Temperature-dependent binding of IgG1 to a human high affinity Fc receptor.

We have measured the kinetics of binding and unbinding of human IgG1 to a human high affinity Fc receptor (FcgammaI) at several temperatures. The association rate constant (kappaf) and the dissociation rate (kr) of this complex was determined with 125I-IgG1 monomer and FcgammaI on U937 cells. At 37 degrees C, kappaf = 2.7 x 10(5) M(-1) s(-1) and kappar = 4.5 x 10(-4) s(-1). Both rates decreased with decreasing temperature. However, the equilibrium association constant, Ka, increased with decreasing temperature. From the temperature dependence of Ka we determined that the binding of IgF1 to FcgammaI is driven largely by enthalpic forces and that a small but positive entropic contribution to free energy leads to a tighter complex at lower temperature.

Cloning in a bacteriophage lambda vector for the display of binding proteins on filamentous phage.

We have combined the efficiency and ease of use of bacteriophage lambda vectors with the power of phage display screening technology to create SurfZAP. The use of bacteriophage lambda allows the construction of large lambda expression libraries, which are rapidly and efficiently converted to stable plasmid libraries by mass excision. In SurfZAP, clones are expressed as fusions with amino acids 198-406 of the M13 minor coat protein (cpIII) and are displayed on the surface of filamentous phage. When produced with helper phage proteins, the fusion proteins are incorporated into the surface of phagemid particles. We demonstrate the utility of biopanning by isolating tetanus toxoid-binding mouse Fab clones from SurfZAP libraries. Approximately 10-100-fold enrichment of specific clones was observed after each panning round. The ability to create a large library of genotypes and screen the phenotypes by activity may be a potent methodology for basic research and drug discovery.

A genetically engineered human IgG with limited flexibility fully initiates cytolysis via complement.

A causal link between antigen induced conformational change and functional activity has been previously suggested for the triggering of immunoglobulin effector functions. To test this hypothesis an immunoglobulin with greatly restricted Fab flexibility has been created by site-directed mutagenesis. Serine119, a residue at the elbow in the first heavy chain constant domain of a mouse-human chimeric immunoglobulin, was replaced by cysteine119. The resulting mutant immunoglobulin had an additional intramolecular disulfide bond connecting the two heavy chains. This newly introduced covalent bond between antigen binding arms gave rise to a compact 'tethered' conformation which displayed lowered segmental flexibility as determined by nanosecond fluorescence depolarization anisotropy. However, the ability of this tethered immunoglobulin to initiate lysis of target cells via the complement cascade was unimpaired. Therefore, it is likely that allosteric or distortive mechanisms of conformational change induced complement activation which require unhindered 'twist' and/or 'waggle' motions of Fab are untenable.

Dynamic conformations compared for IgE and IgG1 in solution and bound to receptors.

Dynamic conformations of two distinct immunoglobulin (Ig) isotypes, murine IgE and human IgG1, were examined with fluorescence resonance energy transfer measurements. The IgE mutant epsilon/C gamma 3* and the IgG1 mutant gamma/C gamma 3* each bind [5-(dimethylamino)naphthalen-1-yl]sulfonyl (DNS) in two identical antigen binding sites at the amino (N)-terminal ends of the Ig in the Fab segments. Eosin-DNS bound in these Fab sites served as the acceptor probe in these studies. Both Ig have a carboxy (C)-terminal domain (C gamma 3*) which contains genetically introduced cysteine residues. Modification of these cysteine sulfhydryls with fluorescein maleimide provided donor probes near the C-terminal ends of the Ig in the Fc segment. Energy transfer between the C-terminal and N-terminal ends was compared for these two Ig in solution and when they were found to their respective high-affinity receptors on plasma membranes: IgE-Fc epsilon RI on RBL cell membranes and IgG1-Fc gamma RI on U937 cell membranes. Previous energy-transfer measurements with these probes yielded an average end-to-end distance of 71 A for IgE in solution and 69 A for IgE bound to Fc epsilon RI, indicating that in both situations IgE is bent such that the axes of the Fab segments and the axis of the Fc segment do not form a planar Y-shape [Zheng, Shopes, Holowka, & Baird (1991) Biochemistry 30, 9125]. In the current study we found the average end-to-end distance for IgG1 in solution is 75 A and greater than or equal to 85 A for IgG1 bound to Fc gamma RI, suggesting an average bend conformation for IgG1 as well. The contributions of segmental flexibility to the average distances were assessed directly by measuring the efficiency of energy transfer as a function of variations in donor quantum yield caused by a collisional quencher and using these data to extract a Gaussian distribution of end-to-end distances. The distribution average (rho) and half-width (hw) were determined to be as follows: rho = 75 A, hw = 24 A for IgE in solution; rho = 71 A, hw = 12 A for IgE bound to Fc epsilon RI; and rho = 100 A, hw = 88 A for IgG in solution.(ABSTRACT TRUNCATED AT 400 WORDS)

A genetically engineered human IgG mutant with enhanced cytolytic activity.

A mutant chimeric anti-5-dimethylaminonaphthalene-1-sulfonyl human Ig gamma that exhibited augmented effector function was constructed. Utilizing directed mutagenesis, a serine residue near the carboxyl terminus of the human IgG1 H chain (Ser444) was replaced by a cysteine. Novel intermolecular disulfide bonds between Cys444 residues linked pairs of Ig "tail-to-tail" to form covalent dimers ((H2L2)2). These dimers were 200-fold more efficient, compared with monomeric human IgG1, at antibody-dependent complement-mediated cytolysis of hapten-bearing erythrocytes. The ability to enhance the cytolytic activity of an mAb by genetic engineering may be of value in immunotherapy.

Bacteriophage cloning and Escherichia coli expression of a human IgM Fab.

We have combined the molecular biology methods of the polymerase chain reaction and recombinant DNA cloning in bacteriophage lambda to express a human IgM Fab in Escherichia coli using genes derived from an Epstein-Barr virus transformed cell line. This method comprises three cDNA amplifications and a single cloning step, culminating in the stable overexpression of mammalian heterodimeric recombinant protein in a prokaryotic host.

Characterization of new rat anti-mouse IgE monoclonals and their use along with chimeric IgE to further define the site that interacts with Fc epsilon RII and Fc epsilon RI.

Three rat monoclonal antibodies specific for mouse IgE (C12B9, 23G3, and B1E3) were established by using monoclonal anti-DNP mouse IgE (mIgE) as immunogen. These antibodies, as well as a fourth, (R1E4) were characterized. It was found that one antibody (C12B9) recognizes an allotypic determinant (Igh-7a) found on the C epsilon chain of mIgE. Antibody cross-blocking studies and epitope mapping studies using recombinant mIgE indicated that 3 antibodies (C12B9, R1E4 and 23G3) were directed against the C epsilon 3 domain while one (B1E3) was directed against the C epsilon 4 domain. A highly specific sandwich RIA for mIgE was developed using these antibodies. Use of these monoclonal anti-mIgE antibodies in conjunction with recombinant chimeric mIgE-human IgG1 molecules, demonstrated that the C epsilon 3 domain is important in the binding of mIgE to the murine B cell Fc epsilon RII as well as to the murine mast cell F epsilon RI. The presence of the C epsilon 4 domain influenced the binding of the recombinant IgE to the Fc epsilon RII; in contrast to the C epsilon 4 domain had no effect on binding to the Fc epsilon RI.

Conformations of IgE bound to its receptor Fc epsilon RI and in solution.

Previous resonance energy transfer studies suggested that murine immunoglobulin E (IgE) is bent near the junction of its Fc and Fab segments when bound to its high-affinity receptor (Fc epsilon RI) on RBL cells. To examine further the conformations of IgE, both bound to this receptor and in solution, a mutant recombinant IgE (epsilon/C gamma 3*) was prepared that has a cysteine replacing a serine near the C-terminal ends of the heavy chain. The introduced cysteine residues provide a means for specific modification of IgE, and the sulfhydryl groups were selectively labeled with fluorescein-5-maleimide (FM-epsilon/C gamma 3*). This IgE also binds a 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) group in the antigen-binding sites. Resonance energy transfer experiments carried out on receptor-bound FM-epsilon/C gamma 3* yielded a distance of 53 A between fluorescein near the C-terminal end of the Fc segment and amphipathic acceptor probes at the membrane surface. The average distance between this C-terminal fluorescein and acceptor eosin-DNS in the antigen-binding sites at the N-terminal ends of the Fab segments was found to be 69 A. These results combine with those from previous structural studies to provide an unprecedented detailed description of the bent geometry of IgE bound to its receptor on the membrane. Energy transfer measured for FM-epsilon/C gamma 3* in solution between fluorescein near the C-terminal end of the Fc segment and eosin-DNS at the N-terminal ends of the Fab segments indicates that the average distance between these probes is about 71 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human IgG1-murine IgE chimeric Ig. Construction, expression, and binding to human Fc gamma receptors.

We have constructed a set of chimeric Ig by exchanging corresponding H chain C domains between human (hu) IgG1 and murine (m) IgE. We used this set of Ig to dissect the interaction of individual Ig domains with human Fc gamma receptors. Only one of the chimeras, epsilon/C gamma 2,3 (an mIgE with C epsilon 3 and C epsilon 4 replaced by C gamma 2 and C gamma 3 from huIgG1), binds tightly to the human Fc gamma RI on U937 cells. We found that epsilon/C gamma 2,3 has only twofold lower affinity for Fc gamma RI as compared to huIgG1. The gamma/C epsilon 4 (huIgG1 with C epsilon 4 replacing C gamma 3) binds weakly to Fc gamma RI. The other chimeric Ig, epsilon/C gamma 3, epsilon/C gamma 2, and gamma/C epsilon 3, as well as mIgE do not bind detectably to Fc gamma RI. From these data we conclude that the C gamma 2 domain is crucial for binding and contains the majority of the binding site for Fc gamma RI on IgG1. The C gamma 3 domain makes a smaller contribution to the binding, and the C gamma 1 domain and the hinge region have very little effect on the Fc gamma RI-IgG1 interaction. The chimeric epsilon/C gamma 2,3 and huIgG1 both mediate the formation of rosettes between K562 cells and antigen-sensitized E with similar concentration dependences. These results suggest similar ability to bind to Fc gamma RII. The other chimeric Ig do not cause rosettes in this assay system. Hence, both C gamma 2 and C gamma 3 seem to be required for binding to Fc gamma RII, but the C gamma 1-hinge region has no detectable effect.

Mapping the site of interaction between murine IgE and its high affinity receptor with chimeric Ig.

We have investigated the interaction of mouse (m) IgE with its Fc epsilon RI on rat basophilic leukemia cells using a set of chimeric Ig that were constructed by exchanging homologous H chain C domains between human (hu) IgG1 and mIgE. Binding affinities were examined with equilibrium and kinetic measurements, and we found that epsilon/C gamma 3 (mIgE with C epsilon 4 replaced by C gamma 3) was indistinguishable from mIgE. The huIgG1 and the other chimeric Ig, which did not contain both C epsilon 2 and C epsilon 3, did not bind detectably to rat basophilic leukemia cells (Ka less than 10(6) M-1). The ability of these chimeric Ig to stimulate a cellular response (degranulation) in the presence of multivalent Ag was also tested. The epsilon/C gamma 3 was indistinguishable from mIgE in eliciting a high level of degranulation, whereas the other chimeric Ig stimulated no response even when they were preaggregated to enhance their binding avidity. These results demonstrate that C epsilon 4 may be replaced by C gamma 3 without affecting the binding and cell activating properties of mIgE. The lack of binding by the other chimeric Ig indicates that both C epsilon 2 and C epsilon 3 are necessary for the binding interaction.