The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens.

B cells are activated in vivo after the B cell receptors (BCRs) bind to antigens captured on the surfaces of antigen-presenting cells. Antigen binding results in BCR microclustering and signaling; however, the molecular nature of the signaling-active BCR clusters is not well understood. Using single-molecule imaging techniques, we provide evidence that within microclusters, the binding of monovalent membrane antigens results in the assembly of immobile signaling-active BCR oligomers. The oligomerization depends on interactions between the membrane-proximal Cmicro4 domains of the membrane immunoglobulin that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cmicro4 domain failed to cluster and signal, and conversely, Cmicro4 domains alone clustered spontaneously and activated B cells. These results support a unique mechanism for the initiation of BCR signaling in which antigen binding induces a conformational change in the Fc portion of the BCR, revealing an interface that promotes BCR clustering.

Impact of IgA constant domain on HIV-1 neutralizing function of monoclonal antibody F425A1g8.

With the majority of HIV infections resulting from mucosal transmission, induction of an effective mucosal immune response is thought to be pivotal in preventing transmission. HIV-specific IgA, but not IgG, has been detected in the genital tract, seminal fluid, urethral swabs, urine, and vaginal wash samples of HIV-negative sex workers and HIV-status discordant couples. Purified mucosal and plasma IgA from some individuals with highly exposed, persistently seronegative status can neutralize infection and present cross-clade neutralization activity, though present at low levels. We generated a CD4-induced human mAb, F425A1g8, and characterized the impact of its isotype variants on HIV neutralizing activity. The result showed that, in contrast to little neutralization by the F425A1g8 IgG1 in the absence of sCD4, the IgA1 variant of the Ab displayed significant independent neutralization activity against a range of HIV clade B isolates in the absence of sCD4. Studies of the neutralizing function of IgA isotypes, and the functional relationship between different antigenic epitopes and IgA Abs, may also suggest strategies for the intervention of virus transmission and spread within the mucosa of the host, as well as serve to inform the design of vaccine strategies that may be more effective at preventing mucosal transmission. This research clearly suggests that IgA isotype, because of its unique molecular structure, may play an important role in HIV neutralization.

Antibody C region influences TGN1412-like functional activity in vitro.

The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab')(2) and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the "wild-type" IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm-exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab')(2) fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.

Structure of a human IgA1 Fab fragment at 1.55†Šresolution: potential effect of the constant domains on antigen-affinity modulation.

Despite being the most abundant class of immunoglobulins in humans and playing central roles in the adaptive immune response, high-resolution structural data are still lacking for the antigen-binding region of human isotype A antibodies (IgAs). The crystal structures of a human Fab fragment of IgA1 in three different crystal forms are now reported. The three-dimensional organization is similar to those of other Fab classes, but FabA1 seems to be more rigid, being constrained by a hydrophobic core in the interface between the variable and constant domains of the heavy chain (VH-CH1) as well as by a disulfide bridge that connects the light and heavy chains, influencing the relative heavy/light-chain orientation. The crystal structure of the same antibody but with a G-isotype CH1 which is reported to display different antigen affinity has also been solved. The differential structural features reveal plausible mechanisms for constant/variable-domain long-distance effects whereby antibody class switching could alter antigen affinity.

Molecular and functional characterization of cynomolgus monkey IgG subclasses.

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc-FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.

Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1.

Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function.

Inhibition of complement-mediated immune hemolysis by peptides derived from the constant domain of immunoglobulin.

High-dose administration of intravenous immunoglobulin is reported to be useful for inhibiting complement-dependent immune cytolysis. We have found that, among the proposed C1q-binding sites of the Fc portion of human IgG1, only residues 282-292 inhibited pig red blood cell lysis by human serum. Moreover, a hexadecemeric multiple antigen peptide of residues 282-292 from IgG showed significantly greater activity in suppressing complement-mediated immune cytolysis and can be used in place of high-dose intravenous immunoglobulin, which is extracted from donors and thus is expensive.

Phylogeny of immunoglobulin structure and function. XIV. Peptide map and amino acid composition studies of shark antibody light chains.

Light chains from antibodies to the streptococcal A-variant carbohydrate from individual nurse sharks were compared by peptide maps of tryptic digests and amino acid compositions. Although the amino acid compositions of the different chains were quite similar, considerable differences as well as similarities were demonstrable on peptide maps. The peptide maps were interpreted as indicating that shark L chains likely have constant and variable regions as seen in the immunoglobulins of higher animals. Furthermore the unique peptides characteristic of different L chains support the hypothesis that nurse sharks, as a species, possess a relatively large number of different L chain amino acid sequences which are compatible with antibody binding sites to the streptococcal antigen. Hence the repetoire of nurse shark antibody combining sites to this antigen appears to be quite extensive.

H chain C domains influence the strength of binding of IgG for streptococcal group A carbohydrate.

We have produced a panel of murine anti-streptococcal mAbs, expressing identical V domains and different H chain C domains, corresponding to the IgG3, IgG1, and IgG2b subclasses. We have used these mAb to evaluate the role of IgG subclass-specific C region determinants in modulating the interaction between antibody and the bacterial surface. We report, for the first time, that V region-identical murine IgG of different subclasses exhibit substantial differences in binding to specific Ag; IgG3 mAb binds more strongly to streptococci than the IgG1 and IgG2b mAb or IgG3-derived F(ab')2 fragments. Furthermore, the IgG3 mAB binds cooperatively to the bacteria, whereas the IgG1, IgG2b, and IgG3-derived F(ab')2 fragments do not exhibit significant cooperativity, which suggests that differences in Fc region structure can affect antibody binding to multivalent Ag by modulating the potential for cooperative binding. These results suggest a plausible mechanism by which murine IgG3 could be more effective, than other antibodies bearing identical V domains, but of different gamma-subclass, in mediating bacterial immunity.

Biological properties of chimeric antibodies. Interaction with complement.

Genetic engineering allows the production of chimeric immunoglobulins where the same variable region is expressed in conjunction with constant regions of different species. We have compared the capacity of mouse and chimeric (mouse variable region/human constant region) immunoglobulin M (IgM) to trigger complement-dependent lysis and to bind complement component C1. Guinea pig and human C1 were bound more efficiently by mouse IgM than by chimeric IgM, whereas rat and rabbit C1 were bound more efficiently by chimeric IgM. Comparable results were obtained for complement as measured by the lysis of sensitized erythrocytes. These results indicated that differences between the human and mouse mu heavy chain constant regions define structures that are important in the C1-IgM interaction. Furthermore, species-specific differences in C1 also influence this interaction.

Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules.

During biosynthesis, class II major histocompatibility complex molecules are intimately associated with invariant chain (Ii). The Ii-class II association has been shown to block peptide-class II binding and to affect the ultimate conformation of class II expressed on the cell surface. To assess the biochemical basis for the effects of Ii on class II, we have analyzed the biosynthesis of class II in EL4 cells transfected with I-Ad with and without Ii. In these studies, we found that Ii had a profound effect on the biosynthesis of I-Ad. In the absence of Ii, class II could form dimers efficiently, but these dimers appeared to be misfolded and this altered conformation resulted in the loss of some monoclonal antibody epitopes and inefficient transport from the endoplasmic reticulum to the Golgi. In addition, class II that was transported through the Golgi accumulated an abnormally increased molecular mass associated with N-linked glycosylation. Subsequent transfection of Ii into these cells resulted in recovery of normal class II conformation, causing a restoration of monoclonal antibody epitopes, efficient intracellular transport, and normal glycosylation. Together, these data indicate that Ii can have a profound effect on the folding, transport, and modification of class II molecules and suggest that one function of Ii may be to act as a class II-specific chaperone.

Effector functions of a mouse IgG that lacks the entire CH1 domain. C1q binding and complement fixation in the absence of antigen.

C1q binding and complement fixation were examined of a short-chain IgG2a antibody that lacks the entire CH1 domain. This short-chain antibody has been reported to have a low level of constitutive complement-activating activity in the absence of Ag. Two-dimensional SDS/PAGE and cation-exchange chromatography have demonstrated that two types of IgG2a proteins are secreted by the short-chain IgG2a antibody producing cell line. It has been shown that 1) the difference between these two types of the IgG2a proteins is whether the two L chains are linked by a disulfide bridge or not, and 2) C1q-binding and complement-activating activities are expressed only when the inter L chain disulfide bridge does not exist. A molecular model is presented for the active form of the short-chain IgG2a antibody.

Silencing of the immunoglobulin heavy chain locus by removal of all eight constant-region genes in a 200-kb region.

Silencing or removal of individual C (constant)-region genes and/or adjacent control sequences did not generate fully deficient Ig (immunoglobulin)- mice. A reason is that different C genes share many functional tasks and most importantly are individually capable of ensuring lymphocyte differentiation. Nevertheless, incomplete arrests in B-cell development were found, most pronounced at the onset of H-chain expression. Here we show that removal of 200 kb accommodating all C genes, Cmu-Cdelta-Cgamma3-Cgamma1-Cgamma2b-Cgamma2a-Cepsilon-Calpha, stops antibody production. For this two loxP targeting constructs were introduced into the most 5' C gene and the distal alpha 3' enhancer. Cre-loxP-mediated in vivo deletion was accompanied by extensive germ-line mosaicism, which could be separated by breeding. Homozygous C-gene deletion mice did not express Ig H or L chains and flow cytometry revealed a complete block in B-cell development. However, C-gene removal did not affect DNA rearrangement processes following locus activation, as recombination efficacy appears to be similar to what is found in normal mice.

Molecular and serologic analysis of IgG1 deficiency caused by new forms of the constant region of the Ig H chain gene deletions.

Selective IgG1 deficiency is a rare disease. We report a familial form of IgG1 deficiency, in which IgG1 was undetectable in a 5-yr-old girl with a history of asthma and respiratory tract infections. Her father had an IgG1 level that was one-third of the mean amount found in normal healthy controls. The defect in the proband was caused by a homozygous deletion of the structural gene for C gamma 1. A Southern blot analysis demonstrated that the maternal haplotype contained a deletion encompassing C gamma 1, C psi epsilon 1, C alpha 1, C psi gamma, and C gamma 2, whereas the deletion on the paternal haplotype was confined to the C gamma 1 gene. Neither of these deletions has previously been reported. IgG1 normally constitutes the dominant isotype for antibodies directed against protein Ag, including viral proteins. We have analyzed the immune response to a number of different protein and polysaccharide Ag in the patient and her parents. In the proband, antiviral antibodies were restricted to the IgG3 and IgG4 subclasses. However, the total amount of IgG directed against several viruses was below the concentration found in normal seropositive individuals. The father and the paternal grandfather, both with low serum IgG1 levels, also had asthma, thus indicating a possible causal relationship.

Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes.

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-beta. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer's patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-gamma and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.

Conventional and surrogate light chains differentially regulate Ig mu and Dmu heavy chain maturation and surface expression.

Positive selection of precursor (pre-) B cells by Ig membrane mu H chains (mum HC) and counterselection mediated by the truncated HC Dmu depend on the ability of each HC to form a pre-B cell receptor (pre-BCR) signaling complex with the surrogate L chain (SLC) components lambda5 and Vpre-B. To better understand how pre-BCR signaling output is determined by its Ig components and the SLC, we investigated the regulation of pre-BCR surface expression and HC secretory maturation in a new nonlymphoid system. We took this approach as a means to distinguish B-lineage-specific effects from pre-BCR-intrinsic properties that may influence these aspects of pre-BCR homeostasis necessary for signaling. As in pre-B cells, the SLC in nonlymphoid cells supported only a limited degree of mum HC maturation and low pre-BCR surface expression levels compared with conventional LCs, indicating that this was due to an intrinsic property of the SLC. We identified the non-Ig region of lambda5 as harboring the restrictive activity responsible for this phenotype. This property of lambda5 was also evident with Dmu, but the overall SLC- and L chain-dependent requirements for Dmu maturation and surface expression were markedly different from those for mum. Surprisingly, Dmu was modified in an unusual manner that was only dependent on Vpre-B. These results establish a novel function of lambda5 in limiting surface pre-BCR levels and reveal biochemical properties of Ig molecules that may underlie the diverse consequences of pre-BCR signaling in vivo by different HCs.

Role for the third constant domain of the IgG H chain in activation of complement in the presence of C1 inhibitor.

The multidomain architecture of Ig H chains was initially implicated in the variety of functions imposed on each species of Ig. However, the activation of C by IgG is the only function that has been attributed to a single domain of C gamma 2, whereas most of other functions of IgG require both C gamma 2 and C gamma 3 domains. This one domain-one function relationship in the C activation by IgG, too, was questioned recently by the fact that a C gamma 3-less fragment of rabbit IgG, F(acb)2, is definitely less capable of activating C than intact IgG. Here we reexamined capacities of F(acb)2 to bind and activate C1 in the presence and absence of C1 inhibitor (C1-In) in comparison with intact IgG, by using SRBC sensitized with these proteins (EFacb, EIgG). At an ionic strength of 0.065 and 37 degrees C, where C1q was bound equally well by these cells and the dissociation was limited, C1s, presumably in the form of C1r2C1s2, dissociated from EFacb at a rate 7-fold greater than that from EIgG, irrespective of the presence or absence of C1-In. A physiologic concentration of C1-In reduced the rate of C1 activation by EFacb to 5% that by EIgG. The results present evidence that the C gamma 3 domain, too, plays a crucial part in the C1 activation process by stabilizing the zymogenic conformation of C1 and protecting it from the attack by C1 inhibitor.

Duplication of primate and rodent B7-H3 immunoglobulin V- and C-like domains: divergent history of functional redundancy and exon loss.

B7-H3 is a novel protein structurally related to the B7 family of ligands by the presence of a single set of immunoglobulin-V-like and immunoglobulin-C-like (VC) domains. By multiplex PCR, the dominantly expressed form of human B7-H3 was found to be a splice variant containing tandemly duplicated VC domains (VCVC). In contrast, mouse B7-H3 cDNA contained only one single VC form due to an exon structure corresponding to V-(pseudoexon C)-(pseudoexon V)-C. Comparisons of human, monkey, mouse, and hamster genomic B7-H3 reveal that primates, but not rodents, exhibited a higher degree of intramolecular sequence similarity between VC duplications than between molecules. Both VC and VCVC forms of human B7-H3 inhibited CD4(+) T cell proliferation and downregulated cytokine production upon TCR activation. These results suggest independent, but convergent, paths of B7-H3 active domain duplication followed by divergent histories of exon degeneration in rodents and exon maintenance by humans.

Efficient neutralization and disruption of rhinovirus by chimeric ICAM-1/immunoglobulin molecules.

The intercellular adhesion molecule 1 (ICAM-1) is used as a cellular receptor by 90% of human rhinoviruses (HRVs). Chimeric immunoadhesin molecules containing extracellular domains of ICAM-1 and constant regions of immunoglobulins (Igs) were designed in order to determine the effect of increased valency, Ig isotype, and number of ICAM-1 domains on neutralization and disruption of rhinovirus structure. These immunoadhesins include ICAM-1 amino-terminal domains 1 and 2 fused to the hinge and constant domains of the heavy chains of IgA1, IgM, and IgG1 (IC1-2D/IgA, -/IgM, and -/IgG). In addition, all five extracellular domains were fused to IgA1 (IC1-5D/IgA). Immunoadhesins were compared with soluble forms of ICAM-1 containing five and two domains (sICAM-1 and ICI-2D, respectively) in assays of HRV binding, infectivity, and conformation. In prevention of HRV plaque formation, IC1-5D/IgA was 200 times and IC1-2D/IgM and IC1-2D/IgA were 25 and 10 times more effective, respectively, than ICAM-1. The same chimeras were highly effective in inhibiting binding of rhinovirus to cells and disrupting the conformation of the virus capsid, as demonstrated by generation of approximately 65S particles. The results show that the number of ICAM-1 domains and a flexible Ig hinge are important factors contributing to the efficacy of neutralization. The higher efficiency of chimeras that bound bivalently in disrupting HRV was attributed to higher binding avidity. The IC1-5D/IgA immunoadhesin was effective at nanomolar concentrations, making it feasible therapy for rhinovirus infection.