RESUMO
PURPOSE: The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation. METHODS: This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor receptor-2 (HER2) kinetic binding, HER2 signaling, HER2 internalization, synergy with docetaxel chemotherapy, FcγR kinetic binding, primary natural killer and monocyte cell binding, antibody-dependent cellular phagocytosis activity, in vivo xenograft studies, and toxicokinetic parameters. RESULTS: The results contribute to the totality of evidence with respect to functional similarity and support that ABP 980 is similar to trastuzumab in all primary and secondary mechanisms of action. CONCLUSIONS: These results also support the scientific justification of extrapolation to all approved indications of trastuzumab given the established functional similarity of the two products and the same mechanisms of action across all conditions of use.
Assuntos
Antineoplásicos/química , Medicamentos Biossimilares/química , Trastuzumab/química , Animais , Ligação Competitiva , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Cinética , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais , Ligação Proteica , Receptor ErbB-2/química , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches. The downstream molecular mechanisms following ligand-receptor blockade by which these antibodies prevent CGRP signaling through CGRP receptors have not been demonstrated. METHODS: Here we produced tool monoclonal functional antagonist antibodies against CGRP and its canonical receptor and developed a novel cellular model using fluorogen-activated protein technology that allows detection of CGRP receptor internalization by flow cytometry and, for an extended time course, visualization by confocal microscopy. RESULTS: Using this cell model we showed that these antagonist antibodies block both CGRP-induced cAMP signaling and CGRP receptor internalization. At least 10-fold higher concentrations of either antibody are necessary to block CGRP receptor internalization compared with cAMP accumulation in our cell model. CONCLUSION: These data reinforce our understanding of how monoclonal functional antagonist antibodies interfere with CGRP signaling.
Assuntos
Anticorpos Monoclonais/metabolismo , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Células CHO , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/farmacologia , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Transgênicos , Transtornos de Enxaqueca/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietin-Tie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.
Assuntos
Anticorpos Neutralizantes/farmacologia , Células Endoteliais/efeitos dos fármacos , Citometria de Fluxo/métodos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Receptor TIE-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Neutralizantes/uso terapêutico , Bromodesoxiuridina , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Feminino , Glioblastoma/patologia , Humanos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Proteínas Recombinantes de Fusão/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. METHODS: In order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours. RESULTS: Significant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p <0.0001), whereas in mouse bone marrow and in tumor a dose-dependent inhibition was achieved at all three doses (p ≤ 0.00015). These studies demonstrate that AMG 900 inhibits p-Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p <0.0001) in COLO 205 tumors. Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues. CONCLUSION: Phosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.
Assuntos
Aurora Quinases/antagonistas & inibidores , Histonas/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Ftalazinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto , Animais , Aurora Quinases/metabolismo , Biópsia por Agulha Fina , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos Nus , Fosforilação/efeitos dos fármacos , Ftalazinas/sangueRESUMO
Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.
Assuntos
Cinesinas , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Cinesinas/genética , Cinesinas/metabolismo , Mitose/genética , Linhagem Celular , Pontos de Checagem da Fase M do Ciclo CelularRESUMO
P-glycoprotein (P-gp) efflux assay is an integral part of discovery screening, especially for drugs requiring brain penetration as P-gp efflux ratio (ER) inversely correlates with brain exposure. However, significant variability in P-gp ER generated across cell lines can lead to misclassification of a P-gp substrate and subsequently disconnect with brain exposure data. We hypothesized that the ER depends on P-gp protein expression level in the in vitro assay. Quantitative proteomics and immunofluorescence staining were utilized to characterize P-gp protein expression and localization in four recombinant cell lines, over-expressing human or mouse P-gp isoforms, followed by functional evaluation. Efflux data generated in each cell line was compared against available rodent brain distribution data. The results suggested that the cell line with highest P-gp expression (hMDCK-MDR1 sourced from NIH) led to greatest dynamic range for efflux; thus, proving to be the most sensitive model to predict brain penetration. Cell lines with lower P-gp expression exhibited the greatest tendency for compound-dependent in vitro efflux saturation leading to false negative results. Ultimately, P-gp kinetics were characterized using a compartmental model to generate system-independent parameters to resolve such discrepancy. This study highlights the need for careful choice of well characterized P-gp in vitro tools and utility of modeling techniques to enable appropriate interpretation of the data.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Descoberta de Drogas , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Camundongos , ProteômicaRESUMO
BACKGROUND: Activating mutations in Kit receptor tyrosine kinase or the related platelet-derived growth factor receptor (PDGFR) play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST). METHODS: This study investigated the activity of motesanib, an inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR; and Kit, against primary activating Kit mutants and mutants associated with secondary resistance to imatinib. Single- and double-mutant isoforms of Kit were evaluated for their sensitivity to motesanib or imatinib in autophosphorylation assays and in Ba/F3 cell proliferation assays. RESULTS: Motesanib inhibited Kit autophosphorylation in CHO cell lines expressing primary activating mutations in exon 9 (AYins503-504, IC50 = 18 nM) and exon 11 (V560 D, IC50 = 5 nM; Delta552-559, IC50 = 1 nM). Motesanib also demonstrated activity against kinase domain mutations conferring imatinib resistance (V560D/V654A, IC50 = 77 nM; V560D/T670I, IC50 = 277 nM; Y823 D, IC50 = 64 nM) but failed to inhibit the imatinib-resistant D816V mutant (IC50 > 3000 nM). Motesanib suppressed the proliferation of Ba/F3 cells expressing Kit mutants with IC50 values in good agreement with those observed in the autophosphorylation assays. CONCLUSIONS: In conclusion, our data suggest that motesanib possesses inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations.
Assuntos
Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Indóis/farmacologia , Mutação/genética , Niacinamida/análogos & derivados , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Western Blotting , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Feminino , Tumores do Estroma Gastrointestinal/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Niacinamida/farmacologia , Oligonucleotídeos , FosforilaçãoRESUMO
IP-10 secretion is induced by pro-inflammatory cytokines and mediates the migration of CXCR3+ cells. Its elevation in clinical samples has been associated with multiple inflammatory diseases and its antagonism has been reported to be effective in several animal models of inflammatory disease. We generated a mouse anti-mouse IP-10 monoclonal antibody (mAb; Clone 20A9) that specifically bound murine IP-10 with high affinity and inhibited in vitro IP-10 induced BaF3/mCXCR3 cell migration with an IC(50) of approximately 4 nM. The 20A9 mAb was completely absorbed in vivo and had dose proportional pharmacokinetic exposure with a serum half life of 2.4-6 days. The 20A9 mAb inhibited IP-10 mediated T-cell recruitment to the airways, indicating that it is effective in vivo. However, administration of the 20A9 mAb had no significant effect on disease in mouse models of delayed type hypersensitivity, collagen induced arthritis, cardiac allograft transplantation tolerance, EAE or CD4+ CD45RBHi T-cell transfer-induced IBD. These data suggest that the 20A9 mAb can antagonize IP-10 mediated chemotaxis in vitro and in vivo and that this is insufficient to cause a therapeutic benefit in multiple mouse models of inflammatory disease.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL10/antagonistas & inibidores , Quimiocina CXCL10/imunologia , Animais , Anticorpos Monoclonais/farmacocinética , Artrite Experimental/patologia , Artrite Experimental/terapia , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Movimento Celular/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/terapia , Feminino , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/imunologia , Inflamação/patologia , Inflamação/terapia , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Camundongos SCID , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Resultado do TratamentoRESUMO
The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRalpha displayed chondrogenic activity in the presence of TGFbeta3 and expressed cartilage-specific genes in 7 to 16 day pellet cultures. Depositions of cartilage matrix and type II collagen were detected by day 14. TGFbeta-stimulated chondrogenesis was synergistically enhanced by PDGF-BB, resulting in a larger cartilage particle filled with a cartilaginous area containing type II collagen, with a surface cell layer expressing type I collagen. In contrast, noggin inhibited both the TGFbeta- and TGFbeta+PDGF-stimulated cartilage formation, suggesting that a BMP-dependent pathway is involved. In fact, replacement of TGFbeta3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFbeta3 and PDGF-BB, followed by the incubation with BMP4 alone, resulted in a cartilage particle lacking type I collagen in the matrix and the surface layer, which suggests hyaline cartilage formation. Furthermore, such hyaline cartilage particles were mineralized. These studies indicate that the PDGFRalpha+ and/or flk-1+ cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.
Assuntos
Embrião de Mamíferos/citologia , Animais , Becaplermina , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrogênese , Técnicas de Cocultura , Colágeno/metabolismo , Meios de Cultura Livres de Soro/farmacologia , Citometria de Fluxo , Mesoderma/metabolismo , Camundongos , Oligonucleotídeos/química , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntington's disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than full-length huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeat-containing huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Calpaína/metabolismo , Linhagem Celular , Corpo Estriado/citologia , Mapeamento de Epitopos , Deleção de Genes , Proteína Huntingtina , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismoRESUMO
We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3(+) eosinophils increased in the blood and lymph nodes of the transgenic mice by 50- and 300-fold, respectively. Eosinophils also increased 8- to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19(+) B cells increased 2- to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4(+) T lymphocytes increased 2-fold in both blood and spleen. High serum levels of the cytokines IL-2, IL-4, IL-5, granulocyte colony-stimulating factor, eotaxin, and interferon gamma were observed. Consistent with B-lymphocyte increases, serum immunoglobulin (Ig) M, IgG, and IgE were significantly elevated. Antigenic challenge of the transgenic mice with keyhole limpet hemocyanin (KLH) resulted in a decrease in anti-KLH IgG accompanied by increases of anti-KLH IgA and IgE. In situ hybridization of transgenic tissues revealed that IL-17Rh1 (IL-17BR/Evi27), a receptor that binds IL-17E, is up-regulated. Taken together, these data indicate that IL-17E regulates hematopoietic and immune functions, stimulating the development of eosinophils and B lymphocytes. The fact that hIL-17E overexpression results in high levels of circulating eosinophils, IL-4, IL-5, eotaxin, and IgE suggests that IL-17E may be a proinflammatory cytokine favoring Th2-type immune responses.