The Fab Conformations in the Solution Structure of Human Immunoglobulin G4 (IgG4) Restrict Access to Its Fc Region: IMPLICATIONS FOR FUNCTIONAL ACTIVITY*

Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser²²²) and a hinge mutant IgG4(Pro²²²) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s_20,w⁰ of 6.6–6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration R_g was unchanged with concentration in 50–250 mm NaCl buffers, whereas the neutron R_g values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser²²²) with extended hinge structures. The IgG4(Pro²²²) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications.     

Selective determination of human immunoglobulin G by flow‐injection chemiluminescence

A novel flow‐injection chemiluminescence method was developed for the selective determination of human immunoglobulin G (IgG) in the presence of thiomersal by changing the flow rates of peristaltic pump. The study was based on the independence and additivity of the CL signals of human IgG and thiomersal in the galangin–potassium permanganate–polyphosphoric acid system. In meantime, two equations relating to the concentrations of mixing solutions of human IgG and thiomersal vs the CL intensity were established and solved, on the basis of which the content of thiomersal included in samples was simultaneously determined too. The enhanced CL intensity was in proportion to concerntrations in the range 8.0 × 10^?7 to 8.0 × 10^?5 g/mL for human IgG and 1.0 × 10^?7 to 2.0 × 10^?6 g/mL for thiomersal with the detection limits of 5.0 × 10^?7 g/mL for human IgG and 6.0 × 10^?8 g/mL for thiomersal, respectively. The relative standard deviation for 1.0 × 10^?5 g/mL human IgG was 0.8% and for 2.0 × 10^?7 g/mL thiomersal it was 2.0% (n = 10). The proposed method was applied to determine three synthetic samples with recoveries of 91.5–109.5%. In addition, the possible chemiluminescence mechanisms are discussed as well. Copyright © 2010 John Wiley & Sons, Ltd.     

Specificity and distribution of antigenic determinants on the polyoma virus capsid and nature of the reaction of immunoglobulin G antibody with the capsid surface.

Antisera to the LP and SP34 strains of polyoma virus have been prepared and their reactions with purified virions studied by double diffusion in agar and direct assay of antibody binding. One or more common antigenic determinants appear on the capsids of both strains. The form of this determinant varies slightly on each of the strains tested. The SP34 strain also carries its own strain-specific antigenic determinant. Both strains of virus were able to bind 150 anti-LP IgG³ molecules per virion and 50 anti-SP IgG molecules per virion. The slow rate of dissociation of bound IgG antibody (k_dissociation = 2 × 10⁻⁶ s⁻¹), and the rapid rate of antibody binding (k_dissociation = 2 × 10⁷m⁻¹ s⁻¹), suggest that IgG antibody is bound to the capsid surface through two antigen-antibody bonds. 50 anti-SP IgG molecules per capsid, divalently bound, completely inhibit the binding of 150 anti-LP IgG molecules, and vice versa. Consideration of the symmetry and molecular dimensions of the IgG molecule and the polyoma virus capsid leads to a model of the divalent interaction of IgG antibody with the common antigenic determinant(s). In this model, one species of antibody binds divalently to opposed subunits of a hexamer morphological unit. The other species of antibody binds divalently to the subunits on either side of the point of tangency of any two morphological units.     

Abundant Intracellular IgG in Enterocytes and Endoderm Lacking FcRn

FcRn, a non-classical MHCI molecule, transports IgG from mother to young and regulates the rate of IgG degradation throughout life. Brambell proposed a mechanism that unified these two functions, saying that IgG was pinocytosed nonspecifically by the cell into an FcRn-expressing endosome, where, at low pH, it bound to FcRn and was exocytosed. This theory was immediately challenged by claims that FcRn specificity for ligand could be conferred at the cell surface in neonatal jejunum. Assessing Brambell''s hypothesis we found abundant nonspecifically endocytosed IgG present in the cytoplasm of FcRn^−/− enterocytes. Further, IgG was present in the intercellular clefts and the cores of FcRn^+/+ but not FcRn^−/− jejunum. FcRn specificity for ligand could be determined within the cell.     

Porcine IgG: structure,genetics, and evolution

Eleven genomic porcine Cγ gene sequences are described that represent six putative subclasses that appear to have originated by gene duplication and exon shuffle. The genes previously described as encoding porcine IgG1 and IgG3 were shown to be the IgG1^a and IgG1^b allelic variants of the IGHG1 gene, IgG2a and IgG2b are allelic variants of the IGHG2 gene, while “new” IgG3 is monomorphic, has an extended hinge, is structurally unique, and appears to encode the most evolutionarily conserved porcine IgG. IgG5^b differs most from its putative allele, and its C_H1 domain shares sequence homology with the C_H1 of IgG3. Four animals were identified that lacked either IgG4 or IgG6. Alternative splice variants were also recovered, some lacking the C_H1 domain and potentially encoding heavy chain only antibodies. Potentially, swine can transcribe >20 different Cγ chains. A comparison of mammalian Cγ gene sequences revealed that IgG diversified into subclasses after speciation. Thus, the effector functions for the IgG subclasses of each species should not be extrapolated from “same name subclasses” in other species. Sequence analysis identified motifs likely to interact with Fcγ receptors, FcRn, protein A, protein G, and C1q. These revealed IgG3 to be most likely to activate complement and bind FcγRs. All except IgG5^a and IgG6^a should bind to FcγRs, while all except IgG6^a and the putative IgG5 subclass proteins should bind well to porcine FcRn, protein A, and protein G. An erratum to this article can be found at     

IgG and IgM levels in dairy cows during the periparturient period

In dairy cows, the incidence of infectious diseases during the periparturient period is high. The most common diseases ante partum (a.p.) and post partum (p.p.) are mastitis and puerperal toxicaemia, puerperal septicaemia, and chronic endometritis, respectively. Studies suggest that this is related to an immunosuppressed status during this period.Therefore, the aim of this study was to determine the periparturient immune status characterized by concentrations of IgG and IgM in peripheral blood and colostrum samples of dairy cows and to assess in detail whether variations in immunoglobulin levels may be related to age and status of productivity. In addition, a possible correlation between the course of immunoglobulin levels and lymphocyte concentrations was assessed.Eighteen clinically healthy German Holstein and Red Holstein dams were selected for this study and sampled regularly between the 8^th week a.p. and the 4^th week p.p. IgG and IgM levels were determined using two novel competitive ELISAs.Results demonstrated a dramatic decrease of serum IgG and IgM levels beginning at the 8^th week and 4^th week a.p., respectively, both reaching trough at parturition. The IgG level recovered by the 4^th week p.p., while IgM concentrations remained low. The extent of IgG reduction seemed to be dependent on the initial IgG concentration when the cow was dried-off (8^th week a.p.). In contrast to IgM, the degree of IgG reduction correlated significantly with the IgG concentrations in the colostrum. Furthermore, a cross-correlation between the IgG levels and the lymphocyte counts was detectable (P < 0.01).In conclusion, the antepartal decline of blood IgG and IgM levels as well as the low periparturient IgG levels could reflect a “physiological phenomenon” of dairy cattle. If the phenomenon is associated with an unstable immune system, it must be assessed in future studies. Nonetheless, a sensitive immune system could explain the high incidence for infectious diseases during this period.     

M-like,immunoglobulin-binding protein ofStreptococcus pyogenes type M15

An M-like protein fromStreptococcus pyogenes type M15 strain EF1949 (EMML15) was cloned inEscherichia coli and sequenced. Recombinant EMML15 protein revealed a unique binding pattern for human IgG subclasses not described previously. Comparative analysis of the EMML15 amino acid sequence with those of other M-like proteins of opacity factor positive (OF⁺) serotypes and protein H, an IgG receptor from OF^– serotype M1, showed that IgG-binding proteins with common binding of IgG3 were closely related and distinct from streptococcal IgG receptors not binding IgG3. Thus, the Ig-binding proteins fromS. pyogenes were subdivided into two main categories according to binding pattern, protein structure, and gene location.     

Kinetic analysis of the binding of immunoglobulins IgG1 and IgG2 to bovine mammary cells

Previous reports were confirmed that specific binding sites exist on bovine mammary cells near parturition presumably involved in the transfer of immunoglobulins IgG1 and IgG2 across the mammary gland at the time of colostrum formation. Determination of the kinetic parameters of these binding sites using ¹²⁵I-labeled IgG1 and IgG2 immunoglobulins indicated the presence of sites with association constants (K_a) of about 5 · 10⁸?10 · 10⁸ M^?1 for both subclasses during normal lactation with about 9000 and 3000 sites per cell for each, respectively. The number of IgG1 sites tended to increase as the time of parturition approached. In addition, a new group of sites numbering about 5000 per cell with very strong binding of IgG1 (K_a about 45 · 10⁸ M^?1) appeared on the cells about a week before parturition. The numbers and affinity of the IgG1 and IgG2 binding sites bear a relationship to the approximate 7:1 ratio of these immunoglobulin subclasses found in colostrum and normal milk and to the time of maximum colostrum formation. The results support the premise that a highly selective transport mechanism exists in the bovine mammary epithelial cell for the transfer of IgG1 and IgG2 immunoglobulins from blood to the lacteal secretions.     

Activation of T lymphocytes by the Fc portion of immunoglobulin

T lymphocytes are stimulated to release T-cell-replacing factors in response to Fc fragments of human IgG. Lyt 1⁺23^? T cells are directly triggered to factor production by Fc subfragments, derived from intact Fc fragments by macrophage-dependent enzymatic cleavage. These factor(s) replace T cell function in two Fc-mediated immune responses; induction of polyclonal antibody synthesis, and potentiation of anti-SRBC responses.     

Exploring the Energy Profile of Human IgG/Rat Anti-human IgG Interactions by Dynamic Force Spectroscopy

Interactions between antibody and antigen molecules play essential roles in biological recognition processes as well as medical diagnosis. Therefore, an understanding of the underlying mechanism of antibody?Cantigen interactions at the single molecular level would be beneficial. In the present study, human immunoglobulin (IgG) tethered cantilevers and rat anti-human IgG functionalized gold surfaces were fabricated by using self-assembled monolayers method. Dynamic force spectroscopy was employed to characterize the interactions between human (IgG) and rat anti-human IgG at the single-molecule level. The unbinding forces were determined to be 44.6?±?0.8, 65.8?±?3.0, 108.1?±?4.1, 131.1?±?11.2, 149.5?±?4.7, 239.5?±?3.1 and 294.7?±?7.7?pN with ramping loading rates of 514, 1,127, 3,058, 7,215, 15,286, 31,974 and 50,468?pN?s^-1, respectively. In addition, the unbinding forces were found to be increasing with the logarithm of apparent loading rates in a linear way. Fitting data group resulted in two distinct linear parts, suggesting there are two energy barriers. The corresponding distances in the bound and transition states (x _?? ) and the dissociation rates (K _off ) were calculated to be 0.129?±?0.006?nm, 3.986?±?0.162?s^?1 for the outer barrier and 0.034?±?0.001?nm, 36.754?±?0.084?s^?1 for the inner barrier. Such findings hold promise of screening novel drugs and discerning different unbinding modes of biological molecules.     

Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s⁰_20,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier R_G values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.     

A radioimmunoassay for the Gm(b0) allotype of human immunoglobulins

A radioimmunoassay for the human allotype Gm(b⁰) which provides a sensitive and quantitative measurement of the level of this IgG3 genetic marker has been developed. The assay system can detect 15 nanograms of Gm(b⁰) IgG3 protein and is not inhibited by immunoglobulins of other allotypes and isotypes. Using this assay, good correlation was found between IgG3 and Gm(b⁰) levels in homozygous Gm(f, b⁰) sera and gene dosage effects could be confirmed. The correlation between Gm(b⁰) levels and IgG3 in Negroid Gm(a, b⁰) sera was not as good. This reduced correlation has been attributed to antigen differences in the IgG3 Gm markers characteristic of some Negroid Gm(a, b⁰) sera.     

Influence of the bispecific antibody IgG subclass on T cell redirection

ABSTRACT

T cell redirection mediated by bispecific antibodies (BsAbs) is a promising cancer therapy. Dual antigen binding is necessary for potent T cell redirection and is influenced by the structural characteristics of a BsAb, which are dependent on its IgG subclass. In this study, model BsAbs targeting CD19xCD3 were generated in variants of IgG1, IgG2, and IgG4 carrying Fc mutations that reduce FcγR interaction, and two chimeric IgG subclasses termed IgG1:2 and IgG4:2, in which the IgG1- or IgG4-F(ab)₂ are grafted on an IgG2 Fc. Molecules containing an IgG2 or IgG4-F(ab)₂ domain were confirmed to be the most structurally compact molecules. All BsAbs were shown to bind both of their target proteins (and corresponding cells) equally well. However, CD19xCD3 IgG2 did not bind both antigens simultaneously as measured by the absence of cellular clustering of T cells with target cells. This translated to a reduced potency of IgG2 BsAbs in T-cell redirection assays. The activity of IgG2 BsAbs was fully restored in the chimeric subclasses IgG4:2 and IgG1:2. This confirmed the major contribution of the F(ab)₂ region to the BsAb’s functional activity and demonstrated that function of BsAbs can be modulated by engineering molecules combining different Fc and F(ab)₂ domains.

Abbreviations: ADCC: Antibody-dependent cellular cytotoxicity; AlphaScreen^TM: Amplified Luminescent Proximity Homogeneous Assay Screening; ANOVA: Analysis of variance; BiTE: bispecific T-cell engager; BSA: bovine serum albumin; BsAb: bispecific antibody; cFAE: controlled Fab-arm exchange; CDC: complement-dependent cellular cytotoxicity; CIEX: cation-exchange; CIR: chimeric immune receptor; DPBS: Dulbecco’s phosphate-buffered saline; EC₅₀ value: effective concentration to reach half-maximum effect; EGFR: epidermal growth factor receptor; EI: expansion index (RA_t=x/RA_t=0); FACS: fluorescence-activated cell sorting; FVD: fixable viability dye; HI-HPLC: hydrophobic interaction HPLC; HI-FBS: heat-inactivated fetal bovine serum; HPLC: high-pressure liquid chromatography; IC₅₀ value: effective concentration to reach half-maximum inhibition; IQ: Inhibition Quotient; IS: immunological synapse; MES: 2-(N-morpholino)ethanesulfonic acid; R-PE: recombinant phycoerythrin; RA: red area in μm²/well; RD: receptor density; RFP: red fluorescent protein; R_g: radius of gyration; RSV: respiratory syncytial virus; SAXS: small-angle x-ray scattering; scFv: single-chain variable fragment; SD: standard deviation; SPR: surface plasmon resonance; WT: wild-type     

An innovative approach for the characterization of the isoforms of a monoclonal antibody product

Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the complex nature of the antibody molecules and allow the manufactures to set their own specifications for lot release, provided the safety and efficacy of the products are established in animal models prior to clinical trials. During the manufacture of a mAb product, we observed lot-to-lot variability in the isoform content and, although the variability is within the set specifications for lot release, made attempts to gain mechanistic insight by isolating and characterizing the individual isoforms. Matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography (LC)/mass spectrometry (MS)/MS analyses of the isolated isoforms indicate that this variability is caused by sialic acid content, as well as truncation of C-terminal lysine of the individual isoforms. Sialidase and carboxypeptidase treatment of the product confirm the observations made by MALDI and LC/MS/MS.Key words: IgG1, isoforms, charge heterogeneity, monoclonal antibody, glycosylation, silaic acidMonoclonal antibodies (mAbs) are used as medical agents to treat a variety of diseases including cancer, cardiovascular diseases and blood disorders.^1–3 Although a few IgG2 (e.g., panitumumab, denosumab) and IgG4 antibody molecules are in the market, most of the approved products are IgG1 molecules. IgG1 antibodies are glycoproteins with a conserved N-glycosylation site at Asn 297. Glycosylation influences the biological functions, such as antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) of the antibodies. The oligosaccharides present in the IgG1 molecules are heterogeneous due to the presence of various sugar residues, including sialic acid, galactose, N-acetylglucasmine and fucose residues. Molecular alterations in antibodies can take place at every stage of manufacturing: upstream and downstream processing, formulation and storage. These alterations can take place enzymatically or non-enzymatically and may produce charge or size heterogeneity. Deamidation, proteolytic fragmentation, oxidation, disulfide bond shuffling and glycosylation are the most common modifications that occur during the production of protein therapeutics.^4–7 These modifications can reduce the biological activity and may induce immunogenicity in patients. Hence, the regulatory agencies require a comprehensive characterization of the structural integrity, purity and stability of the protein therapeutics.⁸To date, eight chimeric, humanized and human IgG1 mAbs have been approved in the United States, Europe, as well as other countries, for the treatment of several types of cancers.^9–12 One such molecule produced at ImClone has two N-glycosylation sites and at least six to eight isoforms with isoelectric points (pIs) between 7.9–8.9 are present in this product. Although techniques such as ion exchange chromatography (IEX) and capillary isoelectic focusing (IEF) are available for the separation and characterization of charge varients,^13,14 we were not successful in separating the individual isoforms with these techniques from the IgG1 product used in this investigation. The peaks from IEX showed the presence of multiple bands on IEF. Hence, an alternative approach was used to isolate each isoform of this IgG1 product, and we demonstrated the involvement of sialic acid and C-terminal lysine as the root causes for lot-to-lot variation observed during the production of this molecule. The method is fast and very effective in separating isoforms with a difference in the pI values < 0.1.     

Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

Our study compares the status of human seminal plasma immunoglobulin G (IgG) and IgA secretory component (SC) fucosylation between infertile leukocytospermic and normal, fertile normozoospermic patients. The seminal IgG and SC are decorated with AAL-reactive core fucose, and antennary UEA- and LTA-reactive fucose of Lewis^y and Lewis^x structures, respectively. However, a correlation between IgG core fucosylation and IgG concentration (r?=??0.52; p?<?0.0003) was observed. The IgG present in leukocytospermic samples is characterized by lower expression of core fucose than in the normal group (0.82?±?0.3 AU and 1.2?±?0.3 AU, respectively; p?<?0.002). In seminal plasma the SC is present in two forms: 78-kDa and 63-kDa. The present study has also shown a higher AAL and LTA specific reactivity of glycans expressed in 63-kDa SC, in comparison to 78-kDa SC, in the normal group. In leukocytospermia, the values of specific lectin reactivity for core fucose, fucose α(1-2)- and α(1-3)- linked, were similar for both SC bands. Moreover, the present study has shown that in leukocytospermic samples the mean concentrations of IgG and S-IgA are twice as high (131.68?±?102.6 mg/l and 36?±?27 mg/l, respectively) as in the normal group (67.68?±?29.2 mg/l; p?<?0.02, and 19?±?18 mg/l, p?<?0.019, respectively). The analysis of IgG and SC fucosylation status and the determination of IgG and S-IgA concentrations in seminal plasma might constitute a valuable diagnosis tools for the evaluation of male infertility associated with leukocytospermia with accompanying inflammation.     

The Solution Structures of Two Human IgG1 Antibodies Show Conformational Stability and Accommodate Their C1q and FcγR Ligands

The human IgG1 antibody subclass shows distinct properties compared with the IgG2, IgG3, and IgG4 subclasses and is the most exploited subclass in therapeutic antibodies. It is the most abundant subclass, has a half-life as long as that of IgG2 and IgG4, binds the FcγR receptor, and activates complement. There is limited structural information on full-length human IgG1 because of the challenges of crystallization. To rectify this, we have studied the solution structures of two human IgG1 6a and 19a monoclonal antibodies in different buffers at different temperatures. Analytical ultracentrifugation showed that both antibodies were predominantly monomeric, with sedimentation coefficients s_20,w⁰ of 6.3–6.4 S. Only a minor dimer peak was observed, and the amount was not dependent on buffer conditions. Solution scattering showed that the x-ray radius of gyration R_g increased with salt concentration, whereas the neutron R_g values remained unchanged with temperature. The x-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions were unchanged in different buffers to indicate conformational stability. Constrained atomistic scattering modeling revealed predominantly asymmetric solution structures for both antibodies with extended hinge structures. Both structures were similar to the only known crystal structure of full-length human IgG1. The Fab conformations in both structures were suitably positioned to permit the Fc region to bind readily to its FcγR and C1q ligands without steric clashes, unlike human IgG4. Our molecular models for human IgG1 explain its immune activities, and we discuss its stability and function for therapeutic applications.     

Solution structure of deglycosylated human IgG1 shows the role of CH2 glycans in its conformation

The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is composed of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG; however, their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. After deglycosylation using peptide:N-glycosidase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s⁰_20,w of IgG1 decreased from 6.45 S by 0.16–0.27 S. This change was attributed to the reduction in mass after glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. Although the radius of gyration (R_G) was unchanged, the cross-sectional radius of gyration (R_XS-1) increased by 0.1 nm, and the commonly occurring distance peak M2 of the distance distribution curve P(r) increased by 0.4 nm. These changes revealed that the Fab-Fc separation in IgG1 was perturbed after deglycosylation. To explain these changes, atomistic scattering modeling based on Monte Carlo simulations resulted in 123,284 and 119,191 trial structures for glycosylated and deglycosylated IgG1 respectively. From these, 100 x-ray and neutron best-fit models were determined. For these, principal component analyses identified five groups of structural conformations that were different for glycosylated and deglycosylated IgG1. The Fc region in glycosylated IgG1 showed a restricted range of conformations relative to the Fab regions, whereas the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the Fcγ receptor in deglycosylated IgG1.     

Reactivities of Shark 19S and 7S IgM Antibodies to <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

LOWER vertebrates such as sharks can synthesize humoral antibodies in response to antigenic stimulation with a wide variety of antigens¹. Physicochemical studies have shown that sharks can synthesize both 19S and 7S immunoglobulins and that these two proteins belong to the same immunoglobulin class, which seems to be structurally homologous to IgM as defined for higher animals. Thus the shark immunoglobulins have been designated 19S IgM and 7S IgM^2–4. Because the predominant immunoglobulin (IgG) of most mammals is absent from sharks, the shark monomeric (7S) IgM might be functionally analogous to IgG. One example of the functional differences between IgM and IgG antibodies is the greater reactivity of the former in agglutination and bactericidal reactions^5,6. We have isolated and characterized functionally the relatively high levels of agglutinating antibodies which the nurse shark, Gingly-mostoma cirratum, synthesizes in response to Salmonella typhimurium “O” antigens.     

5th Annual Monoclonal Antibodies Conference: March 24–25, 2009, London,UK

The conference, which was organized by Visiongain and held at the BSG Conference Center in London, provided an excellent opportunity for participants to exchange views on the development, production and marketing of therapeutic antibodies, and discuss the current business environment. The conference included numerous interactive panel and group discussions on topics such as isotyping for therapeutic antibodies (panel chair: Nick Pullen, Pfizer), prospects for fully human monoclonal antibodies (chair: Christian Rohlff, Oxford BioTherapeutics), perspectives on antibody manufacturing and development (chair: Bo Kara, Avecia), market impact and post-marketing issues (chair: Keith Rodgers, Bodiam Consulting) and angiogenesis inhibitors (chair: David Blakey, AstraZeneca).

• MAbs. 2009 Jul-Aug; 1(4): 308.
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• March 24, 2009 Day 1

2009 Jul-Aug; 1(4): 308.

March 24, 2009 Day 1

Mari HerigstadAuthor information Copyright and License information DisclaimerVisiongain; London, UKCorresponding author.Correspondence to: Mari Herigstad; Visiongain; BSG House; 226-236 City Road; London EC1V 2QU UK; Email: moc.liamg@datsgireh.iramCopyright © 2009 Landes BioscienceThe first day was dedicated to discussion of antibody development and engineering, as well as debate on use of various types of antibodies. The session was chaired by David Blakey (AstraZeneca). The day opened with an overview on global trends in the antibody development and probabilities of approval success for human and humanized monoclonal antibodies (mAbs). The speakers then provided insights into the engineering and development of new therapeutic antibodies. Prospects for novel antibody formats, and assessment of immunogenicity, stability and aggregation risks in the development of therapeutic antibodies through use of in vivo and in silico methods were reviewed.Global trends in antibody development were discussed by Janice Reichert (Tufts Center for the Study of Drug Development and Editor-in-Chief, mAbs). Dr. Reichert emphasized the increased focus on mAbs as therapeutic agents. Of the therapeutic proteins entering clinical study each year, the majority are mAbs. Major pharmaceutical firms are acquiring biotechnology companies to enter this market and new solutions to problems of immunogenicity, stability, affinity, specificity and production are being developed. The research on clinical pipelines undertaken at Tufts CSDD allows calculation of metrics such as clinical development and approval times and probabilities of approval success. Insights gained from these results are important for strategic planning.The cumulative approval success rate for humanized mAbs was 16% for candidates entering clinical study during 1988 and 2008, and 29% for candidates entering clinical study during 1988 and 1997.¹ A conservative estimate of the success rate for humanized monoclonal antibodies would be somewhere in between, at approximately 20%. The trend, however, is toward fully human monoclonal antibodies. There are currently two marketed human mAbs, with another four in regulatory review. The cumulative US approval success rate for human antibodies is currently low, but will rise to 18% if the four in regulatory review are approved.In terms of therapeutic categories, oncology mAbs comprises approximately 50% of the total. Of 228 oncology mAbs that have entered clinical study since 1988, 56% are currently in clinical development. By comparison, 125 immunological mAb therapeutics have entered clinical study since 1990, of which 54% are currently in clinical development. The cumulative success rate for humanized oncology and immunological mAbs is 15% and 20%, respectively. Other therapeutic categories are being considered, including infectious disease. Sixteen anti-infective mAbs are currently in clinical study and one anti-infective mAb (palivizumab) has been approved to date. Oncology and immunology mAbs exhibit similar patterns for phase lengths and transition probabilities. The phase transition probability for phase 1 to 2 is high, followed by a lower phase 2–3 transition probability due to a proof-of-concept barrier. The transition probability for phase 3 to approval is comparable to that of phase 1 to 2.¹Other interesting trends include an increasing emphasis on antibody fragments.² Fragments may be easier and less costly to produce, but have shorter circulating half-life compared to full size antibodies and no effector functions unless this is added. Also worth noting is the growing prevalence of modified versions of mAbs (glycosylation and Fc region engineering) and improvements on circulation half-life through PEGylation.^3,4 Production methods as well as development and approval pathways for mAbs are well established and marketing approvals are set to increase if success rates are consistent with previous rates. This, together with competitive R&D times and potentially large markets, makes mAbs attractive for development as therapeutics.Julian Burke (Genetix) presented a clinical update on the selection of cell lines for antibody expression and protein production. A hybridoma is a hybrid cell that has been engineered to produce a desired antibody in large amounts. ClonePix FL is an antigen based system for in vitro detection and selection of hybridomas. The system incorporates plating hybridomas into a 3D cell matrix-a method which was first described 25 years ago.⁵ Whilst this method is not new, the novel aspect of the ClonePix system lies in the screening and collection of only those clones secreting a specific antibody. There are two options for screening hybridomas: immunoglobin G (IgG) secretion assays and antigenspecific assays. Unlike IgG secretion assays, antigen-specific assays isolate only antigen-specific clones with the desired IgG isotype. The system can also optimize production through detection of the highest producing cell lines. This approach allows the production of 10,000 clones in three weeks compared to the conventional approach which produces approximately 1,000 clones in two months. After a few days growth post-selection, isolated clones can be rapidly re-screened for cell-line stability. This stability test can be run in parallel with the scaling up of clones, thus making the process highly time efficient. To summarize, ClonePix minimizes the labor requirement, shortens the process timeline and permits parallel interrogation of multiple antigens.Masa Fujiwara (Chiome Bioscience) described the generation of antibodies using a novel antibody-generation technology called the ADLib (Autonomously Diversifying Library) System. This is a selection technology system based on cell-cell interactions and surface-displayed antigens in their native conformations. The system provides high-affinity antibody generation against difficult antigens such as self/human/homologous antigens, GPCRs, sugars/lipids, haptens and pathogens.Dr. Fujiwara explained how the ADLib system can be used to generate specific monoclonal antibodies using a chicken B-cell line (DT40) that undergoes gene conversion at immunoglobulin loci. This gene conversion is enhanced by treatment of the cells with trichostatin A, a histone deacetylase inhibitor. DT40 cells that are specific to the target antigen are obtained through ‘fishing’ the ADLib library with antigens conjugated with magnetic beads. This selection process and the subsequent screening for specificity can be completed in approximately one week.^6,7 As such, one of ADLib''s attractive features is the system''s ability to develop diverse monoclonal antibodies within weeks, not months.Optimization of antibodies, with a focus on structure-function relationships, was discussed by Bryan Edwards (MedImmune). Complementary determining regions (CDRs) are found in the variable domains of antibodies and confer the antigen specificity of the molecule. The CDR regions show high levels of natural sequence variability and are targeted during somatic hypermutation to generate higher affinity antibodies to the antigen. When considering strategies for in vitro optimization of antibodies, the CDR regions are typically targeted for mutagenesis. Three CDRs (CDR1, CDR2 and CDR3) are found on both the heavy and light chain regions of an antibody, and the highest level of natural sequence variability is found in the heavy chain CDR3 domain.Dr. Edwards described the optimization of two lead antibody candidates, where the heavy and light chain CDR3 domains were randomized at all amino acid positions and higher affinity variants isolated by both phage and ribosome display. Further gains in antibody potency were then obtained by combining the beneficial amino changes introduced into the heavy and light chain CDR3 domains. Additional sequence space was also explored outside of the CDR3 regions by the generation of error-prone libraries, and subsequent selection by ribosome display. Lead antibody candidates were improved several thousand-fold in potency through a combination of these approaches.By studying the solved crystal structures of Fab:antigen complexes, Dr. Edwards explained that not all CDR regions make direct contact with the target antigen and that amino acids in the framework regions can also contribute to antibody specificity. Moreover, beneficial changes introduced during optimization are not always due to the introduction of new contacts with the target antigen. Several amino acid changes can indirectly improve the potency of the antibody despite being some distance from the antigen binding site. It has been postulated that these amino acid changes improved affinity by reducing the free energy of the antibody:antigen complex, for example by stabilizing CDR loop conformations or by improving the stability of the VH-VL interface.Pavel Bondarenko (Amgen) presented data on the structure and function of disulfide isoforms of the human IgG2 subclass. There are five different known human antibody isotypes; IgA, IgD, IgE, IgM and IgG, of which IgG is the isotype that provides the majority of immune responses against pathogens. IgG antibodies have predictable properties, controlled function and long circulation half-life. Due to these properties, IgG antibodies are the most common therapeutic modalities. There are four human IgG subclasses, IgG1, IgG2, IgG3 and IgG4, of which IgG1 is the most abundant.One therapeutic function of monoclonal IgG antibodies involves binding Fab regions to target receptors, which blocks ligand-receptor interaction. Additional functions include initiating cell destruction through the attraction of immune complexes by the Fc and hinge region on the antibody. Due to their lower affinity for Fc receptors than IgG1s, IgG2s show reduced propensity for activating immune responses. This may be beneficial in some therapeutic aspects.Scientists at Amgen have recently discovered that structural heterogeneity is a naturally occurring feature of human IgG2 antibodies.^8,9 These distinct IgG2 forms are due to differences in the disulfide connectivity at the hinge region. There are three human IgG2 isoforms; IgG2-A, IgG2-B and IgG2-A/B. IgG2-A is defined by structurally independent Fab domains and hinge region. In IgG2-B, on the other hand, both Fab regions are covalently linked to the hinge. IgG2-A/B is an arrangement in which only one Fab arm is covalently linked to the hinge through disulfide bonds.The disulfide isoforms may show differences in potency. Against a cell-surface receptor, IgG1 and IgG2-A forms of a mAb were shown to have approximately similar potency and both had greater potency that IgG2-B. The difference between the IgG2 isotopes was attributed to the greater flexibility of IgG2-A and its ability to bind with both Fab regions. IgG2 disulfide exchange is facilitated by the close proximity of cysteine residues at the hinge region of IgG2. The mutation of a single cysteine residue in the IgG2 hinge region resulted in a loss of disulfide heterogeneity.¹⁰ Also, redox treatments with a cysteine/cystamine mixture have been shown to cause enrichment of both IgG2-A and IgG2-B.⁹ Human IgG2 isoforms are dynamic and exhibit disulfide rearrangement in both blood and cell culture.¹¹ Initially, IgG2 exists as IgG2-A, and is then rapidly converted to the asymmetric IgG2-A/B, followed by a slower conversion to IgG2-B. The biological relevance of IgG2 isoforms and in vivo conversion is currently being studied.⁹Andrew Popplewell (UCB New Medicines) provided an introduction to the prospects for therapeutic antibody fragments. Out of the 22 currently FDA approved monoclonal antibody therapeutics, three are antibody fragment therapeutics. Antibody fragment formats include Fab regions, single chain variable domains (scFv) and variable loops on the heavy and light chain (dAbs).Antibody fragments are highly flexible formats that may be combined to form new multivalent or multi-specific structures. Compared to full-length IgGs, fragments may have improved biodistribution, tissue penetration,^12,13 target access, or potentially better safety profiles due to the lack of Fc regions. Antibody fragments can also be expressed in microbial systems. They also show shorter serum persistence, which, depending on use, can be either disadvantageous or advantageous. However, antibody fragment circulation time can be modified either through PEGylation,^3,4,14 or through use of serum proteins as carriers.¹⁵ Either strategy may alter the pharmacokinetic properties of fragments, allowing the infrequent therapeutic dosing commonly used for full-length IgG therapy.Stability is a major factor for the successful commercialization of antibody-based drugs. Fabs are generally more stable to thermal or physical stress compared to IgGs, and this stability is not affected by PEGylation or by antibody species origin (e.g. mouse, rat, human). ScFvs and dAbs typically exhibit reduced stability compared to Fabs, however advances in technology may contribute to improve stability in these fragments.Dr. Popplewell emphasized that a more complete understanding of biophysical properties and improved stability engineering are required before antibody fragments can reach their full potential. Full-length IgG1s offer active immune cell recruitment, and may thus be better suited for certain therapeutic uses, such as oncology treatments. The development of products with enhanced Fc functionality, the option of using inactive Fcs, and improvements in yields from mammalian cell expression systems are providing further options for the full length IgG format. Dr. Popplewell summarized by pointing out that the intended mechanism of action should guide the choice of format.The challenge of predicting immunogenicity in a potential drug was discussed by Phillipe Stas (Algonomics). This is a difficult, yet important, process because early and precise immunogenicity assessments can reduce the number of drugs that fail to demonstrate efficacy in clinical trials. In order to generate an accurate risk profile of the potential immunogenicity for a given drug, two questions in particular must be addressed. First, the probability of observing an immunogenic response must be analyzed. Second, the severity of the observed immunogenicity needs to be considered.Neutralizing antibody responses can neutralize not only the therapeutic protein, but also its endogenous counterpart; the latter may induce severe side-effects in the patient.¹⁶ Risk factors for immunogenicity include the degree of ‘non-self’ of the antibody, the dosing of the drug (acute versus repeated), route of administration (intravenous versus subcutaneous) and other drug characteristics such as clearance rate of the drug. The patient''s immune status and the properties of the disease (i.e., severity and availability of concomitant immunosuppressants) should be included in a risk analysis.The different drug development stages offer opportunities to use different strategies for immunogenicity assessment. In the clinical phase, the general approach is to conduct antidrug antibodies (ADA) screening on individuals exposed to the drug. However, efforts are now geared toward assessing immunogenicity at earlier stages. The generation of ADA is dependent on the presence of T-cell epitopes. These can be measured in the preclinical setting using in-vitro T-cell assays. Prior to this stage, in silico methods may be used to identify T-cell epitopes. One such T-cell epitope screening tool is Algonomics'' platform Epibase®, which can rapidly analyze and predict the potential immunogenicity of therapeutic protein leads. An in silico approach to T-cell identification can offer relatively inexpensive mapping of epitopes from a wide genetic background.¹⁷ Also, perhaps more importantly, the combined use of in vitro and in silico tools allows for a much more accurate and less time-consuming assessment of expected immunogenicity in a drug.In silico methods in the development of therapeutic antibodies were reviewed by Jesús Zurdo (Lonza Biologics). Dr. Zurdo focused on assessment of stability and aggregation risks. In addition to increased production costs, aggregation reduces product stability, increases immunogenicity and may also elevate the toxicity. AggreSolve is an in silico protein analysis platform that can be applied to predict and overcome protein stability and aggregation issues. The Aggresolve platform assesses protein aggregation propensity and identifies aggregation ‘hot-spots.’ The platform can also predict sequence changes that are likely to reduce aggregation propensity. This library of potential substitutions can then be used to re-engineer antibodies with elevated stability and fewer aggregation problems. Compared to wild-type, selected re-engineered molecules achieve significantly reduced aggregation levels whilst retaining biological activity. Furthermore, protein stabilisation using re-engineering methods can also translate into elevated antibody productivity.