ABSTRACT
Construction of a series of chimeric antibodies (murine variable region and human constant region) derived from the murine antibody BIRR1, which recognizes intercellular adhesion molecule 1 (ICAM-1), has revealed differences in the relative binding abilities of the chimeric antibody to antigen. The chimeric antibodies show a ranking of their ability to compete with BIRR1 for antigen on the surface of cells with the order BIRR1 = cIgG1 (100%) > cIgG4 (30%) > cIgG2 (10%) as demonstrated by solid-phase competitive enzyme-linked immunosorbent assay. Papain digestion yielded Fab fragments that were purified to homogeneity. Competitive enzyme-linked immunosorbent assay showed that the chimeric and murine Fab binding constants were equivalent. A solution-phase binding assay (analyzed by size exclusion high performance liquid chromatography) between the intact mAbs and recombinant soluble ICAM-1 further established that the binding constants involving the Fab arms of the two antibodies were equivalent. In summary, the murine and chimeric anti-ICAM-1 antibodies bind cellular ICAM-1 with equivalent affinities but with differing avidities.
Subject(s)
Antibodies, Monoclonal/immunology , Binding Sites, Antibody , Immunoglobulin Isotypes/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/isolation & purification , Base Sequence , CHO Cells , Cell Adhesion Molecules/immunology , Cricetinae , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Isotypes/genetics , Immunoglobulin Isotypes/isolation & purification , Intercellular Adhesion Molecule-1 , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , Recombinant Fusion ProteinsABSTRACT
OKT3 is a murine monoclonal antibody which recognizes an epitope on the epsilon-subunit within the human CD3 complex. OKT3 possesses potent immunosuppressive properties in vivo and has been proven effective in the treatment of renal, heart and liver allograft rejection. Despite its efficacy, significant problems remain associated with OKT3 therapy, i.e. T-cell activation and the anti-murine antibody response. To address the problem of the anti-murine antibody response we have constructed humanized versions of OKT3. One of the humanized derivatives, gOKT3-7 incorporating the OKT3 complementarity determining regions plus a small number of alterations to the human framework, has an affinity of 1.4 x 10(9) M-1 compared with 1.2 x 10(9) M-1 for the murine OKT3. A humanized antibody (gOKT3-1) incorporating only the CDRs from OKT3 was found to be functionally inactive, confirming the requirement for nonCDR substitutions. gOKT3-7 retains the ability of mOKT3 to induce T cell proliferation in vitro and appears to be a good candidate for further development for in vivo therapy.
Subject(s)
CD3 Complex/immunology , Muromonab-CD3/genetics , Amino Acid Sequence , Animals , Antibody Affinity , Base Sequence , DNA Primers/genetics , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , In Vitro Techniques , Lymphocyte Activation , Mice , Molecular Sequence Data , Muromonab-CD3/immunology , Muromonab-CD3/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , Species Specificity , T-Lymphocytes/immunologyABSTRACT
A genetic system controlling lymphocyte alloantigens of the horse is described. Alloantisera to paternal histocompatibility antigens induced as a result of pregnancy in mares were used in an antibody-mediated complement-dependent microcytotoxicity assay to define 15 Equine Leukocyte Antigen (ELA) specificities using cluster analysis. In this study 369 sera were screened for alloantibody using lymphocytes from 10 randomly selected, unrelated horses. A high proportion (83%) of these sera were found to be positive for antibody to lymphocyte alloantigens. After initial cluster analysis, 120 of the most discriminating sera were tested against a further 400 horses. The phenotypic distribution of the ELA antigens in 304 randomly selected horses and their segregation behavior in a family study of 161 offspring and their sires and dams indicated that 13 of the alloantigens behaved as members of a single allelic series, provisionally named locus ELA-A. These 13 alloantigens accounted for approximately 90% of the genes at this locus. Large differences in the frequencies of some of the ELA-A specificities were found between the Standardbred and Thoroughbred breeds of horses. Rare 'blank' alleles which were predicted by the estimated cumulative gene frequency of known alleles at ELA-A were identified in informative families. The ELA system is probably the Major Histocompatibility Complex of the horse, although the evidence for this is not yet conclusive. The high incidence of sensitization to ELA antigens in pregnant mares suggests that the horse may be an interesting model for investigations of the maternal immunological response to fetal histocompatibility antigens.
