RESUMO
Therapeutic monoclonal antibodies (mAbs) represent a milestone in pharmacological development. Their superiority is based on the combination of high specificity, low toxicity, and long half-life that characterizes biologics. If biologics have Achilles' heel, it is their potential immunogenicity. To better understand the impact of the size of immune complexes of mAbs on anti-drug antibody (ADA) dependent adverse reactions in Macaca fascicularis, we developed an efficient high-throughput size exclusion chromatography- (SEC-) based methodology that enables analysis of the size, size distribution, and ratio of free and ADA-complexed mAb in serum allowing for assessment of formation and clearance of circulating ADA-mAb immune complexes (CIC).
Assuntos
Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Cromatografia em Gel/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Isoanticorpos/imunologia , Isoanticorpos/metabolismo , Macaca fascicularis , Ligação Proteica , Coloração e Rotulagem , Toxicologia/métodosRESUMO
Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.
Assuntos
Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Mutação/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Camelídeos Americanos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Interleucina-6/imunologia , Modelos Imunológicos , Modelos Moleculares , Proteínas Recombinantes/química , Alinhamento de SequênciaRESUMO
PURPOSE: Ovarian cancer is often diagnosed at an advanced stage with dissemination in the peritoneal cavity. Most patients achieve clinical remission after surgery and chemotherapy, but approximately 70% eventually experience recurrence, usually in the peritoneal cavity. To prevent recurrence, intraperitoneal (i.p.) targeted α therapy has been proposed as an adjuvant treatment for minimal residual disease after successful primary treatment. In the present study, we calculated absorbed and relative biological effect (RBE)-weighted (equivalent) doses in relevant normal tissues and estimated the effective dose associated with i.p. administration of (211)At-MX35 F(ab')2. METHODS AND MATERIALS: Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of (211)At-MX35 F(ab')2. Potassium perchlorate was given to block unwanted accumulation of (211)At in thyroid and other NIS-containing tissues. Mean absorbed doses to normal tissues were calculated from clinical data, including blood and i.p. fluid samples, urine, γ-camera images, and single-photon emission computed tomography/computed tomography images. Extrapolation of preclinical biodistribution data combined with clinical blood activity data allowed us to estimate absorbed doses in additional tissues. The equivalent dose was calculated using an RBE of 5 and the effective dose using the recommended weight factor of 20. All doses were normalized to the initial activity concentration of the infused therapy solution. RESULTS: The urinary bladder, thyroid, and kidneys (1.9, 1.8, and 1.7 mGy per MBq/L) received the 3 highest estimated absorbed doses. When the tissue-weighting factors were applied, the largest contributors to the effective dose were the lungs, stomach, and urinary bladder. Using 100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. CONCLUSION: Intraperitoneal (211)At-MX35 F(ab')2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective dose potentially corresponds to a risk of treatment-induced carcinogenesis, optimization may still be valuable.
Assuntos
Anticorpos Monoclonais/farmacocinética , Astato/farmacocinética , Imunoconjugados/farmacocinética , Fragmentos Fab das Imunoglobulinas/metabolismo , Neoplasias Ovarianas/radioterapia , Neoplasias Peritoneais/radioterapia , Radioimunoterapia/métodos , Partículas alfa/uso terapêutico , Elétrons/uso terapêutico , Feminino , Mucosa Gástrica/metabolismo , Humanos , Rim/diagnóstico por imagem , Rim/metabolismo , Pulmão/diagnóstico por imagem , Pulmão/metabolismo , Recidiva Local de Neoplasia , Neoplasia Residual , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/secundário , Terapia com Prótons , Dosagem Radioterapêutica , Eficiência Biológica Relativa , Medição de Risco , Estômago/diagnóstico por imagem , Glândula Tireoide/diagnóstico por imagem , Glândula Tireoide/metabolismo , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Bexiga Urinária/diagnóstico por imagem , Bexiga Urinária/metabolismoRESUMO
The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.
Assuntos
Acetilcolinesterase/metabolismo , Acetilcolinesterase/química , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Domínio Catalítico , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Clonagem Molecular , Regiões Determinantes de Complementaridade , Ativação Enzimática , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse HIV isolates. Importantly, this delivery strategy resulted in a rapid increase (i.e., in as little as 48 h) in Fab levels when compared with protein-based immunization. The active generation of functional Fab molecules in vivo has important conceptual and practical advantages over conventional ex vivo generation, purification and passive delivery of biologically active antibodies. Further study of this technique for the rapid generation and delivery of immunoglobulin and immunoglobulin like molecules is highly relevant and timely.
Assuntos
Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , HIV-1/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Plasmídeos/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Terapia Biológica/métodos , Feminino , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/genética , Infecções por HIV/terapia , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
The antitumour effect of thymidylate synthase inhibitors such as raltitrexed (RTX) may be reversed by salvage of thymidine (Thd). Since thymidine phosphorylase (TP) depletes Thd, the potential for tumour-selective depletion of Thd using antibody-mediated delivery of TP to tumours was investigated. In vitro studies demonstrated that 25 x 10(-3) units/ml TP depleted extracellular Thd (3 microM) and restored sensitivity to the growth inhibitory effects of RTX in Lovo and HT29 cell lines. Thymidine concentrations in xenograft tumours were inversely proportional to the activity of TP in the tumour, and the presence of a subcutaneous Lovo xenograft reduced plasma Thd concentrations from 0.92 +/- 0.07 to 0.37 +/- 0.04 microM. Intravenous administration of native TP enzyme depleted plasma Thd to 5 nM, but following rapid elimination of TP, plasma Thd returned to pretreatment values. There was no effect on tumour TP or Thd. Conjugation of TP to the A5B7 F(ab)2 antibody fragment, which targets carcinoembryonic antigen (CEA) expressed on colorectal cell-lines such as Lovo, did result in selective accumulation of TP in the tumour. However, there was no tumour-selective depletion of Thd and there did not appear to be any potential benefit of combining antibody-targeted TP with RTX.
Assuntos
Quinazolinas/uso terapêutico , Tiofenos/uso terapêutico , Timidina Fosforilase/metabolismo , Timidina/metabolismo , Timidilato Sintase/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Antígeno Carcinoembrionário/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Células HT29 , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Injeções Intravenosas , Camundongos , Camundongos Nus , Quinazolinas/farmacologia , Reprodutibilidade dos Testes , Tiofenos/farmacologia , Timidina Fosforilase/administração & dosagem , Timidina Fosforilase/imunologia , Timidilato Sintase/metabolismoRESUMO
Sepsis is a considerable health problem and a burden on the health care system. Endotoxin, or lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria, is responsible for more than 50% of the sepsis cases and is, therefore, a legitimate target for therapeutic approaches against sepsis. In this study, we selected and characterized a llama single-chain antibody fragment (VHH) directed to Neisseria meningitidis LPS. The VHH, designated VHH 5G, showed affinity to purified LPS as well as to LPS on the surfaces of the bacteria. Epitope mapping using a panel of N. meningitidis mutants revealed that VHH 5G recognizes an epitope in the inner core of LPS, and as expected, the VHH proved to have broad specificity for LPS from different bacteria. Furthermore, this VHH blocked binding of LPS to target cells of the immune system, resulting in the inhibition of LPS signaling in whole blood. Moreover, it was found to remove LPS efficiently from aqueous solutions, including serum. The selected anti-LPS VHH is a leading candidate for therapies against LPS-mediated sepsis.
Assuntos
Camelídeos Americanos/imunologia , Fragmentos Fab das Imunoglobulinas/fisiologia , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/imunologia , Transdução de Sinais/imunologia , Animais , Sítios de Ligação de Anticorpos , Ligação Competitiva/imunologia , Células Cultivadas , Escherichia coli/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Neisseria meningitidis/imunologia , Ligação Proteica/imunologiaRESUMO
Liposomes are potent drug delivery systems that protect the drug from degradation, improve its pharmacokinetic properties, and deliver a high drug payload. Although several liposomal formulations of chemotherapeutic drugs have been approved for cancer therapy, drug delivery by these liposomes is accomplished mainly by passive means, for example, by enhanced permeability and retention. Antibodies that generate target cell-specific immunoliposomes for improved and targeted drug delivery have been extensively tested. In the past five to ten years, tremendous progress has been made to improve the efficacy of both liposomes and of antibodies. With one immunoliposomal formulation currently being manufactured in a GMP-compliant process for preclinical studies, it is envisaged that various other immunoliposomes will follow.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Lipossomos/administração & dosagem , Lipossomos/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Anticorpos/química , Anticorpos/imunologia , Anticorpos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Lipossomos/metabolismo , Neoplasias/tratamento farmacológicoRESUMO
Engineering the permanent formation of a receptor-ligand complex has a number of potential applications in chemistry and biology, including targeted medical imaging and therapy. Starting from the crystal structure of the rare-earth-DOTA binding antibody 2D12.5 (Corneillie, T. M., Fisher, A. J., and Meares, C. F. (2003) J. Am. Chem. Soc. 125, 15039-15048), we used the site-directed incorporation of cysteine nucleophiles at the periphery of the antibody's binding site, paired with the chemical design of a weakly electrophilic ligand, to produce a receptor-ligand pair that associates efficiently and permanently. Protein residues proximal to the ligand's side chain were identified for engineering cysteine mutants. Fab fragments incorporating a cysteine at position 54, 55, or 56 of the heavy chain (complementarity determining region 2) were designed from the structure and then cloned, expressed in Drosophila S2 cells, and tested for reactivity with mildly electrophilic DOTA-yttrium ligands. All showed permanent binding activity, indicating that there is some tolerance for the location of the reactive mutant on the protein surface near the binding site. The G54C Fab mutant displayed the highest expression levels and permanent binding activity in initial experiments and was produced in high yield for further study. Upon examining the behavior of the G54C mutant with a small set of electrophilic ligands, differences in reactivity were observed which indicated that the substituents near the electrophilic atom can be important determinants of permanent binding. The G54C mutant permanently attaches to Y(3+) complexes of (S)-2-(4-acrylamidobenzyl)-DOTA with a half-time of approximately 13 min at 37 degrees C, making it potentially useful for in vivo pretargeting applications.
Assuntos
Cisteína/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Engenharia de Proteínas/métodos , Animais , Sítios de Ligação de Anticorpos/fisiologia , Linhagem Celular , Cisteína/genética , Drosophila , Fragmentos Fab das Imunoglobulinas/genética , Ligantes , Ligação Proteica/fisiologia , Transfecção/métodos , Ítrio/metabolismoRESUMO
We have obtained encouraging responses in recent Phase I studies evaluating (131)I-labeled human/murine chimeric 81C6 anti-tenascin monoclonal antibody (ch81C6) administered into surgically-created tumor resection cavities in brain tumor patients. However, because the blood clearance is slow, hematologic toxicity has been higher than seen with murine 81C6 (mu81C6). In the current study, a series of paired-label experiments were performed in athymic mice bearing subcutaneous D-245 MG human glioma xenografts to compare the biodistribution of the fragment ch81C6 F(ab')(2) labeled using Iodogen to a) intact ch81C6, b) mu81C6, and c) ch81C6 F(ab')(2) labeled using N-succinimidyl 3-[(131)I]iodobenzoate. Tumor retention of radioiodine activity for the F(ab')(2) fragment was comparable to that for intact ch81C6 for the first 24 h and to that for mu81C6 for the first 48 h; as expected, blood and other normal tissue levels declined faster for ch81C6 F(ab')(2.) Radiation dosimetry calculations suggest that (131)I-labeled ch81C6 F(ab')(2) may warrant further evaluation as a targeted radiotherapeutic for the treatment of brain tumors.
Assuntos
Anticorpos Monoclonais/farmacocinética , Glioma/metabolismo , Radioisótopos do Iodo/farmacocinética , Radioimunoterapia/métodos , Animais , Anticorpos Monoclonais/uso terapêutico , Carga Corporal (Radioterapia) , Avaliação Pré-Clínica de Medicamentos , Estudos de Viabilidade , Glioma/radioterapia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Radioisótopos do Iodo/uso terapêutico , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Especificidade de Órgãos , Doses de Radiação , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêutico , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
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.
Assuntos
Alérgenos/química , Anticorpos Monoclonais/química , Epitopos/química , Imunoglobulina E/química , Fragmentos Fab das Imunoglobulinas/química , Proteínas de Plantas/química , Poaceae/imunologia , Pólen/química , Alérgenos/imunologia , Alérgenos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/genética , Basófilos/metabolismo , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Dicroísmo Circular , Reações Cruzadas , Mapeamento de Epitopos , Epitopos/imunologia , Epitopos/metabolismo , Liberação de Histamina/imunologia , Humanos , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Poaceae/química , Pólen/imunologia , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Zea mays/química , Zea mays/imunologiaRESUMO
The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcepsilonRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 A resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients' IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 A2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients' serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.
Assuntos
Alérgenos/metabolismo , Anticorpos Monoclonais/metabolismo , Epitopos Imunodominantes/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Proteínas de Plantas/metabolismo , Pólen/imunologia , Rinite Alérgica Sazonal/metabolismo , Alérgenos/química , Alérgenos/imunologia , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Antígenos de Plantas , Simulação por Computador , Reações Cruzadas , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Fragmentos Fab das Imunoglobulinas/química , Camundongos , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Rinite Alérgica Sazonal/imunologia , Rosales , ÁrvoresRESUMO
The immunological and pharmacokinetic properties of a new, further purified, pasteurized preparation of equine F(ab')2 (VIPERFAV) against Vipera aspis, Vipera berus, and Vipera ammodytes venom were compared with the current equine F(ab')2 preparation (IPSER Europe). Affinity constants of the V. aspis-specific F(ab')2 were determined using biosensor technology and found to be in the range of 10(8) M-1 for the four antigenic fractions of V. aspis toxins and for both F(ab')2 preparations. The improvement of 51% in the specific activity (LD50 mg-1) of the new F(ab')2 was in close agreement with the 1.8-fold increase in the immunoreactive fraction of the new preparation. In vivo investigations of venom immunocomplexation by F(ab')2 in rabbits confirmed the ability of F(ab')2 to neutralize and redistribute toxin venom. Infusion of a stoichiometric molar ratio (i.e., 1 mg kg-1) of the new antivenom induced a 2.3-fold elevation of the plasma venom concentration with a Tmax observed 8 h after F(ab')2 administration and a decline in the terminal half-life from 31.92 +/- 4.49 h to 16.73 +/- 4.34 h, in contrast, for the venom alone. The area under the curve was 1.4-fold greater in the VIPERFAV group than in the IPSER Europe group during the post-F(ab')2 infusion period. Increasing the F(ab')2 dose to 3 mg kg-1 increased by 27% the percent of venom bound to F(ab')2. Finally, the greater the venom distribution, the smaller and less pronounced the plasma redistribution. These results demonstrate that the purification and pasteurization steps involved in the preparation of the new F(ab')2 have no deleterious influence on F(ab')2 affinity but, on the contrary, improve the protective efficacy. Alteration of viper venom kinetics by specific F(ab')2 antivenom was also shown to be dependent on the interval between of F(ab')2 administration and venom bite and on the specific F(ab')2 dose administered.
Assuntos
Antivenenos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Venenos de Víboras/imunologia , Animais , Antivenenos/metabolismo , Antivenenos/farmacologia , Área Sob a Curva , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Infusões Intravenosas , Dose Letal Mediana , Masculino , Camundongos , Coelhos , Venenos de Víboras/administração & dosagem , Venenos de Víboras/antagonistas & inibidores , Venenos de Víboras/farmacocinéticaRESUMO
Bet v 1 and homologous proteins represent major cross-reactive allergens for more than 95% of tree pollen-, fruit-, and vegetable-allergic individuals. To study the interaction of Bet v 1 and the immune system, we characterized a Bet v 1-specific mAb, Bip 1. Soluble rBip 1 Fabs were expressed in Escherichia coli and purified by affinity chromatography using immobilized Bet v 1. Bip 1 Fabs displayed a cross-reactivity to homologous allergens comparable with that of IgE Abs from allergic patients. Preincubation of Bet v 1 with Bip 1 led to an up to fivefold increase of allergic patients' IgE binding to Bet v 1. This enhancement in IgE binding may be interpreted as stabilization of a Bet v 1 state, in which certain IgE epitopes are better applicable. It also shows that allergic patients possess IgE Abs directed against different Bet v 1 conformations. The modulation of Ab binding to a given Ag by other Abs was observed also for human Bet v 1-specific IgG Abs, and may represent a novel mechanism for the regulation of specific humoral immune responses in a complex network.
Assuntos
Alérgenos/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Imunoglobulina E/metabolismo , Proteínas de Plantas/imunologia , Pólen/imunologia , Alérgenos/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Antígenos de Plantas , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , DNA Complementar/genética , Escherichia coli/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Técnicas In Vitro , Dados de Sequência Molecular , Estrutura Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Rinite Alérgica Sazonal/imunologia , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
Reduction of radioactivity levels in nontarget tissues such as the liver and kidney constitutes a problem to be resolved in diagnostic and therapeutic applications of radiolabeled monoclonal antibodies (mAbs). A new radioiodination reagent with an ester bond to liberate m-iodohippuric acid from covalently conjugated proteins, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (MIH), was recently developed. MIH liberated m-iodohippuric acid from galactosylneoglycoalbumin in murine liver, and the radiometabolite was rapidly eliminated from the liver into urine as an intact structure. In this study, intact IgG and Fab fragment of a mAb against osteogenic sarcoma were radioiodinated with MIH to further assess the applicability of MIH to radioimmunoimaging and therapy. For comparison, a mAb radioiodinated with N-succinimidyl iodobenzoate (SIB) and indium-111 (111In)-labeled mAbs with diethylenetriaminepentaacetic dianhydride (cDTPA) or 1-[4-[(5-maleimidopentyl)amino]benzyl]-ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) were used. Size-exclusion HPLC analysis and cell binding assays indicated the preservation of both structure and antigen binding affinity of radioiodinated MIH-OST7 (IgG). In biodistribution studies in mice, [125I]MIH-OST7 (IgG) showed faster systemic clearance of radioactivity after 24 h postinjection than did [131I]SIB- and [111In]EMCS-Bz-EDTA-OST7 (IgG). [125I]MIH-OST7 (IgG) also exhibited much lower radioactivity levels in nontarget tissues such as the liver and kidney, with higher radioactivity levels in the blood up to 72 h postinjection when compared with [111In]cDTPA-OST7 (IgG). Radioactivity excreted from the mice was found in the urine as m-iodohippuric acid, following administration of [125I]MIH-OST7 (IgG). In athymic mice bearing osteogenic sarcoma, [131I]MIH-OST7 (IgG) indicated higher tumor-to-nontarget ratios of radioactivity at both 24 and 48 h postinjection than [125I]SIB-OST7 (IgG). Although both radioiodinated OST7s showed similar radioactivity levels in the target at 24 h postinjection, a small but significant decrease in the target radioactivity level was observed with [131I]MIH-OST7 (IgG) at 48 h postinjection. In addition, [131I]MIH-OST7 (Fab) showed very rapid cleavage of the ester bond both in vivo and in vitro. These findings indicated that while MIH may be a useful reagent for radioimmunoimaging using IgG, mAb, its application to smaller molecular weight mAbs and radioimmunotherapy would be hindered due to the labile characteristics of the ester bond in plasma. Thus, while the present study reinforced the usefulness of metabolizable linkages for reducing nontarget radioactivity levels, a development of plasma-stable metabolizable linkages is also warranted for radioimmunotherapy and for smaller molecular weight polypeptides.
Assuntos
Anticorpos/química , Hipuratos , Indicadores e Reagentes , Radioimunodetecção/métodos , Compostos de Trialquitina , Animais , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Radioisótopos do Iodo , Camundongos , Ácido Pentético , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Fab fragments from two different monoclonal antibodies (BH151 and HC45) which bind to the same antigenic region of the influenza hemagglutinin were crystallized as complexes with the hemagglutinin. The complexes crystallize in PEG 600, pH 6.0, and PEG 2000, pH 8.5, respectively. Both crystals belong to space group P321, with very similar unit cell dimensions.
Assuntos
Anticorpos Monoclonais/química , Cristalografia por Raios X , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Orthomyxoviridae/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Bromelaínas/química , Bromelaínas/metabolismo , Cristalização , Eletroforese em Gel de Poliacrilamida , Hemaglutininas Virais/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismoRESUMO
A 22-year-old man presented to our emergency department after an intentional overdose of a homemade foxglove extract. Clinical symptoms with symptomatic bradyarrhythmia and ECG changes were consistent with cardiac glycoside poisoning. Treatment with digoxin-specific Fab fragments resulted in transient clinical and ECG improvement. Serum immunoassay demonstrated a digitoxin-like glycoside. The serum levels showed no evidence of altered elimination or distribution with Fab therapy despite temporary improvements in the clinical course. The use of Fab did not result in a shortened clinical course in this episode of foxglove poisoning, as one would expect in the setting of commercial glycoside product poisoning.
Assuntos
Digitalis , Digoxina/imunologia , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Plantas Medicinais , Plantas Tóxicas , Intoxicação/terapia , Adulto , Digitoxina/sangue , Meia-Vida , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Masculino , Extratos Vegetais/intoxicação , Tentativa de SuicídioRESUMO
The ability to deliver radiation selectively to lymphohematopoietic tissues may have utility in conditions treated by myeloablative regimens followed by bone marrow transplantation. Since the CD45 antigen is the most broadly expressed of hematopoietic antigens, we examined the biodistribution of radiolabeled anti-CD45 monoclonal antibodies in normal mice. Trace 125I or 131I-labeled monoclonal antibodies 30G12 (rat IgG2a), 30F11 (rat IgG2b), and F(ab')2 fragments of 30F11 were injected i.v. at doses of 5 to 1000 micrograms. For both intact antibodies, a higher percentage of injected dose/g (% ID/g tissue) in blood was achieved with higher antibody doses. However, as the dose of antibody was increased, the % ID/g in the target organs of spleen, marrow, and lymph nodes decreased. At doses between 5 and 10-micrograms, % ID/g in these tissues exceeded that in lung, the normal organ with the highest concentration of radiolabel. In contrast, thymus was the only hematopoietic organ in which the % ID/g increased with increasing antibody dose, although at high dose the % ID/g was still far below that achieved in the other hematopoietic organs. Antibody 30F11 F(ab')2 fragments were cleared more quickly than intact antibody from blood and from both target and nontarget organs, although the relationship between increasing antibody dose and decreasing % ID/g in spleen, marrow, and lymph nodes was observed. The time-activity curves for each dose of antibody were used to calculate estimates of radiation absorbed dose to each organ. At the 10-micrograms dose of 30G12, the spleen was estimated to receive a radiation dose that was 13 times more than lung, the lymph nodes 3 to 4 times more, and the bone marrow 3 times more than lung. For each antibody fragment dose, the radiation absorbed dose per MBq 131I administered was lower because the residence times of the fragments were shorter than those of the intact antibody. Thus these estimates suggested that the best "therapeutic ratio" of radiation delivered to target organ as compared to lung was achieved with lower doses of intact antibody. We have demonstrated that radiolabeled anti-CD45 monoclonal antibodies can deliver radiation to lymphohematopoietic tissues with relative selectivity and that the relative uptake and retention in different hematolymphoid tissues change with increasing antibody dose.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos CD/imunologia , Antígenos de Histocompatibilidade/imunologia , Radioisótopos do Iodo/farmacocinética , Tecido Linfoide/metabolismo , Animais , Medula Óssea/metabolismo , Medula Óssea/efeitos da radiação , Feminino , Fragmentos Fab das Imunoglobulinas/metabolismo , Radioisótopos do Iodo/sangue , Antígenos Comuns de Leucócito , Leucócitos/metabolismo , Leucócitos/efeitos da radiação , Linfonodos/metabolismo , Linfonodos/efeitos da radiação , Tecido Linfoide/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos AKR , Doses de Radiação , Baço/metabolismo , Baço/efeitos da radiação , Timo/metabolismo , Timo/efeitos da radiação , Distribuição TecidualRESUMO
The complex of bromelain agglutinators and their homologous Fab fragments is dissociated by gel chromatography under certain conditions. When albumin is present as a source of thiol groups, Fab fragments previously treated with N-ethylmaleimide (NEM) will dissociate from the agglutinators (fluid phase). If anti-Rh Fab fragments are bound to Rh-positive erythrocytes, the agglutinates are not dissociated by thiols (cellular phase). Prior to erythrocyte sensitization, the agglutinator site on Fab fragments can be blocked by thiol-disulfide exchange. Once the Fab fragments are coated on erythrocytes, the agglutinator site is more readily available than it was prior to sensitization, as evidenced by inhibition with 0.01 M NEM. The differences between the bonding characteristics of the fluid phase and the cellular phase and the influence of mercaptoalbumin on the agglutinator-Fab complex suggest that the agglutinators are not antibodies.