Variable region domain exchange influences the functional properties of IgG.

In the present study we have characterized a family of anti-dansyl Abs with the variable region of the heavy chain on human Ckappa and the variable region of the light chain on different human gamma constant regions (creating inside-out molecules). Although fully assembled molecules were secreted, this variable region exchange slowed the kinetics of Ab assembly. Although the variable region exchange does not lead to a detectable change in the microenvironment of the combining site, it did alter the kinetic parameters of binding to immobilized Ag, slowing both the on and off rates. When effector functions were evaluated, inside-out IgG1 and IgG3 were more effective in complement-mediated cytolysis than their wild-type counterparts. Variable region domain exchange may be one approach to obtaining Abs of identical specificity with altered binding characteristics.

Dimerization of CD4-C kappa chimeric molecules leads to loss of CD4 epitopes.

Structural similarities between two members of the immunoglobulin superfamily were explored by making chimeric immunoglobulin/CD4 antigen molecules. A crossover in the middle of the originally proposed J kappa homology unit of the first domain of the CD4 molecule was used to construct a chimeric molecule having human and mouse CD4 antigen sequence through the first 108 amino acids and murine J kappa and C kappa sequence thereafter. This molecule was expressed in the presence and absence of an immunoglobulin heavy chain. The resulting proteins were assayed for the expression of CD4 epitopes that should be present based on epitope mapping data. Monomeric, homodimeric, and heavy chain/light chain tetrameric forms of the recombinant protein were secreted and were all detectable with anti-kappa reagents. CD4 antibodies precipitated only the form of the CD4-C kappa light chain protein which appears as a monomer by polyacrylamide gel electrophoresis. Neither the homodimer nor the heavy chain/light chain tetramer were detected with CD4 monoclonal antibodies. An engineered gene having this CD4 antigen first domain joined to the human IgG1 constant region, when coexpressed with a mouse lambda light chain, also failed to express detectable CD4 epitopes. The structural implications of the presence or absence of CD4 epitopes on these proteins is discussed.

Mechanisms of anti-CD4-mediated depletion and immunotherapy. A study using a set of chimeric anti-CD4 antibodies.

A family of rat-mouse chimeric anti-murine CD4 antibodies was used to study the mechanisms of anti-CD4-mediated depletion and immunotherapy. The chimeric antibodies retain identical affinity and specificity as the therapeutically effective prototype antibody, rat GK1.5, but are of different mouse isotypes. GK1.5 gamma 1, GK1.5 gamma 2a, and GK1.5 gamma 2b are significantly more effective at CD4+ cell depletion than rat GK1.5 when low doses of antibody are administered. In contrast, no depletion is seen with GK1.5 gamma 3 at any dose. Depletion of CD4+ cells in vivo is not correlated with either the ability of the antibody to mediate C-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity in vitro, implying that additional antibody-mediated cytotoxic mechanisms occur in vivo. The chimeric antibodies were used to investigate the mechanism of GK1.5-mediated immunotherapy in a prototypic model of T cell-mediated autoimmunity, experimental allergic encephalomyelitis. Mice treated with a single dose of 100 micrograms of either GK1.5, GK1.5 gamma 1, or GK1.5 gamma 2a showed significant recovery within 72 h. In contrast, mice treated with 100 micrograms of GK1.5 gamma 3 showed only marginal improvement within the first 72 h and regressed within 5 days of treatment initiation. These data suggest that anti-CD4-mediated immunotherapy of murine experimental allergic encephalomyelitis is correlated with depletion of CD4+ cells.

Electron microscopic study of ring-shaped, bivalent hapten, bivalent antidansyl monoclonal antibody complexes with identical variable domains but IgG1, IgG2a and IgG2b constant domains.

We have studied by electron microscopy a fascinating series of antidansyl monoclonal antibodies developed by Dangl et al. (Cytometry 2, 395-401, 1982) which have the same variable domain but different constant domains. Three of the four subclasses of mouse IgG were represented, IgG1, IgG2a and IgG2b. Previously, Oi et al. (Nature 307, 136-140, 1984) had examined the flexibilities of these antibodies by time-resolved fluorescence depolarization and found that IgG1 was least flexible, IgG2a was intermediate and IgG2b was the most flexible. In this communication we examine the conformations of circular complexes formed between these antibodies and a bivalent hapten, bis-dansyl cadaverine. The circular complexes were predominantly composed of two antibodies linked into a ring by two bivalent haptens, and are referred to as dimers, since only the antibody molecules are seen with the electron microscope. A few trimers and an occasional tetramer were also present in these preparations. For the least flexible IgG1, almost all (greater than 99%) of the circular dimers were "open-hinge" complexes with a hinge angle between the Fab arms of 100-120 degrees. For the intermediate IgG2a, most of the dimers were "open-hinge" complexes, but a larger percentage, 4 to 5%, had closed hinges with a hinge angle approaching 0 degrees. For the most flexible IgG2b, over 40% of the dimers were "closed-hinge" complexes. A model is proposed to explain these differences based upon orientation of the hapten in the combining site and differences in hinge structure.

Influence of the hinge region on complement activation, C1q binding, and segmental flexibility in chimeric human immunoglobulins.

We have characterized a series of genetically engineered chimeric human IgG3 and IgG4 anti-dansyl (DNS) antibodies with identical antibody-combining sites but different hinge region amino acid compositions to determine how the hinge region influences Fab fragment segmental flexibility, C1q binding, and complement activation. Our data support the correlation between "upper hinge" length and Fab segmental flexibility; moreover, we confirm that a hinge region is essential for C1q binding and complement activation. However, the hinge length by itself is not sufficient for complement activity in IgG molecules. We have demonstrated that the IgG4 hinge, which imparts restricted segmental flexibility, reduces the ability of IgG3 molecules to activate complement. We also find that the IgG3 hinge region, which imparts greater segmental motion, is not sufficient to create complement activation activity in IgG4 anti-DNS antibodies. Finally, we conclude that (i) segmental motion is correlated with "upper hinge" length, (ii) hinge length and segmental flexibility is not enough to alter complement binding and activation, and (iii) segmental flexibility does not correlate with proficiency to activate the complement cascade.

Comparison of rat and rat-mouse chimeric anti-murine CD4 antibodies in vitro. Chimeric antibodies lyse low-density CD4+ cells.

GK1.5, a rat anti-mouse CD4 mAb, is effective in the treatment of several autoimmune syndromes, induces tolerance to co-administered Ag, and prolongs allograft survival. We have constructed a family of molecules with GK1.5 V regions and mouse gamma 1, gamma 2a, gamma 2b, or gamma 3 constant regions to investigate the mechanisms underlying the effectiveness of GK1.5. The rat-mouse chimeric antibodies are specific for murine CD4 and have identical binding curves as rat GK1.5 on CD4+ T cells. The chimeric GK1.5 gamma 2a, GK1.5 gamma 2b, and GK1.5 gamma 3 antibodies are more efficient than rat GK1.5 at C-mediated cytotoxicity. This is attributed to the enhanced capacity of the chimeric antibodies, compared to rat GK1.5, to lyse CD4+ cells with a low cell surface Ag density. This observation may have important implications for therapy.

Interaction of IgE with its high-affinity receptor. Structural basis and requirements for effective cross-linking.

Structural interactions between IgE and its high-affinity receptor have been investigated with the methods of fluorescence resonance energy transfer and genetic engineering. The results indicate that IgE has a bent conformation when bound to receptor on the cell surface and that the site of interaction is contained in the C epsilon 2 and C epsilon 3 domains; the C-terminal domain, C epsilon 4, is not required for binding. Cross-linking of IgE-receptor complexes is required for signal transduction across the plasma membrane. Binding studies with defined bivalent ligands indicate that structural and/or kinetic features determine the functional effectiveness of the cross-linked states.

Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen-combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.

Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobulin heavy chain enhancer.

Transgenic rabbits with the rabbit c-myc oncogene fused with the rabbit immunoglobulin heavy chain enhancer region (E mu) DNA were developed by microinjecting pronuclei of single cell zygotes with the gene construct and implanting the microinjected eggs into pseudopregnant females. At age 17-20 days, 3 of 21 offspring born to seven females were found to have peripheral blood leukocyte counts of greater than 100,000 per mm3. Histology analyses showed extensive lymphocytic infiltration in the liver and kidney, indicating that these rabbits had a malignant lymphocytic leukemia. Genomic DNA analyses of thymus and peripheral blood lymphocytes revealed that the leukemic rabbits were transgenic and that both immunoglobulin heavy and kappa light chain genes were rearranged in the leukemic cells; thus, the leukemic cells are of B-cell lineage. One to four heavy and light chain gene rearrangements were observed, suggesting that the B-cell tumors were oligoclonal. Stable tissue culture cell lines from the bone marrow and peripheral blood of one of the transgenic rabbits have been developed. The development of B-cell leukemias in rabbits with the E mu-myc transgene contrasts with the development of B-cell lymphomas in mice carrying a similar transgene. The lymphomas in mice develop in adults and are monoclonal in origin. The leukemias in rabbits develop in juveniles, less than 3 weeks after birth, and appear oligoclonal in origin. The leukemias seem to develop in rabbit at a specific stage of development, and we suggest that a normal developmental signal may be involved in the oncogenesis.

Genetically engineered immunoglobulins reveal structural features controlling segmental flexibility.

We have carried out nanosecond fluorescence polarization studies of genetically engineered immunoglobulins to determine the structural features controlling their segmental flexibility. The proteins studied were hybrids of a relatively rigid isotype (mouse IgG1) and a relatively flexible one (mouse IgG2a). They have identical light chains and heavy chain variable regions and have the same combining sites for epsilon-dansyl-L-lysine, a fluorescent hapten. The fluorescence of the bound dansyl chromophore was excited at 348 nm with subnanosecond laser pulses, and the emission in the nanosecond time range was measured with a single-photon-counting apparatus. The emission anisotropy kinetics of the hybrid antibodies revealed that segmental flexibility is controlled by the heavy chain constant region 1 (CH1) as well as by the hinge. In contrast, the CH2 and CH3 domains did not influence segmental flexibility. The hinge and CH1 domains must be properly matched to allow facile movement of the Fab units. Studies of hybrids of IgG1 and IgG2a within CH1 showed that the loop formed by residues 131-139 is important in controlling segmental flexibility. X-ray crystallographic studies by others of human IgG1 have shown that this loop makes several van der Waals contacts with the hinge.

Secretion of a chimeric T-cell receptor-immunoglobulin protein.

To produce sufficient quantities of soluble T-cell receptor protein for detailed biochemical and biophysical analyses we have explored the use of immunoglobulin--T-cell receptor gene fusions. In this report we describe a chimeric gene construct containing a T-cell receptor alpha-chain variable (V) domain and the constant (C) region coding sequences of an immunoglobulin gamma 2a molecule. Cells transfected with the chimeric gene synthesize a stable protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. We show that the determinant recognized by the anticlonotypic antibody A2B4.2 resides on the V alpha domain of the T-cell receptor. The chimeric protein associates with a normal lambda light chain to form an apparently normal tetrameric (H2L2, where H = heavy and L = light) immunoglobulin molecule that is secreted. Also of potential significance is the fact that a T-cell receptor V beta gene in the same construct is neither assembled nor secreted with the lambda light chain, and when expressed with a C kappa region it does not assemble with the chimeric V alpha C gamma 2a protein mentioned above. This indicates that not all T-cell receptor V regions are similar enough to immunoglobulin V regions for them to be completely interchangeable.

Genetically engineered antibody molecules and their application.

Immunoglobulin genes can be efficiently expressed following transfection into myeloma cells. Using protoplast fusion, transfection frequencies greater than 10(-3) can be achieved. Compatible plasmids containing two different selectible markers are used to simultaneously deliver heavy and light chain genes to the same cell. To produce molecules with differing specificities the rearranged and expressed variable regions can be cloned from the appropriate hybridoma. In some cases, variable regions from cDNAs can be inserted into the expression vectors. It is possible to manipulate the immunoglobulin genes and produce novel antibody molecules. Antibodies have been produced in which the variable regions from mouse antibodies have been joined to human constant regions. In addition, antibodies with altered constant regions have been produced. These genetically engineered antibodies provide a unique set of reagents to study structure-function relationships within the molecule. They also can potentially be used in the diagnosis and therapy of human disease.

Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells.

We have produced a series of hybrid IgG1.IgG2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differences between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse gamma 1 and gamma 2a gene segments. Crossovers occurred throughout these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H2L2 tetramers and properly glycosylated. In addition, all 11 immunoglobulins have identical antigen combining sites specific for the fluorescent hapten epsilon-dansyl-L-lysine. Protein A binding was used as a probe of the structural integrity of the Fc portion of these variant antibodies. Differences in protein A binding between IgG1 and IgG2a appear to be due to amino acid differences at positions 252 (Thr----Met) and 254 (Thr----Ser) of the heavy chain (EU numbering).

A human-mouse chimeric immunoglobulin gene with a human variable region is expressed in mouse myeloma cells.

We have constructed and obtained expression of a chimeric human-mouse immunoglobulin gene after transfection into mouse myeloma cells. A human VDJH gene segment was joined to a mouse C kappa gene in the plasmid vector pSV2-gpt, and the construct was transfected into J558L cells by protoplast fusion. Analyses of six transformants by RIA and SDS-PAGE indicated that the chimeric protein was synthesized in large amounts in five. A kappa-specific transcript was observed by Northern blot analysis. Four out of five clones were stable producers of this chimeric chain over a period of 10 mo.

Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains.

We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line S107, which produces an IgA (kappa) anti-phosphocholine antibody. The heavy chain variable region exon was joined to human IgG1 or IgG2 heavy chain constant region genes, and the light chain variable region exon from the same myeloma was joined to the human kappa light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (kappa) or IgG (kappa) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice.

Expression of a VHC kappa chimaeric protein in mouse myeloma cells.

The heavy (H) and light (L) chains of antibodies consist of variable (V) and constant (C) regions. The V regions of the heavy and light chains form the antibody combining site. To determine whether a V region could be functional when joined to a polypeptide other than its own C region, we constructed a chimaeric gene encoding the V region of a mouse heavy chain and the C region of a mouse kappa light chain ( VHC kappa). The heavy-chain gene is derived from an A/J mouse hybridoma cell line 36-65 whose antibody product (gamma 1, kappa) is specific for the hapten azophenylarsonate. We report here that, when introduced into a mouse myeloma cell line, the chimaeric gene is expressed and a protein of the expected molecular weight is secreted into the medium. As light chains tend to dimerize we expected that the VHC kappa protein might associate with light chain from the cell line 36-65 to form an antibody-binding molecule. Affinity binding experiments and Ka determination indicate that this is the case. Dimers of this type offer a novel and interesting alternative to existing antibody-binding molecules.