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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.

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.

Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.

A monoclonal chimeric antibody ch.MK1 was generated by immunizing F004 mice expressing human instead of murine IgG1/kappa immunoglobulin constant regions. The novel antibody specifically binds cell surface-expressed human neural cell adhesion molecule (NCAM) as shown by immunoprecipitation, flow cytometry and cytospins. Functional analysis revealed nearly complete absence of complement-dependent cytolysis in ch.MK1 and in all other anti-NCAM antibodies tested for reference (UJ13a, ERIC1, 123C3, ch.5A2, B159), indicating an unexpected and group-specific property of anti-NCAM antibodies. As a most plausible mechanism, posttranslational modification of NCAM by complement-inhibiting polysialic acid is discussed. The antibody ch.MK1 demonstrated significant in vivo activity against NCAM-positive neuroblastoma in SCID mice in presence of human peripheral blood mononuclear cell. In absence of human peripheral blood mononuclear cell no distinct antitumor activity of the antibody alone was observed. In ch.MK1 the cellular component of the immune system seems to be the dominant effector mechanism, whereas complement-dependent cytolysis seems not to be necessarily required for antitumor activity. These observations help us to understand immunotherapeutic mechanisms of native anti-NCAM antibodies and may additionally contribute to the understanding of results of currently ongoing clinical studies with conjugated anti-NCAM antibodies.

The clinical success of therapeutic antibodies is demonstrated by the number of antibody therapeutics that have been brought to market and the increasing number of therapeutic antibodies in development. Recombinant antibodies are molecular-targeted therapeutic agents and represent a major new class of drugs. However, it is still very important to optimize and maximize the clinical efficacy of therapeutic antibodies, in part to help lower the cost of therapeutic antibodies by potentially reducing the dose or the duration of treatment. Clinical trials using therapeutic antibodies fully lacking core fucose residue in the Fc oligosaccharides are currently underway, and their remarkable physiological activities in humans in vivo have attracted attention as next-generation therapeutic antibody approaches with improved efficacy. Thus, an industrially applicable antibody production process that provides consistent yields of fully non-fucosylated antibody therapeutics with fixed quality has become a key goal in the successful development of next-generation therapeutic agents. In this article, we review the current technologies for production of therapeutic antibodies with control of fucosylation of the Fc N-glycans.

In this study, we discussed the necessity of human IgG1 Cgamma1 domain for recombinant antibody using computer-aided homology modeling method and experimental studies. The heavy (VH) and light (VL) chain variable regions of 1-28, a murine IgM-type anti-CD20 mAb, were ligated by linker peptide (Gly4Ser)3 to form the single-chain Fv fragment (scFv). Then, the engineered antibody (LH1-3) was generated by fusing scFv with the entire IgG1 heavy constant regions. The 3-D structure of LH1-3 was modeled using computer-aided homology modeling method and the binding activity of LH1-3 was evaluated theoretically. Compared to the 3-D structure of the Fv fragment of the parent antibody, the conformation of the active pocket of LH1-3 was remained because of the rigid support of Cgamma1. Further experimental results of flow cytometry showed that the engineered anti-CD20 antibody possessed specifically binding activity to CD20-expressing target cells. The anti-CD20 antibody fragments could also mediate complement-dependent cytotoxicity (CDC) of human B-lymphoid cell lines. Our study highlights some interests and advantages of a methodology based on the homology modeling and analysis of molecular structural properties.

Humanized antibodies (Abs) are effective drugs against a variety of diseases such as cancer, autoimmune diseases, transplant rejection and others. The most powerful technology to develop humanized Abs is the use of mice that produce humanized Abs. By modifying the genetic background of F004 mice a new mouse substrain was developed for optimized "one step" generation of chimeric humanized monoclonal Abs. The new mice (F004-Jen) demonstrated improved fertility still expressing the human locus at the same level as the parental F004 mouse. The value of these mice for the generation of chimeric Abs was exemplified for a panel of chimeric Abs against the human neural cell adhesion molecule (NCAM): The fully chimeric human IgG1/kappa Ab Ch.MK1 bound to NCAM expressing cells with a K(D)=4.3-8.7 x 10(-8) M and was functionally active as demonstrated by depleting NCAM expressing cells. We also demonstrated that chimeric IgG1/kappa Abs can be induced by hybridoma class switching of IgM producing hybridoma cells, providing an alternative way to chimeric Abs. The present data highlight F004-Jen mice as an efficient tool for "one step" generation of chimeric Abs.

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge., ((c) 2002 Elsevier Science Ltd.)

The C(H)3 domain of antibodies is characterized by two antiparallel beta-sheets forming a disulfide-linked sandwich-like structure. At acidic pH values and low ionic strength, C(H)3 becomes completely unfolded. The addition of salt transforms the acid-unfolded protein into an alternatively folded state exhibiting a characteristic secondary structure. The transition from native to alternatively folded C(H)3 is a fast reaction. Interestingly, this reaction involves the formation of a defined oligomer consisting of 12-14 subunits. Association is completely reversible and the native dimer is quantitatively reformed at neutral pH. This alternatively folded protein is remarkably stable against thermal and chemical denaturation and the unfolding transitions are highly cooperative. With a t(m) of 80 degrees C, the stability of the alternatively folded state is comparable to that of the native state of C(H)3. The defined oligomeric structure of C(H)3 at pH 2 seems to be a prerequisite for the cooperative unfolding transitions., (Copyright 2001 Academic Press.)

AF2 is a high affinity murine Ab possessing potent neutralizing activity against human IFN-gamma. In carrying out the modifications to humanize this Ab, we discovered that an initial version displayed affinity for IFN-gamma that was slightly less than that of AF2, but exhibited IFN-gamma-neutralizing activity that was severely diminished. Characterization via site-directed mutagenesis revealed that the majority of this loss in IFN-gamma-neutralizing activity was due to altering the V(H) framework residue at position 11. V(H) position 11 is distal to the binding surface of the Ab; however, it, along with residues 110 and 112, have been identified as forming the socket of a molecular ball-and-socket joint between the V and C domains of the Ig Fab, which influences the elbow angle between these domains. To determine whether disrupting the structure of this joint was the basis for reduced IFN-gamma-neutralizing capacity, we altered residue 148 of C(H1), which with residue 149 comprises the corresponding ball portion of the joint. Changing this single C(H1) domain residue diminished the ability of the Ab to neutralize IFN-gamma to a level similar to that observed with the V(H) alteration. Thus, an intact ball-and-socket joint between the V and C domains in AF2 is required for potent neutralization of IFN-gamma. These results suggest the importance of the elbow angle between Ig V and C domains in Ab activity, and support the hypothesis that this joint can be an important functional element of Ab structure.

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

The pFc' fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc' fragments were separated from F(ab')2 fragments by affinity chromatography. The pFc' fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable-length peptide libraries. Interacting phage-displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc' binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography.

Two sets of monoclonal antibodies (mAbs) probably reacting with two different epitopes in the CH3 domain of the human IgG4 molecule were studied. We observed that the commercially available mAb HP 6011 inhibited the antigen binding of the three mutually inhibitable mAbs, 40-A2, 41-E8 and 43-F11 (40-series), made by us. However, the 40-series mAbs, including those with similar affinity such as mAb HP6011, were not able to inhibit mAb HP 6011. When the 40-series mAbs were preincubated with IgG4, the mAb HP 6011 could partially displace these antibodies. This one-way inhibition indicates that upon binding mAb HP 6011 changes the antigenic structure of the IgG4 molecule by disrupting the epitope for the 40-series mAbs. A steric hindrance of this epitope by mAb HP 6011 is more unlikely, since the small Fab fragment of mAb HP 6011 also inhibited the reaction of the 40-series mAbs.

We have constructed four different deletion mutants of a chimeric mouse-human IgG3 anti-(4-hydroxy-3-nitrophenyl)acetyl/(5-iodo-4 hydroxy-3 nitrophenyl) acetyl (NP/NIP) antibody lacking one or more of the four exons coding for the hinge region. The mutant variants all retained intact hinge region epitopes since they all reacted with IgG3 hinge-specific antibodies. Surprisingly, all the deletion mutants bound C1q equally well or even better than the wild type. Thus the high C1q binding activity of IgG3 compared to IgG1 is apparently not due to the total length of the IgG3 hinge, which is 62 amino acids, nor is it due to the length of the upper hinge which is the stretch from the end of CH1 to the first inter-heavy chain disulfide bond.

Idiotypes are believed to be due to the structural conformation of the variable region of immunoglobulins (Ig). We have found an idiotype (C3-24) that requires both variable and constant regions of the heavy chain to be expressed. C3-24 Id is associated with both the T15 variable region from anti-phosphorylcholine (PC) antibodies and the constant region for the alpha-heavy chain. High titer anti-PC serum from a variety of inbred strains of different Ig haplotypes failed to express C3-24 Id. However, when IgA but not IgG or IgM fractions were isolated from a pool of anti-PC serum from BALB/c mice, more than 70% of the molecules expressed C3-24 Id. The high frequency of the expression of C3-24 Id in IgA anti-PC hybridoma proteins from mice of different Ig haplotypes and in the IgA fraction of normal anti-PC antibodies from BALB/c and presumably other strains of mice suggests that idiotypic determinants produced by the three-dimensional product of VH and CH regions may not be unusual.