RESUMEN
Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Arenavirus/inmunología , Fiebres Hemorrágicas Virales/inmunología , Polisacáridos/inmunología , Animales , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Humanos , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.
Asunto(s)
Interleucina-6/química , Interleucina-6/metabolismo , Receptores de Interleucina-6/química , Receptores de Interleucina-6/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Receptor gp130 de Citocinas/química , Receptor gp130 de Citocinas/metabolismo , Femenino , Prueba de Complementación Genética , Humanos , Interleucina-6/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Células 3T3 NIH , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Ratas , Receptores de Interleucina-6/genética , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell-targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell-targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell-targeted approach predicated on the induction of B cell anergy.
Asunto(s)
Enfermedades Autoinmunes/prevención & control , Linfocitos B/inmunología , Antígenos CD79/inmunología , Anergia Clonal/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Autoinmunidad/inmunología , Femenino , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones NoqueadosRESUMEN
Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.
Asunto(s)
Inflamación/inmunología , Macrófagos/inmunología , Receptores de IgG/inmunología , Receptor Toll-Like 4/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Células CHO , Línea Celular , Cricetulus , Dimerización , Femenino , Humanos , Inflamación/metabolismo , Macrófagos/citología , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de IgG/metabolismo , Receptor Toll-Like 4/química , Receptor Toll-Like 4/metabolismo , Células U937RESUMEN
In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.
Asunto(s)
Anticuerpos Biespecíficos , Antígeno B7-H1 , Antígeno CD47 , Fragmentos Fab de Inmunoglobulinas , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Humanos , Antígeno CD47/inmunología , Antígeno CD47/química , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Antígeno B7-H1/inmunología , Antígeno B7-H1/química , Antígeno B7-H1/antagonistas & inhibidores , Cristalografía por Rayos X , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/inmunología , Modelos MolecularesRESUMEN
BACKGROUND: T-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA). METHODS: We present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). RESULTS: NILK-2301 binds CD3É on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the "LALAPA" mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice. CONCLUSIONS: In summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.
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Adenocarcinoma , Anticuerpos Biespecíficos , Neoplasias Pancreáticas , Humanos , Animales , Ratones , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Línea Celular Tumoral , Inmunoterapia , Complejo CD3 , Antígeno Carcinoembrionario , Proteínas Ligadas a GPIRESUMEN
IL-6-mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6-mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.
Asunto(s)
Artritis Experimental/inmunología , Autoinmunidad/inmunología , Interleucina-6/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Artritis Experimental/metabolismo , Separación Celular , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica , Interleucina-6/metabolismo , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Receptores de Interleucina-6/inmunología , Receptores de Interleucina-6/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
BACKGROUND: CD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS: The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments. RESULTS: We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS: The study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).
RESUMEN
Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined. Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (FcγR) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely FcγR dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity. These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development.
Asunto(s)
Anticuerpos Antivirales/farmacología , Antivirales/farmacología , Anticuerpos Anti-VIH/farmacología , Receptores Fc/efectos de los fármacos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/inmunología , Epítopos/efectos de los fármacos , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , Inmunoglobulina G/efectos de los fármacos , Inmunoglobulina G/inmunología , Ratones Endogámicos C57BL , Receptores de IgG/efectos de los fármacos , Receptores de IgG/inmunologíaRESUMEN
Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.
Asunto(s)
Clonación Molecular/métodos , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Animales , Biotina/genética , Biotina/metabolismo , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Interleucinas/biosíntesis , Interleucinas/genética , Ratones , Ratas , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/genética , Proteínas Recombinantes/genéticaRESUMEN
CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 "antigen sink". A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8+ T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.
RESUMEN
Bispecific antibodies (bsAbs) are often composed of several polypeptide chains that have to be expressed adequately to enable optimal assembly and yield of the bsAb. κλ bodies are a bispecific format with a native IgG structure, composed of two different light chains that pair with a common heavy chain. Introduction of non-optimal codons into the sequence of a particular polypeptide is an effective strategy for down modulating its expression. Here we applied this strategy but restricted the modification of the codon content to the constant domain of one light chain. This approach facilitates parallel optimization of several bsAbs by using the same modified constant domains. Partial sequence de-optimization reduced expression of the targeted polypeptide. Stable cell pools could be isolated displaying increased bispecific antibody titers as well as changes in the abundance of undesired by-products that require elimination during downstream processing. Thus, modulating the relative expression of polypeptides can have a significant impact on bsAb titer and product related impurities; which are important factors for large scale manufacturing for clinical supply.
RESUMEN
When production of bispecific antibodies requires the co-expression and assembly of three or four polypeptide chains, low expression of one chain can significantly limit assembly and yield. κλ bodies, fully human bispecific antibodies with native IgG structure, are composed of a common heavy chain and two different light chains, one kappa and one lambda. No engineering is applied to force pairing of the chains, thus both monospecific and bispecific antibodies are secreted in the supernatant. In this context, stoichiometric expression of the two light chains allows for maximal assembly of the bispecific antibody. In this study, we selected a κλ body with suboptimal characteristics due to low kappa chain expression. Codon optimization to increase expression of the kappa chain did not improve bispecific yield. Surprisingly, progressive introduction of non-optimal codons into the sequence of the lambda chain resulted in lowering its expression for an optimal tuning of the relative distribution of monospecific and bispecific antibodies. This codon de-optimization led to doubling of the κλ body yield. These results indicate that assembly of different proteins into a recombinant complex is an interconnected process and that reducing the expression of one polypeptide can actually increase the overall yield.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Ingeniería de Proteínas/métodos , Animales , Codón , Humanos , Inmunoglobulina G/biosíntesis , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/genéticaRESUMEN
Fusion to an IgG Fc region is an established strategy to extend the half-life of therapeutic proteins. Most Fc fusion proteins, however, do not achieve the long half-life of IgGs. Based on findings that scFv-Fc fusion proteins exhibit a shorter half-life than the corresponding IgG molecules, we performed a comparative study of different antibody-derived Fc fusion proteins. We could confirm that fusion of single-chain Fv (scFv) and single-chain diabody (scDb) molecules to an Fc region yields in fusion proteins with substantially extended half-lives compared with the single-chain versions. However, even fusion proteins with a size similar to that of IgG, e.g., scDb-Fc, did not have a half-life as long as an IgG molecule. Binding to the neonatal Fc receptor (FcRn) under acidic and neutral conditions was similar for IgG and all Fc fusion proteins. However, we observed differences between IgG and the Fc fusion proteins for dissociation of FcRn-bound proteins induced by shifting from acidic to neutral pH, reflecting the physiological release mechanism, further supporting a contribution of the kinetics of pH-dependent release from FcRn to the pharmacokinetic properties of IgG and Fc fusion proteins.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulina G/farmacología , Proteínas Recombinantes de Fusión , Animales , Células CHO , Cricetinae , Cricetulus , Femenino , Células HEK293 , Humanos , Ratones , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Hu 15C1 is a potent anti-human Toll-like receptor 4 (TLR4) neutralizing antibody. To better understand the molecular basis of its biological activity, we used a multidisciplinary approach to generate an accurate model of the Hu 15C1-TLR4 complex. By combining site-directed mutagenesis, in vitro antibody evolution, affinity measurements and X-ray crystallography of Fab fragments, we identified key interactions across the Hu 15C1-TLR4 interface. These contact points were used as restraints to predict the structure of the Fab region of Hu 15C1 bound to TLR4 using computational molecular docking. This model was further evaluated and validated by additional site-directed mutagenesis studies. The predicted structure of the Hu 15C1-TLR4 complex indicates that the antibody antagonizes the receptor dimerization necessary for its activation. This study exemplifies how iterative cycles of antibody engineering can facilitate the discovery of components of antibody-target interactions.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Complejo Antígeno-Anticuerpo/ultraestructura , Sitios de Unión de Anticuerpos/inmunología , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Receptor Toll-Like 4/inmunología , Secuencia de Aminoácidos , Animales , Complejo Antígeno-Anticuerpo/inmunología , Células CHO , Línea Celular , Técnicas de Visualización de Superficie Celular , Simulación por Computador , Cricetinae , Cricetulus , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Alineación de Secuencia , Especificidad de la Especie , Resonancia por Plasmón de SuperficieRESUMEN
Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.
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Anticuerpos Biespecíficos/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Pruebas de Neutralización , Biblioteca de Péptidos , Linfocitos T/inmunologíaRESUMEN
The quality of the target antigen is very important in order to generate a good antibody, in particular when binding to a conformational epitope is desired. The use of mammalian cells for recombinant protein expression provides an efficient machinery for the correct folding and posttranslational modification of proteins. In this chapter, we describe a process to rapidly generate semi-stable human cell lines secreting a recombinant protein of interest into the culture medium. Simple disposable bioreactors that can be used in any standard cell culture laboratory enable the production of recombinant protein in the multi-milligram range. The protein can be readily purified from the culture supernatant by immobilized metal affinity chromatography. In addition, by inserting a tag recognized by a co-expressed biotin ligase, the protein can be biotinylated during the secretion process. This greatly facilitates the immobilization of the protein for assay development or for antibody isolation using in vitro selection technologies.
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Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Animales , Antígenos/genética , Antígenos/metabolismo , Reactores Biológicos , Cromatografía de Afinidad , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
Magnetic microspheres represent an interesting alternative to conventional chromatography resins in automated high-throughput protocols replacing centrifugation and filtration by magnetic separation. Some magnetic microspheres have unique features like high magnetite content and non-porous surface which allows them to migrate very fast in magnetic fields while binding target molecules with a low unspecific adsorption. Here, we describe the use of protein A or protein G-coated magnetic microspheres to purify quickly monoclonal antibodies from crude serum-free supernatants without the need of preliminary clarification or purification step. Using this method, multiple samples can be processed in parallel, a high level of purity can be obtained, and the purified IgG maintain their biological activity.
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Anticuerpos Monoclonales/aislamiento & purificación , Proteínas Bacterianas/química , Microesferas , Proteína Estafilocócica A/químicaRESUMEN
In order to treat Toll like receptor 4 (TLR4)-mediated diseases, we generated a potent antagonistic antibody directed against human TLR4, Hu 15C1. This antibody's potency can be modulated by engaging not only TLR4 but also Fcγ receptors (FcγR), a mechanism that is driven by avidity and not cell signaling. Here, using various formats of the antibody, we further dissect the relative contributions of the Fv and Fc portions of Hu 15C1, discovering that the relationship to potency of the different antibody arms is not linear. First, as could be anticipated, we observed that Hu 15C1 co-engages up to 3 receptors on the same plasma membrane, i.e., 2 TLR4 molecules (via its variable regions) and either FcγRI or FcγRIIA (via the Fc). The Kd of these interactions are in the nM range (3 nM of the Fv for TLR4 and 47 nM of the Fc for FcγRI). However, unexpectedly, neutralization experiments revealed that, due to the low level of cell surface TLR4 expression, the avidity afforded by engagement through 2 Fv arms was significantly limited. In contrast, the antibody's neutralization capacity increases by 3 logs when able to exploit Fc-FcγR interactions. Taken together, these results demonstrate an unforeseen level of contribution by FcγRs to an antibody's effectiveness when targeting a cell surface protein of relatively low abundance. These findings highlight an exploitable mechanism by which FcγR-bearing cells may be more powerfully targeted, envisioned to be broadly applicable to other reagents aimed at neutralizing cell surface targets on cells co-expressing FcγRs.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Afinidad de Anticuerpos/inmunología , Receptores de IgG/inmunología , Receptor Toll-Like 4/inmunología , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Células CHO , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Modelos Inmunológicos , Unión Proteica/inmunología , Receptores de IgG/metabolismo , Resonancia por Plasmón de Superficie , Receptor Toll-Like 4/metabolismo , Células U937RESUMEN
Plasmacytoid dendritic cells (pDC) rapidly and massively produce type I IFN and other inflammatory cytokines in response to foreign nucleic acids, thereby indirectly influencing T-cell responses. Moreover, antigen (Ag)-presenting pDCs directly regulate T-cell differentiation. Depending on the immune environment, pDCs exhibit either tolerogenic or immunogenic properties. Here, we show that CpG-activated pDCs promote efficient Th17 differentiation. Indeed, Th17 responses are defective in mice selectively lacking MHCII on pDCs upon antigenic challenge. Importantly, in those mice, the frequency of Th17 cells infiltrating solid tumors is impaired. As a result, the recruitment of infiltrating leukocytes in tumors, including tumor-specific cytotoxic T lymphocytes (CTL), is altered and results in increased tumor growth. Importantly, following immunization with tumor Ag and CpG-B, MHCII-restricted Ag presentation by pDCs promotes the differentiation of antitumor Th17 cells that induce intratumor CTL recruitment and subsequent regression of established tumors. Our results highlight a new role for Ag presenting activated pDCs in promoting the development of Th17 cells and impacting on antitumor immunity.