RESUMO
PURPOSE: Although programmed death-ligand 1 (PD-L1) antibody-based therapy has improved the outcome of patients with cancer, acquired resistance to these treatments limits their clinical efficacy. FS118 is a novel bispecific, tetravalent antibody (mAb2) against human lymphocyte activation gene-3 (LAG-3) and PD-L1 with the potential to reinvigorate exhausted immune cells and overcome resistance mechanisms to PD-L1 blockade. Here, using FS118 and a murine surrogate, we characterized the activity and report a novel mechanism of action of this bispecific antibody. EXPERIMENTAL DESIGN: This study characterizes the binding activity and immune function of FS118 in cell lines and human peripheral blood mononuclear cells and further investigates its antitumor activity and mechanism of action using a surrogate murine bispecific antibody (mLAG-3/PD-L1 mAb2). RESULTS: FS118 demonstrated simultaneous binding to LAG-3 and PD-L1 with high affinity and comparable or better activity than the combination of the single component parts of the mAb2 in blocking LAG-3- and PD-L1-mediated immune suppression and enhancing T-cell activity. In syngeneic tumor mouse models, mLAG-3/PD-L1 mAb2 significantly suppressed tumor growth. Mechanistic studies revealed decreased LAG-3 expression on T cells following treatment with the mouse surrogate mLAG-3/PD-L1 mAb2, whereas LAG-3 expression increased upon treatment with the combination of mAbs targeting LAG-3 and PD-L1. Moreover, following binding of mLAG-3/PD-L1 mAb2 to target-expressing cells, mouse LAG-3 is rapidly shed into the blood. CONCLUSIONS: This study demonstrates a novel benefit of the bispecific approach over a combination of mAbs and supports the further development of FS118 for the treatment of patients with cancer.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos CD/metabolismo , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Afinidade de Anticorpos , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Camundongos , Ligação Proteica , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
PURPOSE: With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. EXPERIMENTAL DESIGN: A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. RESULTS: We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8+ T-cell activation assays in a PD-L1-dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8+ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. CONCLUSIONS: By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.
Assuntos
Anticorpos Biespecíficos/fisiologia , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular Tumoral/transplante , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HEK293 , Humanos , Leucócitos Mononucleares , Macaca fascicularis , Camundongos , Cultura Primária de Células , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Biologic treatment options such as tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of inflammatory diseases, including rheumatoid arthritis. Recent data suggest, however, that full and long-lasting responses to TNF inhibitors are limited because of the activation of the pro-inflammatory TH17/interleukin (IL)-17 pathway in patients. Therefore, dual TNF/IL-17A inhibition is an attractive avenue to achieve superior efficacy levels in such diseases. Based on the marketed anti-TNF antibody adalimumab, we generated the bispecific TNF/IL-17A-binding FynomAb COVA322. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. COVA322 was characterized in detail and showed a remarkable ability to inhibit TNF and IL-17A in vitro and in vivo. Through its unique mode-of-action of inhibiting simultaneously TNF and the IL-17A homodimer, COVA322 represents a promising drug candidate for the treatment of inflammatory diseases. COVA322 is currently being tested in a Phase 1b/2a study in psoriasis ( ClinicalTrials.gov Identifier: NCT02243787).
Assuntos
Interleucina-17/antagonistas & inibidores , Psoríase/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/imunologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Feminino , Humanos , Interleucina-17/imunologia , Masculino , Camundongos , Psoríase/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
FS102 is a HER2-specific Fcab (Fc fragment with antigen binding), which binds HER2 with high affinity and recognizes an epitope that does not overlap with those of trastuzumab or pertuzumab. In tumor cells that express high levels of HER2, FS102 caused profound HER2 internalization and degradation leading to tumor cell apoptosis. The antitumor effect of FS102 in patient-derived xenografts (PDXs) correlated strongly with the HER2 amplification status of the tumors. Superior activity of FS102 over trastuzumab or the combination of trastuzumab and pertuzumab was observed in vitro and in vivo when the gene copy number of HER2 was equal to or exceeded 10 per cell based on quantitative polymerase chain reaction (qPCR). Thus, FS102 induced complete and sustained tumor regression in a significant proportion of HER2-high PDX tumor models. We hypothesize that the unique structure and/or epitope of FS102 enables the Fcab to internalize and degrade cell surface HER2 more efficiently than standard of care antibodies. In turn, increased depletion of HER2 commits the cells to apoptosis as a result of oncogene shock. FS102 has the potential of a biomarker-driven therapeutic that derives superior antitumor effects from a unique mechanism-of-action in tumor cells which are oncogenically addicted to the HER2 pathway due to overexpression.
Assuntos
Apoptose/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/farmacologia , Neoplasias/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proliferação de Células , Humanos , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptor ErbB-2/imunologia , Transdução de Sinais , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fynomers are small binding proteins derived from the human Fyn SH3 domain. Using phage display technology, Fynomers were generated inhibiting the activity of the proinflammatory cytokine interleukin-17A (IL-17A). One specific Fynomer called 2C1 inhibited human IL-17A in vitro with an IC50 value of 2.2 nm. Interestingly, when 2C1 was genetically fused to the Fc part of a human antibody via four different amino acid linkers to yield bivalent IL-17A binding proteins (each linker differed in length), the 2C1-Fc fusion protein with the longest linker displayed the most potent inhibitory activity. It blocked homodimeric IL-17A with an IC50 value of 21 pm, which corresponds to a hundredfold improved IC50 value as compared to the value obtained with monovalent Fynomer 2C1. In contrast, the 2C1-Fc fusion with the shortest linker showed only an â¼8-fold improved IC50 value of 260 pm. Furthermore, in a mouse model of acute inflammation, we have shown that the most potent 2C1-Fc fusion protein is able to efficiently inhibit IL-17A in vivo. With their suitable biophysical properties, Fynomer-Fc fusion proteins represent new drug candidates for the treatment of IL-17A mediated inflammatory conditions such as psoriasis, psoriatic arthritis, or rheumatoid arthritis.
Assuntos
Fragmentos Fc das Imunoglobulinas/genética , Interleucina-17/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10 (-4) s (-1) , and to selectively inhibit chymase activity with an IC 50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.
Assuntos
Proteínas de Transporte/metabolismo , Quimases/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Domínio Catalítico , Quimases/química , Quimases/genética , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Modelos Moleculares , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-fyn/genéticaRESUMO
Energy landscape theory is a powerful tool for understanding the structure and dynamics of complex molecular systems, in particular biological macromolecules. The primary sequence of a protein defines its free-energy landscape and thus determines the folding pathway and the rate constants of folding and unfolding, as well as the protein's native structure. Theory has shown that roughness in the energy landscape will lead to slower folding, but derivation of detailed experimental descriptions of this landscape is challenging. Simple folding models show that folding is significantly influenced by chain entropy; proteins in which the contacts are local fold quickly, owing to the low entropy cost of forming stabilizing, native contacts during folding. For some protein families, stability is also a determinant of folding rate constants. Where these simple metrics fail to predict folding behaviour, it is probable that there are features in the energy landscape that are unusual. Such general observations cannot explain the folding behaviour of the R15, R16 and R17 domains of alpha-spectrin. R15 folds approximately 3,000 times faster than its homologues, although they have similar structures, stabilities and, as far as can be determined, transition-state stabilities. Here we show that landscape roughness (internal friction) is responsible for the slower folding and unfolding of R16 and R17. We use chimaeric domains to demonstrate that this internal friction is a property of the core, and suggest that frustration in the landscape of the slow-folding spectrin domains may be due to misdocking of the long helices during folding. Theoretical studies have suggested that rugged landscapes will result in slower folding; here we show experimentally that such a phenomenon directly influences the folding kinetics of a 'normal' protein, that is, one with a significant energy barrier that folds on a relatively slow, millisecond-second, timescale.
Assuntos
Entropia , Fricção , Dobramento de Proteína , Espectrina/química , Espectrina/metabolismo , Cinética , Modelos Químicos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , ViscosidadeRESUMO
The 15th, 16th, and 17th repeats of chicken brain alpha-spectrin (R15, R16, and R17, respectively) are very similar in terms of structure and stability. However, R15 folds and unfolds 3 orders of magnitude faster than R16 and R17. This is unexpected. The rate-limiting transition state for R15 folding is investigated using protein engineering methods (Phi-value analysis) and compared with previously completed analyses of R16 and R17. Characterisation of many mutants suggests that all three proteins have similar complexity in the folding landscape. The early rate-limiting transition states of the three domains are similar in terms of overall structure, but there are significant differences in the patterns of Phi-values. R15 apparently folds via a nucleation-condensation mechanism, which involves concomitant folding and packing of the A- and C-helices, establishing the correct topology. R16 and R17 fold via a more framework-like mechanism, which may impede the search to find the correct packing of the helices, providing a possible explanation for the fast folding of R15.
Assuntos
Dobramento de Proteína , Espectrina/química , Espectrina/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known. Here we investigated the effect of a folded neighbouring domain on the folding pathway of spectrin R16 (the 16th alpha-helical repeat from chicken brain alpha-spectrin) by using the two-domain construct R1516. The R16 folds faster and unfolds more slowly in the presence of its folded neighbour R15 (the 15th alpha-helical repeat from chicken brain alpha-spectrin). An extensive Phi-value analysis of the R16 domain in R1516 was completed to compare the transition state of the R16 domain alone with that of the R16 domain in a multidomain construct. The results indicate that the folding pathways are the same. This result validates the current approach of breaking up larger proteins into domains for the study of protein folding pathways.
Assuntos
Dobramento de Proteína , Estrutura Terciária de Proteína , Animais , Galinhas , Humanos , Cinética , Mutação , Estrutura Terciária de Proteína/genética , Espectrina/química , Espectrina/genéticaRESUMO
Multidomain proteins account for over two-thirds of the eukaryotic genome. Although there have been extensive studies into the biophysical properties of isolated domains, few have investigated how the domains interact. Spectrin is a well-characterized multidomain protein with domains linked in tandem array by contiguous helices. Several of these domains have been shown to be stabilized by their neighbors. Until now, this stabilization has been attributed to specific interactions between the natural neighbors, however we have recently observed that nonnatural neighboring domains can also induce a significant amount of stabilization. Here we investigate this nonnative stabilizing effect. We created spectrin-titin domain pairs of both spectrin R16 and R17 with a single titin I27 domain at either the N- or the C-terminus and found that spectrin domains are significantly stabilized, through slowed unfolding, by nonnative interactions at the C-terminus only. Of particular importance, we show that specific interactions between natural folded neighbors at either terminus confer even greater stability by additionally increasing the folding rate constants. We demonstrate that it is possible to distinguish between natural stabilizing interactions and nonspecific stabilizing effects through examination of the kinetics of well chosen mutant proteins. This work adds to the complexity of studying multidomain proteins.
Assuntos
Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Espectrina/química , Espectrina/metabolismo , Animais , Galinhas , Conectina , Humanos , Cinética , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
There have been relatively few detailed comprehensive studies of the folding of protein domains (or modules) in the context of their natural covalently linked neighbors. This is despite the fact that a significant proportion of the proteome consists of multidomain proteins. In this review we highlight some key experimental investigations of the folding of multidomain proteins to draw attention to the difficulties that can arise in analyzing such systems. The evidence suggests that interdomain interactions can significantly affect stability, folding, and unfolding rates. However, preliminary studies suggest that folding pathways are unaffected-to this extent domains can be truly considered to be independent folding units. Nonetheless, it is clear that interactions between domains cannot be ignored, in particular when considering the effects of mutations.
RESUMO
Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent. Furthermore, recent studies indicate that multidomain proteins have evolved mechanisms to minimize the problems of interdomain misfolding.
Assuntos
Evolução Molecular , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas/classificaçãoRESUMO
Approximately 75% of eukaryotic proteins contain more than one so-called independently folding domain. However, there have been relatively few systematic studies to investigate the effect of interdomain interactions on protein stability and fewer still on folding kinetics. We present the folding of pairs of three-helix bundle spectrin domains as a paradigm to indicate how complex such an analysis can be. Equilibrium studies show an increase in denaturant concentration required to unfold the domains with only a single unfolding transition; however, in some cases, this is not accompanied by the increase in m value, which would be expected if the protein is a truly cooperative, all-or-none system. We analyze the complex kinetics of spectrin domain pairs, both wild-type and carefully selected mutants. By comparing these pairs, we are able to demonstrate that equilibrium data alone are insufficient to describe the folding of multidomain proteins and to quantify the effects that one domain can have on its neighbor.
Assuntos
Dobramento de Proteína , Espectrina/química , Espectrina/metabolismo , Cinética , Modelos Moleculares , Mutação/genética , Estrutura Terciária de Proteína , Espectrina/genéticaRESUMO
Spectrin domains are three-helix bundles, commonly found in large tandem arrays. Equilibrium studies have shown that spectrin domains are significantly stabilized by their neighbors. In this work we show that domain:domain interactions can also have profound effects on their kinetic behavior. We have studied the folding of a tandem pair of spectrin domains (R1617) using a combination of single- and double-jump stopped flow experiments (monitoring folding by both circular dichroism and fluorescence). Mutant proteins were also used to investigate the complex folding kinetics. We find that, although the domains fold and unfold individually, there is a single rate-determining step for both folding and unfolding of the protein. This is consistent with the equilibrium observation of cooperative folding of the entire two-domain protein. The results may have important biological implications. Not only will the protein fold more efficiently during cotranslational folding, but the ability of the multidomain protein to withstand thermal unfolding in the cell will be dramatically increased. This study suggests that caution has to be exercised when extrapolating from single domains to larger proteins with a number of independently folding modules arranged in tandem. The multidomain protein spectrin is certainly more than "the sum of its parts".
Assuntos
Modelos Químicos , Modelos Moleculares , Espectrina/química , Sítios de Ligação , Simulação por Computador , Cinética , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Espectrina/ultraestruturaRESUMO
Most protein domains are found in multi-domain proteins, yet most studies of protein folding have concentrated on small, single-domain proteins or on isolated domains from larger proteins. Spectrin domains are small (106 amino acid residues), independently folding domains consisting of three long alpha-helices. They are found in multi-domain proteins with a number of spectrin domains in tandem array. Structural studies have shown that in these arrays the last helix of one domain forms a continuous helix with the first helix of the following domain. It has been demonstrated that a number of spectrin domains are stabilised by their neighbours. Here we investigate the molecular basis for cooperativity between adjacent spectrin domains 16 and 17 from chicken brain alpha-spectrin (R16 and R17). We show that whereas the proteins unfold as a single cooperative unit at 25 degrees C, cooperativity is lost at higher temperatures and in the presence of stabilising salts. Mutations in the linker region also cause the cooperativity to be lost. However, the cooperativity does not rely on specific interactions in the linker region alone. Most mutations in the R17 domain cause a decrease in cooperativity, whereas proteins with mutations in the R16 domain still fold cooperatively. We propose a mechanism for this behaviour.
Assuntos
Dobramento de Proteína , Espectrina/química , Sequência de Aminoácidos , Animais , Galinhas , Clonagem Molecular , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Sais , Soluções , Espectrina/genética , TermodinâmicaRESUMO
The study of proteins with the same architecture, but different sequence has proven to be a valuable tool in the protein folding field. As a prelude to studies on the folding mechanism of spectrin domains we present the kinetic characterisation of the wild-type forms of the 15th, 16th, and 17th domains of chicken brain alpha-spectrin (referred to as R15, R16 and R17, respectively). We show that the proteins all behave in a two-state manner, with different kinetic properties. The folding rate varies remarkably between different members, with a 5000-fold variation in folding rate and 3000-fold variation in unfolding rate seen for proteins differing only 1 kcal mol(-1) in stability. We show clear evidence for significant complexity in the energy landscape of R16, which shows a change in amplitude outside the stopped-flow timescale and curvature in the unfolding arm of the chevron plot. The accompanying paper describes the characterisation of the folding pathway of this domain.