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1.
PLoS One ; 19(6): e0301223, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38837964

RESUMO

New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries-two synthetic and two immunized-against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.


Assuntos
Antagonistas do Receptor A2 de Adenosina , Receptor A2A de Adenosina , Humanos , Animais , Receptor A2A de Adenosina/metabolismo , Receptor A2A de Adenosina/imunologia , Células HEK293 , Camundongos , Antagonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Macaca fascicularis , Biblioteca de Peptídeos
2.
MAbs ; 14(1): 2002236, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34967699

RESUMO

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Técnicas de Visualização da Superfície Celular , Imunoglobulina G/imunologia , Biblioteca de Peptídeos , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , COVID-19/metabolismo , COVID-19/prevenção & controle , COVID-19/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Epitopos , Feminino , Interações Hospedeiro-Patógeno , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Mesocricetus , SARS-CoV-2/patogenicidade , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/farmacologia , Células Vero
3.
MAbs ; 13(1): 1893425, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33706686

RESUMO

G protein-coupled receptors (GPCRs) are a group of seven-transmembrane receptor proteins that have proven to be successful drug targets. Antibodies are becoming an increasingly promising modality to target these receptors due to their unique properties, such as exquisite specificity, long half-life, and fewer side effects, and their improved pharmacokinetic and pharmacodynamic profiles compared to peptides and small molecules, which results from their more favorable biodistribution. To date, there are only two US Food and Drug Administration-approved GPCR antibody drugs, namely erenumab and mogamulizumab, and this highlights the challenges encountered in identifying functional antibodies against GPCRs. Utilizing Twist's precision DNA writing technologies, we have created a GPCR-focused phage display library with 1 × 1010 diversity. Specifically, we mined endogenous GPCR binding ligand and peptide sequences and incorporated these binding motifs into the heavy chain complementarity-determining region 3 in a synthetic antibody library. Glucagon-like peptide-1 receptor (GLP-1 R) is a class B GPCR that acts as the receptor for the incretin GLP-1, which is released to regulate insulin levels in response to food intake. GLP-1 R agonists have been widely used to increase insulin secretion to lower blood glucose levels for the treatment of type 1 and type 2 diabetes, whereas GLP-1 R antagonists have applications in the treatment of severe hypoglycemia associated with bariatric surgery and hyperinsulinomic hypoglycemia. Here we present the discovery and creation of both antagonistic and agonistic GLP-1 R antibodies by panning this GPCR-focused phage display library on a GLP-1 R-overexpressing Chinese hamster ovary cell line and demonstrate their in vitro and in vivo functional activity.


Assuntos
Anticorpos Monoclonais/farmacologia , Glicemia/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Controle Glicêmico , Hipoglicemiantes/farmacologia , Incretinas/farmacologia , Biblioteca de Peptídeos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Sítios de Ligação de Anticorpos , Biomarcadores/sangue , Glicemia/metabolismo , Células CHO , Cricetulus , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Ensaios de Triagem em Larga Escala , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacocinética , Incretinas/genética , Incretinas/metabolismo , Incretinas/farmacocinética , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Domínios e Motivos de Interação entre Proteínas , Ratos Sprague-Dawley
5.
Cancer Immunol Immunother ; 65(10): 1169-75, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27506529

RESUMO

In this study, HB22.7, an anti-CD22 monoclonal antibody, was used for specific, targeted delivery of monomethyl auristatin E (MMAE) to non-Hodgkin lymphoma (NHL). MMAE was covalently coupled to HB22.7 through a valine-citrulline peptide linker (vc). Maleimide-functionalized vcMMAE (mal-vcMMAE) was reacted with thiols of the partially reduced mAb. Approximately 4 molecules of MMAE were conjugated to HB22.7 as determined by residual thiol measurement and hydrophobic interaction chromatography-HPLC (HIC-HPLC). HB22.7-vcMMAE antibody-drug conjugate (ADC) retained its binding to Ramos NHL cells and also exhibited potent and specific in vitro cytotoxicity on a panel of B cell NHL cell lines with IC50s of 20-284 ng/ml. HB22.7-vcMMAE also showed potent efficacy in vivo against established NHL xenografts using the DoHH2 and Granta 519 cell lines. One dose of the ADC induced complete and persistent response in all DoHH2 xenografts and 90 % of Granta xenografts. Minimal toxicity was observed. In summary, HB22.7-vcMMAE is an effective ADC that should be evaluated for clinical translation.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/efeitos dos fármacos , Imunoterapia/métodos , Imunotoxinas/uso terapêutico , Linfoma não Hodgkin/terapia , Oligopeptídeos/uso terapêutico , Animais , Anticorpos Monoclonais/química , Apoptose , Linfócitos B/imunologia , Linhagem Celular Tumoral , Feminino , Imunotoxinas/química , Linfoma não Hodgkin/imunologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Oligopeptídeos/química , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Autoimmun Rev ; 13(4-5): 560-4, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24418298

RESUMO

Uncompensated autoantibody-mediated red blood cell (RBC) consumption is the hallmark of autoimmune hemolytic anemia (AIHA). Classification of AIHA is pathophysiologically based and divides AIHA into warm, mixed or cold-reactive subtypes. This thermal-based classification is based on the optimal autoantibody-RBC reactivity temperatures. AIHA is further subcategorized into idiopathic and secondary with the later being associated with a number of underlying infectious, neoplastic and autoimmune disorders. In most cases AIHA is confirmed by a positive direct antiglobulin test (DAT). The standard therapeutic approaches to treatment of AIHA include corticosteroids, splenectomy, immunosuppressive agents and monoclonal antibodies.


Assuntos
Anemia Hemolítica Autoimune/diagnóstico , Corticosteroides/uso terapêutico , Anemia Hemolítica Autoimune/induzido quimicamente , Anemia Hemolítica Autoimune/tratamento farmacológico , Anemia Hemolítica Autoimune/imunologia , Anticorpos Monoclonais/uso terapêutico , Teste de Coombs , Humanos , Imunossupressores/uso terapêutico , Esplenectomia
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