RESUMEN
Despite the reasonably long half-life of immunoglogulin G (IgGs), market pressure for higher patient convenience while conserving efficacy continues to drive IgG half-life improvement. IgG half-life is dependent on the neonatal Fc receptor (FcRn), which among other functions, protects IgG from catabolism. FcRn binds the Fc domain of IgG at an acidic pH ensuring that endocytosed IgG will not be degraded in lysosomal compartments and will then be released into the bloodstream. Consistent with this mechanism of action, several Fc-engineered IgG with increased FcRn affinity and conserved pH dependency were designed and resulted in longer half-life in vivo in human FcRn-transgenic mice (hFcRn), cynomolgus monkeys, and recently in healthy humans. These IgG variants were usually obtained by in silico approaches or directed mutagenesis in the FcRn-binding site. Using random mutagenesis, combined with a pH-dependent phage display selection process, we isolated IgG variants with improved FcRn-binding, which exhibited longer in vivo half-life in hFcRn mice. Interestingly, many mutations enhancing Fc/FcRn interaction were located at a distance from the FcRn-binding site validating our random molecular approach. Directed mutagenesis was then applied to generate new variants to further characterize our IgG variants and the effect of the mutations selected. Since these mutations are distributed over the whole Fc sequence, binding to other Fc effectors, such as complement C1q and FcγRs, was dramatically modified, even by mutations distant from these effectors' binding sites. Hence, we obtained numerous IgG variants with increased FcRn-binding and different binding patterns to other Fc effectors, including variants without any effector function, providing distinct "fit-for-purpose" Fc molecules. We therefore provide evidence that half-life and effector functions should be optimized simultaneously as mutations can have unexpected effects on all Fc receptors that are critical for IgG therapeutic efficacy.
RESUMEN
Ovarian cancer has the highest mortality rate among gynecologic malignancies. The monoclonal antibody 12G4 specifically recognizes the human Müllerian inhibiting substance type II receptor (MISRII) that is strongly expressed in human granulosa cell tumors (GCT) and in the majority of human epithelial ovarian cancers (EOC). To determine whether MISRII represents an attractive target for antibody-based tumor therapy, we first confirmed by immunohistochemistry with 12G4 its expression in all tested GCT samples (4/4) and all, but one, EOC human tissue specimens (13/14). We then demonstrated in vitro the internalization of 12G4 in MISRII(high)COV434 cells after binding to MISRII and its ability to increase the apoptosis rate (FACS, DNA fragmentation) in MISRII(high)COV434 (GCT) and MISRII(medium)NIH-OVCAR-3 (EOC) cells that express different levels of MISRII. A standard (51)Cr release assay showed that 12G4 mediates antibody-dependent cell-meditated cytotoxicity. Finally, in vivo assessment of 12G4 anti-tumor effects showed a significant reduction of tumor growth and an increase of the median survival time in mice xenografted with MISRII(high)COV434 or MISRII(medium)NIH-OVCAR-3 cells and treated with 12G4 in comparison to controls treated with an irrelevant antibody. Altogether, our data indicate that MISRII is a new promising target for the control of ovarian GCTs and EOCs. A humanized version of the 12G4 antibody, named 3C23K, is in development for the targeted therapy of MISRII-positive gynecologic cancers.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Neoplasias Ováricas/inmunología , Receptores de Péptidos/inmunología , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Femenino , Tumor de Células de la Granulosa/inmunología , Tumor de Células de la Granulosa/metabolismo , Tumor de Células de la Granulosa/terapia , Humanos , Inmunohistoquímica , Inmunoterapia/métodos , Ratones Desnudos , Microscopía Fluorescente , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/terapia , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/terapia , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen™) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling(®) platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/metabolismo , Inmunoterapia/métodos , Ingeniería de Proteínas/métodos , Receptores Fc/metabolismo , Animales , Anticuerpos Monoclonales/genética , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Técnicas de Visualización de Superficie Celular , Citotoxicidad Inmunológica/genética , Glicosilación , Semivida , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoglobulina G/genética , Inmunoterapia/tendencias , Ratones , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Mutación/genética , Receptores Fc/genética , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/inmunología , Receptores de IgG/metabolismoRESUMEN
The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Reacciones Cruzadas/inmunología , Mapeo Epitopo , Epítopos/inmunología , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/química , Afinidad de Anticuerpos , Antígenos Bacterianos/química , Toxinas Bacterianas/química , Biología Computacional , Edema/inmunología , Mapeo Epitopo/métodos , Epítopos/química , Inmunoglobulina G/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Anticuerpos de Cadena Única/inmunologíaRESUMEN
BACKGROUND: So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate. RESULTS: A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells. CONCLUSIONS: Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology.
Asunto(s)
Anticuerpos Monoclonales , Neoplasias de la Mama/inmunología , Mucina-1/inmunología , Animales , Línea Celular Tumoral , Epítopos , Femenino , Humanos , Ratones , Biblioteca de Péptidos , Anticuerpos de Cadena Única , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo-immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo-immunotherapy. We generated a chimeric anti-CD20 monoclonal antibody (mAb), EMAB-6, which has a low fucose content. Apoptosis and complement activities for EMAB-6 were similar to those seen for rituximab. By contrast, EMAB-6 mAb showed improved Fcgamma receptor IIIA (FcgammaRIIIA)/CD16 binding and FcgammaRIIIA-dependent effector functions. It induced a higher in vitro antibody-dependent cellular cytotoxicity against CLL cells and a higher FcgammaRIIIA-mediated interleukin-2 production by FcgammaRIIIA(+) Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB-6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB-6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.
Asunto(s)
Antígenos CD20/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Receptores de IgG/inmunología , Anciano , Anciano de 80 o más Años , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Apoptosis/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Interleucina-2/biosíntesis , Células Asesinas Naturales/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Masculino , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
Many tumors express CD95L (CD178, FasL, APO-1L) and may thus kill tumor-infiltrating lymphocytes, a phenomenon called tumor counterattack. However, presently it is not clear whether tumor counterattack is a relevant immune escape mechanism. To characterize the effect of CD95L expression of tumor cells on tumor-specific T cells, we established an in vitro system with TCR tg T cells and a model tumor antigen. Preactivated antitumor T cells were able to kill CD95L(-) and CD95L(+) tumor cells. CD95L(+) tumor cells killed activated T cells in vitro and inhibited the expansion of cytotoxic antitumor T cells in mixed lymphocyte tumor reactions. In vivo CD95L expression led to delayed tumor growth or complete tumor rejection. Neutrophils were not responsible for the delayed growth of the CD95L(+) tumors tested. In mice with neutrophils deficient for important cytotoxicity mechanisms (p47phox(-/-) or iNOS(-/-) mice), CD95L(+) tumors grew similarly as in wild-type mice. Incidence and growth rate of CD95L(+) tumors in mice injected with a neutrophil-depleting or an isotype control antibody was the same. In CD95-deficient lpr mice, tumor growth was not altered as compared to wild-type mice. Taken together, CD95L mediated tumor counterattack in vitro, but led to neutrophil-independent tumor rejection in vivo.
Asunto(s)
Citotoxicidad Inmunológica , Glicoproteínas de Membrana/inmunología , Neoplasias Experimentales/inmunología , Linfocitos T Citotóxicos/inmunología , Receptor fas/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Proteína Ligando Fas , Femenino , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , NADPH Oxidasas , Neutrófilos/metabolismo , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa de Tipo II , Fosfoproteínas/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Many tumors express the death ligand CD95L (CD178, APO-1L, FasL) and can kill activated T cells in vitro. This may enable the tumor cells to suppress anti-tumor immune responses, a phenomenon called "tumor counterattack". Preliminary evidence of tumor counterattack in human tumors exists. However, CD95L-expressing tumors are rapidly rejected in mice. In order to clarify this controversial situation we investigated whether the level or the time point of CD95L expression might be critical factors determining tumor counterattack versus tumor rejection. We generated CD95-resistant tumor cell lines expressing different levels of CD95L (LKC-CD95L). In nude mice the CD95L expression level had no influence on the growth of the CD95L(+) tumors. In contrast, a CD95L(-) control tumor cell line (LKC) grew much faster. In addition, we generated a CD95-resistant cell line in which CD95L was induced via the tet system (LKCR-tetCD95L). Induction of CD95Lin established tumors in nude and NOD/SCID mice led to rapid rejection of the tumors. Induction of lower CD95L expression levels delayed tumor rejection only marginally. These results demonstrate that rejection of CD95L-expressing tumors in mice is not a result of overexpression and does not depend on the presence of CD95L at the onset of tumor progression.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Trasplante de Neoplasias/inmunología , Neoplasias Experimentales/inmunología , Animales , Apoptosis , Citotoxicidad Inmunológica , Proteína Ligando Fas , Femenino , Rechazo de Injerto , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/patología , Células Tumorales CultivadasRESUMEN
Once again, genomics is about to change drug development. Following its major impact on target discovery and assay development, which increased the number of compounds at early stages of the process, genomics is now zeroing in on the prediction of potential toxicological problems of compounds. Toxicogenomics is the analysis of toxicological processes at the transcriptome level of a target organ or cell. By simultaneously monitoring the effect of a compound on the transcription levels of hundreds to thousands of genes, toxicogenomics can provide an enormous amount of data. This data bears information on the way in which compounds act at the molecular level, reaching far beyond the mere conclusion of whether or not a particular toxicological outcome is elicited. By compiling transcription profiles for well-known toxicants, we are beginning to learn how to analyze this novel type of data in the context of mechanistic and predictive toxicology.