RESUMO
The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex, and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH mAb that can potentiate the regulatory function of FH. This Ab could serve as a potential new drug for aHUS patients and alternative to C5 blockade by eculizumab. However, it is unclear whether this Ab can potentiate FH mutant variants in addition to wild-type (WT) FH. In this study, the functionality and potential of the agonistic Ab in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating Ab and similarly, the decay-accelerating activity of all mutants is increased. The potentiating anti-FH Ab is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels on a human HAP-1 cell line and on sheep erythrocytes. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.
Assuntos
Anticorpos/metabolismo , Síndrome Hemolítico-Urêmica Atípica/genética , Complemento C3b/metabolismo , Fator H do Complemento/imunologia , Genótipo , Animais , Linhagem Celular , Ativação do Complemento , Fator H do Complemento/agonistas , Fator H do Complemento/genética , Predisposição Genética para Doença , Humanos , Camundongos , Mutação/genética , Polimorfismo Genético , Ligação ProteicaRESUMO
The binding of programmed death ligand 1 protein (PD-L1) to its receptor programmed death protein 1 (PD-1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD-L1/PD-1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD-L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD-L1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD-1 binding interface, consistent with a desired PD-1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD-L1 variants confirmed that the region identified by HDX-MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD-L1-mediated immune evasion. This is the first report of an epitope for any antibody targeting PD-L1 and demonstrates the power of combining orthogonal epitope mapping techniques.
Assuntos
Anticorpos Monoclonais/química , Antígeno B7-H1/imunologia , Epitopos/análise , Medição da Troca de Deutério/métodos , Mapeamento de Epitopos/métodos , Humanos , Espectrometria de Massas/métodos , ProteóliseRESUMO
Purpose: Factor H (FH, encoded by CFH) prevents activation of the complement system's alternative pathway (AP) on host tissues. FH impedes C3 convertase (C3bBb) formation, accelerates C3bBb decay, and is a cofactor for factor I (FI)-catalyzed C3b cleavage. Numerous CFH variants are associated with age-related macular degeneration (AMD), but their functional consequences frequently remain undetermined. Here, we conduct functional comparisons between a control version of FH (not AMD linked) and 21 AMD-linked FH variants. Methods: Recombinantly produced, untagged, full-length FH versions were assayed for binding to C3b and decay acceleration of C3bBb using surface-plasmon resonance, FI-cofactor activity using a fluorescent probe of C3b integrity, suppression of C5b-9 assembly on an AP-activating surface, and inhibition of human AP-mediated lysis of sheep erythrocytes. Results: All versions were successfully purified despite below-average yields for Arg2Thr, Arg53Cys, Arg175Pro, Arg175Gln, Ile221Val, Tyr402His, Pro503Ala, Arg567Gly, Gly1194Asp, and Arg1210Cys. Compared to control FH, Arg2Thr, Leu3Val, Ser58Ala, Asp90Gly, Asp130Asn, Gln400Lys, Tyr402His, Gly650Val, Ser890Ile, and Thr965Met showed minimal functional differences. Arg1210C, Arg53His, Arg175Gln, Gly1194Asp, Pro503Ala, Arg53Cys, Arg576Gly, and Arg175Pro (in order of decreasing efficacy) underperformed, while Ile221Val, Arg303Gln, and Arg303Trp were "marginal." We newly identified variants toward the center of the molecule, Pro503Ala and Arg567Gly, as potentially pathogenic. Conclusions: Our approach could be extended to other variants of uncertain significance and to assays for noncanonical FH activities, aiming to facilitate selection of cohorts most likely to benefit from therapeutic FH. This is timely as recombinant therapeutic FH is in development for intravitreal treatment of AMD in patients with reduced FH functionality.
Assuntos
Fator H do Complemento , Degeneração Macular , Animais , Humanos , Aceleração , Fator H do Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento , Proteínas do Sistema Complemento , Degeneração Macular/genética , OvinosRESUMO
PURPOSE: GEM103 is a recombinantly produced full-length version of the human complement factor H (CFH) under clinical investigation for treatment of age-related macular degeneration (AMD) in individuals carrying an AMD risk-associated genetic variant of CFH. This study aimed to investigate the complement pathway-related functions of GEM103 in comparison with those of native human CFH. METHODS: Key biological activities of GEM103 and human serum-derived CFH (sdCFH) were compared using four independent functional assays. Assays of C3b binding and C3 convertase decay-accelerating activity (DAA) were performed by surface plasmon resonance (SPR). Cofactor activity (CA) was measured using 8-anilinonaphthalene-1-sulfonic acid as a fluorescent probe of C3b integrity. The abilities of GEM103 and sdCFH to protect sheep erythrocytes from hemolysis by CFH-depleted normal human serum were assessed colorimetrically. RESULTS: In multiple SPR-based assays of C3b binding and DAA, the performance of GEM103 was consistently comparable to that of sdCFH across a range of matching concentrations. The EC50 ± SD in the fluorescence-based fluid-phase CA assay was 0.21 ± 0.06 µM for GEM103 compared to 0.20 ± 0.09 µM for sdCFH. In hemolysis assays, the EC50 value of 0.33 ± 0.16 µM for GEM103 versus 0.46 ± 0.06 µM for sdCFH were not significantly different (p = 0.81). CONCLUSIONS: GEM103, a recombinant CFH developed by Gemini Therapeutics, shows activity profiles comparable to sdCFH in all complement-related assays employed in this study, suggesting that GEM103 is equivalent to the native glycoprotein in terms of its in vitro functional activity. These results support further study of GEM103 as a potential therapy for AMD.
Assuntos
Fator H do Complemento , Degeneração Macular , Animais , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Hemólise , Humanos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Polimorfismo de Nucleotídeo Único , OvinosRESUMO
Leptin plays a central role in the homeostasis of body weight through its regulatory effects on appetite and energy expenditure, yet in trials as a therapeutic agent for the treatment of obesity in humans it has been disappointing. The poor clinical efficacy of leptin results from its short circulating half-life, low potency and poor solubility, necessitating large and frequent doses to obtain even modest clinical benefit. Engineered Fc-leptin immunofusins, consisting of the Fc fragment of an immunoglobulin gamma chain followed by leptin, exhibit improved pharmacological properties with very consistent and potent biological activities. Furthermore, in extending the circulating half-life of the protein in vivo from a few minutes for leptin to many hours for Fc-leptin, these proteins have the potential to reduce drastically the dosage and frequency of administration required to obtain clinical benefit. The results of this study show that the engineered leptin immunofusins described here have significantly enhanced pharmacological properties in comparison with the recombinant leptin that was used in clinical trials. As such, they could represent an important step towards a therapeutically superior form of leptin if the disappointing performance of leptin in early clinical trials was due to its poor pharmacological properties rather than any conceptual weakness in the strategy of using leptin for the treatment of obesity and its related disorders.
Assuntos
Leptina/genética , Leptina/uso terapêutico , Obesidade/tratamento farmacológico , Engenharia de Proteínas , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Primers do DNA , Esquema de Medicação , Humanos , Leptina/administração & dosagem , Leptina/farmacocinética , Camundongos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Erythropoietin (Epo) is a cytokine that controls the production of red blood cells (RBCs). Epo acts continuously on RBC precursors to prevent apoptosis, so it is important to maintain high levels of Epo activity when treating anemic patients. We describe here modified human Epo [Epo(NDS)] with mutations His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that result in the rearrangement of the disulfide bonding pattern from Cys29-Cys33 to Cys29-Cys88 and that, in the context of an Fc-Epo(NDS) fusion protein, lead to significantly improved properties. Fc-Epo was secreted from NS/0 myeloma cells as about 35% high molecular weight aggregates, was unstable upon removal of N-linked oligosaccharides and showed poor pharmacokinetics and little efficacy in mice. In contrast, a corresponding Fc-Epo(NDS) was secreted almost exclusively as a unit dimer, was relatively stable to removal of N-linked oligosaccharides, had much improved pharmacokinetic properties and had a significantly improved effect on RBC production. These results indicate that rearrangement of the disulfide bonding pattern in a therapeutic protein can have a significant effect on pharmacokinetics and, potentially, the dosing schedule of a protein drug.
Assuntos
Eritropoetina/química , Eritropoetina/farmacocinética , Engenharia de Proteínas/métodos , Receptores Fc/química , Sequência de Aminoácidos , Animais , Apoptose , Bovinos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Códon , Simulação por Computador , Cisteína/química , Dimerização , Dissulfetos/química , Eritropoetina/genética , Glicosilação , Humanos , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oligossacarídeos/química , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Ratos , Receptores Fc/genética , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Transfecção , Tripsina/químicaRESUMO
IL-12 is a cytokine which showed anti-tumor effects in clinical trials, but also produced serious toxicity. We describe a fusion protein, huBC1-IL12, designed to achieve an improved therapeutic index by specifically targeting IL-12 to tumor and tumor vasculature. huBC-1 is a humanized antibody that targets a cryptic sequence of the human ED-B-containing fibronectin isoform, B-FN, present in the subendothelial extracellular matrix of most aggressive tumors. B-FN is oncofetal and angiogenesis-associated, and is undetectable in most normal adult tissues. The original murine BC-1 antibody has been used successfully for immunoscintigraphy to image brain tumor mass in glioblastoma patients. In huBC1-IL12, each of the IgG heavy chains is genetically fused to the N-terminus of the IL-12 p35 subunit, which in turn is disulfide-bonded to the p40 subunit, resulting in a hexameric molecule of MW of approximately 300 kDa. Since human IL-12 has no biological activity in mice, we produced huBC1-muIL12 as a surrogate molecule for animal tumor models. Despite the relatively poor PK profile of this molecule in mice and the apparent drawbacks of xenogeneic models in SCID mice, which lack T and B cells, one cycle of treatment with huBC1-muIL12 was efficacious in the PC3mm2, A431, and HT29 subcutaneous tumor models and PC3mm2 lung metastasis model. This molecule also was found to have surprisingly low toxicity in immunocompetent mice. A fusion protein that contains human IL-12 (huBC1-huIL12), which is a suitable molecule for investigation as a therapeutic, has also been produced. This protein has been shown to have a longer serum half-life than huBC1-muIL12 in mice, and retains both antigen binding and IL-12 activity in in vitro assays.