RESUMEN
The antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs have been researched for more than 30â¯years, currently only a limited number of bispecific antibodies have achieved regulatory approval. Of the few successful examples of industrially manufacturing a bispecific antibody, the "common light chain format" is an elegant technology that simplifies the purification of a whole IgG-type bispecific antibody. Using this IgG format, the bispecific function can be introduced while maintaining the natural molecular shape of the antibody. In this article, we will first introduce the outline, prospects, and limitations of the common light chain format. Then, we will describe the identification and optimization process for ERY974, an anti-glypican-3â¯×â¯anti-CD3ε T cell-redirecting bispecific antibody with a common light chain. This format includes one of Chugai's proprietary technologies, termed ART-Ig technology, which consists of a method to identify a common light chain, isoelectric point (pI) engineering to purify the desired bispecific IgG antibody from byproducts, and Fc heterodimerization by an electrostatic steering effect. Furthermore, we describe some tips for de-risking the antibody when engineering a T cell redirecting antibody.
Asunto(s)
Anticuerpos Biespecíficos , Inmunoglobulina G , Cadenas Ligeras de Inmunoglobulina , Ingeniería de Proteínas/métodos , Animales , Complejo CD3/inmunología , Glipicanos/inmunología , Humanos , RatonesRESUMEN
Scratching is an important factor exacerbating skin lesions through the so-called itch-scratch cycle in atopic dermatitis (AD). In mice, interleukin (IL)-31 and its receptor IL-31 receptor A (IL-31RA) are known to play a critical role in pruritus and the pathogenesis of AD; however, study of their precise roles in primates is hindered by the low sequence homologies between primates and mice and the lack of direct evidence of itch sensation by IL-31 in primates. We showed that administration of cynomolgus IL-31 induces transient scratching behaviour in cynomolgus monkeys and by that were able to establish a monkey model of scratching. We then showed that a single subcutaneous injection of 1 mg/kg nemolizumab, a humanized anti-human IL-31RA monoclonal antibody that also neutralizes cynomolgus IL-31 signalling and shows a good pharmacokinetic profile in cynomolgus monkeys, suppressed the IL-31-induced scratching for about 2 months. These results suggest that the IL-31 axis and IL-31RA axis play as critical a role in the induction of scratching in primates as in mice and that the blockade of IL-31 signalling by an anti-human IL-31RA antibody is a promising therapeutic approach for treatment of AD. Nemolizumab is currently under investigation in clinical trials.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Interleucinas/farmacología , Prurito/inducido químicamente , Receptores de Interleucina/metabolismo , Células A549 , Animales , Células CHO , Línea Celular , Cricetulus , ADN Complementario/metabolismo , Humanos , Cinética , Macaca fascicularis , Masculino , Ratones , Prurito/metabolismo , Transducción de Señal , Piel/inmunología , Piel/patología , Enfermedades de la Piel/inmunología , Enfermedades de la Piel/patologíaRESUMEN
ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Factor IXa/inmunología , Factor X/inmunología , Hemofilia A/complicaciones , Hemorragia/complicaciones , Hemorragia/prevención & control , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Línea Celular , Modelos Animales de Enfermedad , Hemorragia/patología , Humanos , Articulaciones/efectos de los fármacos , Articulaciones/patología , Macaca fascicularis , Masculino , RatonesRESUMEN
Bispecific antibodies (BsAbs) can bind to two different antigens, enabling therapeutic concepts that cannot be achieved with monoclonal antibodies. Immuno-competent mice are essential for validating drug discovery concepts, necessitating the development of surrogate mouse BsAbs. In this study, we explored the potential of FAST-IgTM, a previously reported BsAb technology, for mouse BsAb production. We investigated charge-based orthogonal Fab mutations to facilitate the correct assembly of heavy and light chains of mouse antibodies and employed knobs-into-holes mutations to facilitate the heterodimerization of heavy chains. We combined five anti-CD3 and two anti-HER2 antibodies in mouse IgG1 and IgG2a subclasses. These 20 BsAbs were analyzed using mass spectrometry or ion exchange chromatography to calculate the percentages of BsAbs with correct chain pairing (BsAb yields). Using FAST-Ig, 19 out of the 20 BsAbs demonstrated BsAb yields of 90% or higher after simple protein A purification from transiently expressed antibodies in Expi293F cells. Importantly, the mouse BsAbs maintained their fundamental physicochemical properties and affinity against each antigen. A Jurkat NFAT-luciferase reporter cell assay demonstrated the combined effects of epitope, affinity, and subclasses. Our findings highlight the potential of FAST-Ig technology for efficiently generating mouse BsAbs for preclinical studies.
RESUMEN
Efficient production of bispecific antibodies (BsAbs) in single mammalian cells is essential for basic research and industrial manufacturing. However, preventing unwanted pairing of heavy chains (HCs) and light chains (LCs) is a challenging task. To address this, we created an engineering technology for preferential cognate HC/LC and HC/HC paring called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology - Immunoglobulin), and applied it to NXT007, a BsAb for the treatment of hemophilia A. We introduced charged amino-acid substitutions at the HC/LC interface to facilitate the proper assembly for manufacturing a standard IgG-type BsAb. We generated CH1/CL interface-engineered antibody variants that achieved > 95% correct HC/LC pairing efficiency with favorable pharmacological properties and developability. Among these, we selected a design (C3) that allowed us to separate the mis-paired species with an unintended pharmacological profile using ion-exchange chromatography. Crystal structure analysis demonstrated that the C3 design did not affect the overall structure of both Fabs. To determine the final design for HCs-heterodimerization, we compared the stability of charge-based and knobs into hole-based Fc formats in acidic conditions and selected the more stable charge-based format. FAST-Ig was also applicable to stable CHO cell lines for industrial production and demonstrated robust chain pairing with different subclasses of parent BsAbs. Thus, it can be applied to a wide variety of BsAbs both preclinically and clinically.
Asunto(s)
Anticuerpos Biespecíficos , Hemofilia A , Animales , Ingeniería de Proteínas/métodos , Línea Celular , Dimerización , MamíferosRESUMEN
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.
Asunto(s)
Enfermedad Celíaca , Glútenes , Ratones , Animales , Humanos , Conejos , Glútenes/química , Anticuerpos Neutralizantes , Antígenos HLA-DQ , Péptidos/química , Epítopos/química , Ratones TransgénicosRESUMEN
A conventional antibody targeting a soluble antigen in circulation typically requires a huge dosage and frequent intravenous administration to neutralize the antigen. This is because antigen degradation is reduced by the formation of antigen-antibody immune complexes, which escape from lysosomal degradation using neonatal Fc receptor (FcRn)-mediated recycling. To address this, we developed an antigen-sweeping antibody that combines pH-dependent antigen binding and Fc engineering to enhance Fc receptor binding. The sweeping antibody actively eliminates the plasma antigens by increasing the cellular uptake of the immune complex and dissociating the antigens in the acidic endosome for degradation. Strong antigen sweeping can reduce the dosage, potentially achieve higher efficacy, and expand the scope of antigen space available for targeting by antibodies. In this study, to further improve the sweeping efficacy, we developed a novel antibody Fc variant by enhancing Fcγ receptor IIb (FcγRIIb) binding and modulating charge characteristics for increased cellular uptake of the immune complex, together with enhancing FcRn binding for efficient salvage of the antigen-free antibodies. Our Fc variant achieved strong antigen sweeping in cynomolgus monkeys with antibody pharmacokinetics comparable to a wild-type human IgG1 antibody. The positive-charge substitutions enhanced uptake of the immune complex by FcγRIIb-expressing cells in vitro, which was completely inhibited by an anti-FcγRIIb antibody. This suggests that the strong in vivo sweeping efficacy improved by the charge engineering is more likely achieved by FcγRIIb-dependent uptake of the immune complex rather than nonspecific uptake. We expect this novel Fc engineering can maximize the antigen sweeping efficacy even in humans and create novel therapeutic antibodies that meet unmet medical needs for patients.
Asunto(s)
Complejo Antígeno-Anticuerpo , Antígenos , Animales , Humanos , Concentración de Iones de Hidrógeno , Fragmentos Fc de Inmunoglobulinas , Macaca fascicularisRESUMEN
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.
Asunto(s)
COVID-19 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/terapia , Humanos , Glicoproteínas de Membrana , Pruebas de Neutralización , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas del Envoltorio ViralRESUMEN
Myostatin, a member of the transforming growth factor-ß superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via "sweeping antibody technology", GYM329 reduces or "sweeps" myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Fuerza Muscular/efectos de los fármacos , Enfermedades Musculares/fisiopatología , Miostatina/inmunología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Modelos Animales de Enfermedad , Femenino , Factores de Diferenciación de Crecimiento/metabolismo , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular/patología , Atrofia Muscular/fisiopatología , Tamaño de los Órganos , Transducción de SeñalRESUMEN
Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physicochemical stability, expression in host cells and pharmacokinetics, and the basic methodologies of antibody engineering involved. By simultaneously optimizing the antibody molecule in light of these properties, it should prove possible to shorten the research and development period necessary to identify a highly qualified clinical candidate and consequently accelerate the start of the clinical trial.
Asunto(s)
Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Ingeniería de Proteínas , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Expresión Génica , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Mutación , Estabilidad Proteica , Proteínas RecombinantesRESUMEN
Monoclonal antibody (mAb) drugs offer a number of valuable treatments. Many newly developed mAb drugs include artificial modification of amino acid sequences from human origin, which may cause higher immunogenicity to induce anti-drug antibodies (ADA). If the immunogenicity of a new candidate can be understood in the nonclinical phase, clinical studies will be safer and the success rate of development improved. Empirically, in vitro immunogenicity assays with human cells have proved to be sufficiently sensitive to nonhuman proteins, but not to human/humanized mAb. To detect the weaker immunogenicity of human-based mAb, a more sensitive biomarker for in vitro assays is needed. The in vitro study here developed a proliferation assay (TH cell assay) using flow cytometry analysis that can detect a slight increase in proliferating TH cells. Samples from 218 donors treated with a low-immunogenic drug (etanercept) were measured to determine a positive threshold level. With this threshold, positive donor percentages among PBMC after treatment with higher-immunogenicity mAb drugs were noted, that is, 39.5% with humanized anti-human A33 antibody (hA33), 27.3% with abciximab, 25.9% with adalimumab, and 14.8% with infliximab. Biotherapeutics with low immunogenicity yielded values of 0% for basiliximab and 3.7% for etanercept. These data showed a good comparability with previously reported incidences of clinical ADA with the evaluated drugs. Calculations based on the data here showed that a TH cell assay with 40 donors could provide statistically significant differences when comparing low- (etanercept) versus highly immunogenic mAb (except for infliximab). Based on the outcomes here, for screening purposes, a practical cutoff point of 3/20 positives with 20 donors was proposed to alert immunogenicity of mAb drug candidates.
Asunto(s)
Anticuerpos Monoclonales Humanizados/efectos adversos , Bioensayo/métodos , Productos Biológicos/efectos adversos , Inmunidad Celular/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/inmunología , Productos Biológicos/administración & dosificación , Productos Biológicos/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Evaluación Preclínica de Medicamentos/métodos , Etanercept/administración & dosificación , Etanercept/efectos adversos , Etanercept/inmunología , Voluntarios Sanos , Hemocianinas/administración & dosificación , Hemocianinas/inmunología , Humanos , Cultivo Primario de Células , Valores de Referencia , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/genética , Activación de Complemento/efectos de los fármacos , Complemento C5/antagonistas & inhibidores , Ingeniería de Proteínas/métodos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Afinidad de Anticuerpos , Activación de Complemento/inmunología , Complemento C5/inmunología , Complemento C5/aislamiento & purificación , Simulación por Computador , Descubrimiento de Drogas/métodos , Endosomas/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Concentración de Iones de Hidrógeno , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/inmunología , Macaca fascicularis , Ratones , Ratones Transgénicos , Mutagénesis , Receptores Fc/genética , Receptores Fc/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Factores de TiempoRESUMEN
Monoclonal antibodies are currently the most attractive therapeutic modality in a broad range of disease areas, including infectious diseases, autoimmune diseases, and oncology. Fc engineering is one attractive application to maximize the value or overcome the drawbacks of monoclonal antibodies for therapeutic use. With the Fc region, antibodies bind to several types of receptors, such as Fc gamma receptors, a complement receptor, and a neonatal Fc receptor. Through this interaction with the receptors, antibodies demonstrate unique functions, such as antibody-dependent cellular cytotoxicity, antibody- dependent cellular phagocytosis, complement dependent cytotoxicity, agonistic activity, and endosomal recycling. Fc engineering technology is conducted mainly to maximize the receptor-mediated functions of antibodies. Moreover, Fc engineering of the two heavy chains to facilitate heterodimerization is indispensable for generating IgG-like bispecific antibodies that are asymmetric. Fc engineering is also conducted to avoid the undesired properties, such as cytokine release and protease-mediated cleavage of the hinge region, of wild-type antibodies, as well as providing additional functions. Thus, Fc engineering technology is an attractive approach for maximizing the potency and convenience of therapeutic antibodies. This review will cover a variety of Fc engineering technologies that improve the functions of therapeutic antibodies.
RESUMEN
Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T(M)) of the C(H)2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T(M) of the C(H)2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.
Asunto(s)
Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Fragmentos Fc de Inmunoglobulinas/genética , Ingeniería de Proteínas/métodos , Receptores de IgG/metabolismo , Sustitución de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos , Línea Celular , Fucosa/metabolismo , Variación Genética , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Unión ProteicaRESUMEN
Since the first generation of humanized IgG1 antibodies reached the market in the late 1990s, IgG antibody molecules have been extensively engineered. The success of antibody therapeutics has introduced severe competition in developing novel therapeutic monoclonal antibodies, especially for promising or clinically validated targets. Such competition has led researchers to generate so-called second or third generation antibodies with clinical differentiation utilizing various engineering and optimization technologies. Parent IgG antibodies can be engineered to have improved antigen binding properties, effector functions, pharmacokinetics, pharmaceutical properties and safety issues. Although the primary role of the antibody variable region is to bind to the antigen, it is also the main source of antibody diversity and its sequence affects various properties important for developing antibody therapeutics. Here we review recent research activity in variable region engineering to generate superior antibody therapeutics.