RESUMO
The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella-containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane-bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP-bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63-containing late endosomes. Nischarin is recruited to the SCV in a Rab14-dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners--Rac1, Rab14 and Rab9 GTPases--reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.
Assuntos
Endossomos/microbiologia , Receptores de Imidazolinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Vacúolos/microbiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Transporte Biológico , Western Blotting , Endossomos/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Receptores de Imidazolinas/genética , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Lisossomos/microbiologia , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Salmonella/microbiologia , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in â¼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.
Assuntos
Síndrome de Bardet-Biedl/fisiopatologia , Proteínas de Transporte/metabolismo , Centrossomo/metabolismo , Cílios/fisiologia , Transdução de Sinais/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Análise de Variância , Animais , Síndrome de Bardet-Biedl/metabolismo , Imunofluorescência , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Espectrometria de Massas , Membranas/crescimento & desenvolvimento , Imagem com Lapso de Tempo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Peixe-ZebraRESUMO
The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3- and AP-1-bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3-positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3-deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.
Assuntos
Endossomos/fisiologia , Lisossomos/fisiologia , Proteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Complexo 3 de Proteínas Adaptadoras , Subunidades beta do Complexo de Proteínas Adaptadoras , Animais , Linhagem Celular , Membrana Celular/fisiologia , Endossomos/ultraestrutura , Cinética , Lisossomos/ultraestrutura , Transporte Proteico , Fator de Transcrição AP-1/metabolismo , Rede trans-Golgi/fisiologia , Rede trans-Golgi/ultraestruturaRESUMO
The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane. The complex may also function in the nervous system, but its precise role is unknown. We have investigated exocyst function in Drosophila with mutations in one member of the complex, sec5. Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand. In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted. Using a trafficking assay, we found impairments in the membrane addition of newly synthesized proteins. In contrast, synaptic vesicle fusion was not impaired. Thus, Sec5 differentiates between two forms of vesicle trafficking: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not. In this regard, sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways.
Assuntos
Exocitose/fisiologia , Neurônios/metabolismo , Neurotransmissores/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Membrana Celular/metabolismo , Mapeamento Cromossômico , Drosophila , Mutagênese/fisiologia , Neuritos/fisiologia , Junção Neuromuscular/fisiologia , Neurônios/ultraestrutura , Transporte Proteico/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismoRESUMO
Synaptic activity causes reductions in cleft [Ca(2+)] that may impact subsequent synaptic efficacy. Using modified patch-clamp techniques to record from single neocortical nerve terminals, we report that physiologically relevant reductions of extracellular [Ca(2+)] ([Ca(2+)](o)) activate voltage-dependent outward currents. These outward currents are carried by a novel nonselective cation (NSC) channel that is indirectly inhibited by various extracellular agents (rank order potency, Gd(3+) > spermidine > Ca(2+) > Mg(2+), typical for [Ca(2+)](o) receptors). The identification of a Ca(2+) sensor-NSC channel pathway establishes the existence of a mechanism by which presynaptic terminals can detect and respond to reductions in cleft [Ca(2+)]. Activation of NSC channels by falls in [Ca(2+)](o) would be expected during periods of high activity in the neocortex and may modulate the excitability of the presynaptic terminal.
Assuntos
Cálcio/metabolismo , Canais Iônicos/fisiologia , Neocórtex/fisiologia , Terminações Pré-Sinápticas/fisiologia , Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Cátions Bivalentes/farmacologia , Espaço Extracelular/fisiologia , Fluorometria , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Microscopia Eletrônica , Neocórtex/citologia , Neurotransmissores/metabolismo , Técnicas de Patch-Clamp , Receptores de Detecção de Cálcio/fisiologia , Sinaptossomos/metabolismo , Sinaptossomos/fisiologiaRESUMO
Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a reactive cysteine residue into proteins with multiple disulfide bonds is often a challenging task as it may interfere with structural and functional properties of the protein. Here we developed a phage display-based biochemical assay, PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) to rapidly screen reactive thiol groups on antibody fragments without interfering with their antigen binding, using trastuzumab-Fab (hu4D5Fab) as a model system. The solvent accessibility values for all the amino acid residues in the hu4D5Fab were calculated using available crystal structure information. Serine, alanine and valine residues with highest solvent accessibility values were selected and tested to compare structure-based design with the PHESELECTOR biochemical method. Cysteine substitutions at partially solvent-accessible alanine or valine residues exhibited better thiol reactivity values than substitutions at serine residues. The poor correlation between fractional solvent accessibility and thiol reactivity of the engineered hu4D5Fab variants indicated the value of PHESELECTOR biochemical assay to identify reactive thiol groups on the antibody-Fab surface. Mass spectrometric analysis of biotinylated ThioFab (Fab with engineered cysteine) variants confirmed that conjugation occurred only at the engineered cysteine thiols of either light or heavy chains. ThioFabs with engineered cysteine residues in the constant domains (CL and CH(1)) should allow universal application for site-specific conjugation of antibody-Fabs.
Assuntos
Anticorpos Monoclonais/química , Cisteína/química , Fragmentos Fab das Imunoglobulinas/química , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Biotina/química , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Cisteína/genética , Cisteína/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Regulação da Expressão Gênica/genética , Engenharia Genética , Variação Genética , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Maleimidas/química , Espectrometria de Massas/métodos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Propriedades de SuperfícieRESUMO
Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/antagonistas & inibidores , Imunoconjugados/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos/química , Anticorpos/imunologia , Anticorpos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/imunologia , Células HEK293 , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacocinética , Células MCF-7 , Macaca fascicularis , Camundongos Nus , Camundongos SCID , Ratos Sprague-Dawley , Receptores de Esteroides/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP-Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.
Assuntos
Proteínas de Transporte/metabolismo , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Receptores da Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Endocitose/efeitos dos fármacos , Endocitose/genética , Endossomos/imunologia , Endossomos/fisiologia , Células HeLa , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas rab de Ligação ao GTP/análise , Proteínas rab4 de Ligação ao GTP/metabolismoRESUMO
Rab GTPases are localized to various intracellular compartments and are known to play important regulatory roles in membrane trafficking. Here, we report the subcellular distribution and function of Rab14. By immunofluorescence and immunoelectron microscopy, both endogenous as well as overexpressed Rab14 were localized to biosynthetic (rough endoplasmic reticulum, Golgi, and trans-Golgi network) and endosomal compartments (early endosomal vacuoles and associated vesicles). Notably overexpression of Rab14Q70L shifted the distribution toward the early endosome associated vesicles, whereas the S25N and N124I mutants induced a shift toward the Golgi region. A similar, although less pronounced, redistribution of the transferrin receptor was also observed in cells overexpressing Rab14 mutants. Impairment of Rab14 function did not however affect transferrin uptake or recycling kinetics. Together, these findings suggest that Rab14 is involved in the biosynthetic/recycling pathway between the Golgi and endosomal compartments.
Assuntos
Endossomos/fisiologia , Complexo de Golgi/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Anticorpos/imunologia , Linhagem Celular , Membrana Celular/fisiologia , Endossomos/ultraestrutura , Expressão Gênica , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/análise , Humanos , Espaço Intracelular/ultraestrutura , Camundongos , Microscopia de Fluorescência , Mutação Puntual/genética , Transporte Proteico/fisiologia , Ratos , Receptores da Transferrina/análise , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/genéticaRESUMO
ErbB2 is a transmembrane tyrosine kinase whose surface overexpression is linked to tumorigenesis and poor prognosis in breast cancer patients. Two models have emerged that account for the high surface distribution of ErbB2. In one model, the surface pool is dynamic and governed by a balance between endocytosis and recycling, whereas in the other it is retained, static, and excluded from endocytosis. These models have contrasting implications for how ErbB2 exerts its biological function and how cancer therapies might down-regulate surface ErbB2, such as the antibody trastuzumab (Herceptin) or the Hsp90 inhibitor geldanamycin. Little is known, however, about how these treatments affect ErbB2 endocytic trafficking. To investigate this issue, we examined breast carcinoma cells by immunofluorescence and quantitative immunoelectron microscopy and developed imaging and trafficking kinetics assays using cell surface fluorescence quenching. Surprisingly, trastuzumab does not influence ErbB2 distribution but instead recycles passively with internalized ErbB2. By contrast, geldanamycin down-regulates surface ErbB2 through improved degradative sorting in endosomes exclusively rather than through increased endocytosis. These results reveal substantial dynamism in the surface ErbB2 pool and clearly demonstrate the significance of endosomal sorting in the maintenance of ErbB2 surface distribution, a critical feature of its biological function.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Endocitose , Neoplasias/metabolismo , Neoplasias/patologia , Quinonas/farmacologia , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Benzoquinonas , Transporte Biológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Lactamas Macrocíclicas , Lisossomos/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , TrastuzumabRESUMO
Synaptic vesicle fusion is driven by the formation of a four-helical bundle composed of soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors (SNAREs). Exactly how the structural interactions that lead to the formation of this complex relate to neurotransmitter release is not well understood. To address this question, we used a strategy to "rescue" synaptic transmission after proteolytic cleavage of the synaptosome-associated protein of 25 kDa (SNAP-25) by botulinum neurotoxin E (BoNtE). Transfection of CA3 hippocampal pyramidal cells with BoNtE-resistant SNAP-25 restored synaptic transmission. Additional mutations that alter the interaction between SNAP-25 C-terminal coil and the other SNARE coils dramatically reduce transmitter release probability but leave the kinetics of synaptic responses unaltered. These data indicate that at synapses, SNARE interactions are necessary for fusion but are not the rate-limiting step of neurotransmission.
Assuntos
Fusão de Membrana/fisiologia , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animais , Biolística , Toxinas Botulínicas/farmacologia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Genes Reporter , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Substâncias Macromoleculares , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Proteínas SNARE , Relação Estrutura-Atividade , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma , TransfecçãoRESUMO
Phorbol esters, activators of protein kinase C (PKC), have been shown to enhance synaptic transmission. One potential downstream target of PKC in the presynaptic terminal is the soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) SNAP-25, which has a PKC phosphorylation site in its C-terminal coil centered at serine 187 (S187/Ser187). We examined the role of S187 in hippocampal synaptic transmission. After proteolytic cleavage of native SNAP-25 by botulinum neurotoxin E (BoNT/E), synaptic transmission was restored in a subset of transfected CA3 pyramidal cells with a toxin-resistant form of SNAP-25 containing unaltered S187 (Swt), S187 mutated to alanine (SA) or S187 mutated to glutamate (SE). We observed that phorbol-12,13-diacetate (PDAc, 10 microM) induced potentiation of neurotransmission to a similar degree for both Swt and SA (2.4-fold and 3.1-fold increase, respectively). Furthermore, basal levels of transmission mediated by SE were reduced relative to that of Swt (failure rates of 72% and 41%, respectively). Together, these data suggest that phosphorylation of SNAP-25 S187 does not mediate the observed enhancement of neurotransmission by phorbol esters at hippocampal synapses.
Assuntos
Hipocampo/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso , Ésteres de Forbol/farmacologia , Serina/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Regulação para Cima , Animais , Hipocampo/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Fosforilação , Serina/genética , Transmissão Sináptica/genética , Proteína 25 Associada a Sinaptossoma , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.
Assuntos
Anticorpos/imunologia , Imunização , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Plasmídeos , Vacinas de DNA , Animais , Linhagem Celular , DNA Complementar/imunologia , DNA Complementar/farmacologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmídeos/imunologia , Plasmídeos/farmacologia , Estrutura Secundária de Proteína , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologiaRESUMO
Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/síntese química , Imunoconjugados/farmacologia , Maitansina/análogos & derivados , Engenharia de Proteínas , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , TrastuzumabRESUMO
The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
Assuntos
Anticorpos/sangue , Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoglobulina G/química , Engenharia de Proteínas , Aminobenzoatos/química , Aminobenzoatos/imunologia , Animais , Anticorpos/química , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Cisteína/química , Humanos , Imunoconjugados/administração & dosagem , Imunoglobulina G/imunologia , Macaca fascicularis , Maleimidas/química , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/imunologia , Maitansina/química , Maitansina/imunologia , Camundongos , Camundongos Nus , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/imunologia , Conformação Proteica , Ratos , Relação Estrutura-Atividade , Compostos de Sulfidrila/química , TrastuzumabRESUMO
PURPOSE: Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate. EXPERIMENTAL DESIGN AND RESULTS: To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) molecule was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 molecules per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti-cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equivalent doses (µg/m(2)) and retained efficacy at equivalent antibody doses (mg/kg). An improved safety profile of >2-fold was observed in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6,000 µg DM1/m(2)) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6,000 µg DM1/m(2)). CONCLUSIONS: The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clinical development of HER2-positive targeted breast cancer therapies.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Maitansina/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Compostos de Sulfidrila/farmacologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Humanos , Macaca fascicularis , Maitansina/química , Camundongos , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/química , Compostos de Sulfidrila/química , TrastuzumabRESUMO
Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.