RESUMEN
OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), is a potent immunosuppressive agent used in clinical transplantation to treat allograft rejection. Two major drawbacks of this therapy are the systemic release of several cytokines due to cross-linking mediated by the mAb between T cells and FcgammaR-bearing cells and the human anti-mouse antibody (HAMA) response. To overcome these side effects, three chimeric OKT3 single chain variable fragment (scFv) IgM antibodies, scOKT3-gamma DeltaIgM wt, scOKT3-gamma DeltaIgM C575S and scOKT3-gamma DeltaIgM VAEVD, were generated. They consist of the light and heavy variable binding domains of OKT3 mAb as well as the CH3 and CH4 domains of different human IgM variants linked with a human IgG3 hinge region to provide more flexibility and stability. Like the native IgM, scOKT3-gamma DeltaIgM antibodies are able to form polymeric structures, which lead to an increase in binding affinity and immunosuppressive potential compared with the parental OKT3 mAb. However, independently of their polymerization, all scOKT3-gamma DeltaIgM constructs do not induce any significant T cell proliferation or cytokine release (IL-2, TNF-alpha and IFN-gamma) in in vitro assays, while their CD3-modulating properties are retained. These results suggest that the use of scOKT3-gamma DeltaIgM antibodies may offer significant advantages over the OKT3 mAb in improving clinical immunosuppressive treatment.
Asunto(s)
Fragmentos de Inmunoglobulinas/farmacología , Inmunoglobulina M/farmacología , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Muromonab-CD3/farmacología , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/efectos de los fármacos , Adulto , Secuencia de Aminoácidos , Línea Celular , Activación de Complemento/efectos de los fármacos , Complemento C1q/metabolismo , Estabilidad de Medicamentos , Femenino , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina M/inmunología , Datos de Secuencia Molecular , Muromonab-CD3/inmunología , Linfocitos T/inmunologíaRESUMEN
Cloning the correct genes that code for antibody-variable domains from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from a myeloma cell line. For the rapid functional evaluation of recombinant antibody fragments against cell-surface antigens, we established an efficient expression and screening system using phagemid antibodies and fixed cells. VL and VH-polymerase chain reaction (PCR) products, amplified from hybridoma cDNA, were cloned into the phagemid vector pSEX81. After transduction into E. coli and phage rescue, clones were tested for antigen binding using a phage-enzyme-linked immunosorbent assay (ELISA) procedure with whole cells fixed to ELISA wells. This procedure facilitated the successful cloning of a functional anti-CD20, single-chain antibody from hybridoma cDNA. The CD20 B-lymphocyte surface antigen expressed by B-cell lymphomas is an attractive target for cancer treatment using immunoconjugates or bi-specific antibodies.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos CD20/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos Monoclonales/uso terapéutico , Linfocitos B/inmunología , Biotecnología , Separación Celular , Clonación Molecular , Citometría de Flujo , Humanos , Hibridomas/inmunología , Inmunoconjugados/uso terapéutico , Región Variable de Inmunoglobulina/genética , Inmunoterapia , Linfoma de Células B/inmunología , Linfoma de Células B/terapiaRESUMEN
We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1-5x10(7) independent clones giving a final combined library of 4x10(9) members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Biblioteca de Péptidos , Donantes de Sangre , Biblioteca de Genes , Genes de Inmunoglobulinas , Humanos , Inmunoglobulina G/genética , Inmunoglobulina M/genética , Reacción en Cadena de la PolimerasaRESUMEN
Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4 x 10(7)) of single chain antibodies (scFv). The affinities of both the anti-estradiol and antiprogesterone scFv were approximately 10(8) M(-1). Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response.