RESUMO
Therapeutic antibody development requires discovery of an antibody molecule with desired specificities and drug-like properties. For toxicological studies, a therapeutic antibody must bind the ortholog antigen with a similar affinity to the human target to enable relevant dosing regimens, and antibodies falling short of this affinity design goal may not progress as therapeutic leads. Herein, we report the novel use of mammalian recombination signal sequence (RSS)-directed recombination for complementarity-determining region-targeted protein engineering combined with mammalian display to close the species affinity gap of human interleukin (IL)-13 antibody 731. This fully human antibody has not progressed as a therapeutic in part because of a 400-fold species affinity gap. Using this nonhypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including the highest affinity antibody reported to date (34 fM). Resolution of a cocrystal structure of the optimized antibody with the cynomolgus monkey (or nonhuman primate) IL-13 protein revealed that the RSS-derived mutations introduced multiple successive amino-acid substitutions resulting in a de novo formation of a π-π stacking-based protein-protein interaction between the affinity-matured antibody heavy chain and helix C on IL-13, as well as an introduction of an interface-distant residue, which enhanced the light chain-binding affinity to target. These mutations synergized binding of heavy and light chains to the target protein, resulting in a remarkably tight interaction, and providing a proof of concept for a new method of protein engineering, based on synergizing a mammalian display platform with novel RSS-mediated library generation.
Assuntos
Anticorpos , Interleucina-13 , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/imunologia , Afinidade de Anticorpos , Humanos , Interleucina-13/genética , Interleucina-13/imunologia , Macaca fascicularis , Mamíferos , Recombinação GenéticaRESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid ß (Aß). Alzheimer's disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aß pathology. Here, we show that TREM2 engagement by the mAb hT2AB as surrogate ligand activates microglia in 5XFAD transgenic mice that accumulate Aß and express either the common TREM2 variant (TREM2CV) or TREM2R47H scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control hIgG1 exposed four distinct trajectories of microglia activation leading to disease-associated (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia types. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia activation trajectories. Moreover, Cyc-M and IFN-R microglia were more abundant in female than male TREM2CV-5XFAD mice, likely due to greater Aß load in female 5XFAD mice. A single systemic injection of hT2AB replenished Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which these trajectories had already reached maximum capacity. Moreover, hT2AB induced shifts in gene expression patterns in all microglial pools without affecting representation. Repeated treatment with a murinized hT2AB version over 10 d increased chemokines brain content in TREM2R47H-5XFAD mice, consistent with microglia expansion. Thus, the impact of hT2AB on microglia is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.
Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Glicoproteínas de Membrana/genética , Microglia/metabolismo , Receptores Imunológicos/genética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Proliferação de Células , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Cinética , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microglia/classificação , Microglia/efeitos dos fármacos , Microglia/patologia , Mutação , Ligação Proteica , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Fatores SexuaisRESUMO
Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs.
Assuntos
Anticorpos Monoclonais/farmacologia , Técnicas Biossensoriais/métodos , Receptor de Morte Celular Programada 1/imunologia , China , Desenvolvimento de Medicamentos , Epitopos/imunologia , União Europeia , Ensaios de Triagem em Larga Escala , Humanos , Receptor de Morte Celular Programada 1/química , Ligação Proteica , Ressonância de Plasmônio de Superfície , Estados UnidosRESUMO
OBJECTIVE: Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE. METHODS: Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti-IL-21R treatment regimens. RESULTS: IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti-IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease. CONCLUSION: Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Modelos Animais de Doenças , Progressão da Doença , Imunidade Humoral/imunologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Interleucina-21/antagonistas & inibidores , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Autoimunidade/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Feminino , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina G/sangue , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Endogâmicos NZB , Receptores de Interleucina-21/efeitos dos fármacos , Receptores de Interleucina-21/imunologia , Ovinos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Resultado do TratamentoRESUMO
Measuring the affinity of a therapeutic antibody to its antigen, expressed in its native form on a cell surface, is an important aspect to understanding its in vivo potency. Measured affinities can also help in selecting the best antibody for therapy. The on-cell binding affinity of antibodies was determined in the past by labelling the antibody using radioactive, fluorescent, or other probes. Labelling the antibody could potentially modify the structure of the antibody and hence could alter its original affinity. Here, we describe a label-free method to measure the affinity of antibodies to their target antigens that are expressed on the surface of cells and the number of active antigen molecules present on a given cell. In addition, this method can also be used to measure the affinity of a ligand to its receptor on a cell.
Assuntos
Afinidade de Anticorpos/imunologia , Antígenos/imunologia , Membrana Celular/imunologia , HumanosRESUMO
Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.
Assuntos
Anemia Ferropriva/tratamento farmacológico , Anticorpos Neutralizantes/farmacologia , Eritrócitos/efeitos dos fármacos , Hemoglobinas/biossíntese , Ferro/sangue , Anemia Ferropriva/sangue , Anemia Ferropriva/patologia , Animais , Anticorpos Neutralizantes/biossíntese , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Eritrócitos/metabolismo , Eritrócitos/patologia , Eritropoese/efeitos dos fármacos , Feminino , Hematínicos/farmacologia , Humanos , Inflamação/prevenção & controle , Macaca fascicularis , Masculino , CamundongosRESUMO
Calcium entry into T cells following antigen stimulation is crucial for nuclear factor of activated T cells (NFAT)-mediated T cell activation. The movement of calcium is mediated by calcium release-activated calcium (CRAC) channels. There are two key components of this channel: Orai1 is the pore-forming subunit located in the plasma membrane, and stromal interaction molecule 1 (STIM1) functions as a Ca(2+) sensor in the endoplasmic reticulum. A subset of human patients carry mutations in either STIM1 or Orai1 that affect protein function or expression, resulting in defective store-operated Ca(2+) influx and CRAC channel function, and impaired T cell activation. These patients suffer from a hereditary form of severe combined immune deficiency syndrome, highlighting the importance of the CRAC channel for T lymphocyte function in humans. Since autoreactive T cells play an important role in the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and organ transplantation, Orai1 becomes an attractive therapeutic target for ameliorating autoimmune disease. We developed a novel approach to inhibiting CRAC function by generating high-affinity fully human monoclonal antibodies to human Orai1. These antibodies inhibited ICRAC current, store-operated Ca(2+) influx, NFAT transcription, and cytokine release. These fully human antibodies to human Orai1 may represent a novel therapeutic approach for the treatment of autoimmunity.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/imunologia , Equorina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/biossíntese , Anticorpos Monoclonais/biossíntese , Western Blotting , Quimera , Citocinas/sangue , Mapeamento de Epitopos , Epitopos/efeitos dos fármacos , Citometria de Fluxo , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Cinética , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC/biossíntese , Fatores de Transcrição NFATC/genética , Proteína ORAI1 , Técnicas de Patch-Clamp , Polimorfismo de Nucleotídeo Único , RatosRESUMO
The role of gamma amino butyric acid A receptors/neurons of the hypothalamic, endocrine and alimentary systems in the food intake seen in hunger was studied in 20 h food-deprived rats. Food deprivation decreased blood glucose, serum insulin and produced hyperphagia. The hyperphagia was inhibited by subcutaneous or ventromedial hypothalamic administration of gamma amino butyric acid A antagonists picrotoxin or bicuculline. Although results of blood glucose was variable, insulin level was increased by picrotoxin or bicuculline. In contrast, lateral hypothalamic administration of these agents failed to reproduce the above changes. Subcutaneous administration of picrotoxin or bicuculline increased gastric content, decreased gastric motility and small bowel transit. In contrast, ventromedial or lateral hypothalamic administration of picrotoxin or bicuculline failed to alter the gastric content but decreased the small bowel transit. The results of alimentary studies suggest that gamma amino butyric acid neurons of both ventromedial and lateral hypothalamus selectively regulate small bowel transit but not the gastric content. It may be concluded that ventromedial hypothalamus plays a dominant role in the regulation of food intake and that picrotoxin or bicuculline inhibited food intake by inhibiting gamma amino butyric acid receptors of the ventromedial hypothalamus, increasing insulin level and decreasing the gut motility.
RESUMO
A rapid screening method has been developed to determine binding affinities for protein-ligand interactions using the Gyrolab workstation, a commercial microfluidic platform developed to accurately and precisely quantify proteins in solution. This method was particularly suited for assessing the high-affinity interactions that have become typical of therapeutic antibody-antigen systems. Five different commercially available antibodies that bind digoxin and a digoxin-bovine serum albumin (BSA) conjugate with high affinity were rigorously evaluated by this method and by the more conventional kinetic exclusion assay (KinExA) method. Binding parameter values obtained using Gyrolab were similar to those recovered from KinExA. However, the total experimental time for 20 binding affinity titrations, with each titration covering 12 data points in duplicate, took approximately 4h by the Gyrolab method, which reduced the experimental duration by more than 10-fold when compared with the KinExA method. This rapid binding analysis method has significant applications in the screening and affinity ranking selection of antibodies from a very large pool of candidates spanning a wide range of binding affinities from the low pM to µM range.
Assuntos
Anticorpos/metabolismo , Microfluídica , Soroalbumina Bovina/metabolismo , Animais , Anticorpos/imunologia , Complexo Antígeno-Anticorpo , Bovinos , Digoxina/imunologia , Digoxina/metabolismo , Cinética , Ligantes , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Soroalbumina Bovina/imunologiaRESUMO
Affinity measurements of antigen-antibody interactions are generally performed using known concentrations of purified or recombinant materials. In addition, many technologies that measure affinity require the interacting components to be present in at least microgram quantities. Specifically, if the antigen is either available only in low quantities or unable to be purified, or if the quantity is unknown, then the measurement of affinity can be very difficult. Using the Kinetic Exclusion Assay (KinExA) technology, here we describe a method that overcomes the requirement for large amounts of purified and known quantities of antigen. We used this method to precisely measure the affinity of fully human anti-human interleukin 13 (IL13) monoclonal antibodies to IL13 produced in native form from primary T cells derived from a variety of species, including human. These antigens were available only in the limited quantities present in the conditioned cell culture medium, and the affinity was measured directly without further purification.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Interleucina-13/imunologia , Animais , Anticorpos Monoclonais/análise , Células Cultivadas , Humanos , Interleucina-13/análise , Cinética , Linfócitos T/imunologiaRESUMO
A simple method that allows affinity measurements of antibodies to integral membrane proteins is described. Kinetic Exclusion Assay was used to determine the concentration of free antibody that remains in solution after equilibrium has been established between the antibody and the cell-surface-expressed antigen, from which the equilibrium dissociation constant (Kd) was determined. It eliminates the requirement for soluble antigen and modifications such as radio-labeling or fluorescent labeling of the antibody. For one of the cell-surface-expressed antigens, it was determined that the affinity of the antibody to the cell-surface-expressed antigen was similar to that of the purified, soluble form of the antigen. In addition to the simplicity of the approach, the method provides a true measure of the affinity/avidity of the antibody to the native form of cell-surface-expressed targets, including antigens that cannot be produced in soluble forms, and to unknown cell surface antigens.
Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Animais , Reações Antígeno-Anticorpo , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , HumanosRESUMO
The high specificity and affinity of monoclonal antibodies make them attractive as therapeutic agents. In general, the affinities of antibodies reported to be high affinity are in the high picomolar to low nanomolar range and have been affinity matured in vitro. It has been proposed that there is an in vivo affinity ceiling at 100 pM and that B cells producing antibodies with affinities for antigen above the estimated ceiling would have no selective advantage in antigen-induced affinity maturation during normal immune responses. Using a transgenic mouse producing fully human antibodies, we have routinely generated antibodies with sub-nanomolar affinities, have frequently rescued antibodies with less than 10 pM affinity, and now describe the existence of an in vivo generated anti-hIL-8 antibody with a sub-picomolar equilibrium dissociation constant. This confirms the prediction that antibodies with affinities beyond the proposed affinity ceiling can be generated in vivo. We also describe the technical challenges of determining such high affinities. To further understand the importance of affinity for therapy, we have constructed a mathematical model to predict the relationship between the affinity of an antibody and its in vivo potency using IL-8 as a model antigen.