RESUMEN
Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.
Asunto(s)
Alérgenos/química , Anticuerpos Monoclonales/química , Epítopos/química , Inmunoglobulina E/química , Fragmentos Fab de Inmunoglobulinas/química , Proteínas de Plantas/química , Poaceae/inmunología , Polen/química , Alérgenos/inmunología , Alérgenos/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos/genética , Basófilos/metabolismo , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Dicroismo Circular , Reacciones Cruzadas , Mapeo Epitopo , Epítopos/inmunología , Epítopos/metabolismo , Liberación de Histamina/inmunología , Humanos , Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/genética , Inmunoglobulina E/metabolismo , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Poaceae/química , Polen/inmunología , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Zea mays/química , Zea mays/inmunologíaRESUMEN
BACKGROUND: The monoclonal antibody (mAb) TP-3 binds selectively to human and canine osteosarcoma (OS) cells and is therefore a potential candidate for use as a targeting agent in radioimmunoimaging and therapy of OS metastases. However, intact murine mAbs have several drawbacks such as large size, delayed blood clearance and high immunogenicity, all of which can be overcome by genetic engineering. OBJECTIVES: To construct and express bivalent and multivalent TP-3 scFv fragments from the mammalian expression vector, pLNO. This vector has unique restriction sites for simple cassette cloning of any individual variable (V) and constant (C) genes and has previously been used for expression of intact chimeric TP-3 mAbs and Fab fragments. Furthermore, it is also suitable for expression of any modified V region, such as a scFv fragment, fused to any modified C region or to non-immunoglobulin protein sequences. STUDY DESIGN: Six different constructs were made; three scFv-CH3 fragments that differed in the design of linker between the scFv fragment and the IgG CH3 domain. These constructs were also made with the IgM secretory tailpiece (microtp) attached to the C terminus. RESULTS: All constructs were secreted as bivalent antibody fragments with a molecular weight of about 100 kDa. A band corresponding to a dimer appeared in all the supernatants from TP-3 scFv-CH3 producing cells, whether microtp was present or not, whereas higher orders of multimers were not seen. However, pulse chase analyses of the cells revealed that a small fraction of higher order polymers was formed from genes including the fragment encoding microtp and that microtp conferred retention both to monomers and intermediate polymers. The recombinant TP-3 antibody fragments were shown to bind human OS cells. CONCLUSION: Recombinant mAb fragments can be designed and cloned into the mammalian expression vector, pLNO. This vector is flexible in the sense that the genes encoding such fragments can be expressed from either cDNA or from genomic DNA. A microtp attached to the CH3 domain in these fragments was sufficient to drive polymerization, however inefficiently and intracellular retention of both monomers and intermediate polymers was observed.
Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Inmunoglobulina M/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Componente Secretorio/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Neoplasias Óseas/inmunología , Dimerización , Perros , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Genes de Inmunoglobulinas , Ingeniería Genética , Vectores Genéticos , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Osteosarcoma/inmunología , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
We have developed new cassette expression vectors for the cloning of any intact V-region gene followed by any C-region gene. Both the heavy-and light chain vectors harbor a strong hCMV promoter, restriction site cassettes for cloning of both V- and C-region genes, transcription termination signals, fl-ori for single stranded DNA (ssDNA) synthesis, selection marker for Neomycin and SV40 ori for transient expression. The vectors accept VH and VL chain genes obtained by RT-PCR. Reamplification of the V genes is then performed with a new set of primers which are designed specifically for each individual V gene. Cloning into the vectors is aided by restriction sites located just outside the V-gene coding region, thus keeping the V-genes intact. The vectors also contain cloning sites for the exchange of genomic C-genes so that the resulting Ig genes may code for complete antibodies, antibody fragments or fusion proteins. A simple subcloning step permits the expression of both heavy and light chain genes from one single vector, thus avoiding co-transfection of the two vectors. The usefulness of the vectors was confirmed by construction of mouse-human chimeric antibodies. The V-genes were derived from a hybridoma cell line, TP-3, and was combined with human C kappa, C gamma 3 and C gamma 1 genes as well as with CH1 gamma 3. High yields of recombinant antibody products in NSO cells were obtained. Transient expression was also demonstrated.
Asunto(s)
Vectores Genéticos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Animales , Anticuerpos Monoclonales/inmunología , Células COS , Clonación Molecular , Expresión Génica , Humanos , Hibridomas , Fragmentos Fab de Inmunoglobulinas/inmunología , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Pentameric IgM and dimeric IgA both contain disulfide bonds between cysteines located in the secretory tailpieces of the heavy chains. To compare the influences of the mu and alpha tailpieces on the polymeric structure, we have replaced amino acids in the tailpiece of the human mu-chain with amino acids found in the corresponding positions in the human alpha tailpiece. We show that an IgM with an alpha tailpiece (IgM L561H, Y562V, L566V, S569A, D570E, T571V, and A572D) as well as IgM L561H, Y562V, and IgM A572D have a size distribution similar to that of wild-type IgM. However, one IgM mutant with a mu/alpha hybrid tailpiece (IgM L566V, S569A, D570E, T571V, and A572D) is secreted as a mixture of mainly hexamers, pentamers, tetramers, and dimers. The tetramers and dimers are specifically formed and secreted at the expense of pentamers and hexamers; no alterations in polymerization or secretion rates were observed. We have also incorporated the mu, alpha, and hybrid mu/alpha tailpieces to a human IgG3 or IgGL309C mutant. The IgG-tailpiece mutants are poorly secreted, but the secreted fractions contain multimeric molecules. Each of the mutants that contain both the L309C mutation and a secretory tailpiece forms mainly hexamers; however, small differences in polymer distribution exist for the different tailpieces. Comparison of the influence of different tailpieces on IgM and IgG polymeric structures suggests that the function of a specific tailpiece is dependent on other parts of the heavy chain, which can vary for different isotypes.
Asunto(s)
Inmunoglobulina A Secretora/biosíntesis , Inmunoglobulina A Secretora/farmacología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/metabolismo , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/farmacología , Componente Secretorio/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biopolímeros/biosíntesis , Biopolímeros/metabolismo , Línea Celular , Clonación Molecular , Humanos , Inmunoglobulina A Secretora/genética , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/genética , Ratones , Datos de Secuencia Molecular , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , Componente Secretorio/genéticaRESUMEN
Fc gamma receptor (Fc gamma R) phagocytosis and respiratory burst were induced by chimeric mouse-human anti-(4-hydroxy-5-iodo-3-nitrophenyl) acetyl IgG3 antibodies with mutations in hinge and/or in CH1 region. IgG3 mutants with different hinge length ranging from 47 to 0 amino acids, an IgG3 molecule with an artificial hinge of just one cysteine residue (HM-1), and two hybrid IgG3 molecules with IgG4 hinge or IgG4 CH1-hinge were tested. Using the monocytic cell line U937 as effector cells, the mutated IgG3 molecules were very similar, revealing high activity, while the IgG3/IgG4 hybrids revealed a slightly reduced activity. However, the hingeless (0-h) mutant was negative, except after interferon-gamma stimulation when it became slightly positive. Interestingly, HM-1 was as active as the IgG3 mutants. With polymorphonuclear leucocytes (PMN) as effector cells we obtained some day-to-day variations, but all the IgG3 mutants were highly active, with the two shortest hinge mutants somewhat less active. The IgG3/IgG4 hybrid molecules revealed an intermediate activity, while IgG4 wild-type and the 0-h mutant were negative. However, the HM-1 molecule revealed an activity similar to that of the IgG3 mutants. The phagocytic activity of U937 was inhibited by monomeric IgG, indicating the importance of Fc gamma RI. In contrast, with PMN both blockage of Fc gamma RII and cleavage of Fc gamma RIII were required to significantly reduce the phagocytosis and respiratory burst, thus showing that both receptors contribute to the effect. These results demonstrate that the extended IgG3 hinge region is not necessary for a high phagocytic activity and that the major structural importance of the hinge is to connect the two heavy chains in this region.
Asunto(s)
Inmunoglobulina G/química , Cadenas gamma de Inmunoglobulina/química , Neutrófilos/inmunología , Fagocitosis , Receptores de IgG/fisiología , Estallido Respiratorio , Disulfuros , Humanos , Técnicas In Vitro , Proteínas Recombinantes de Fusión/química , Transducción de Señal , Relación Estructura-ActividadRESUMEN
A matched set of chimeric mouse-human NP-antibodies were studied for the capacity to induce cell mediated cytotoxicity (ADCC) by normal peripheral blood NK/K cells. The target cells were sheep red blood cells (SRBC) sensitized with the haptens NP or NIP. All four IgG subclasses and several IgG3 variants with altered hinge were tested for ADCC activity. The hierarchy of the ADCC capacity among the subclasses was found to be IgG3 greater than IgG1 greater than IgG4 greater than IgG2. The superiority of IgG3 was only revealed at low effector cell:target cell ratio. The ADCC activity was for the most part unaltered by shortening the hinge region of IgG3 from 62 to 15 amino acids. Also, when the hinge region of IgG3 was mutated to become identical to that of IgG4, the ADCC activity was mainly unchanged. However, an IgG3 variant with deletion of all four hinge exons showed a depressed ADCC activity compared to the wild type. The IgG subclass pattern of complement-mediated lysis (CML) and ADCC is different and the capacity to induce CML and ADCC is changed differently by hinge region modification. Thus CML and ADCC have different structural requirements in the Fc region of IgG.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Inmunoglobulina G/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos/inmunología , Quimera , Clonación Molecular , Pruebas Inmunológicas de Citotoxicidad , Relación Dosis-Respuesta Inmunológica , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Técnicas In Vitro , Células Asesinas Naturales/inmunología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Ácido NucleicoRESUMEN
We have altered the amino acid sequence of the hinge and the first constant domain (CH1) of mouse/human chimeric IgG3 antibodies by site-directed mutagenesis, so as to make the sequences identical to those of IgG4. All the mutant antibodies with altered hinge region were more active in complement activation and complement-mediated lysis than native IgG3. The mutations in CH1, however, did not alter the activity. This demonstrates the importance of the hinge region in modulating this effector function. The results show that the primary structure of neither CH1 nor the hinge of IgG4 is responsible for the lack of complement activation shown by this subclass.