of innate immunity in the central nervous system (CNS); they orchestrate a robust defense in response to both exogenous and endogenous danger signals. Recently, toll-like receptor 4 (TLR4) has emerged as a therapeutic target for the treatment of CNS-related diseases such as sepsis and chronic pain. We herein report a chemical biology approach by using a rationally designed peptide inhibitor to disrupt the TLR4-MD2 association, thereby blocking TLR4 signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Linhagem Celular , Biologia Computacional , Antígeno 96 de Linfócito , Camundongos , Modelos Moleculares , Peptídeos/síntese química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Receptor 4 Toll-Like/química
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