Novel blocking human IgG directed against the pentapeptide repeat motifs of Neisseria meningitidis Lip/H.8 and Laz lipoproteins

Ab-initiated, complement-dependent killing contributes to host defenses against invasive meningococcal disease. Sera from nonimmunized individuals vary widely in their bactericidal activity against group B meningococci. We show that IgG isolated from select individuals can block killing of group B meningococci by human sera that are otherwise bactericidal. This IgG also reduced the bactericidal efficacy of Abs directed against the group B meningococcal protein vaccine candidates factor H-binding protein currently undergoing clinical trials and Neisserial surface protein A. Immunoblots revealed that the blocking IgG was directed against a meningococcal Ag called H.8. Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when binding of blocking Ab to meningococci was inhibited using either synthetic peptides corresponding to H.8 or a nonblocking mAb against H.8. Furthermore, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab')(2) fragments were ineffective. Blocking required IgG glycosylation because deglycosylation with peptide:N-glycanase eliminated blocking. C4b deposition mediated by an anti-factor H-binding protein mAb was reduced by intact blocking IgG, but not by peptide:N-glycanase-treated blocking IgG, suggesting that blocking resulted from inhibition of classical pathway of complement. In conclusion, we have identified H.8 as a meningococcal target for novel blocking Abs in human serum. Such blocking Abs may reduce the efficacy of select antigroup B meningococcal protein vaccines. We also propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.     

Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain

IgG antibodies (Abs) and fragments of IgG Abs are becoming major biotherapeutics to treat an assortment of human diseases. Commonly prepared fragments of IgGs include Fc, Fab, and F(ab')2 fragments, all of which can be made using the sulfhydryl protease papain, although prolonged digestion times and/or excessive amounts of papain typically result in further cleavage of the Fc domain into smaller fragments. During our attempts to use papain to isolate Fc fragments from different IgG monoclonal Abs, it was observed that prior removal of Fc glycans resulted in a faster rate of papain-mediated degradation of the Fc domain. Subsequent time-course experiments comparing glycosylated and deglycosylated versions of IgG antibodies showed that the majority of molecules in a deglycosylated IgG sample were converted into Fab, Fc, and smaller Fc fragments in less than one hour, whereas the original glycosylated IgG required more than two hours to convert into a comparable amount of Fab and Fc fragments. Furthermore, whereas papain digestion converted almost all of a deglycosylated Fc fragment into smaller fragments of approximately 10 and approximately 12 kDa within 4 h, more than 40% of a glycosylated Fc fragment remained intact even after 24 h of digestion. These results indicate that the presence of CH(2) domain glycans in either IgGs or purified Fc fragments increases resistance to papain digestion. Increased sensitivity of non-glycosylated Fc domains to papain is consistent with the Fc domains lacking a defined structure, as exemplified by their inability to bind Fcgamma receptors, since misfolded proteins are often degraded by proteases because of increased accessibility of their proteolytic cleavage sites. Based on these observations it is possible to use papain sensitivity as a means of assessing proper Fc structure of IgG molecules.     

Involvement of Fc gamma RI (CD64) in the mechanism of HIV-1 inhibition by polyclonal IgG purified from infected patients in cultured monocyte-derived macrophages

The aim of this study was to investigate the mechanism of HIV-1 neutralization using monocyte-derived macrophages (MDM) in comparison to PBMC as target cells. For this purpose, we analyzed neutralizing activities of different human polyclonal IgG samples purified from sera of HIV-1-infected individuals using a single cycle infection assay. We found an increase of the neutralizing titer when macrophages vs PBMC were used as target cells. Moreover, polyclonal IgG from HIV-1-infected patients that are not able to neutralize virus when PBMC are used as target cells strongly inhibit MDM infection. Similar results were obtained with neutralizing mAbs. To explore the participation of FcgammaRs in HIV-1 inhibition, F(ab')(2) and Fab of these Igs were produced. Results indicated that both F(ab')(2) and Fab are less effective to inhibit virus replication in MDM. Moreover, competition experiments with Fc fragments of IgG from healthy donors or with purified monoclonal anti-human FcgammaRs Ab strengthen the participation of the FcgammaRs, and in particular of FcgammaRI (CD64) in HIV-1 inhibition on MDM. Mechanisms by which HIV-specific IgG inhibit virus replication in cultured macrophages are proposed and the benefit of inducing such Abs by vaccination is discussed.     

Ligand-loaded but not free complement receptors for C3b/C4b and C3d co-cap with cross-linked B cell surface IgM and IgD

We have performed experiments to investigate possible physical interactions between C receptors (CR) and surface Ig (sIg) on the B cell plasma membrane. These molecules were found to be independent, non-linked, B cell surface structures, because capping CR1, CR2, sIgM, or sIgD with a specific antibody did not affect the distribution of the remainder of these molecules. Both CR1 and CR2, if bound by antibodies that did not independently cap CR, however, became associated with cross-linked sIg because CR that have been bound by intact anti-CR antibodies or their Fab fragments co-capped with sIgM or sIgD that had been bound by divalent anti-IgM or anti-IgD antibody. CR1 that had bound C3b similarly co-capped with sIg when sIg was cross-linked. Ligand-bound or even cross-linked CR did not associate with non-cross-linked sIg because sIgD, bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with CR that had been cross-linked by a sandwich of mouse anti-CR antibody and goat anti-mouse Ig. Other surface molecules, such as B1 and HLA-DR Ag, when bound by specific antibodies, did not cap with cross-linked sIg, and sIgD, when bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with cross-linked sIgM. Interactions between CR and sIg were not mediated by an association with IgG FcR because co-capping of CR and sIg was observed when F(ab')2 fragments of both anti-CR and anti-Ig antibodies were used. These results demonstrate that B cell surface CR can become associated with sIg, but only if sIg is cross-linked and CR is bound by anti-CR antibody or has bound its natural ligand.     

A fluorescence study on the mobility of surface antigens of untreated tumor cells and of tumor cells undergoing cell-mediated lysis

Heterologous (rabbit) antibodies were raised against murine P-815 mastocytoma cells of DBA/2 origin. Antisera and IgG preparations were highly cytotoxic, whereas Fab fragments thereof lost all activity. Fab fragments also showed a much lower avidity than IgG, both for tumor and normal DBA/2 and C57 spleen cells as measured by the release of iodinated Fab and IgG. Both preparations bound specifically to P-815 cells since they were capable of inhibiting T cell-mediated target cell lysis. The binding of IgG and monovalent Fab fragments was studied by fluorescence. Rhodamine-coupled IgG bound homogeneously in the cold and quickly formed patches upon warming but did not form caps even after prolonged incubation at 37 degrees C. Rhodamine-coupled Fab fragments also bound homogeneously. Their distribution was unaltered after incubation at 37 degrees C even when tumor cells formed uropod-like tails. Fab fragments, however, could be induced to cap with a second and third antibody layer. P-815 cells labeled with rhodamine-coupled Fab fragments were incubated with cytolytic T cells (CTL). The conjugates formed between CTL and fluorescent target cells were observed. No gross redistribution of surface antigens on target cells was observed even at late stages of the lytic process. CTL, therefore, do not seem to operate via a redistribution of surface antigens.     

The murine B cell repertoire is severely selected against endogenous cellular prion protein

Abs to the prion protein (PrP) can protect against experimental prion infections, but efficient Ab responses are difficult to generate because PrP is expressed on many tissues and induces a strong tolerance. We previously showed that immunization of wild-type mice with PrP peptides and CpG oligodeoxynucleic acid overcomes tolerance and induces cellular and humoral responses to PrP. In this study, we compared Ab and T cell repertoires directed to PrP in wild-type and PrP knockout (Prnp o/o) C57BL/6 mice. Animals were immunized with mouse PrP-plasmid DNA or with 30-mer overlapping peptides either emulsified in CFA or CpG/IFA. In Prnp o/o mice, Abs raised by PrP-plasmid DNA immunization recognized only N-terminal PrP peptides; analyses of Ab responses after PrP peptide/CFA immunization allowed us to identify six distinct epitopes, five of which were also recognized by Abs raised by PrP peptides/CpG. By contrast, in wild-type mice, no Ab response was detected after PrP-plasmid DNA or peptide/CFA immunization. However, when using CpG, four C-terminal peptides induced Abs specific for distinct epitopes. Importantly, immune sera from Prnp o/o but not from wild-type mice bound cell surface PrP. Abs of IgG1 and IgG2b subclasses predominated in Prnp o/o mice while the strongest signals were for IgG2b in wild-type mice. Most anti-PrP Th cells were directed to a single epitope in both Prnp o/o and wild-type mice. We conclude that endogenous PrPC expression profoundly affects the Ab repertoire as B cells reactive for epitopes exposed on native PrPC are strongly tolerized. Implications for immunotherapy against prion diseases are discussed.     

A phage‐displayed single‐chain Fab library optimized for rapid production of single‐chain IgGs

Phage‐displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full‐length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate‐limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage‐displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage‐displayed synthetic single‐chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single‐chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage‐derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell‐surface receptors, and scIgGs behave the same as conventional IgGs.     

Regional and segmental flexibility of antibodies in interaction with antigens of different size

The interaction of antibodies (Abs) with protein antigens (Ags) of different size, such as hen egg white lysozyme, ovalbumin, and bovine serum albumin, was examined using analytical ultracentrifugation, electrospray ionization time-of-flight mass spectrometry, and surface plasmon resonance in order to estimate regional and segmental Ab flexibility. When both Abs and Ags were free in solution, sedimentation equilibrium and surface plasmon resonance analyses showed the formation of an Ag(2)Ab(1) complexes regardless of Ag size, suggesting that the Fab arms were able to move to avoid interference between Ags bound to Ab combining sites. The Ag(2)Ab(1) complex, as well as the Ag(1)Ab(1) complex, was observed by MS. However, when Abs were immobilized on the surface of a sensor chip through the Fc region, the stoichiometry of the Ag-Ab complex was dependent on the Ag size; Ag(2)Ab(1) forming with hen egg white lysozyme and Ag(1)Ab(1) with ovalbumin and bovine serum albumin. These results indicated that immobilization of the Fc region reduces the dynamic range of the Fab arms and results in interference from the first Ag bound to either combining site, which in turn prevents the binding of the second Ag to the other combining site. Our results allow us to propose that the Fab arms of B-cell receptors whose Fc regions are immobilized on cell surface have a reduced dynamic range.     

Ontogeny of proteolytic immunity: IgM serine proteases

We report the chemical activity of immunoglobulin micro and kappa/lambda subunits expressed on the surface of B cells and in secreted IgM antibodies (Abs) found in the preimmune repertoire. Most of the nucleophilic reactivity of B cells measured by formation of covalent adducts of a hapten amidino phosphonate diester was attributed to micro and kappa/lambda subunits of the B cell receptor. Secreted IgM Abs displayed superior nucleophilic reactivity than IgG Abs. IgM Abs catalyzed the cleavage of model peptide substrates at rates up to 344-fold greater than IgG Abs. Catalytic activities were observed in polyclonal IgM Abs from immunologically na?ve mice and humans without immunological disease, as well as monoclonal IgM Abs to unrelated antigens. Comparison of several IgM Abs indicated divergent activity levels and substrate preferences, with the common requirement of a basic residue flanking the cleavage site. Fab fragments of a monoclonal IgM Ab expressed catalytic activity, confirming the V domain location of the catalytic site. The catalytic reaction was inhibited by the covalently reactive hapten probe and diisopropylfluorophosphate, suggesting a serine protease-like mechanism. These observations indicate the existence of serine protease-like BCRs and secreted IgM Abs as innate immunity components with potential roles in B cell development and Ab effector functions.     

Human anti-IgG1 hinge autoantibodies reconstitute the effector functions of proteolytically inactivated IgGs

A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab')(2) possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab')(2)s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab')(2), as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab')(2). Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab')(2) in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.     

Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge

Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.     

Analysis of the interaction of antibodies with immunoglobulin idiotypes on neoplastic B lymphocytes: implications for immunotherapy

We have examined the reactions of a panel of nine monoclonal anti-idiotype antibodies with the surface immunoglobulin in situ on guinea pig L2C leukemic lymphocytes. Equilibrium binding constants were shown to range between 10(7) and 10(8) M-1 for univalent Fab' gamma fragments and between 10(8) and 10(9) M-1 for intact IgG. Saturation of the cell surface binding sites was achieved with 2.9 X 10(5) Fab' gamma molecules/cell and 1.2 X 10(5) IgG molecules/cell for each antibody, a result that is consistent with a bivalent mode of interaction for the IgG. Despite these overall similarities in binding characteristics antibodies showed striking differences in their ability to clear Ig from the cell surface by antigenic modulation in vitro. This suggested differences in the readiness with which the antibodies cross-linked neighboring surface Ig molecules. Such an interpretation was supported by differences in the times required to achieve bivalent binding at 0 degree C, and in the rates at which labeled antibody dissociated from the cell surface in the presence or absence of an excess of unlabeled antibody. The data are consistent with there being two functionally distinct types of anti-idiotype antibody: those that form predominantly intra-Ig bridges, with each antibody Fab being linked to an Fab on one target molecule ("monogamous" binding) and not favoring modulation; and those that form predominantly inter-Ig bridges ("bigamous" binding) and favor modulation. The nature of interaction is presumably dictated by the orientation of the particular idiotope concerned. This distinction could be of great importance in the therapeutic use of anti-idiotype to ablate B cell neoplasms.     

Receptor modulation by Fc gamma RI-specific fusion proteins is dependent on receptor number and modified by IgG.

The high-affinity IgG receptor, FcgammaRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcgammaRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcgammaRI causes receptor recycling, while Abs that cross-link FcgammaRI cause rapid down-modulation of surface FcgammaRI. Because studies performed in the absence of ligand may not be representative of FcgammaRI modulation in vivo, we investigated the ability of FcgammaRI-cross-linking Abs and non-cross-linking derivatives to modulate FcgammaRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcgammaRI on the human myeloid cell line, U937, and its high FcgammaRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcgammaRI expression and correlated with internalization of both wH22xeGFP and FcgammaRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcgammaRI, was unable to modulate FcgammaRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcgammaRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcgammaRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.     

Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.     

MuSK Myasthenia Gravis IgG4 Disrupts the Interaction of LRP4 with MuSK but Both IgG4 and IgG1-3 Can Disperse Preformed Agrin-Independent AChR Clusters

A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 MuSK antibodies will also contribute to the reduced AChR density and neuromuscular dysfunction in myasthenia patients with MuSK antibodies.     

Different adaptations of IgG effector function in human and nonhuman primates and implications for therapeutic antibody treatment

Safety of human therapeutic Abs is generally assessed in nonhuman primates. Whereas IgG1 shows identical FcγR interaction and effector function profile in both species, fundamental differences in the IgG2 and IgG4 Ab subclasses were found between the two species. Granulocytes, the main effector cells against IgG2- and IgG4-opsonized bacteria and parasites, do not express FcγRIIIb, but show higher levels of FcγRII in cynomolgus monkey. In humans, IgG2 and IgG4 adapted a silent Fc region with weak binding to FcγR and effector functions, whereas, in contrast, cynomolgus monkey IgG2 and IgG4 display strong effector function as well as differences in IgG4 Fab arm exchange. To balance this shift toward activation, the cynomolgus inhibitory FcγRIIb shows strongly increased affinity for IgG2. In view of these findings, in vitro and in vivo results for human IgG2 and IgG4 obtained in the cynomolgus monkey have to be cautiously interpreted, whereas effector function-related effects of human IgG1 Abs are expected to be predictable for humans.     

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.     

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.     

Crystal structure of the middle domain of human poly(A)-binding protein-interacting protein 1

This communication describes SAXS data based global structures of tetravalent antibody CD4–IgG2 and its dimeric to pentameric complexes with gp120s. Comparison of models brought forth that while the two CD4s grafted on each arm remain tightly packed in the unliganded antibody, they enable binding of first two gp120s preferentially to the same Fab arm in an asymmetric manner. Retention of residues in the CD4–Fab linker earlier reasoned to enable bi-fold collapse of gp120-bound soluble CD4, and observed asymmetry of the (CD4–IgG2)/(gp120)₂ complex suggest that encoded flexibility in CD4–Fab linker is a critical structure–function factor for this broad spectrum neutralizing antibody.     

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTβR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti- LTβR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a full-length anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.