ABSTRACT
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
Subject(s)
CD3 Complex , Intracellular Signaling Peptides and Proteins , Lung Neoplasms , Membrane Proteins , Small Cell Lung Carcinoma , T-Lymphocytes , Tumor Necrosis Factor Receptor Superfamily, Member 9 , Small Cell Lung Carcinoma/immunology , Small Cell Lung Carcinoma/pathology , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/therapy , Small Cell Lung Carcinoma/metabolism , Humans , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/drug therapy , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , CD3 Complex/immunology , Animals , Mice , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Membrane Proteins/immunology , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Cell Line, Tumor , Lymphocyte Activation/immunology , Female , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Xenograft Model Antitumor AssaysABSTRACT
In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.
Subject(s)
Celiac Disease , Glutens , Mice , Animals , Humans , Rabbits , Glutens/chemistry , Antibodies, Neutralizing , HLA-DQ Antigens , Peptides/chemistry , Epitopes/chemistry , Mice, TransgenicABSTRACT
To combat infectious diseases, vaccines are considered the best prophylactic strategy for a wide range of the population, but even when vaccines are effective, the administration of therapeutic antibodies against viruses could provide further treatment options, particularly for vulnerable groups whose immunity against the viruses is compromised. Therapeutic antibodies against dengue are ideally engineered to abrogate binding to Fcγ receptors (FcγRs), which can induce antibody-dependent enhancement (ADE). However, the Fc effector functions of neutralizing antibodies against SARS-CoV-2 have recently been reported to improve post-exposure therapy, while they are dispensable when administered as prophylaxis. Hence, in this report, we investigated the influence of Fc engineering on anti-virus efficacy using the anti-dengue/Zika human antibody SIgN-3C and found it affected the viremia clearance efficacy against dengue in a mouse model. Furthermore, we demonstrated that complement activation through antibody binding to C1q could play a role in anti-dengue efficacy. We also generated a novel Fc variant, which displayed the ability for complement activation but showed very low FcγR binding and an undetectable level of the risk of ADE in a cell-based assay. This Fc engineering approach could make effective and safe anti-virus antibodies against dengue, Zika and other viruses.
ABSTRACT
The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/therapy , Humans , Membrane Glycoproteins , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Envelope ProteinsABSTRACT
Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. In order to neutralize the increased C5, an even higher dosage of the antibody would be required. This antigen accumulation can be suppressed by giving the antibody a pH-dependent C5-binding property so that C5 is released from the antibody in the acidic endosome and then trafficked to the lysosome for degradation, while the C5-free antibody returns back to plasma. We recently demonstrated that a pH-dependent C5-binding antibody, SKY59, exhibited long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration. Here we report the details of the antibody engineering involved in generating SKY59, from humanizing a rabbit antibody to improving the C5-binding property. Moreover, because the pH-dependent C5-binding antibodies that we first generated still accumulated C5, we hypothesized that the surface charges of the ICs partially contributed to a slow uptake rate of the C5-antibody ICs. This idea motivated us to engineer the surface charges of the antibody. Our surface-charge engineered antibody consequently exhibited a high capacity to sweep C5 and suppressed the C5 accumulation in vivo by accelerating the cycle of sweeping: uptake of ICs into cells, release of C5 from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, our engineered anti-C5 antibody, SKY59, is expected to provide significant benefits for patients with complement-mediated disorders.
Subject(s)
Antibodies, Monoclonal/genetics , Complement Activation/drug effects , Complement C5/antagonists & inhibitors , Protein Engineering/methods , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibody Affinity , Complement Activation/immunology , Complement C5/immunology , Complement C5/isolation & purification , Computer Simulation , Drug Discovery/methods , Endosomes/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Hydrogen-Ion Concentration , Immune System Diseases/drug therapy , Immune System Diseases/immunology , Macaca fascicularis , Mice , Mice, Transgenic , Mutagenesis , Receptors, Fc/genetics , Receptors, Fc/immunology , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Time FactorsABSTRACT
Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.
Subject(s)
Antibodies, Neutralizing/immunology , Complement C5/antagonists & inhibitors , Complement C5/immunology , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Complement C5/chemistry , Crystallography, X-Ray , Hemoglobinuria, Paroxysmal/drug therapy , Humans , Macaca fascicularis , Protein Binding , Protein ConformationABSTRACT
The p7 protein from the hepatitis C virus (HCV) is a 63 amino acid long polypeptide that is essential for replication, and is involved in protein trafficking and proton transport. Therefore, p7 is a possible target for antivirals. The consensus model for the channel formed by p7 protein is a hexameric or heptameric oligomer of α-helical hairpin monomers, each having two transmembrane domains, TM1 and TM2, where the N-terminal TM1 would face the lumen of this channel. A reported high-throughput functional assay to search for p7 channel inhibitors is based on carboxyfluorescein (CF) release from liposomes after p7 addition. However, the rationale for the dual ability of p7 to serve as an ion or proton channel in the infected cell, and to permeabilize membranes to large molecules like CF is not clear. We have recreated both activities in vitro, examining the conformation present in these assays using infrared spectroscopy. Our results indicate that an α-helical form of p7, which can transport protons, is not able to elicit CF release. In contrast, membrane permeabilization to CF is observed when p7 contains a high percentage of ß-structure, or when using a C-terminal fragment of p7, encompassing TM2. We propose that the reported inhibitory effect of some small compounds, e.g., rimantadine, on both CF release and proton transport can be explained via binding to the membrane-inserted C-terminal half of p7, increasing its rigidity, in a similar way to the influenza A M2-rimantadine interaction.
Subject(s)
Fluorescent Dyes/metabolism , Protons , Viral Proteins/chemistry , Viral Proteins/metabolism , Biological Transport , Electron Transport , Gene Expression , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Mass Spectrometry , Protein Conformation , Protein Interaction Domains and Motifs , Proton Pumps/chemistry , Proton Pumps/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Proteins/geneticsABSTRACT
The small hydrophobic (SH) protein is encoded by the human respiratory syncytial virus. Its absence leads to viral attenuation in the context of whole organisms, and it prevents apoptosis in infected cells. Herein, we have examined the structure of SH protein in detergent micelles and in lipid bilayers, by solution NMR and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. We found that SH protein has a single α-helical transmembrane domain and forms homopentamers in several detergents. In detergent micelles, the transmembrane domain is flanked N-terminally by an α-helix that forms a ring around the lumen of the pore and C-terminally by an extended ß-turn. SH protein was found in the plasma membrane of transiently expressing HEK 293 cells, which showed pH-dependent (acid-activated) channel activity. Channel activity was abolished in mutants lacking both native His residues, His(22) and His(51), but not when either His was present. Herein, we propose that the pentameric model of SH protein presented is a physiologically relevant conformation, albeit probably not the only one, in which SH contributes to RSV infection and replication. Viroporins are short (â¼100 amino acids) viral membrane proteins that form oligomers of a defined size, act as proton or ion channels, and in general enhance membrane permeability in the host. However, with some exceptions, their precise biological role of their channel activity is not understood. In general, viroporins resemble poorly specialized proteins but are nevertheless critical for viral fitness. In vivo, viruses lacking viroporins usually exhibit an attenuated or weakened phenotype, altered tropism, and diminished pathological effects. We have chosen to study the SH protein, 64 amino acids long, found in the human respiratory syncytial virus because of the effect of RSV on human health and the lack of adequate antivirals. We show that SH protein forms oligomers that behave as ion channels when activated at low pH. This study adds SH protein to a growing group of viroporins that have been structurally characterized. Although the precise biological role of this pentameric channel is still unknown, this report is nevertheless essential to fill some of the many gaps that exist in the understanding of SH protein function.
Subject(s)
Ion Channels/metabolism , Respiratory Syncytial Virus, Human/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Protein Multimerization/physiology , Spectroscopy, Fourier Transform InfraredABSTRACT
The use of blue native polyacrylamide gel electrophoresis (BN-PAGE) has been reported in the literature to retain both water-soluble and membrane protein complexes in their native hetero-oligomeric state and to determine the molecular weight of membrane proteins. However, membrane proteins show abnormal mobility when compared with water-soluble markers. Although one could use membrane proteins as markers or apply a conversion factor to the observed molecular weight to account for the bound Coomassie blue dye, when one just wants to assess homo-oligomeric size, these methods appear to be too time-consuming or might not be generally applicable. Here, during detergent screening studies to identify the best detergent for achieving a monodisperse sample, we observed that under certain conditions membrane proteins tend to form ladders of increasing oligomeric size. Although the ladders themselves contain no indication of which band represents the correct oligomeric size, they provide a scale that can be compared with a single band, representing the native homo-oligomeric size, obtained in other conditions of the screen. We show that this approach works for three membrane proteins: CorA (42 kDa), aquaporin Z (25 kDa), and small hydrophobic (SH) protein from respiratory syncytial virus (8 kDa). In addition, polydispersity results and identification of the most suitable detergent correlate optimally not only with size exclusion chromatography (SEC) but also with results from sedimentation velocity and equilibrium experiments. Because it involves minute quantities of sample and detergent, this method can be used in high-throughput approaches as a low-cost technique.
Subject(s)
Electrophoresis, Polyacrylamide Gel/economics , Electrophoresis, Polyacrylamide Gel/methods , Membrane Proteins/analysis , Membrane Proteins/chemistry , Cloning, Molecular , Molecular Weight , Particle Size , SolubilityABSTRACT
The small hydrophobic (SH) protein from the human respiratory syncytial virus (hRSV) is a glycoprotein of approximately 64 amino acids with one putative alpha-helical transmembrane domain. Although SH protein is important for viral infectivity, its exact role during viral infection is not clear. Herein, we have studied the secondary structure, orientation, and oligomerization of the transmembrane domain of SH (SH-TM) in the presence of lipid bilayers. Only one oligomer, a pentamer, was observed in PFO-PAGE. Using polarized attenuated total reflection-Fourier transform infrared (PATR-FTIR) spectroscopy, we show that the SH-TM is alpha-helical. The rotational orientation of SH-TM was determined by site-specific infrared dichroism (SSID) at two consecutive isotopically labeled residues. This orientation is consistent with that of an evolutionary conserved pentameric model obtained from a global search protocol using 13 homologous sequences of RSV. Conductance studies of SH-TM indicate ion channel activity, which is cation selective, and inactive below the predicted pK(a) of histidine. Thus, our results provide experimental evidence that the transmembrane domain of SH protein forms pentameric alpha-helical bundles that form cation-selective ion channels in planar lipid bilayers. We provide a model for this pore, which should be useful in mutagenesis studies to elucidate its role during the virus cycle.
Subject(s)
Ion Channels/chemistry , Respiratory Syncytial Virus, Human/metabolism , Retroviridae Proteins, Oncogenic/chemistry , Amino Acid Sequence , Cations/chemistry , Electrophoresis, Polyacrylamide Gel , Humans , Hydrogen Bonding , Lipid Bilayers/chemistry , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Spectroscopy, Fourier Transform InfraredABSTRACT
Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin alpha and fertilin beta that plays an important role in sperm-egg fusion. Fertilin alpha and beta are members of the ADAM family, and contain each one transmembrane alpha-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane alpha-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate.