Wild-type Antibody Research Articles

ChIP-on-Chip Analysis Using Dominant Negative Form of PAX5 Fusion Gene Revealed Genes Associated with Tumorigenesis Were Directly Regulated by PAX5 in a Human B Cell Line, NALM6

PAX5 is a transcriptional factor playing an important role in B-cell development. Overexpression of PAX5 induced by translocation to the enhancer region of immunoglobulin heavy chain gene occurs in non-Hodgkin lymphomas (NHL), suggesting that PAX5 can be also associated with development of NHL. To identify genes associated with tumorigenesis in malignancies overexpressing PAX5, we performed ChIP-on-chip analysis using PAX5 specific antibody. Non-specifically immunoprecipitated DNA by antibodies can cause false positive results using ChIP-on-chip analysis (background). To reduce the background in ChIP-on chip analysis, we used a dominant negative form of PAX5 and a wild-type PAX5 specific antibody for our ChIP-on-chip analysis. We have previously found a PAX5 chimeric protein expressed in acute lymphoblastic leukemia in which the C-terminal end of PAX5 was replaced by C20ORF112 protein (Kawamata N et al, Proc Natl Acad Sci U S A. Aug. 12, 2008). We have also found that this chimeric protein behaved in a dominant negative fashion over the wild-type PAX5 and suppressed expression of target genes of wild-type PAX5. PAX5 chimeric protein can compete with wild-type PAX5 for binding on the promoter region of direct down-stream target genes. To identify the genes directly regulated by PAX5 in human B-cells, we transfected the dominant-negative form of PAX5 chmeric protein, PAX5-C20ORF112 (PAX5-C20S) into NALM6 human B-cell leukemia cells which constitutively express abundant PAX5. Transfected cells were collected and chromatin immunoprecipitation (ChIP) assay was performed using PAX5 C-terminal specific antibody which can recognize only wild-type PAX5, but not the chimeric PAX5 protein, PAX5C20S. As a control, we also performed ChIP assay using NALM6 cells transfected with an empty vector. Immunoprecipitated DNA was recovered and amplified using the whole genome amplification technique. The DNAs were hybridized with oligonucleotide probes containing the promoter regions of the human genome. The levels of hybridized DNA were quantified and genes directly bound by PAX5 were identified. Comparison between NALM6 cells transfected with the empty vector and PAX5C20S significantly reduced the background and allowed identification of genes directly regulated by PAX5 in NALM6, including BUB1B, SSSCA1, CEP68, and BAG1. BUB1B, CEP68 and SSSCA1 are proteins involved in mitosis; BAG1 is a protein associated with apoptosis. Dysregulation of these genes by overexpressed PAX5 may be associated with development of B-cell malignancies.

Engineered Antibodies of IgG1/IgG3 Mixed Isotype with Enhanced Cytotoxic Activities

Enhancement of multiple effector functions of an antibody may be a promising approach for antibody therapy. We have previously reported that fucose removal from Fc-linked oligosaccharides greatly enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies. Here, we report a unique approach to enhance complement-dependent cytotoxicity (CDC), another important effector function of antitumor antibodies, by using engineered constant region of human IgG1/IgG3 chimeric isotypes. We systematically shuffled constant domains of IgG1 and IgG3 to generate a comprehensive set of mixed chimeric isotypes of anti-CD20 antibodies. Among these, the variant 1133, consisting of the CH1 and the hinge each from IgG1 and the Fc from IgG3, was unexpectedly found to exhibit markedly enhanced CDC that exceeded wild-type levels. However, it lacked protein A-binding capacity, an important feature for the industrial production. To eliminate this deficiency, a portion in COOH-terminal CH3 domain of 1133 was substituted with IgG1, resulting in full recovery of protein A binding without compromising the enhanced CDC and ADCC activities. The CDC-enhancing effect using a chimeric isotype was also shown in CD52 antigen/antibody system. The ADCC activity of the variants was also maximized by the absence of fucose from its carbohydrate structure, a phenomenon that has previously been observed for wild-type antibodies. Enhanced cytotoxicity of a variant was confirmed in a cynomolgus monkey model. These findings suggest that the variant antibodies with IgG1/IgG3 chimeric constant regions and nonfucosylated oligosaccharides that possess dual-enhanced cytotoxic functions may be an improvement for the next generation of therapeutic antitumor antibodies.

The Effect of a Point Mutation on the Stability of IgG4 as Monitored by Analytical Ultracentrifugation

There is presently considerable interest in the state of aggregation and biophysical integrity of antibody preparations, and recent advances in the analysis of data from the analytical ultracentrifuge renders it a powerful probe of these stability phenomena, under both storage and freeze-thaw conditions. Solutions of a wild-type IgG4 antibody and a single amino acid hinge mutant IgG4m (serine residue 241 converted to proline) were exposed to different accelerated stress conditions, namely (i) elevated temperature storage for various periods (up to 59 days at 37 degrees C) or (ii) a series of freeze-thaw cycles (storage at -80 degrees C then incubation at 20 degrees C for 1 h under different conditions). Analysis using the nondisruptive probe of sedimentation velocity in the analytical ultracentrifuge indicated that for both antibodies the monomer was always the most common species present whatever storage regime had been used. Sedimentation coefficient distribution analysis showed that other higher oligomer species and half-antibodies were present, and appeared to be not in chemical equilibrium with each other. Solution heterogeneity was found to increase considerably with treatment for both native and hinge-mutant antibodies although the latter appeared to be more resistant to freeze-thaw-induced aggregation.

Regulation of p53 Nuclear Export through Sequential Changes in Conformation and Ubiquitination

Wild-type p53 is a conformationally labile protein that undergoes nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 nuclear export by exposing or activating a nuclear export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation. The cytoplasmic localization of mutant p53s required the C-terminal NES and an intact ubiquitination pathway. Mutant p53 ubiquitination occurred at lysines in both the DNA-binding domain (DBD) and C terminus. Interestingly, Lys to Arg mutations that inhibited ubiquitination restored nuclear localization to mutant p53 but had no apparent effect on p53 conformation. Further studies revealed that wild-type p53, like mutant p53, is ubiquitinated by MDM2 in both the DBD and C terminus and that ubiquitination in both regions contributes to its nuclear export. MDM2 binding can induce a conformational change in wild-type p53, but this conformational change is insufficient to promote p53 nuclear export in the absence of MDM2 ubiquitination activity. Taken together, these results support a stepwise model for mutant and wild-type p53 nuclear export. In this model, the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination. The addition of ubiquitin to DBD and C-terminal lysines then promotes nuclear export via the C-terminal NES.

MDM2 Binding Induces a Conformational Change in p53 That Is Opposed by Heat-shock Protein 90 and Precedes p53 Proteasomal Degradation

p53 protein conformation is an important determinant of its localization and activity. Changes in p53 conformation can be monitored by reactivity with wild-type conformation-specific (pAb-1620) or mutant conformation-specific (pAb-240) p53 antibodies. Wild-type p53 accumulated in a mutant (pAb-240 reactive) form when its proteasome-dependent degradation was blocked during recovery from stress treatment and in cells co-expressing p53 and MDM2. This suggests that conformational change precedes wild-type p53 degradation by the proteasome. MDM2 binding to the p53 N terminus could induce a conformational change in wild-type p53. Interestingly, this conformational change was opposed by heat-shock protein 90 and did not require the MDM2 RING-finger domain and p53 ubiquitination. Finally, ubiquitinated p53 accumulated in a pAb-240 reactive form when p53 degradation was blocked by proteasome inhibition, and a p53-ubiquitin fusion protein displayed a mutant-only conformation in MDM2-null cells. These results support a model in which MDM2 binding induces a conformational change that is opposed by heat-shock protein 90 and precedes p53 ubiquitination. The covalent attachment of ubiquitin may "lock" p53 in a mutant conformation in the absence of MDM2-binding and prior to its degradation by the proteasome.

Application of humanized FcRn mouse model for in vivo testing of therapeutic antibodies (131.37)

Abstract IgG antibodies modified for high binding affinity for their receptor FcRn have emerged as a major treatment for a wide array of both neoplastic and autoimmune diseases. The goal of this study was to analyze the effect of human IgG1 antibodies with enhanced in vitro binding to FcRn on their in vivo half-life in mice deficient in mouse FcRn and expressing human FcRn. Two mutants (N434A and T307A/E380A/N434A) of the humanized monoclonal antibody Hu4D5, directed against epidermal growth factor receptor 2 (p185 HER2), show increased pH-dependent binding to human FcRn in vitro. They also have an extended half-life in vivo compared to wild-type antibody in FcRn transgenic mice but not in mice expressing endogenous mouse FcRn. When injected into FcRn-humanized mice at a concentration sufficient to partially saturate human FcRn, the mutants with an extended serum half-life were most effective in reducing the half-life of a tracer antibody. Finally, we use a serum-transfer model of arthritis and FcRn-humanized mice to test whether mutant antibodies could have enhanced therapeutic potential by promoting accelerated clearance of arthritogenic antibodies. Results indicate that the engineered mutant T307A/E380A/N434A is most effective in vivo in ameliorating the inflammatory arthritic lesions. In summary, FcRn-humanized mice are a promising animal model for human IgG therapeutic development.

Monoclonal Antibody Clearance: IMPACT OF MODULATING THE INTERACTION OF IgG WITH THE NEONATAL Fc RECEPTOR

The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations, which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-alpha (TNFalpha)). The T250Q/M428L anti-TNFalpha variant displayed an approximately 40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNFalpha variant (P257I/Q311I) whose binding kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for murine FcRn was increased approximately 500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNFalpha T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When administered intravenously to mice, the T250Q/M428L anti-TNFalpha variant displayed improved pharmacokinetics, characterized by an approximately 2-fold slower clearance than the wild-type antibody. The discrepancy between these data and previously reported benefits in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing the disposition or elimination of monoclonal antibodies.

Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin

Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.

Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease

The MHC class I-like Fc receptor FcRn plays an essential role in extending the half-life (t(1/2)) of IgG antibodies and IgG-Fc-based therapeutics in the circulation. The goal of this study was to analyze the effect of human IgG1 (hIgG1) antibodies with enhanced in vitro binding to FcRn on their in vivo t(1/2) in mice expressing human FcRn (hFcRn). Mutants of the humanized monoclonal Herceptin antibody (Hu4D5-IgG1), directed against human epidermal growth factor receptor 2 (p185 (HER2)), show altered pH-dependent binding to hFcRn in vitro. Two engineered IgG1 mutants (N434A and T307A/E380A/N434A) showed a considerably extended t(1/2) in vivo compared with wild-type antibody in mice expressing an hFcRn transgene (Tg) but not in mice expressing the endogenous mouse FcRn. The efficiency of hFcRn-mediated protection was dependent on hFcRn Tg copy number. Moreover, when injected into FcRn-humanized mice at a concentration sufficient to partially saturate hFcRn, the engineered IgG1 mutants with an extended serum t(1/2) were most effective in reducing the t(1/2) of a tracer hIgG1 antibody. Finally, administration of mutant with high binding to hFcRn ameliorated arthritis induced by passive transfer with human pathogenic plasma. These results indicate that Fc regions modified for high binding affinity to hFcRn increases serum persistence of therapeutic antibodies, that the same approach can be exploited as an anti-autoimmune therapy to promote the clearance of endogenous pathogenic IgG and that FcRn-humanized mice are a promising surrogate for hIgG therapeutic development.

Effects of mutation at the D‐JH junction on affinity, specificity, and idiotypy of anti‐progesterone antibody DB3

The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.

Protection of mice against Human respiratory syncytial virus by wild-type and aglycosyl mouse–human chimaeric IgG antibodies to subgroup-conserved epitopes on the G glycoprotein

Monoclonal antibodies (mAbs) to conserved epitopes on the G glycoprotein of human respiratory syncytial virus (HRSV) subgroup A fail to neutralize the virus in cell culture in the absence of complement, but are protective in rodent models of infection. They may have potential as prophylactic agents in human infants. In order to investigate the role of Fc-dependent pathways in protection by one such antibody, 1C2, the V(H) and V(L) genes were isolated by RT-PCR and assembled with human kappa light-chain and human gamma1 heavy-chain constant-region genes to form two mouse-human chimaeras, which were expressed in NS0 cells. One of the chimaeras carried a wild-type gamma1 chain, whilst the other had an aglycosyl mutation in the C(H)2 domain rendering the antibody defective in complement activation and FcgammaR binding. Whilst both chimaeric antibodies exhibited similar avidity for HRSV in ELISA, only the fully glycosylated wild type was capable of neutralizing the virus in the presence of complement. In mice passively immunized with either murine or wild-type gamma1 chimaeric antibody, no virus could be recovered from the lungs 4 days after intranasal inoculation of HRSV. In mice immunized with the aglycosyl gamma1 chimaera, however, virus was present in the lungs following challenge, although virus titres were significantly reduced compared with controls (P < 0.005). These results indicate that the protective effect of this antibody is mediated by both Fc-dependent and Fc-independent pathways.

Intravascular survival of red cells coated with a mutated human anti-D antibody engineered to lack destructive activity

AbstractAlloimmune feto-maternal destruction of blood cells is thought to be mediated by binding of alloantibodies to Fc receptors on effector cells. Blocking the antigen using inert antibodies might prolong cell survival. We have performed a “proof of principle” study in volunteers to measure the intravascular survival of autologous red cells coated with human recombinant IgG antibody containing a novel constant region, G1Δnab, devoid of in vitro cytotoxic activity. RhD-positive red blood cells (RBCs), labeled with chromium-51 or technetium-99m, were separately coated to equal levels with wild-type IgG1 or G1Δnab anti-D antibody (Fog-1). After re-injection, there was complete, irreversible clearance of IgG1-coated RBCs by 200 minutes, concomitant with appearance of radiolabel in plasma. Gamma camera imaging revealed accumulation in spleen and, at higher coating levels, in liver. In contrast, clearance of G1Δnab-coated cells was slower, incomplete, and transient, with whole blood counts falling to 7% to 38% injected dose by about 200 minutes before increasing to 12% to 67% thereafter. There was no appearance of plasma radiolabel and no hepatic accumulation. These findings suggest that G1Δnab-coated RBCs were not hemolysed but temporarily sequestered in the spleen and that our approach merits investigation in larger studies.

Improved Effector Functions of a Therapeutic Monoclonal Lewis Y-Specific Antibody by Glycoform Engineering

The aim of the present study was to produce glycosylation variants of the therapeutic Lewis Y-specific humanized IgG1 antibody IGN311 to enhance cell-killing effector function. This was achieved via genetic engineering of the glycosylation machinery of the antibody-producing host. Antibody genes were transiently cotransfected with acetyl-glycosaminyltransferase-III genes into human embryonic kidney-EBV nuclear antigen cells. A control wild-type antibody, IGN311wt, was expressed in the same host using identical expression vectors, but without cotransfection of genes for acetyl-glycosaminyltransferase-III expression. Both expression products were purified to homogeneity and characterized. The glyco-engineered expression product (IGN312-Glyco-I) showed a remarkably homogenous N-linked glycosylation pattern consisting of one major hybrid-type, non-fucosylated and agalactosylated form carrying a bisecting GlcNAc-group. Wild-type expression product (IGN311wt) on the other hand was glycosylated by a multitude of different core-fucosylated complex-type structures of variable degrees of galactosylation. Target affinity of the glyco-engineered antibody as well as heavy and light chain assembly were not affected by acetyl-glycosaminyltransferase-III expression. In vitro experiments showed a approximately 10-fold increase of antibody-dependent cellular cytotoxicity of the glyco-engineered antibody using different Lewis Y-positive target cancer cell lines (SK-BR-3, SK-BR-5, OVCAR-3, and Kato-III). Complement-mediated cytotoxicity of IGN312-Glyco-I was 0.4-fold reduced using SK-BR-5 as target cell line. The reduction of complement activation could be prevented and even converted into a slight increase of activity by using a different molecular-biological approach directing the glycosylation towards increased levels of complex N-linked oligosaccharides of bisected, non-fucosylated type, as a result of cotransfection of mannosidase II together with acetyl-glycosaminyltransferase-III.

Fine Tuning of the Specificity of an Anti-progesterone Antibody by First and Second Sphere Residue Engineering

The specificity of anti-progesterone P15G12C12G11 antibody was improved by combination of in vitro scanning saturation mutagenesis and error-prone PCR. The most evolved mutant is able to discriminate against 5beta- or 5alpha-dihydroprogesterone, 23 and 15 times better than the starting antibody, while maintaining the affinity for progesterone that remains in the picomolar range. The high level of homology with anti-progesterone monoclonal antibody DB3 allowed the construction of three-dimensional models of P15G12C12G11 based on the structures of DB3 in complex with various steroids. These models together with binding data, derived from site-directed mutagenesis, were used to build a phage library in which five first sphere positions in complementarity-determining regions 2H and 3L were varied. Variants selected by an initial screening in competition against a large excess of 5beta- or 5alpha-dihydroprogesterone were characterized by a convergent amino acid signature different from that of the wild-type antibody and had lower cross-reactivity. Binding properties of this first set of mutants were further improved by the addition of second sphere mutations selected independently from an error-prone library. The three-dimensional models of the best variant show changes in the antigen binding site that explain well the increase in selectivity. The improvements are partly linked to a change in the canonical class of the light chain third hypervariable loop.

WT1 expression in angiogenic tumours of the skin

To determine the expression of WT1 in endothelial proliferations and tumours. Endothelial cells are derived from angioblasts which differentiate into bone marrow stem cells (BMSC). BMSC are characterized by the constitutive expression of the WT1 gene and we have postulated that its expression may be maintained during the differentiation of angioblasts to endothelial cells. The expression of WT1 was studied in human umbilical vein-derived (HUVEC) and brain microvascular endothelial cells (HBME) as well as in a Kaposi sarcoma (KS) cell line in vitro. Forty-two human skin biopsy samples of endothelial proliferations and tumours were analysed for the protein expression of WT1 using the monoclonal antibodies for wt-WT1 (6F-H2) and its 17AA+ variant (2C12). WT1 expression was detectable in HUVEC and KS cells and all WT1 splice variants examined (17AA+/- KTS+/-) were detectable in KS cells, while the 17AA+/- and KTS- variants were present in HUVEC. Immunohistochemical analysis of the 42 human skin biopsy samples revealed cytoplasmic WT1 expression using wild-type specific antibody (6FH2) in microvessels, which is maintained during neoangiogenesis (inflammation, haemorrhage, peritumoral angiogenesis). Around one-third of haemangiomas (3/10) and non-HIV-Kaposi sarcomas (7/18) expressed the WT1 protein in the cytoplasm of tumour cells compared with its frequent expression in angiosarcomas (7/8) using the same antibody (6FH2). The nuclear 17AA+ isoform of WT1 was detectable at protein level in a small proportion of KS cases exclusively (3/7). Our data suggest that WT1 protein expression is maintained during angiogenesis and malignant transformation of endothelial cells and can be considered as a new endothelial marker.

Identification of functionally important residues in the pyridoxal-5'-phosphate-dependent catalytic antibody 15A9.

Antibody 15A9 is unique in its ability to catalyze the transamination reaction of hydrophobic D-amino acids with pyridoxal-5'-phosphate (PLP). Both previous chemical modification studies and a three dimensional (3-D) homology model indicated the presence of functionally important tyrosine residues in the antigen-binding cavity of antibody 15A9. To gain further insight into the hapten, ligand binding, and catalytic mechanism of 15A9, all tyrosine residues in the complementarity-determining regions (CDRs) and the single arginine residue in CDR3 of the light chain were subject to an alanine scan. Substitution of Tyr(H33), Tyr(L94), or Arg(L91) abolished the catalytic activity and reduced the affinity for PLP and N(a)-(5'-phosphopyridoxyl)-amino acids, which are close analogues of covalent PLP-substrate adducts. The Tyr(H100b)Ala mutant possessed no detectable catalytic activity, while its affinity for each ligand was essentially the same as that of the wild-type antibody. The binding affinity for the hapten was drastically reduced by a Tyr(L32)Ala mutation, suggesting that the hydroxyphenyl group of Tyr(L32) participates in the binding of the extended side chain of the hapten. The other Tyr --> Ala substitutions affected both binding and catalytic activity only to a minor degree. On the basis of the information obtained from the mutagenesis study, we docked N(alpha)-(5'-phosphopyridoxyl)-D-alanine into the antigen-binding site. According to this model, Arg(L91) binds the alpha-carboxylate group of the amino acid substrate and Tyr(H100b) plays an essential role in the catalytic mechanism of antibody 15A9 by facilitating the Calpha/C4' prototropic shift. In addition, the catalytic apparatus of antibody 15A9 revealed several mechanistic features that overlap with those of PLP-dependent enzymes.

Engineered Human IgG Antibodies with Longer Serum Half-lives in Primates

The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.

Differential binding to human FcγRIIa and FcγRIIb receptors by human IgG wildtype and mutant antibodies

We are investigating the interactions of recombinant human IgG antibodies with Fc receptors to enable selection of a constant region giving minimal depletion of antigen-bearing cells. Eight variant constant regions were made by substituting motifs between human IgG subclasses in the lower hinge region and/or a specially close loop of the CH2 domain. Mutations in the lower hinge region were shown to eliminate FcγRI binding and monocyte activation [Eur. J. Immunol. 29 (1999) 2613]. Here, we detail interactions with FcγRIIa of the 131R and 131H allotypes and FcγRIIb. Lower hinge mutations caused large reductions in binding whereas modification of residues 327, 330 and 331 had less dramatic effects. However, like the wildtype IgG subclass binding hierarchies, the effect of the mutations varied between different receptors. We identified IgG1 variants which react with the activating receptor, FcγRIIa, at least 10-fold less efficiently than wildtype IgG1 but whose binding to the inhibitory receptor, FcγRIIb, is only four-fold reduced. Manipulation of interactions with FcγRIIb separately from those with activating receptors provides potential for designing antibodies with novel and effective combinations of attributes. In addition, insight is gained into the evolution of functional differences in human IgG subclasses.

Development and implementation of an electrochemiluminescence immunoassay for the determination of an angiogenic polypeptide in dog and rat plasma

A quantitative method based on electrochemiluminescence immunoassay for the determination of the angiogenic agent aFGF-S117 has been developed and validated. Two polyclonal antibodies specific to aFGF-S117 and a wild-type aFGF antibody were selected for the analysis. The assay was based on the non-competitive sandwich immunoassay principle in which the drug is trapped with a biotinylated antibody that is immobilized on a streptavidin magnetic particle. The drug is then sandwiched with a ruthenium chelated second antibody. The assay demonstrates good accuracy and reproducibility at plasma concentration of 0.5 ng/ml.

Structural Consequences of Target Epitope-directed Functional Alteration of an Antibody: THE CASE OF ANTI-HEN LYSOZYME ANTIBODY, HyHEL-10

Decreased affinity of an antibody for a mutated epitope in an antigen can be enhanced and reversed by mutations in certain antibody residues. Here we describe the crystal structures of (a) the complex between a naturally mutated proteinaceous antigen and an antibody that was mutated and selected in vitro, and (b) the complex between the normal antigen and the mutated antibody. The mutated and selected antibody recognizes essentially the same epitope as in the wild-type antibody, indicating successful target site-directed functional alteration of the antibody. In comparing the structure of the mutated antigen-mutant antibody complex with the previously established structure of the wild-type antigen-wild-type antibody complex, we found that the enhanced affinity of the mutated antibody for the mutant antigen originated not from improvements in local complementarity around the mutated sites but from subtle and critical structural changes in nonmutated sites, including an increase in variable domain interactions. Our findings indicate that only a few mutations in the antigen-binding region of an antibody can lead to some structural changes in its paratopes, emphasizing the critical roles of the plasticity of loops in the complementarity-determining region and also the importance of the plasticity of the interaction between the variable regions of immunoglobulin heavy and light chains in determining the specificity of an antibody.