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1.
Anal Chem ; 91(20): 12724-12732, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31478640

RESUMO

Antibody-drug conjugates (ADCs) have become a major class of oncology biopharmaceuticals. Traditional ADCs have a stochastic distribution of cytotoxic drugs attached at several different sites on the antibody. The heterogeneous nature of stochastic ADCs results in a complex compositional analysis. To improve on traditional ADC technology, we have developed a chemical conjugation platform termed "AJICAP" for the site-specific modification of native antibodies using a class of IgG Fc affinity reagents. Here we report further investigation focusing on several analyses of a first-generation AJICAP-ADC (Angew. Chem., Int. Ed. 2019, 58, 5592-5597). For drug-antibody ratio (DAR) determination, we examined and compared six different analytical methods. To the best of our knowledge, this is the first report of a comparison of analytical techniques to measure the DAR for ADCs produced by a site-specific technology such as AJICAP. Furthermore, a rapid analytical process for confirmation of the site selectivity of AJICAP conjugation was established by SEC-Q-TOF-MS. The analytical strategy reported here can be applied to the DAR determination of site-specific ADCs.


Assuntos
Imunoconjugados/análise , Espectrometria de Massas/métodos , Antineoplásicos/química , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Fragmentos Fc das Imunoglobulinas/química , Oligopeptídeos/química , Trastuzumab/química
2.
Mol Pharm ; 15(6): 2384-2390, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29757653

RESUMO

AGS62P1 is an antibody drug conjugate (ADC) composed of a human IgG1κ monoclonal antibody against FLT3 (FMS-like tyrosine kinase 3) with a p-acetyl phenylalanine (pAF) residue inserted at position 124 of each heavy chain linked to the proprietary microtubule disrupting agent AGL-0182-30 via an alkoxyamine linker that forms an oxime upon conjugation to the antibody. AGS62P1 is currently in Phase I human clinical trials for acute myelogenous leukemia (AML). The identified primary metabolite of an oxime-linked ADC is presented for the first time. AGS62P1 metabolism was assessed in xenograft tumor-bearing mice and rats treated with the ADC using liquid chromatography and mass spectrometry-based methods described herein. In this study, we identified the metabolite of AGS62P1 as pAF-AGL-0185-30, which contains a fragment resulting from the catabolism of the antibody component of the ADC and hydrolysis of the terminal amide portion of the linker-payload. We demonstrated that the metabolite of AGS62P1 is tolerated in rats above 1.5 mg/kg and above 0.334 mg/kg in cynomolgus monkeys when given as a single dose. Furthermore, we established in vitro that pAF-AGL-0185-30 does not significantly inhibit hERG or cytochrome P450 family enzymes (CYPs).


Assuntos
Antineoplásicos/metabolismo , Imunoconjugados/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Canal de Potássio ERG1/metabolismo , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Leucemia Mieloide Aguda/patologia , Macaca fascicularis , Camundongos , Camundongos SCID , Oximas/química , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Bioorg Med Chem ; 22(7): 2303-10, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24588962

RESUMO

The ribonucleotide reductase (RNR) enzyme is a heteromer of RRM1 and RRM2 subunits. The active enzyme catalyzes de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. Complexity in the generation of physiologically relevant, active RRM1/RRM2 heterodimers was perceived as limiting to the identification of selective RRM1 inhibitors by high-throughput screening of compound libraries and led us to seek alternative methods to identify lead series. In short, we found that gemcitabine, as its diphosphate metabolite, represents one of the few described active site inhibitors of RRM1. We herein describe the identification of novel 5'-amino gemcitabine analogs as potent RRM1 inhibitors through in-cell phenotypic screening.


Assuntos
Desoxicitidina/análogos & derivados , Proteínas Supressoras de Tumor/antagonistas & inibidores , Linhagem Celular Tumoral , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ribonucleosídeo Difosfato Redutase , Relação Estrutura-Atividade , Gencitabina
4.
J Med Chem ; 66(19): 13809-13820, 2023 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-37729617

RESUMO

Boron neutron capture therapy (BNCT) is a re-emerging binary cellular level cancer intervention that occurs through the interaction of a cancer-specific 10boron (10B) drug and neutrons. We created a new 10B drug, 3-borono-l-tyrosine (BTS), that improves on the characteristics of the main historical BNCT drug 4-borono-l-phenylalanine (BPA). BTS has up to 4 times greater uptake in vitro than BPA and increased cellular retention. Like BPA, BTS uptake is mediated by the l-type amino acid transporter-1 (LAT1) but is less sensitive to natural amino acid competition. BTS can be formulated and bolus dosed at much higher levels than BPA, resulting in 2-3 times greater boron delivery in vivo. Fast blood clearance and greater tumor boron delivery result in superior tumor-to-blood ratios. BTS boron delivery appears to correlate with LAT1 expression. BTS is a promising boron delivery drug that has the potential to improve modern BNCT interventions.


Assuntos
Aminoácidos , Terapia por Captura de Nêutron de Boro , Linhagem Celular Tumoral , Boro , Terapia por Captura de Nêutron de Boro/métodos , Solubilidade , Fenilalanina/química , Compostos de Boro/química
5.
Exp Lung Res ; 37(4): 227-38, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21309737

RESUMO

Idiopathic interstitial pneumonias are a group of idiopathic interstitial lung diseases of which idiopathic pulmonary fibrosis (IPF) is the lesion of usual interstitial pneumonia. Although the pathogenic mechanisms remain incompletely understood, disease-specific changes in blood, a readily accessible biospecimen, have not been fully characterized. To identify biomarkers from blood and sera, the immune status of IPF patients and control subjects without structural lung disease was quantified by measuring cell surface markers, mRNA levels, and serum proteins. Statistically significant differences in cellular and molecular markers were observed between the 2 groups. The cytokine receptor IL-17RB was significantly higher in CD14+ peripheral blood mononuclear cells (PBMCs) from IPF patients, whereas expression of the chemokine receptor CXCR4 was lower. Gene expression analyses identified 18 differentially expressed genes out of 195 selected. Of these, EMR1, CCR3, UPAR, FCGR2A, OPN, CEACAM3, CD16a, CD18, CD11b, LTF, and LCN2 were up-regulated, whereas IL-17RB, IL-10, PDGFA, CD301/Clec10a, CD25/IL-2RA, IL-23p19, and IL-15 were down-regulated in IPF. Differentially regulated genes were in the functional areas of inflammation and cell signaling. Serum levels of UPAR and OPN were higher in IPF. These observations reveal significant differences in cell and molecular markers involved in monocyte/macrophage activation and migration, and suggest a role for IL-17RB in IPF.


Assuntos
Fibrose Pulmonar Idiopática/patologia , Macrófagos/patologia , Monócitos/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Movimento Celular , Humanos , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Receptores de Interleucina/fisiologia , Receptores de Interleucina-17
6.
Artigo em Inglês | MEDLINE | ID: mdl-32036254

RESUMO

Antibody-drug conjugates (ADCs) have become major biopharmaceutical drugs in the field of oncology. Traditional ADCs possess a stochastic distribution of cytotoxic payloads linked to several different amino acid residues of the antibody. This heterogeneous nature of stochastic ADCs results in a complex conjugation-site characterization. To improve upon traditional ADC technology, we have developed a chemical conjugation platform, termed AJICAP™, for site-specific modification of native antibodies using a class of IgG Fc-affinity reagents (Yamada et al., 2019). Here, we report further investigation focusing on peptide mapping of the AJICAP™-ADC to confirm the exact conjugation position of the first generation AJICAP™-ADC. Neutral pH pretreatment for peptide mapping prevented undesired PTMs such as succinimide ring hydrolysis. Mirroring comparison using the purified ADC visibly indicated that Lys248 in the Fc region was conjugated to the drug-linker. MS/MS analysis also provided evidence to support Lys248 conjugation. Finally, extracted ion-chromatogram methodology suggested the site-specificity of AJICAP™ conjugation. Purified ADCs by preparative HIC-HPLC showed clear visual results and more than 93% sequence coverage by a single enzymatic digestion. The analytical strategy described herein demonstrated a robust analytical methodology for revealing the conjugation site of ADCs.


Assuntos
Antineoplásicos/química , Imunoconjugados/química , Antineoplásicos/farmacologia , Sítios de Ligação , Imunoconjugados/farmacologia , Fragmentos Fc das Imunoglobulinas/química , Lisina/química , Oligopeptídeos/química , Mapeamento de Peptídeos , Ligação Proteica , Succinimidas/química , Trastuzumab/química
7.
Mol Cancer Ther ; 16(9): 1866-1876, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28522588

RESUMO

Neutropenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an in vitro assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay. Results showed that HSCs had similar sensitivity to vcMMAE-ADCs and mcMMAF-ADCs; however, vcMMAE-ADCs were more cytotoxic to differentiating neutrophils than the same antibody conjugated to mcMMAF. This inhibitory effect was not mediated by internalization of ADC either by macropinocytosis or FcγRs. Our results suggested that extracellular proteolysis of the cleavable valine-citrulline linker is responsible for the cytotoxicity to differentiating neutrophils. Mass spectrometry analyses indicated that free MMAE was released from vcMMAE-ADCs in the extracellular compartment when they were incubated with differentiating neutrophils or neutrophil conditioned medium, but not with HSC-conditioned medium. Using different protease inhibitors, our data suggested that serine, but not cysteine proteases, were responsible for the cleavage. In vitro experiments demonstrated that the purified serine protease, elastase, was capable of releasing free MMAE from a vcMMAE-ADC. Here we propose that ADCs containing protease cleavable linkers can contribute to neutropenia via extracellular cleavage mediated by serine proteases secreted by differentiating neutrophils in bone marrow. Mol Cancer Ther; 16(9); 1866-76. ©2017 AACRSee related article by Zhao et al., p. 1877.


Assuntos
Antineoplásicos/efeitos adversos , Imunoconjugados/efeitos adversos , Mielopoese/efeitos dos fármacos , Neutropenia/sangue , Neutropenia/etiologia , Neutrófilos/efeitos dos fármacos , Animais , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Camundongos , Neutrófilos/metabolismo , Pinocitose , Receptores de IgG/metabolismo , Serina Proteases/metabolismo
8.
PLoS One ; 9(11): e111714, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25375241

RESUMO

Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.


Assuntos
Replicação do DNA , Histonas/metabolismo , Proteínas Quinases/metabolismo , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Desoxirribonucleotídeos/metabolismo , Humanos , Fosforilação , RNA Interferente Pequeno/metabolismo , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleotídeos/metabolismo , Proteínas Supressoras de Tumor/genética
9.
Biomark Insights ; 7: 87-104, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22837640

RESUMO

BACKGROUND: Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible. OBJECTIVE: Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment. METHODS: The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma. RESULTS: Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids. CONCLUSIONS AND CLINICAL RELEVANCE: These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination.

10.
Clin Transl Gastroenterol ; 3: e10, 2012 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-23238132

RESUMO

OBJECTIVES: Interleukin-23 (IL-23) has emerged as a new therapeutic target for the treatment of inflammatory bowel disease (IBD). As biomarkers of disease state and treatment efficacy are becoming increasingly important in drug development, we sought to identify efficacy biomarkers for anti-IL-23 therapy in Crohn's disease (CD). METHODS: Candidate IL-23 biomarkers, downstream of IL-23 signaling, were identified using shotgun proteomic analysis of feces and colon lavages obtained from a short-term mouse IBD model (anti-CD40 Rag2(-/-)) treated preventively with monoclonal antibodies (mAbs) to the IL-23 receptor (IL-23R). The biomarkers were then measured in an IBD T-cell transfer model treated therapeutically with a mAb to IL-23 (p19), confirming their association with IBD. To assess the clinical relevance of these markers, we assessed their concentrations in clinical serum, colon tissue, and feces from CD patients. RESULTS: We identified 57 proteins up or downregulated in diseased animals that returned to control values when the mice were treated with mAbs to IL-23R. Among those, S100A8, S100A9, regenerating protein 3ß (REG), REG3γ, lipocalin 2 (LCN2), deleted in malignant tumor 1 (DMBT1), and macrophage migration inhibitory factor (MIF) mRNA levels correlated with disease score and dose titration of mAbs to IL-23R or IL-23(p19). All biomarkers, except DMBT1, were also downregulated after therapeutic administration of mAbs to IL-23(p19) in a T-cell transfer IBD mouse model. In sera from CD patients, we confirmed a significant upregulation of S100A8/A9 (43%), MIF (138%), pancreatitis-associated protein (PAP, human homolog of REG3ß/γ; 49%), LCN2 (520%), and CCL20 (1280%), compared with control samples, as well as a significant upregulation of S100A8/A9 (887%), PAP (401%), and LCN2 (783%) in human feces from CD patients compared with normal controls. CONCLUSIONS: These studies identify multiple protein biomarkers downstream of IL-23 that could be valuable tools to assess the efficacy of this new therapeutic agent.Clinical and Translational Gastroenterology (2012) 3, e10; doi:10.1038/ctg.2012.2; published online 16 February 2012.

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