RESUMO
Activating interferon responses with STING agonists (STINGa) is a current cancer immunotherapy strategy, and therapeutic modalities that enable tumor-targeted delivery via systemic administration could be beneficial. Here we demonstrate that tumor cell-directed STING agonist antibody-drug-conjugates (STINGa ADCs) activate STING in tumor cells and myeloid cells and induce anti-tumor innate immune responses in in vitro, in vivo (in female mice), and ex vivo tumor models. We show that the tumor cell-directed STINGa ADCs are internalized into myeloid cells by Fcγ-receptor-I in a tumor antigen-dependent manner. Systemic administration of STINGa ADCs in mice leads to STING activation in tumors, with increased anti-tumor activity and reduced serum cytokine elevations compared to a free STING agonist. Furthermore, STINGa ADCs induce type III interferons, which contribute to the anti-tumor activity by upregulating type I interferon and other key chemokines/cytokines. These findings reveal an important role for type III interferons in the anti-tumor activity elicited by STING agonism and provide rationale for the clinical development of tumor cell-directed STINGa ADCs.
Assuntos
Imunidade Inata , Imunoconjugados , Interferons , Proteínas de Membrana , Animais , Proteínas de Membrana/agonistas , Proteínas de Membrana/imunologia , Imunidade Inata/efeitos dos fármacos , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Imunoconjugados/farmacologia , Imunoconjugados/administração & dosagem , Interferons/metabolismo , Interferon lambda , Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Interferon Tipo I/imunologia , Citocinas/metabolismo , Células Mieloides/imunologia , Células Mieloides/efeitos dos fármacos , Imunoterapia/métodos , Camundongos Endogâmicos C57BL , Receptores de IgG/agonistas , Receptores de IgG/metabolismo , Receptores de IgG/imunologiaRESUMO
Antibody-drug conjugates (ADC) achieve targeted drug delivery to a tumor and have demonstrated clinical success in many tumor types. The activity and safety profile of an ADC depends on its construction: antibody, payload, linker, and conjugation method, as well as the number of payload drugs per antibody [drug-to-antibody ratio (DAR)]. To allow for ADC optimization for a given target antigen, we developed Dolasynthen (DS), a novel ADC platform based on the payload auristatin hydroxypropylamide, that enables precise DAR-ranging and site-specific conjugation. We used the new platform to optimize an ADC that targets B7-H4 (VTCN1), an immune-suppressive protein that is overexpressed in breast, ovarian, and endometrial cancers. XMT-1660 is a site-specific DS DAR 6 ADC that induced complete tumor regressions in xenograft models of breast and ovarian cancer as well as in a syngeneic breast cancer model that is refractory to PD-1 immune checkpoint inhibition. In a panel of 28 breast cancer PDXs, XMT-1660 demonstrated activity that correlated with B7-H4 expression. XMT-1660 has recently entered clinical development in a phase I study (NCT05377996) in patients with cancer.
Assuntos
Antineoplásicos , Neoplasias da Mama , Imunoconjugados , Humanos , Feminino , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Anticorpos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immunoglobulin G (IgG) antibodies are an integral part of the adaptive immune response that provide a direct link between humoral and cellular components of the immune system. Insights into relationships between the structure and function of human IgGs have prompted molecular engineering efforts to enhance or eliminate specific properties, such as Fc-mediated immune effector functions. Human IgGs have an N-glycosylation site at Asn297, located in the second heavy chain constant region (CH2). The composition of the Fc glycan can have substantial impacts on Fc gamma receptor(FcγR) binding. The removal of the glycan through enzymatic deglycosylation or mutagenesis of the N-linked glycosylation site has been reported to "silence" FcγR-binding and effector functions, particularly with assays that measure monomeric binding. However, interactions between IgGs and FcγRs are not limited to monomeric interactions but can be influenced by avidity, which takes into account the sum of multimeric interactions between antigen-engaged IgGs and FcγRs. We show here that under in vitro conditions, which allowed avidity binding, aglycosylated IgGs can bind to one of the FcγRs, FcγRI, and mediate effector functions. These studies highlight how the valency of a molecular interaction (monomeric binding versus avidity binding) can influence antibody/FcγR interactions such that avidity effects can translate very low intrinsic affinities into significant functional outcomes.
Assuntos
Afinidade de Anticorpos , Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Animais , Antígenos CD/imunologia , Ligação Competitiva , Proliferação de Células , Glicosilação , Células HEK293 , Humanos , Imunoglobulina G/química , Macrófagos/imunologia , Macrófagos/fisiologia , Camundongos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Fagocitose/imunologia , Ligação Proteica , Receptores de IgG/química , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Over recent years, much progress has been made in the identification and characterization of factors involved in the biosynthesis of integral membrane proteins of the helix-bundle type. In addition, our knowledge of membrane protein structure and the forces stabilizing helix-helix interactions in a lipid environment is expanding rapidly. However, it is still not clear how a membrane protein folds into its final form in vivo, nor what constraints there are on the folded structure that results from the mechanistic details of translocon-mediated assembly rather than simply from the thermodynamics of protein-lipid interactions.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Growth differentiation factor 15 (GDF15), a distant member of the transforming growth factor (TGF)-ß family, is a secreted protein that circulates as a 25-kDa dimer. In humans, elevated GDF15 correlates with weight loss, and the administration of GDF15 to mice with obesity reduces body weight, at least in part, by decreasing food intake. The mechanisms through which GDF15 reduces body weight remain poorly understood, because the cognate receptor for GDF15 is unknown. Here we show that recombinant GDF15 induces weight loss in mice fed a high-fat diet and in nonhuman primates with spontaneous obesity. Furthermore, we find that GDF15 binds with high affinity to GDNF family receptor α-like (GFRAL), a distant relative of receptors for a distinct class of the TGF-ß superfamily ligands. Gfral is expressed in neurons of the area postrema and nucleus of the solitary tract in mice and humans, and genetic deletion of the receptor abrogates the ability of GDF15 to decrease food intake and body weight in mice. In addition, diet-induced obesity and insulin resistance are exacerbated in GFRAL-deficient mice, suggesting a homeostatic role for this receptor in metabolism. Finally, we demonstrate that GDF15-induced cell signaling requires the interaction of GFRAL with the coreceptor RET. Our data identify GFRAL as a new regulator of body weight and as the bona fide receptor mediating the metabolic effects of GDF15, enabling a more comprehensive assessment of GDF15 as a potential pharmacotherapy for the treatment of obesity.
Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/genética , Obesidade/metabolismo , Redução de Peso/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Ingestão de Alimentos/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Fator 15 de Diferenciação de Crescimento/farmacologia , Humanos , Macaca fascicularis , Camundongos , Camundongos Knockout , Redução de Peso/genéticaRESUMO
Receptor-like protein tyrosine phosphatases (RPTPs) are type I integral membrane proteins. Together with protein tyrosine kinases, RPTPs regulate the phosphotyrosine levels in the cell. Studies of two RPTPs, CD45 and PTPalpha, have provided strong evidence that dimerization leads to inactivation of the receptors, and that the dimerization of PTPalpha involves interactions in the transmembrane domain (TMD). Using the TOXCAT assay, a genetic approach for analyzing TM interactions in Escherichia coli membranes, we show that the TMD of RPTPs interact in the membrane, albeit to different extents. Using fusion proteins of TMDs, we also observe an equilibrium between monomer and dimer in sodium dodecyl sulfate (SDS) micelles. Through a mutational study of the DEP1 TMD, we demonstrate that these interactions are specific. Taken together, our results define a subset of the RPTP family in which TM homodimerization may act as a mediator of protein function.
Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Bioensaio , Membrana Celular/metabolismo , Análise Mutacional de DNA , Dimerização , Escherichia coli/metabolismo , Humanos , Micelas , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Dodecilsulfato de Sódio/químicaRESUMO
TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.
Assuntos
Antígenos CD/metabolismo , Interleucina-2/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Genes Reporter , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Ativação Linfocitária/imunologia , Proteínas de Membrana/química , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
The folding of alpha-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable alpha-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.
Assuntos
Proteínas de Membrana/química , Aquaporinas/química , Proteínas de Bactérias/química , Bacteriorodopsinas/química , Sítios de Ligação , Proteínas de Escherichia coli/química , Ligantes , Bicamadas Lipídicas/química , Modelos Moleculares , Canais de Potássio/química , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Plasma cell membrane glycoprotein-1, or ectonucleotide pyrophosphatase/phosphodieterase (PC-1/ENPP1) has been shown to inhibit insulin signaling in cultured cells in vitro and in transgenic mice in vivo when overexpressed. Furthermore, both genetic polymorphism and increased expression of PC-1 have been reported to be associated with type 2 diabetes in humans. Thus it was proposed that PC-1 inhibition represents a potential strategy for the treatment of type 2 diabetes. However, it has not been proven that suppression of PC-1 expression or inhibition of its function will actually improve insulin sensitivity. We show in the current study that transient overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells, while knockdown of PC-1 with siRNA significantly increases insulin-stimulated Akt phosphorylation in HuH7 human hepatoma cells. Adenoviral vector expressing a short hairpin RNA against mouse PC-1 (PC-1shRNA) was utilized to efficiently knockdown PC-1 expression in the livers of db/db mice. In comparison with db/db mice treated with a control virus, db/db mice treated with the PC-1shRNA adenovirus had approximately 80% lower hepatic PC-1 mRNA levels, approximately 30% lower ambient fed plasma glucose, approximately 25% lower fasting plasma glucose, and significantly improved oral glucose tolerance. Taken together, these results demonstrate that suppression of PC-1 expression improves insulin sensitivity in vitro and in an animal model of diabetes, supporting the proposition that PC-1 inhibition is a potential therapeutic approach for the treatment of type 2 diabetes.
Assuntos
Regulação para Baixo , Insulina/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Adenoviridae/genética , Animais , Glicemia/metabolismo , Linhagem Celular , Jejum , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Transdução de Sinais/genética , Fatores de Tempo , Transfecção , Tirosina/metabolismoRESUMO
Plasma cell membrane glycoprotein-1 or ectonucleotide pyrophosphatase/phosphodiesterase (PC-1/ENPP1) has been shown to inhibit insulin signaling, and its genetic polymorphism or increased expression is associated with type 2 diabetes in humans. Therefore, PC-1 inhibition represents a potential strategy in treating diabetes. Since patients with phosphodiesterase/pyrophosphatase deficient PC-1 manifest abnormal calcification, enhancing insulin signaling by inhibiting PC-1 for the treatment of diabetes will be feasible only if PC-1 phosphodiesterase/pyrophosphatase activity needs not be significantly diminished. However, whether inhibition of insulin receptor signaling by PC-1 is dependent upon its phosphodiesterase/pyrophosphatase activity remains controversial. In this study, the extracellular domain of the human PC-1 in its native form or with a T256A or T256S mutation was overexpressed and purified. Enzymatic assays showed that both mutants have less than 10% of the activity of the wild-type protein. In HEK293 cells stably expressing recombinant insulin receptor or insulin-like growth factor 1 (IGF1) receptor, transient expression of wild-type full length PC-1 (PC-1.FL.WT) but not the T256A or T256S mutants inhibits insulin signaling without affecting IGF1 signaling. Western blot and FACS analysis showed that the wild-type and mutant full length PC-1 proteins are expressed at similar levels in the cells, and were localized to the similar levels on the cell surface. Overexpression of PC-1.FL.WT did not affect insulin receptor mRNA level, total protein and cell surface levels. Together, these results suggest that the inhibition of insulin signaling by PC-1 is somewhat specific and is dependent upon the enzymatic activity of the phosphodiesterase/pyrophosphatase.
Assuntos
Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/metabolismo , Transdução de Sinais , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Mutação , Diester Fosfórico Hidrolases/deficiência , Diester Fosfórico Hidrolases/genética , Transporte Proteico , Pirofosfatases/deficiência , Pirofosfatases/genética , Receptor de Insulina/genética , TransfecçãoRESUMO
The human cannabinoid receptor 1 (CB1) belongs to the G protein-coupled receptor (GPCR) family. Among the members of GPCR family, it has an exceptionally long extracellular N-terminal domain (N-tail) of 116 amino acids but has no typical signal sequence. This poses questions of how the long N-tail affects the biosynthesis of the receptor and of how it is inserted into the endoplasmic reticulum (ER) membrane. Here we have examined the process of membrane assembly of CB1 in the ER membrane and the maturation of the receptor from the ER to the plasma membrane. We find that the long N-tail cannot be efficiently translocated across the ER membrane, causing the rapid degradation of CB1 by proteasomes; this leads to a low level of expression of the receptor at the plasma membrane. The addition of a signal peptide at the N terminus of CB1 or shortening of the long N-tail greatly enhances the stability and cell surface expression of the receptor without affecting receptor binding to a cannabinoid ligand, CP-55,940. We propose that the N-tail translocation is a crucial early step in biosynthesis of the receptor and may play a role in regulating the stability and surface expression of CB1.
Assuntos
Canabinoides/metabolismo , Membranas Intracelulares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores de Droga/química , Receptores de Droga/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Cicloexanóis/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Ligação Proteica/fisiologia , Receptores de CanabinoidesRESUMO
We present an approach that allows rapid determination of the topology of Escherichia coli inner-membrane proteins by a combination of topology prediction and limited fusion-protein analysis. We derive new topology models for 12 inner-membrane proteins: MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA. We estimate that our approach should make it possible to arrive at highly reliable topology models for roughly 10% of the approximately 800 inner-membrane proteins thought to exist in E. coli.