Site of binding of IgG2b and IgG2a by mouse macrophage Fc receptors by using cyanogen bromide fragments

Cyanogen bromide fragments of murine IgG2b and IgG2a immunoglobulins were used to localize the sequences that are bound by specific IgG2b and IgG2a Fc receptors on murine macrophages. One fragment from the CH2 domain of IgG2b bound to the gamma 2b Fc receptor. Two fragments from IgG2a--one one from the CH2 domain, differing by only four amino acids from the homologous IgG2b fragment, and the other from the CH3 domain--specifically bound to the gamma 2a Fc receptor. In both a rosetting assay and a radioactive binding assay, these two fragments from IgG2a competed with intact IgG2a: however, they did not compete with each other. Rather, binding of the fragment from the CH3 domain of IgG2a augmented the binding of the fragment from the CH2 domain of IgG2a but not that of the homologous fragment from IgG2b. The binding of both IgG2a fragments was abolished by trypsin treatment of macrophages. These data suggest that 1) a sequence in the CH2 domain of IgG2b is sufficient for binding to the gamma 2b Fc receptor, 2) sequences from both the CH2 and CH3 domains of IgG2a bind to the gamma 2a Fc receptor, and 3) the binding of sequences from the CH3 domain of IgG2a may induce a conformational change in the gamma 2a Fc receptor that leads to enhanced binding of sequences from the CH2 domain.     

A common theme in interaction of bacterial immunoglobulin-binding proteins with immunoglobulins illustrated in the equine system

The M protein of Streptococcus equi subsp. equi known as fibrinogen-binding protein (FgBP) is a cell wall-associated protein with antiphagocytic activity that binds IgG. Recombinant versions of the seven equine IgG subclasses were used to investigate the subclass specificity of FgBP. FgBP bound predominantly to equine IgG4 and IgG7, with little or no binding to the other subclasses. Competitive binding experiments revealed that FgBP could inhibit the binding of staphylococcal protein A and streptococcal protein G to both IgG4 and IgG7, implicating the Fc interdomain region in binding to FgBP. To identify which of the two IgG Fc domains contributed to the interaction with FgBP, we tested two human IgG1/IgA1 domain swap mutants and found that both domains are required for full binding, with the CH3 domain playing a critical role. The binding site for FgBP was further localized using recombinant equine IgG7 antibodies with single or double point mutations to residues lying at the CH2-CH3 interface. We found that interaction of FgBP with equine IgG4 and IgG7 was able to disrupt C1q binding and antibody-mediated activation of the classical complement pathway, demonstrating an effective means by which S. equi may evade the immune response. The mode of interaction of FgBP with IgG fits a common theme for bacterial Ig-binding proteins. Remarkably, for those interactions studied in detail, it emerges that all the Ig-binding proteins target the CH2-CH3 domain interface, regardless of specificity for IgG or IgA, streptococcal or staphylococcal origin, or host species (equine or human).     

Mapping rheumatoid factor binding sites using genetically engineered, chimeric IgG antibodies.

We are using chimeric IgG antibodies consisting of murine variable regions joined to human constant regions as rheumatoid factor (RF) binding substrates to localize and map IgM RF binding sites on IgG. Using chimeric antibodies in a modified RF ELISA, we showed that RFs from rheumatoid arthritis (RA) and Waldenstrom's macroglobulinemia (WMac) patients differ in their binding specificities for IgG3, although some of these RFs share common specificity for IgG1, IgG2, and IgG4. By shuffling constant region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for WMac-derived RF differentiation of IgG3 and IgG4. By making site-directed mutations in the wild-type IgG3 or IgG4 human gamma constant genes, we showed that His-435 is an essential residue in RF binding to IgG for most WMac RFs. The allotypic polymorphism in IgG3 at 436 is not responsible for differences in previous reports of high-frequency IgG3 binding by WMac RFs. A amino acid loop in the CH2 domain of IgG4 proximal to the CH2-CH3 interface is important in WMac RF binding to IgG; a more distal CH2 loop in CH2 has a more variable effect on WMac RF binding. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating the glycosylation signal Asn-297 to another amino acid. Of all four IgG subclasses, only aglycosylated IgG3 was a better RF binding substrate than its glycosylated subclass counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus

cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma-chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross-hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H-chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H-chains, a mechanism which is not operative in the chicken L-chain locus. The most notable among the chicken Igs is the so-called 7S IgG because its H-chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H-chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8-10 H-chain genes.     

Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea.

The digestion of human IgG1/K myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic immunoglobulin fragments. Fb'2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the light chains, and the CH1 domains and hinge regions of the heavy chains. The smaller fragment, upFc, has now been characterized. After reduction, three peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the heavy chain. Fragment upFc therefore contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulphide loop. Peptic digestion of IgG1/K proteins in 8M-urea therefore provides a method for isolating from gamma1 heavy chains five homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb'2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two gamma1 heavy chains have been shown to differ in sequence at three positions from the sequence reported for protein Eu.     

Baboon immunoglobulin constant region heavy chains: identification of four <Emphasis Type="Italic">IGHG</Emphasis> genes

The increasing use of nonhuman primate models in biomedical research and especially in vaccine development requires the characterization of their immunoglobulin genes and corresponding products. Therefore, we sequenced, cloned and characterized the four immunoglobulin gamma chain constant region genes ( IGHG) present in baboons. These four genes were designated IGHG1, IGHG2, IGHG3 and IGHG4 on the basis of sequence similarities with the four human genes encoding the IgG1, IgG2, IgG3 and IgG4 subclasses and the three known rhesus macaque IGHG genes. Specifically, the baboon IgG1, IgG2, IgG3 and IgG4 sequences exhibit 90.3%, 88.3%, 86.7% and 89.6% amino acid identity to their human counterpart. The percent of amino acid identity of baboon IgG1, IgG2 and IgG3 to the corresponding rhesus macaque sequences is 98.5, 93.1 and 94.4, respectively. Therefore, baboon and rhesus macaque IGHG genes are highly homologous to each other. The majority of differences existing between baboon and human sequences are clustered in the hinge region, with the upper hinge being the most diverse and containing several proline residues. Similar to rhesus macaques, the hinge regions of all baboon IGHG genes consist of a single exon, whereas in humans the IgG3 molecule is encoded by multiple exons. These results confirm the evolutionary instability of the hinge region and indicate that functional properties associated with the hinge regions of baboon and human IgG molecules might differ between the two species.     

Molecular cloning and expression of the mouse high affinity Fc receptor for IgG

Full length cDNA clones encoding the mouse Fc gamma RI were isolated by using redundant oligonucleotide probes based on previously determined amino acid sequence of protein bound to an IgG2a antibody column. Sequence analysis of cDNA clones indicates that mouse Fc gamma RI is a transmembrane glycoprotein that is composed of three disulfide bonded extracellular Ig binding domains unlike Fc gamma RII of man and mouse. These extracellular domains contain five potential sites of N-linked glycosylation; three sites in the first domain and one in each of the second and third domains. In addition a transmembrane region is present followed by a cytoplasmic tail of 84 amino acids. Analysis of the amino acid sequence of the first two extracellular domains of Fc gamma RI indicate that these are highly homologous to the extracellular domains of Fc gamma RII; the third domain is different and shows a lower level of homology to other FcR domains but is clearly related to the Ig super-family. Transfected cells expressing Fc gamma RI were shown to bind immune complexes of rabbit IgG; and monomeric IgG2a bound to transiently transfected cells with an affinity of approximately 5 x 10(7) M-1, i.e. the receptor was of high affinity and therefore was by definition Fc gamma RI. Northern analysis demonstrated that Fc gamma RI mRNA could be detected in the Fc gamma RI+ myeloid cell lines WEH1 3B and J774. Finally, Southern analysis indicated that Fc gamma RI is likely to be encoded by a single copy gene of approximately 9 kb.     

Chimeric Fc receptors identify functional domains of the murine high affinity receptor for IgG.

Chimeric Fc gamma R have been generated between the mouse high affinity receptor for IgG (Fc gamma RI) and the low affinity receptor for IgG (Fc gamma RII) by exchanging the first two domains of the three-domain extracellular structure of Fc gamma RI with the homologous two-domain extracellular structure of Fc gamma RII. Studies of the affinity and specificity of binding of mouse Ig classes to these receptors defined functional regions of Fc gamma RI and showed some surprising results. After removal of the third extracellular domain of Fc gamma RI, the remaining two domains (domains 1 and 2) retained the capacity to bind Ig in the form of immune complexes, however, they bound monomeric IgG2a with a reduced affinity. Surprisingly, these two domains in the absence of the third domain bound not only IgG2a but also IgG1 and IgG2b, i.e., the third domain of Fc gamma RI suppresses the intrinsic capacity of the first two domains to act as a low affinity Fc gamma RII-like molecule. Linking the third extracellular domain of Fc gamma RI to the two extracellular domains of Fc gamma RII resulted in a receptor that retained the specificity and affinity of Fc gamma RII. Thus, the removal of domain 3 from Fc gamma RI resulted in the conversion of Fc gamma RI to an "Fc gamma RII-like" receptor. These findings indicate that domains 1 and 2 of Fc gamma RI form an Ig-binding motif, and although domain 3 is not essential for Fc binding by Fc gamma RI, it plays a crucial role in determining the specific high affinity interaction of Fc gamma RI with IgG2a.     

Impact of a three amino acid deletion in the CH2 domain of murine IgG1 on Fc-associated effector functions

Four murine IgG subclasses display markedly different Fc-associated effector functions because of their differential binding to three activating IgG Fc receptors (FcgammaRI, FcgammaRIII, and FcgammaRIV) and C1q. Previous analysis of IgG subclass switch variants of 34-3C anti-RBC monoclonal autoantibodies revealed that the IgG1 subclass, which binds only to FcgammaRIII and fails to activate complement, displayed the poorest pathogenic potential. This could be related to the presence of a three amino acid deletion at positions 233-235 in the CH2 domain uniquely found in this subclass. To address this question, IgG1 insertion and IgG2b deletion mutants at positions 233-235 of 34-3C anti-RBC Abs were generated, and their ability to initiate effector functions and their pathogenicity were compared with those of the respective wild-type Abs. The insertion of amino acid residues at positions 233-235 enabled the IgG1 subclass to bind FcgammaRIV but did not improve the binding to C1q. Accordingly, its pathogenicity was enhanced but still inferior to that of IgG2b. In contrast, the IgG2b deletion mutant lost its ability to bind to FcgammaRIV and activate complement. Consequently, its pathogenicity was markedly diminished to a level comparable to that of IgG1. Our results demonstrated that the initiation of FcgammaR- and complement-mediated effector functions of IgG2b was profoundly affected by the three amino acid deletion at positions 233-235, but that this natural three amino acid deletion could only partially explain the poor binding of IgG1 to FcgammaRIV and C1q. This indicates the lack in the IgG1 subclass of as yet unknown motifs promoting efficient interaction with FcgammaRIV and C1q.     

Fasciola hepatica: parasite-secreted proteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identification of the immunoglobulin fragments produced

The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.     

Differential mapping of Fc gamma-binding and monoclonal antibody-reactive epitopes on gE, the Fc gamma-binding glycoprotein of herpes simplex virus type 1.

The entire 396 residue extracellular sequence of gE the HSV-1 Fc gamma-binding glycoprotein has been studied to determine epitopes binding to two mAb II-481 and 88S previously demonstrated to react with gE at or near the Fc gamma-binding regions. Overlapping 7-mers constructed from the established sequence were tested with mAb II-481 and 88S along with their Fab fragments. Control mAb of the same IgG 2b subclass as well as whole rabbit and human IgG and Fc were also tested for binding to overlapping linear sequences using the ELISA pin assay to map Fc gamma-binding regions. Six sequences PKTSWRRVS, GLYTLSV, QVASVVLVVQP, PAPPRSWP, CLYHPQLP, and ASTWTSRL were found that constituted major regions binding to the two different mAb of the same specificity. Glycine substitution for each residue within these sequences indicated that arginine 29, tryptophane 70, valine 144, valine 157, arginine 208, histidine 283, and arginine 305 constituted important portions of the II 481 mAb-reactive epitope. Many of the same regions along with one other, GPLHPSW, appeared to be involved in Fc gamma binding. Substitution of glycine for each residue indicated that histidine 67, tryptophane 70, valine 71, valine 157, valine 158, valine 160, valine 161, tryptophane 210, serine 279, cysteine 280, leucine 281, tyrosine 282, histidine 283, proline 284, glutamine 285, proline 287, tryptophane 302, and arginine 305 were important for Fc gamma-binding. Inhibition by gE peptides of rosetting of E sensitized with rabbit IgG antibody around HSV-1-infected cells, as well as inhibition of rosetting using F(ab)2 fragments of rabbit antibodies to these peptides was used to assay relative contributions of all seven regions to Fc gamma-binding activity. Our results provide a tentative map of mAb binding and Fc gamma-reactive sites on gE. mAb and Fc gamma binding of a limited number of individual antigenic amino acids widely distributed among the separate reactive regions suggest that many of the same separate residues contribute both to antigenicity as well as to Fc gamma-binding activity.     

Gene conversion in human immunoglobulin gamma locus shown by unusual location of IgG allotypes

The constant region of the gamma 1, gamma 2 and gamma 3 heavy chains of the human IgG1, IgG2 and IgG3 immunoglobulins carries antigenic determinants or G1m, G2m and G3m allotypes, which are genetic markers of these subclasses. The exceptional presence on gamma 1 and gamma 2 chains of Gm allotypes usually located on the CH3 domain of gamma 3 shows an unexpected clustering of base changes and subsequent identity of short DNA sequences in the CH3 exon of the non-allelic gamma 1, gamma 2 and gamma 3 genes. Such clusters of substitutions are not easily explained on the classical basis of point mutations. A gene conversion, which substituted a segment of the gamma 1 or gamma 2 gene with the homologous region of the non-allelic gamma 3 gene, is more likely. Other examples of possible conversion involving the gamma genes are described. The conservation or the restoration of short sequences produced by the conversion events might be related to the biological properties of the constant region of the heavy chains.     

Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG.

Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII.     

Molecular cloning of rabbit gamma heavy chain mRNA.

A cDNA library of rabbit spleen mRNA was screened for immunoglobulin heavy chain sequences. In this paper we report the nucleotide sequence of two cDNA clones containing part of the constant region of the rabbit gamma heavy chain mRNA. The sequence encodes part of the CH2 domain (amino acids 268 to 340), the entire CH3 domain (amino acids 341 to 447) and the 3' untranslated region. This nucleotide sequence has been compared to the corresponding sequences of mouse gamma 1, gamma 2a and gamma 2b genes. The homologies between rabbit gamma chain gene sequence and each of the mouse gamma chain gene sequences are of the same magnitude order. This comparison shows that the CH2 domains are more homologous to each other than CH3 domains or 3' untranslated sequences. The presence of species specific nucleotide positions suggests that mouse gamma chain genes could have evolved from a common ancestor shortly after the mouse-rabbit species separation. Genomic blot analysis of rabbit liver DNA with the rabbit C gamma probes shows a limited number of related sequences, with little restriction site polymorphism between individual rabbits.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

Human IgG comprises four subclasses with different biological functions. The IgG3 subclass has a unique character, exhibiting high effector function and Fab arm flexibility. However, it is not used as a therapeutic drug owing to an enhanced susceptibility to proteolysis. Antibody aggregation control is also important for therapeutic antibody development. To date, there have been few reports of IgG3 aggregation during protein expression and the low pH conditions needed for purification and virus inactivation. This study explored the potential of IgG3 antibody for therapeutics using anti‐CD20 IgG3 as a model to investigate aggregate formation. Initially, anti‐CD20 IgG3 antibody showed substantial aggregate formation during expression and low pH treatment. To circumvent this phenomenon, we systematically exchanged IgG3 constant domains with those of IgG1, a stable IgG. IgG3 antibody with the IgG1 CH3 domain exhibited reduced aggregate formation during expression. Differential scanning calorimetric analysis of individual amino acid substitutions revealed that two amino acid mutations in the CH3 domain, N392K and M397V, reduced aggregation and increased CH3 transition temperature. The engineered human IgG3 antibody was further improved by additional mutations of R435H to obtain IgG3KVH to achieve protein A binding and showed similar antigen binding as wild‐type IgG3. IgG3KVH also exhibited high binding activity for FcγRIIIa and C1q. In summary, we have successfully established an engineered human IgG3 antibody with reduced aggregation during bioprocessing, which will contribute to the better design of therapeutic antibodies with high effector function and Fab arm flexibility.     

牛1gG高亲和力受体的分子克隆及序列分析

报道编码牛 Ig G高亲和力受体 ( bovine Ig G Fc receptor I,bo FcγR )的全长序列 .从牛肺巨噬细胞 c DNA文库中克隆的该片段全长 1 .4kb,其中的 ORF为 1 0 50 bp,共编码包括信号肽、胞外域、穿膜区和胞内区在内的 349个氨基酸 ,含有 5个潜在的 N-连接糖基化位点 .与人和鼠的 Ig G高亲和力受体 ( hu FcγR 和 mo FcγR )相比 ,其核苷酸同源性分别为 80 %和 69% ,氨基酸同源性分别为 66%和 55% .研究表明 ,人、牛和鼠的 3种 Ig G高亲和力受体的单体 Ig G结合域高度保守     

Identification of residues within the extracellular domain 1 of bovine Fc gamma 2R essential for binding bovine IgG2

Neutrophils and monocytes in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2 (bIgG2), termed bFc gamma 2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass, whose interaction with bFc gamma 2R initiates the killing process. bFc gamma 2R is a transmembrane glycoprotein consisting of two extracellular immunoglobulin-like domains, followed by a 19-amino acid membrane-spanning region and a short cytoplasmic tail. Although related to other mammalian Fc gamma Rs, bFc gamma 2R belongs to a novel gene family that includes the human killer cell inhibitory receptor and Fc alpha RI (CD89) proteins. We have shown previously (Morton, H. C., van Zandbergen, G., van Kooten, C., Howard, C. J., van de Winkel, J. G., and Brandtzaeg, P. (1999) J. Exp. Med. 189, 1715-1722) that like these proteins (and unlike other Fc gamma Rs), bFc gamma 2R binds bIgG2 via the membrane-distal extracellular domain 1 (EC1). In this present study, we introduced mutations into the predicted loop regions of the EC1 domain and assayed the resulting bFc gamma 2R mutants for their ability to bind bIgG2. Our results indicated that the bIgG2 binding site lies within the predicted F-G loop region of the EC1 domain. Furthermore, single amino acid mutational analysis of this region identified Phe-82 and Trp-87 as being critical for bIgG2 binding.     

Engineering a human IgG2 antibody stable at low pH

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid‐induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti‐2,4‐dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti‐DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain—namely, F300Y, V309L, and T339A (IgG2_YLA)—reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen‐binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications.     

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.