RESUMO
Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0 ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.
Assuntos
Imunoconjugados/sangue , Maitansina/sangue , Cromatografia Líquida , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Maitansina/análogos & derivados , Maitansina/metabolismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30-40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells. METHODS: Recombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD). RESULTS: Soluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo. CONCLUSIONS: Our data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Maitansina/farmacologia , Proteínas de Membrana/farmacologia , Animais , Antineoplásicos/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Maitansina/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Recombinantes/biossíntese , Transdução de Sinais/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.
Assuntos
Imunoconjugados/metabolismo , Maitansina/química , Moduladores de Tubulina/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Maitansina/metabolismo , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Relação Estrutura-Atividade , Transplante Heterólogo , Moduladores de Tubulina/metabolismoRESUMO
The major drawbacks of the cytotoxin like DM1 are the off-target effects. Here, the targeting nanovesicles were developed by synthesizing tocopherol-SS-DM1 and conjugating a pH low insertion peptide (pHLIP) to PEGylated phospholipids, in which tocopherol-SS-DM1 improves the drug loading and is glutathione responsive in the cytoplasm, meanwhile, the pH insertion peptide targets the acidic microenvironment of cancer cells. Besides, these nanovesicles can accumulate at the endoplasmic reticulum and show increased cancer therapeutic effects both in vitro and in vivo. These targeting nanovesicles provide a novel formulation for subcellular organelle targeting, a platform for precisely delivery of cytotoxic DM1 to cancer cells, and an alternative strategy for antibody-drug conjugates (ADCs).
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Portadores de Fármacos , Retículo Endoplasmático/metabolismo , Glutationa/metabolismo , Lipídeos/química , Maitansina/farmacologia , Proteínas de Membrana/metabolismo , Nanopartículas , Neoplasias/tratamento farmacológico , Pró-Fármacos/farmacologia , Tocoferóis/farmacologia , Células A549 , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Composição de Medicamentos , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Maitansina/química , Maitansina/metabolismo , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Tocoferóis/química , Tocoferóis/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Covering: 2014-2019We review recent progress on natural products that target cytoskeletal components, including microtubules, actin, intermediate filaments, and septins and highlight their demonstrated and potential utility in the treatment of human disease. The anticancer efficacy of microtubule targeted agents identified from plants, microbes, and marine organisms is well documented. We highlight new microtubule targeted agents currently in clinical evaluations for the treatment of drug resistant cancers and the accumulating evidence that the anticancer efficacy of these agents is not solely due to their antimitotic effects. Indeed, the effects of microtubule targeted agents on interphase microtubules are leading to their potential for more mechanistically guided use in cancers as well as neurological disease. The discussion of these agents as more targeted drugs also prompts a reevaluation of our thinking about natural products that target other components of the cytoskeleton. For instance, actin active natural products are largely considered chemical probes and non-selective toxins. However, studies utilizing these probes have uncovered aspects of actin biology that can be more specifically targeted to potentially treat cancer, neurological disorders, and infectious disease. Compounds that target intermediate filaments and septins are understudied, but their continued discovery and mechanistic evaluations have implications for numerous therapeutic indications.
Assuntos
Actinas/metabolismo , Produtos Biológicos/farmacologia , Citoesqueleto/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Animais , Produtos Biológicos/química , Colchicina/química , Colchicina/metabolismo , Colchicina/farmacologia , Citoesqueleto/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Eucarióticas/citologia , Células Eucarióticas/efeitos dos fármacos , Genoma , Humanos , Maitansina/química , Maitansina/metabolismo , Maitansina/farmacologia , Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/patologia , Taxoides/química , Taxoides/farmacologiaRESUMO
Ansamitocins are extraordinarily potent antitumor agents. Ansamitocin P-3 (AP-3), which is produced by Actinosynnema pretiosum, has been developed as a cytotoxic drug for breast cancer. Despite its importance, AP-3 is of limited applicability because of the low production yield. A. pretiosum strain X47 was developed from A. pretiosum ATCC 31565 by mutation breeding and shows a relatively high AP-3 yield. Here, we analyzed the A. pretiosum X47 genome, which is ~8.13 Mb in length with 6693 coding sequences, 58 tRNA genes, and 15 rRNA genes. The DNA sequence of the ansamitocin biosynthetic gene cluster is highly similar to that of the corresponding cluster in A. pretiosum ATCC 31565, with 99.9% identity. However, RT-qPCR analysis showed that the expression levels of ansamitocin biosynthetic genes were significantly increased in X47 compared with the levels in the wild-type strain, consistent with the higher yield of AP-3 in X47. The annotated complete genome sequence of this strain will facilitate understanding the molecular mechanisms of ansamitocin biosynthesis and regulation in A. pretiosum and help further genetic engineering studies to enhance the production of AP-3.
Assuntos
Actinobacteria/genética , Actinobacteria/metabolismo , Antibióticos Antineoplásicos/metabolismo , Genoma Bacteriano , Maitansina/análogos & derivados , Análise de Sequência de DNA , Vias Biossintéticas/genética , Perfilação da Expressão Gênica , Maitansina/metabolismo , Anotação de Sequência MolecularRESUMO
INTRODUCTION: Our objective was to determine correlations between the tumor uptake and T/B ratios for 89Zr-labeled T-DM1 (89Zr-DFO-T-DM1) in mice with human BC xenografts by microPET/CT and biodistribution studies with HER2 expression and response to treatment with trastuzumab-DM1 (T-DM1). METHODS: The tumor and normal tissue uptake and T/B ratios for 89Zr-DFO-T-DM1 (10⯵g; 7.0â¯MBq) incorporated into a therapeutic dose (60⯵g) were determined by microPET/CT and biodistribution studies at 96â¯h p.i. in NOD/SCID mice with s.c. MDA-MB-231 (5â¯×â¯104 HER2/cell), MDA-MB-361 (5â¯×â¯105 HER2/cell) and BT-474 (2â¯×â¯106 HER2/cell) human BC xenografts. Mice bearing these tumors were treated with T-DM1 (3.6â¯mg/kg every 3â¯weeks) and the tumor doubling time estimated by fitting of tumor volume vs. time curves. A tumor doubling time ratio (TDR) was calculated by dividing the doubling time for T-DM1 and normal saline treated control mice. The clonogenic survival (CS) of BC cells with increasing HER2 expression treated for 72â¯h in vitro with T-DM1 or trastuzumab (0-100⯵g/mL) was compared. Correlations were determined between the T/B ratios for 89Zr-DFO-T-DM1 and HER2 expression, TDR and CS, and between CS and TDR. RESULTS: Uptake of 89Zr-DFO-T-DM1 in MDA-MB-231, MDA-MB-361 and BT-474 tumors was 2.4⯱â¯0.4%ID/g, 6.9⯱â¯2.2%ID/g and 9.8⯱â¯1.1%ID/g, respectively. There was a non-linear but direct correlation between the T/B ratios for 89Zr-DFO-T-DM1 and HER2 expression with the T/B ratio ranging from 4.5⯱â¯0.7 for MDA-MB-231 to 18.2⯱â¯1.8 for MDA-MB-361 and 35.9⯱â¯5.1 for BT-474 xenografts. Tumor intensity on microPET/CT images was proportional to HER2 expression. The standard uptake value (SUV) for the tumors on the images was strongly correlated with the T/B ratio in biodistribution studies. There was a direct linear correlation between the T/B ratio for 89Zr-DFO-T-DM1 and TDR, with TDR ranging from 0.9 for MDA-MB-231 to 1.6 for MDA-MB-361 and 2.1 for BT-474 tumors. The cytotoxicity of T-DM1 in vitro on BC cells was dependent on HER2 expression but T-DM1 was more potent than trastuzumab. There was an inverse correlation between the TDR for mice treated with T-DM1 and CS of BC cells exposed in vitro to T-DM1. CONCLUSIONS: Based on the direct correlations between the T/B ratio for 89Zr-DFO-T-DM1 by PET and HER2 expression and response to T-DM1, our results suggest that PET with 89Zr-DFO-T-DM1 may predict response of HER2-positive BC to treatment with T-DM1. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Our results suggest that PET with 89Zr-DFO-T-DM1 may predict response to treatment with T-DM1 in HER-positive BC.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Maitansina/análogos & derivados , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Radioisótopos , Receptor ErbB-2/metabolismo , Trastuzumab/metabolismo , Trastuzumab/uso terapêutico , Zircônio , Ado-Trastuzumab Emtansina , Animais , Transporte Biológico , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/terapia , Maitansina/sangue , Maitansina/metabolismo , Maitansina/farmacocinética , Maitansina/uso terapêutico , Camundongos , Distribuição Tecidual , Trastuzumab/sangue , Trastuzumab/farmacocinética , Resultado do TratamentoRESUMO
For millenia, plants have been a major source of medications against human and animal diseases. In the case of anticancer agents, a significant number of current agents can trace their source back to nominally plant secondary metabolites, with examples being taxol, vinca alkaloids, camptothecin (CPT), and their modified derivatives. However, it is now becoming apparent that these and other plant-derived materials, plus similar agents from marine sources may well have a microbe in their background. In this short Opinion, evidence for such claims are presented for some of the agents currently in use or in preclinical and clinical trials against cancer.
Assuntos
Antineoplásicos/metabolismo , Produtos Biológicos/metabolismo , Endófitos/metabolismo , Neoplasias/tratamento farmacológico , Plantas/microbiologia , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/uso terapêutico , Bactérias/metabolismo , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/uso terapêutico , Fungos/metabolismo , Humanos , Maitansina/isolamento & purificação , Maitansina/metabolismo , Maitansina/uso terapêutico , Paclitaxel/isolamento & purificação , Paclitaxel/metabolismo , Paclitaxel/uso terapêutico , Poríferos/microbiologia , Rizosfera , Urocordados/microbiologiaRESUMO
Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on ß-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.
Assuntos
Polarização de Fluorescência/métodos , Maitansina/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Ado-Trastuzumab Emtansina , Animais , Antineoplásicos/metabolismo , Sítios de Ligação , Humanos , Ligantes , Macrolídeos/metabolismo , Maitansina/análogos & derivados , Oxazóis/metabolismo , Trastuzumab/metabolismoRESUMO
The type-I polyketide ansamitocin P-3 (AP-3) is a potent antitumor agent. Its production is most likely hampered by the required multiple substrate supplies and complicated post-PKS modifications in Actinosynnema pretiosum subsp. pretiosum ATCC 31280. For titer improvement, gene ansa30, encoding for a glycosyltransferase competing for the N-demethyl-AP-3 (PND-3) intermediate for AP-3 biosynthesis, was initially inactivated. In the mutant NXJ-22, the AP-3 titer was increased by 66% along with an obvious accumulation of PND-3, indicating that the N-methylation is a rate-limiting step. Alternatively, when abundant upstream intermediate 19-chloroproansamitocin was fed into a PKS mutant, 3-O-acylation was further identified along with the N-methylation as the rate-limiting steps. Subsequent overexpression of N-methyltransferase gene asm10 in NXJ-22 resulted in a 93% increase of AP-3 and a corresponding 92% decrease of PND-3. Additional supplementation of L-methionine, the precursor for SAM biosynthesis, substantially decreased the accumulation of PND-3. In parallel, the 3-O-acylation bottleneck was relieved by feeding with L-valine to NXJ-22, resulting in a 126% increase of AP-3. Eventually, a combined asm10 overexpression and supplementation of L-methionine and L-valine resulted in a 5-fold increase of AP-3, from 42 ± 2 mg L-1 to 246 ± 6 mg L-1 , without any noticeable accumulation of PND-3.
Assuntos
Actinobacteria/genética , Actinobacteria/metabolismo , Maitansina/análogos & derivados , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Actinobacteria/enzimologia , Maitansina/análise , Maitansina/metabolismo , Redes e Vias Metabólicas/fisiologia , Mutação/genéticaRESUMO
The selective destruction of tumour cells while sparing healthy tissues is one of the main challenges in cancer therapy. Antibody-drug conjugates (ADCs) are arguably the most rapidly expanding class of targeted cancer therapies. Efficient drug conjugation and release technologies are essential for the development of these new therapeutic agents. In response to the ever-increasing demand for efficient drug release systems, we have developed a new class of ß-galactosidase-cleavable linkers for ADCs. Within this framework, novel payloads comprising a galactoside linker, the monomethyl auristatin E (MMAE) and cysteine-reactive groups were synthesized, conjugated with trastuzumab and evaluated both in vitro and in vivo. The ADCs with galactoside linkers demonstrated superior therapeutic efficacy in mice compared to the marketed trastuzumab emtansine used for the treatment of breast cancer.
Assuntos
Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Imunoconjugados/química , Imunoconjugados/farmacologia , Maitansina/análogos & derivados , Trastuzumab/química , Trastuzumab/farmacologia , beta-Galactosidase/metabolismo , Ado-Trastuzumab Emtansina , Animais , Antineoplásicos Imunológicos/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/uso terapêutico , Maitansina/química , Maitansina/metabolismo , Maitansina/farmacologia , Maitansina/uso terapêutico , Camundongos Nus , Trastuzumab/metabolismo , Trastuzumab/uso terapêuticoRESUMO
Trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) consisting of human epidermal growth factor receptor 2 (HER2)-targeted mAb trastuzumab linked to antimicrotubule agent mertansine (DM1), has been approved for the treatment of HER2-positive metastatic breast cancer. Acquired resistance has been a major obstacle to T-DM1 treatment, and mechanisms remain incompletely understood. In the present study, we established a T-DM1-resistant N87-KR cell line from HER2-positive N87 gastric cancer cells to investigate mechanisms of acquired resistance and develop strategies for overcoming it. Although the kinetics of binding, internalization, and externalization of T-DM1 were the same in N87-KR cells and N87 cells, N87-KR was strongly resistant to T-DM1, but remained sensitive to both trastuzumab and DM1. T-DM1 failed to inhibit microtubule polymerization in N87-KR cells. Consistently, lysine-MCC-DM1, the active T-DM1 metabolite that inhibits microtubule polymerization, accumulated much less in N87-KR cells than in N87 cells. Furthermore, lysosome acidification, achieved by vacuolar H+ -ATPase (V-ATPase), was much diminished in N87-KR cells. Notably, treatment of sensitive N87 cells with the V-ATPase selective inhibitor bafilomycin A1 induced T-DM1 resistance, suggesting that aberrant V-ATPase activity decreases T-DM1 metabolism, leading to T-DM1 resistance in N87-KR cells. Interestingly, HER2-targeted ADCs containing a protease-cleavable linker, such as hertuzumab-vc-monomethyl auristatin E, were capable of efficiently overcoming this resistance. Our results show for the first time that a decrease in T-DM1 metabolites induced by aberrant V-ATPase activity contributes to T-DM1 resistance, which could be overcome by HER2-targeted ADCs containing different linkers, including a protease-cleavable linker. Accordingly, we propose that V-ATPase activity in lysosomes is a novel biomarker for predicting T-DM1 resistance.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Maitansina/análogos & derivados , Neoplasias Gástricas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Ado-Trastuzumab Emtansina , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Antineoplásicos/metabolismo , Western Blotting , Linhagem Celular Tumoral , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Maitansina/metabolismo , Maitansina/farmacologia , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Receptor ErbB-2/biossíntese , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.
Assuntos
Anticorpos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoconjugados/farmacologia , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Animais , Anticorpos/genética , Ligante CD27/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoconjugados/química , Maitansina/metabolismo , Camundongos , Camundongos SCID , Receptores Fc/genéticaRESUMO
Ansamitocins, produced by Actinosynnema pretiosum, are a group of maytansinoid antibiotics that block the assembly of tubulin into functional microtubules. The precursors of ansamitocin biosynthesis are generally derived from the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid cycle. In this study, central carbon flux distributions were analyzed by (13)C-based flux analysis to reveal the contribution of individual central carbon metabolism pathways. To direct more carbon flux into ansamitocin biosynthesis, pentose phosphate (PP) pathway only and the combination of PP pathway and Entner-Doudoroff (ED) pathway were weakened, respectively. Ansamitocin P-3 (AP-3) productions by both kinds of pathways weakened mutant strains were significantly enhanced in chemically defined medium. In order to draw metabolic flux to the biosynthesis of ansamitocins more efficiently, heterologous phosphoglucomutase was subsequently overexpressed based on a mutant strain with combinational regulation of PP pathway and ED pathway. More fluxes were successfully directed into the UDP-glucose synthetic pathway and the AP-3 production was further improved in this case, reaching approximately 185mg/L in fermentation medium. It was demonstrated that eliminating the bypass pathways and favoring the precursor synthetic pathway could effectively improve ansamitocin production by A. pretiosum, suggesting a promising role of metabolic strategy in improving secondary metabolite production.
Assuntos
Actinobacteria/metabolismo , Maitansina/análogos & derivados , Engenharia Metabólica/métodos , Via de Pentose Fosfato/fisiologia , Maitansina/análise , Maitansina/biossíntese , Maitansina/metabolismo , Redes e Vias Metabólicas , FosfoglucomutaseRESUMO
Antibody-drug conjugates (ADCs) are a promising class of cancer therapeutics that combine the specificity of antibodies with the cytotoxic effects of payload drugs. A quantitative understanding of how ADCs are processed intracellularly can illustrate which processing steps most influence payload delivery, thus aiding the design of more effective ADCs. In this work, we develop a kinetic model for ADC cellular processing as well as generalizable methods based on flow cytometry and fluorescence imaging to parameterize this model. A number of key processing steps are included in the model: ADC binding to its target antigen, internalization via receptor-mediated endocytosis, proteolytic degradation of the ADC, efflux of the payload out of the cell, and payload binding to its intracellular target. The model was developed with a trastuzumab-maytansinoid ADC (TM-ADC) similar to trastuzumab-emtansine (T-DM1), which is used in the clinical treatment of HER2+ breast cancer. In three high-HER2-expressing cell lines (BT-474, NCI-N87, and SK-BR-3), we report for TM-ADC half-lives for internalization of 6-14 h, degradation of 18-25 h, and efflux rate of 44-73 h. Sensitivity analysis indicates that the internalization rate and efflux rate are key parameters for determining how much payload is delivered to a cell with TM-ADC. In addition, this model describing the cellular processing of ADCs can be incorporated into larger pharmacokinetics/pharmacodynamics models, as demonstrated in the associated companion paper.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Antineoplásicos/metabolismo , Membrana Celular/metabolismo , Desenho de Fármacos , Maitansina/metabolismo , Trastuzumab/metabolismo , Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Linhagem Celular Tumoral , Humanos , Maitansina/química , Trastuzumab/químicaRESUMO
Ansamitocins isolated from Actinosynnema pretiosum, potent antitumor compounds, belong to the family of maytansinoids, and the antibody-maytansinoid conjugates are currently under different phases of clinical trials. The clinical applications of ansamitocins have stimulated extensive studies to improve their production yields. In this study, we investigated the function of a pathway-specific S treptomyces antibiotic regulatory protein (SARP) family regulator, Asm18, and observed that ectopic overexpression of the asm18 gene increased the production of N-demethyl-4,5-desepoxy-maytansinol (2) to 50 mg/L in the HGF052 + pJTU824-asm18 strain, an increase by 4.7-fold compared to that of the control strain HGF052 + pJTU824. Real-time PCR analysis showed that the overexpression of the asm18 gene selectively increased the transcription levels of the genes involved in the biosynthesis of the starter unit (asm43), polyketide assembly (asmA), post-PKS modification (asm21), as well as the transcription levels of the regulatory gene (asm8), which is a specific LAL-type activator in ansamitocin biosynthesis. With the increase of fermentation titre, seven ansamitocin analogs (1-7) including three new ones (1, 5, and 6) and maytansinol (7) were isolated from the HGF052 + pJTU824-asm18 strain. Our results not only pave the way for further improving the production of ansamitocin analogs but also indicate that the post-PKS modifications of ansamitocin biosynthesis are flexible, which brings a potential of producing maytansinol, the most fascinating intermediate for the synthesis of antibody-maytansinoid conjugates, by optimizing the HGF052 and/or HGF052 + pJTU824-asm18 strains.
Assuntos
Actinobacteria/genética , Actinobacteria/metabolismo , Antineoplásicos/metabolismo , Maitansina/análogos & derivados , Engenharia Metabólica/métodos , Expressão Gênica , Perfilação da Expressão Gênica , Genes Bacterianos , Maitansina/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Ansamitocin P-3 (AP-3), an amacrocyclic lactam compound, is produced by Actinosynnema pretiosum. As a group of maytansinoid antibiotics, ansamitocins have an extraordinary antitumor activity by blocking the assembly of tubulin forming into functional microtubules. The biosynthesis of ansamitocins is initialized by the formation of UDP-glucose (UDPG) which is converted from glucose-1-phosphate (G1P). In this study, we focused on the influence of enhancement of UDPG biosynthesis on the production of ansamitocins in A. pretiosum. The homologous overexpressions of phosphoglucomutase, starch phosphorylase, and UTP-G1P uridylyltransferase, respectively, could largely increase the pool sizes of G1P and UDPG and result in improved AP-3 production. The elevated intracellular glucose-6-phosphate (G6P) level provided by the enhanced glyconeogenesis had, however, no significant effects on the biosynthesis of AP-3. The G6P-G1P-UDPG pathway was therefore systematically engineered by multiple genetic modifications, and a significant increase in AP-3 production was achieved (168 mg/L of AP-3 in flask culture, 40 % higher than the control strain). We also found that the enhancement of starch assimilation pathway could also improve the assembly of AP-3 to some extent. In addition, heterologous gene overexpression from Actinosynnema mirum could result in more AP-3 biosynthesis in comparison to the corresponding homologous overexpression, suggesting an alternative and promising avenue of metabolic engineering strategy for improving AP-3 production.
Assuntos
Actinobacteria/genética , Actinobacteria/metabolismo , Vias Biossintéticas/genética , Maitansina/análogos & derivados , Engenharia Metabólica/métodos , Moduladores de Tubulina/metabolismo , Uridina Difosfato Glucose/biossíntese , Gluconeogênese , Maitansina/metabolismo , Amido/metabolismoRESUMO
Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.
Assuntos
Antineoplásicos Fitogênicos/metabolismo , Imunoconjugados/metabolismo , Maitansina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Antineoplásicos Fitogênicos/administração & dosagem , Linhagem Celular Tumoral , Citoplasma/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Imunoconjugados/administração & dosagem , Lisossomos/metabolismo , Maitansina/administração & dosagemRESUMO
The development of antibody-drug conjugates (ADC), a promising class of anticancer agents, has traditionally relied on the use of antibodies capable of selective internalization in tumor cells. We have recently shown that also noninternalizing antibodies, coupled to cytotoxic drugs by means of disulfide linkers that can be cleaved in the tumor extracellular environment, can display a potent therapeutic activity. Here, we have compared the tumor-targeting properties, drug release rates, and therapeutic performance of two ADCs, based on the maytansinoid DM1 thiol drug and on the F8 antibody, directed against the alternatively spliced Extra Domain A (EDA) domain of fibronectin. The antibody was used in IgG or in small immune protein (SIP) format. In both cases, DM1 was coupled to unpaired cysteine residues, resulting in a drug-antibody ratio of 2. In biodistribution studies, SIP(F8)-SS-DM1 accumulated in the tumor and cleared from circulation more rapidly than IgG(F8)-SS-DM1. However, the ADC based on the IgG format exhibited a higher tumor uptake at later time points (e.g., 33%IA/g against 8%IA/g at 24 hours after intravenous administration). In mouse plasma, surprisingly, the ADC products in IgG format were substantially more stable compared with the SIP format (half-lives >48 hours and <3 hours at 37°C, respectively), revealing a novel mechanism for the control of disulfide-based drug release rates. Therapy experiments in immunocompetent mice bearing murine F9 tumors revealed that SIP(F8)-SS-DM1 was more efficacious than IgG(F8)-SS-DM1 when the two products were compared either in an equimolar basis or at equal milligram doses.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos/farmacologia , Imunoconjugados/farmacologia , Maitansina/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Animais , Anticorpos/metabolismo , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Peso Corporal/efeitos dos fármacos , Células CHO , Linhagem Celular Tumoral , Cromatografia Líquida , Cricetinae , Cricetulus , Liberação Controlada de Fármacos , Eletroforese em Gel de Poliacrilamida , Feminino , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Espectrometria de Massas/métodos , Maitansina/metabolismo , Maitansina/farmacocinética , Camundongos , Microscopia de Fluorescência , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Distribuição Tecidual , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacosRESUMO
The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.