RESUMO
The objective of this work is to compare the three-dimensional structures of "humanized" and mouse-human chimeric forms of a murine monoclonal antibody elicited against human gamma-interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen-binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8,000 by nearly identical microseeding procedures. Their structures were solved by X-ray analyses at 2.9 A resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity-determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N-terminal segment (designated strand 4-1) to be closely juxtaposed to an adjacent strand (4-2) and form hydrogen bonds typical of an antiparallel beta-pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an alpha-helix involving residues 25-32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 A to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space-saving alpha-helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use.
Assuntos
Fragmentos Fab das Imunoglobulinas/química , Interferon gama/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Elétrons , Humanos , Ligação de Hidrogênio , Regiões Constantes de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Cadeias gama de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/química , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: The participation of L-selectin in leukocyte recruitment during inflammation has suggested the use of L-selectin inhibitors as potential anti-inflammatory therapeutics. Blocking monoclonal antibodies could serve as such therapeutic agents, particularly if humanized to reduce their immunogenicity and improve their serum half-life. OBJECTIVES: For this purpose, two mouse monoclonal antibodies, DREG-55 and DREG-200, that block human L-selectin were humanized and characterized. STUDY DESIGN: The resulting humanized antibodies, HuDREG-55 and HuDREG-200, constructed with human IgG4 constant regions, were evaluated for their specificity, affinity and ability to block L-selectin-dependent adhesion in in vitro assays. Their pharmacokinetic behavior in rhesus monkeys was also studied. RESULTS: HuDREG-55 and HuDREG-200 were found to retain the specificity and affinity, within 2-fold, of the parent murine antibodies. HuDREG-55 and HuDREG-200 block L-selectin-dependent adhesion of human lymphocytes to high endothelial venules in frozen sections of lymph nodes. In addition, HuDREG-55 and HuDREG-200 are inhibitory in a novel L-selectin-dependent adhesion assay. This assay utilizes flow cytometry to measure binding of polymerized liposomes containing an analog of sialyl Lewis X, sialyl Lewis X glycoliposomes, to peripheral blood neutrophils and lymphocytes. Studying the pharmacokinetics of HuDREG-55 and HuDREG-200 in rhesus monkeys showed terminal elimination half-lives at 12.0 and 20.3 days, respectively. CONCLUSION: The shorter terminal elimination half-life of HuDREG-55 in rhesus monkeys may be due to the ability of HuDREG-55 but not HuDREG-200 to bind rhesus monkey L-selectin.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos , Selectina L/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Adesão Celular , Clonagem Molecular , Reações Cruzadas , Endotélio Linfático/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Leucócitos/citologia , Leucócitos/metabolismo , Lipossomos/metabolismo , Macaca mulatta , Camundongos , Dados de Sequência Molecular , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Especificidade da Espécie , TransfecçãoRESUMO
The ability to produce monoclonal antibodies (Mabs) in plants offers the opportunity for the development of an inexpensive method of mucosal immunoprotection against sexually transmitted diseases. To investigate the suitability of plant-expressed Mabs for vaginal preventive applications, we compared a humanized anti-herpes simplex virus 2 (HSV-2) Mab expressed in mammalian cell culture with the same antibody expressed in soybean. We found these Mabs to be similar in their stability in human semen and cervical mucus over 24 h, their ability to diffuse in human cervical mucus, and their efficacy for prevention of vaginal HSV-2 infection in the mouse.
Assuntos
Anticorpos Monoclonais/imunologia , Herpes Genital/prevenção & controle , Plantas Geneticamente Modificadas/genética , Vagina/imunologia , Animais , Anticorpos Monoclonais/genética , Modelos Animais de Doenças , Feminino , Herpes Genital/imunologia , Herpesvirus Humano 2/isolamento & purificação , Humanos , Imunidade nas Mucosas , Camundongos , Vagina/virologiaRESUMO
E- and P-selectin (CD62E and CD62P) are cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions and are involved in leukocyte recruitment during inflammation. We previously developed a murine mAb, EP-5C7 (or mEP-5C7), that binds and blocks both E- and P-selectin. When used in humans, murine mAbs have short circulating half-lives and generally induce potent human anti-mouse Ab responses. We therefore engineered a humanized, complementarity determining region-grafted version of mEP-5C7 incorporating human gamma4 heavy and kappa light chain constant regions (HuEP5C7.g4). HuEP5C7.g4 retains the specificity and avidity of mEP-5C7, binding to human E- and P-selectin but not to human L-selectin, and blocking E- and P-selectin-mediated adhesion. Surprisingly, when administered to rhesus monkeys, HuEP5C7.g4 was eliminated from the circulation very rapidly, even faster than the original murine Ab. To isolate the cause of the short serum half-life of HuEP5C7.g4, several Ab variants were constructed. A chimeric IgG4 Ab was made by replacing the humanized V regions with murine V regions. A humanized IgG2 Ab, HuEP5C7.g2, was also made by replacing the human gamma4 with a gamma2 constant region. Results from pharmacokinetic studies in rhesus monkeys demonstrated that the chimeric IgG4 is also rapidly eliminated rapidly from serum, similar to the humanized IgG4 Ab, while the humanized IgG2 Ab displays a long circulation half-life, typical of human Abs.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Especificidade de Anticorpos , Selectina E/imunologia , Selectina-P/imunologia , Proteínas Recombinantes de Fusão/síntese química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Sequência de Bases , Ligação Competitiva/imunologia , Adesão Celular/imunologia , Clonagem Molecular , DNA Complementar/isolamento & purificação , Selectina E/fisiologia , Meia-Vida , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Selectina-P/fisiologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
The murine monoclonal antibody BR55-2 is directed against the tumor-associated antigen Lewis Y oligosaccharide. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. Analysis of binding profiles of lactoseries isomeric structures by BR55-2 suggest that the binding epitope includes the OH-4 and OH-3 groups of the beta-D-galactose unit, the 6-CH3 groups of the two fucose units and the N-acetyl group of the subterminal beta-D-N-acetylglucosamine (beta DGlcNAc). To elucidate the molecular recognition properties of BR55-2 for the Y antigen, BR55-2 was cloned, sequenced and its three-dimensional structure was examined by molecular modeling. The crystal structure of BR96, another anti-Lewis Y antibody, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, and the lectin IV protein in complex with a Lewis b tetrasaccharide core were used as a guide to probe the molecular basis for BR55-2 antigen recognition and specificity. Our modeling study shows that BR55-2 shares similar recognition features for the difucosylated type 2 lactoseries Lewis Y structure observed in the BR96-sugar complex. We observe that a major source of specificity for the Lewis Y structure by anti-Y antibodies emanates from interaction with the beta-D-N-acetylglucosamine residue and the nature of the structures extended at the reducing site of the fucosylated lactosoamine.
Assuntos
Anticorpos Monoclonais/química , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Sítios de Ligação , Análise dos Mínimos Quadrados , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
1. The potential antiplatelet agent use of the whole molecule, F(ab)2 and Fab fragments of humanized antiglycoprotein (GP) IIb/IIIa monoclonal antibody, hC4G1, were investigated in rhesus monkeys. 2. Fab completely inhibited platelet aggregation 1 hr after an i.v. bolus administration of 1 mg/kg without a decrease in platelet count or prolongation of bleeding time, and the duration of inhibition was much shorter than that of F(ab)2. 3. These results suggest that the Fab fragment of hC4G1 may be a more useful antiplatelet agent in patients with acute thromboembolic diseases than the whole molecule or F(ab)2 fragments.
Assuntos
Anticorpos Monoclonais/farmacologia , Fragmentos Fab das Imunoglobulinas/imunologia , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Animais , Tempo de Sangramento , Humanos , Técnicas In Vitro , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas/efeitos dos fármacosRESUMO
ABL 364 is a murine monoclonal IgG3 antibody directed against the Lewis Y carbohydrate antigen (Le(y)) expressed on the surface of many epithelial cell tumors. The antibody mediates cytotoxicity via activation of human complement or human effector cells, and has been evaluated in several clinical trials including two Phase I/II trials in relapsed small cell lung cancer and metastatic breast cancer. To improve the effector functions of the antibody, increase its half-life in circulation, and avoid the human antimouse antibody response, two chimeric and several humanized antibodies were constructed for evaluation. The chimeric IgG1 is more potent than the murine IgG3 in tumor cell lysis via activation of human peripheral mononuclear cells (10-fold), but somewhat less effective in complement-dependent lysis (2-3 fold). The chimeric IgG3 is slightly less potent than the IgG1. A humanized IgG1 was constructed by combining the complementarity-determining regions of the ABL 364 antibody with human framework and constant regions. Several additional variants were subsequently constructed to improve the binding affinity and increase expression of the antibody. Two of the variants, designated I and K, differ by a single amino acid at position 75 of the heavy chain. Both variants have affinity within 2-fold of the chimeric IgG1 antibody and retain the cytolytic activities toward tumor cell lines. However, it was possible to express variant K at a significantly higher level (5- 10-fold) than variant I. Pharmacokinetics of the humanized ABL 364 antibody variant K was compared with that of the parent murine antibody in rhesus monkeys. It was shown that the terminal half-life of the humanized antibody in rhesus monkeys is 14-20 days, with a mean of 16.3 days, while that of the parent murine antibody is only 1.9 days.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacocinética , Sequência de Bases , Células Cultivadas , DNA Complementar/genética , Humanos , Ativação Linfocitária , Macaca mulatta , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Characterization of monoclonal antibodies (MAbs) produced for therapeutic or diagnostic purposes increasingly includes an assessment of their carbohydrate content. Using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD), we have analyzed the PNGase F released oligosaccharides of several IgG preparations including human polyclonal IgG, a humanized monoclonal IgG (MAb M115), and a murine monoclonal IgG (MAb MY9-6) derived respectively from serum, hybridoma cultures, and ascites fluid. The N-linked oligosaccharides released by PNGase F treatment of the above IgGs were found to consist mainly of neutral, fucosylated, biantennary species. Comparison of glycosylation of human polyclonal IgG, MAb M115, and MAb MY9-6 revealed differences in the levels of galactosylation and in the levels as well as the form of sialic acid present. HPAEC/PAD oligosaccharide profiling, combined with the use of enzymes (PNGase F, endoglycosidase F2, endoglycosidase H, neuraminidase, beta-galactosidase, and beta-N-acetylhexosaminidase), and monosaccharide analysis allowed making of tentative structural assignments. By performing monosaccharide analysis directly on PVDF electroblotted heavy and light chain bands separated by SDS-PAGE, it was verified that IgGs used in this study were glycosylated predominantly in their heavy chain.
Assuntos
Anticorpos Monoclonais/análise , Carboidratos/análise , Imunoglobulina G/análise , Amidoidrolases/metabolismo , Sequência de Carboidratos , Química Farmacêutica , Cromatografia por Troca Iônica , Eletroquímica , Glicoproteínas/análise , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Dados de Sequência Molecular , Oligossacarídeos/análise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina AmidaseRESUMO
Polymorphonuclear leukocytes (i.e. neutrophils) significantly mediate damage in myocardial ischemia followed by reperfusion. In the present study, the cardioprotective effects of a humanized form of a monoclonal antibody directed against L-selectin designated monoclonal antibody (mAb) HuDREG-200 were examined in a feline model of 90-min myocardial ischemia followed by 270 min of reperfusion. In preliminary studies, flow cytometric analysis indicated that HuDREG-200 binds to feline neutrophils. In vitro administration of mAb HuDREG-200 significantly inhibited (P < .01) adherence of unstimulated neutrophils to ischemic-reperfused coronary endothelium in a concentration-dependent manner. Humanized DREG-200 (2 mg/kg) administered 10 min before reperfusion significantly attenuated myocardial necrosis compared to an isotype-matched humanized control mAb (HuABL364) which does not bind to L-selectin (14 +/- 3 vs. 29 +/- 3% necrosis/area-at-risk, P < .01), representing a 52% reduction in myocardial necrosis. This myocardial preservation also was related to reduced creatine kinase release and improved recovery of cardiac contractility (i.e. left ventricular dP/dtmax). Moreover, endothelial function, as assessed by relaxation to acetylcholine, also was significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb HuDREG-200 compared to mAb HuABL364 (68 +/- 6 vs. 18 +/- 5, P < .01). Thus, a humanized anti-L-selectin mAb appears to be an effective means of preserving the ischemic myocardium from reperfusion injury and of preserving myocardial contractile function, at least during the early reperfusion period.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Moléculas de Adesão Celular/fisiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Gatos , Adesão Celular , Moléculas de Adesão Celular/imunologia , Reações Cruzadas , Endotélio Vascular/fisiologia , Hemodinâmica , Humanos , Selectina L , Contagem de Leucócitos , Masculino , Neutrófilos/fisiologiaRESUMO
C4G1, a murine mAb reactive with the platelet gpIIb/IIIa integrin, was humanized for potential treatment of thrombosis-related disorders. The variable regions of light- and heavy-chain cDNAs from the C4G1 hybridoma were first cloned and sequenced. Humanized C4G1 Ab of the IgG1 isotype was constructed by combining the complementarity-determining regions of C4G1 with human framework and constant regions. The human framework was chosen to maximize homology with the C4G1 variable region sequence, and a computer model of C4G1 was used to aid design of the final framework sequence. Genetic constructs were also developed to produce Fab and F(ab')2 fragments of the humanized C4G1 Ab. The humanized IgG1 Ab as well as the Fab and F(ab')2 fragments showed equivalent binding affinities to their murine counterparts, indicating no loss in binding affinity during the humanization process. The humanized Ab and its fragments were also shown to inhibit platelet aggregation and to inhibit binding of fibrinogen to gpIIb/IIIa in vitro.
Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas da Membrana de Plaquetas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Especificidade de Anticorpos , Clonagem Molecular , Simulação por Computador , Fibrinogênio/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Camundongos , Dados de Sequência Molecular , Agregação PlaquetáriaRESUMO
Long-term survival rates of patients with acute myelogenous leukemia treated with intensive chemotherapy are 15-20%, despite efforts to develop new treatment strategies. Murine M195 (131I-M195), an anti-CD33, immunoglobulin (Ig) G2a monoclonal antibody has reactivity restricted to early myeloid cells and myeloid leukemic blasts but not hematopoietic progenitors. Previous trials in patients with relapsed or refractory myeloid leukemia showed that 131I-M195 rapidly targeted to the bone marrow and internalized into target cells. This article describes a therapeutic dose escalation study in which 24 patients received from 50 mCi/m2 to 210 mCi/m2 of 131I-M195 in divided doses. Cytoreduction of peripheral cell counts and bone marrow blasts occurred without nonhematopoietic toxicity. Doses of 131I-M195 greater than 135 mCi/m2 were associated with marrow cytoreduction sufficient to necessitate bone marrow transplant. However, 37% of the patients developed human anti-mouse antibody, preventing retreatment. To decrease immunogenicity and improve effector function, chimeric IgG1 and IgG3, and complementarity-determining region-grafted, humanized IgG1 and IgG3 versions of mouse M195 were developed by genetic engineering techniques. The new versions maintained specificity and biologic function, and they were superior to the mouse M195 in their ability to perform antibody-dependent cellular cytotoxicity against leukemia cells. Humanized M195, but not chimeric M195, showed a 4-8.6 times higher avidity than its mouse counterpart. Because effector function of IgG depends to a large extent on Fc clustering, a homodimeric HuG1 also was developed. Homodimeric HuG1 showed an ability to cause additional dramatic improvements in effector functions, as well as an ability to internalize and retain radioisotope in target leukemia cells. Monomeric and dimeric forms of humanized M195 may be advantageous in the therapy of acute myelogenous leukemia.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Radioisótopos do Iodo/uso terapêutico , Leucemia Mieloide Aguda/radioterapia , Radioimunoterapia , Adulto , Animais , Anticorpos Monoclonais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos , Humanos , Radioisótopos do Iodo/administração & dosagem , Camundongos/imunologia , Proteínas Recombinantes de Fusão , Lectina 3 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
M195 is a murine monoclonal antibody that binds to the CD33 antigen and is being tested for the treatment of myeloid leukemia. Surprisingly, a complementarity determining region (CDR)-grafted, humanized M195 antibody displayed a several-fold higher binding affinity for the CD33 antigen than the original murine antibody. Here we show that the increase in binding affinity resulted from eliminating an N-linked glycosylation site at residue 73 in the heavy chain variable region in the course of humanization. Re-introducing the glycosylation site in the humanized antibody reduces its binding affinity to that of the murine antibody, while removing the glycosylation site from the murine M195 variable domain increases its affinity. The removal of variable region carbohydrates may provide a method for increasing the affinity of certain monoclonal antibodies with diagnostic and therapeutic potential.
Assuntos
Anticorpos Monoclonais/química , Afinidade de Anticorpos , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos , Glicoproteínas/imunologia , Região Variável de Imunoglobulina/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Relação Estrutura-AtividadeRESUMO
Seven neutralizing murine monoclonal antibodies specific for the glycoprotein H of human cytomegalovirus were produced and used to construct a topological map of two nonoverlapping antigenic sites that are bridged by a third antigenic site. Neutralization assays with 15 laboratory or clinical human cytomegalovirus strains indicated that the monoclonal antibodies recognize three antigenically variable and three conserved epitopes within the three antigenic sites. The variable-domain genes encoding monoclonal antibodies representing each of the three antigenic sites were cloned and sequenced, and molecular models of their binding sites were generated. Conformational differences in the antibody-binding sites suggested a structural basis for experimentally observed differences in gH epitope recognition.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Citomegalovirus/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/farmacologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/crescimento & desenvolvimento , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Conformação Proteica , Homologia de SequênciaRESUMO
Human-mouse chimeric immunoglobulins G1 and G3 (IgG1 and IgG3) (ChG1, ChG3) and "complementarity-determining region"-grafted, humanized IgG1 and IgG3 (HuG1, HuG3) constructs of the mouse monoclonal antibody (mAb) M195 were characterized. M195 is a murine immunoglobulin G2a (IgG2a), anti-CD33 mAb, specifically reactive with acute myelogenous leukemia cells, that is active as an antileukemia agent in humans. The new mAb constructs maintained specificity and biological function, including rapid internalization after binding to the cell surface, which has been important for delivery of therapeutic isotopes in patients. Although previously reported complementarity-determining region-grafted mAbs had reduced avidities, the HuG1 and HuG3 M195 showed up to an 8.6- and 4-fold higher binding avidity, respectively, than the original murine mAb. All constructs were effective at mediating rabbit complement-mediated cytotoxicity against HL60 targets. Fibroblasts transfected with CD33 genes and expressing high levels of CD33 antigen were also lysed in the presence of human complement, but HL60 cells or fibroblasts with lower CD33 levels were not killed. Thus, the inability of M195 and constructs to kill HL60 targets with human complement is due to the much lower antigen density on HL60 cells compared to CD33+ fibroblasts. Unlike the murine M195, the chimeric and humanized M195 demonstrated antibody-dependent cell-mediated cytotoxicity using human peripheral blood mononuclear cells as effectors. Because the chimeric and humanized M195 have improved avidities as compared to the original M195 and have, in addition, the potential to avoid human anti-mouse antibody responses and to recruit human effector functions, these new constructs may be useful therapeutically, either alone or conjugated to toxins or isotopes, in the treatment of acute myelogenous leukemia.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Animais , Especificidade de Anticorpos , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Ligação Competitiva , Citotoxicidade Imunológica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ensaio Radioligante , Lectina 3 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
Humanized IgG1 M195 (HuG1-M195), a complementarity determining region-grafted recombinant monoclonal antibody, is reactive with CD33, an antigen expressed on myelogenous leukemia cells. M195 is in use in trials for the therapy of acute myelogenous leukemia. Since biological activity of IgG may depend, in part, on multimeric Fab and Fc clustering, homodimeric forms of HuG1-M195 were constructed by introducing a mutation in the gamma 1 chain CH3 region gene to change a serine to a cysteine, allowing interchain disulfide bond formation at the COOH terminal of the IgG. Despite similar avidity, the homodimeric IgG showed a dramatic improvement in the ability to internalize and retain radioisotope in target leukemia cells. Moreover, homodimers were 100-fold more potent at complement-mediated leukemia cell killing and antibody-dependent cellular cytotoxicity using human effectors. Therefore, genetically engineered multimeric constructs of IgG may have advantages relative to those forms that are found naturally.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Linfócitos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular , Membrana Celular/imunologia , Citometria de Fluxo , Humanos , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Leucemia Promielocítica Aguda , Substâncias Macromoleculares , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.
Assuntos
Anticorpos Monoclonais/genética , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Imunoglobulina G/genética , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Sequência de Bases , Clonagem Molecular , Humanos , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Lectina 3 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
Protein engineering has made it possible to combine the binding sites of murine antibodies with human antibody regions. Antibodies constructed in this way have important advantages for therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Engenharia de Proteínas , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Sítios de Ligação de Anticorpos/genética , Humanos , Linfócitos/imunologia , Camundongos , Proteínas Recombinantes/uso terapêuticoRESUMO
Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.
Assuntos
Anticorpos Monoclonais/genética , Antivirais , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Simulação por Computador , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Simplexvirus/imunologia , Células Vero , Proteínas do Envelope Viral/imunologiaRESUMO
Antireceptor antibodies have been developed as a probe to study the cellular receptor for reovirus type 3. Using this probe, a glycoprotein with a molecular weight of 65-67 kilodaltons and a pI of 5.8-6.0 was isolated and identified as the reovirus receptor. This protein was also structurally similar to the affinity-purified beta-adrenergic receptor from calf lung. In this report, we employ [125I]-iodocyanopindolol, a high affinity beta-adrenergic antagonist, to further characterize this protein. We show that R1.1, a murine thymoma cell line, possesses about 2,000 receptors per cell with high affinity for ICYP (kD = 3.3 X 10(-11) M). Competitive inhibition studies suggest that the receptor is of the beta-2 subtype. Solubilized receptor proteins from R1.1 cells bound to the antireceptor antibody were further purified by SDS-PAGE and electroelution from the gel. Five percent of the proteins thus obtained could bind ICYP with high affinity (kD = 1.6 X 10(-10) M). This suggests that the purification procedure produced a collection of forms of this 65- to 67-kilodalton protein, some of which retained the conformation for binding the beta ligands. We also demonstrate that the isolated receptor protein was able to bind ICYP even when the virus binding site was occupied by the anti-idiotype, suggesting that reovirus type 3 and the beta ligands bind to distinct domains on the receptor protein.
Assuntos
Agonistas Adrenérgicos beta/metabolismo , Orthoreovirus Mamífero 3/metabolismo , Pindolol/análogos & derivados , Receptores Adrenérgicos beta/análise , Receptores Virais/análise , Reoviridae/metabolismo , Animais , Sítios de Ligação de Anticorpos , Linhagem Celular , Idiótipos de Imunoglobulinas/imunologia , Radioisótopos do Iodo , Iodocianopindolol , Camundongos , Pindolol/metabolismo , Receptores Adrenérgicos beta/imunologia , Receptores Adrenérgicos beta/isolamento & purificação , Receptores Virais/imunologia , Receptores Virais/isolamento & purificação , Timoma/metabolismoRESUMO
By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.