An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part II: limit of detection and follow-up of patients with small M-proteins.

Background Electrophoretic methods to detect, characterize and quantify M-proteins play an important role in the management of patients with monoclonal gammopathies (MGs). Significant uncertainty in the quantification and limit of detection (LOD) is documented when M-proteins are <10 g/L. Using spiked sera, we aimed to assess the variability in intact M-protein quantification and LOD across 16 laboratories. Methods Sera with normal, hypo- or hyper-gammaglobulinemia were spiked with daratumumab or elotuzumab, with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Laboratories blindly analyzed samples according to their serum protein electrophoresis (SPEP)/isotyping standard operating procedures. LOD and intra-laboratory percent coefficient of variation (%CV) were calculated and further specified with regard to the method (gel/capillary electrophoresis [CZE]), gating strategy (perpendicular drop [PD]/tangent skimming [TS]), isotyping (immunofixation/immunosubtraction [ISUB]) and manufacturer (Helena/Sebia). Results All M-proteins ≥1 g/L were detected by SPEP. With isotyping the LOD was moderately more sensitive than with SPEP. The intensity of polyclonal background had the biggest negative impact on LOD. Independent of the method used, the intra-laboratory imprecision of M-protein quantification was small (mean CV = 5.0%). Low M-protein concentration and high polyclonal background had the strongest negative impact on intra-laboratory precision. All laboratories were able to follow trend of M-protein concentrations down to 1 g/L. Conclusions In this study, we describe a large variation in the reported LOD for both SPEP and isotyping; overall LOD is most affected by the polyclonal immunoglobulin background. Satisfactory intra-laboratory precision was demonstrated. This indicates that the quantification of small M-proteins to monitor patients over time is appropriate, when subsequent testing is performed within the same laboratory.

An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part I: factors impacting limit of quantitation of serum protein electrophoresis.

Background Serum protein electrophoresis (SPEP) is used to quantify the serum monoclonal component or M-protein, for diagnosis and monitoring of monoclonal gammopathies. Significant imprecision and inaccuracy pose challenges in reporting small M-proteins. Using therapeutic monoclonal antibody-spiked sera and a pooled beta-migrating M-protein, we aimed to assess SPEP limitations and variability across 16 laboratories in three continents. Methods Sera with normal, hypo- or hypergammaglobulinemia were spiked with daratumumab, Dara (cathodal migrating), or elotuzumab, Elo (central-gamma migrating), with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Provided with total protein (reverse biuret, Siemens), laboratories blindly analyzed samples according to their SPEP and immunofixation (IFE) or immunosubtraction (ISUB) standard operating procedures. Sixteen laboratories reported the perpendicular drop (PD) method of gating the M-protein, while 10 used tangent skimming (TS). A mean percent recovery range of 80%-120% was set as acceptable. The inter-laboratory %CV was calculated. Results Gamma globulin background, migration pattern and concentration all affect the precision and accuracy of quantifying M-proteins by SPEP. As the background increases, imprecision increases and accuracy decreases leading to overestimation of M-protein quantitation especially evident in hypergamma samples, and more prominent with PD. Cathodal migrating M-proteins were associated with less imprecision and higher accuracy compared to central-gamma migrating M-proteins, which is attributed to the increased gamma background contribution in M-proteins migrating in the middle of the gamma fraction. There is greater imprecision and loss of accuracy at lower M-protein concentrations. Conclusions This study suggests that quantifying exceedingly low concentrations of M-proteins, although possible, may not yield adequate accuracy and precision between laboratories.

Immunoglobulins with Non-Canonical Functions in Inflammatory and Autoimmune Disease States.

Immunoglobulins are known to combine various effector mechanisms of the adaptive and the innate immune system. Classical immunoglobulin functions are associated with antigen recognition and the initiation of innate immune responses. However, in addition to classical functions, antibodies exhibit a variety of non-canonical functions related to the destruction of various pathogens due to catalytic activity and cofactor effects, the action of antibodies as agonists/antagonists of various receptors, the control of bacterial diversity of the intestine, etc. Canonical and non-canonical functions reflect the extreme human antibody repertoire and the variety of antibody types generated in the organism: antigen-specific, natural, polyreactive, broadly neutralizing, homophilic, bispecific and catalytic. The therapeutic effects of intravenous immunoglobulins (IVIg) are associated with both the canonical and non-canonical functions of antibodies. In this review, catalytic antibodies will be considered in more detail, since their formation is associated with inflammatory and autoimmune diseases. We will systematically summarize the diversity of catalytic antibodies in normal and pathological conditions. Translational perspectives of knowledge about natural antibodies for IVIg therapy will be also discussed.

Discovery and bio-optimization of human antibody therapeutics using the XenoMouse® transgenic mouse platform.

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards.

An Algorithm for Building Multi-State Classifiers Based on Collision-Induced Unfolding Data.

Collision-induced unfolding (CIU) has emerged as a valuable method for distinguishing iso-cross-sectional protein ions through their distinct gas-phase unfolding trajectories. CIU shows promise as a high-throughput, structure-sensitive screening technique with potential applications in drug discovery and biotherapeutic characterization. We recently developed a CIU classification workflow to support screening applications that utilized CIU data acquired from a single protein charge state to distinguish immunoglobulin (IgG) subtypes and membrane protein lipid binding. However, distinguishing highly similar protein structures, such as those associated with biotherapeutics, can be challenging. Here, we present an expansion of this classification method that includes CIU data from multiple charge states, or indeed any perturbation to protein structure that differentially affects CIU, into a combined classifier. Using this improved method, we are able to improve the accuracy of existing, single-state classifiers for IgG subtypes and develop an activation-state-sensitive classifier for selected Src kinase inhibitors when data from a single charge state was insufficient to do so. Finally, we employ the combination of multiple charge states and stress conditions to distinguish a highly similar innovator/biosimilar biotherapeutic pair, demonstrating the potential of CIU as a rapid screening tool for drug discovery and biotherapeutic analysis.

TRIM21: a cytosolic Fc receptor with broad antibody isotype specificity.

Antibodies are key molecules in the fight against infections. Although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. This postentry viral defense mechanism requires binding of the antibody to a cytosolic Fc receptor named tripartite motif containing 21 (TRIM21). In contrast to other Fc receptors, TRIM21 shows remarkably broad isotype specificity as it does not only bind IgG but also IgM and IgA. When viral pathogens coated with these antibody isotypes enter the cytosol, TRIM21 is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. In addition, inflammatory signaling is induced. As such, TRIM21 acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. Here, we summarize our current understanding of how TRIM21 orchestrates humoral immunity in the cytosolic environment.

Isotype dependent on-column non-reversible aggregation of monoclonal antibodies.

Monoclonal antibodies of the IgG2 and IgG4 isotype were found to exhibit an increased propensity for displaying two-peak elution profiles during cation exchange chromatography. In some cases, this two-peak elution profile also resulted in the formation of non-reversible mAb aggregates. Comparison of IgG1, IgG2, and IgG4 molecules with the same variable region reveals that the two-peak behaviour is predominantly mediated by the constant region and most likely the lower CH1, hinge and upper CH2 regions of the mAb. Furthermore, comparison of the behaviour of two different IgG4 molecules, reveals that the degree of non-reversible aggregate formation, whilst facilitated by the isotype format, is mediated primarily by the variable region of the molecule. As well as the properties of the mAb molecule itself, the chemistry and structure of the cation exchange resin was also found to have an effect, with the two-peak elution profile being more pronounced with polymer-grafted resins such as Capto S Impact and Eshmuno CPX. These results combined support the theory that binding of IgG2 and IgG4 mAbs to cation exchange resins usually occurs through at least two mechanisms mediated by the structural features of the constant region of IgG2s and IgG4s. One of these mechanisms is not only stronger than the other, but also can lead to a conformational change in the molecule. This conformational change can occur in both variable and constant domains of the antibody. This transitory unfolded state can in turn lead to non-reversible aggregation of some mAb molecules.

Exploring Site-Specific N-Glycosylation of HEK293 and Plant-Produced Human IgA Isotypes.

The full potential of recombinant Immunoglobulin A as therapeutic antibody is not fully explored, owing to the fact that structure-function relationships of these extensively glycosylated proteins are not well understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered Nicotiana benthamiana plants and in human HEK293-6E cells. All three IgA isotypes were purified and subjected to biophysical and biochemical characterization. While no differences in assembly, antigen binding, and glycosylation occupancy were observed, both systems vary tremendously in terms of glycan structures and heterogeneity of glycosylation. Mass-spectrometric analysis of site-specific glycosylation revealed that plant-produced IgAs carry mainly complex-type biantennary N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous N-glycans with high levels of sialylation, core-fucose, and the presence of branched structures. The site-specific analysis revealed major differences between the individual N-glycosylation sites of each IgA subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from N. benthamiana displayed numerous plant-specific modifications. Interestingly, a shift in unfolding of the CH2 domain of plant-produced IgA toward lower temperatures can be observed with differential scanning calorimetry, suggesting that distinct glycoforms affect the thermal stability of IgAs.

Molecular cloning, characterization and expression of immunoglobulin D on pathogen challenge and pathogen associated molecular patterns stimulation in freshwater carp, Catla catla.

The primordial immunoglobulin class, IgD, was the first non-IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (CcIgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of CcIgD exhibited greatest similarity with Ctenopharyngodon idella. qRT-PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post-Aeromonas hydrophila challenge. CcIgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in CcIgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.

Isotype-specific glycosylation analysis of mouse IgG by LC-MS.

With mice being the top model organism in immunology and with Fc glycosylation being increasingly recognized as important modulator of antibody function, the time has come to take a look at the glycosylation of mouse IgG isotypes. Tryptic glycopeptides of mouse IgG1, IgG2, and IgG3 differ in mass and so these three isoforms can be easily discriminated by MS. Commercial IgG contained a rare IgG1 variant but no IgG3, which, however, was found in sera of C57BL/6 and BALB/c mice. These strains deviated with regard to IgG2a and IgG2b alleles. The Ig2a B allele was not observed in any of the four samples investigated. All a/c isotypes contain the same glycopeptide sequence, which deviates from that of IgG2b by containing Leu instead of Ile. The Leu/Ile glycopeptide variants were separated by RP chromatography and the order of elution was determined. The major glycoforms on all isotypes were fucosylated with no and one galactose (GnGnF and GnAF) followed by fully galactosylated AAF and smaller amounts of mono- and disialylated N-glycans. In the commercial serum pool, the relative ratios of glycans differed between isotypes. Sialic acid exclusively occurred as N-glycolylneuraminic acid. Fucosylation was essentially complete. No bisected and no α1,3-galactosylated glycans were found.

Molecular cloning of IgZ heavy chain isotype in Catla catla and comparative expression profile of IgZ and IgM following pathogenic infection.

Immunoglobulins serve as a crucial arm of the adaptive immune system against detrimental pathogenic threats in teleosts. However, whether the novel Ig isotype IgZ is present in the Indian major carp, Catla catla, has not yet been elucidated. The present study reports the presence of IgZ ortholog in C. catla (CcIgZ) and further demonstrates its comparative tissue specific expression with IgM (CcIgM) in response to bacterial and parasitic stimulation. The putative 139 amino acid sequence of IgZ heavy chain cDNA of C. catla showed homology with IgZ constant domains of other teleosts. Phylogenetic analysis of the predicted IgZ transcript sequence clustered with previously identified IgZ heavy chain sequences of Cyprinidae family members. The inductive expression profiles of IgZ and IgM genes were evaluated in immunologically relevant tissues at 24, 48 and 72 hr post infection with Aeromonas hydrophila, Streptococcus uberis and Argulus sp. Both CcIgZ and CcIgM were expressed most strongly in the kidneys of healthy fish. Basal expression of CcIgM transcript was higher than that of CcIgZ in all the examined tissues. Stimulation with bacteria triggered significant increase of IgZ in the intestine (P < 0.001) and spleen (P < 0.01), whereas IgM was relatively up-regulated in blood (P < 0.001) after stimulation with each of the three pathogens assessed. The study is the first to report identification of IgZ in C. catla. Further, it provides insights into the differential expression profiles of IgZ and IgM isotypes against various pathogenic infection in C. catla, which may facilitate better prophylaxis again such infections.

Impact of IgA constant domain on HIV-1 neutralizing function of monoclonal antibody F425A1g8.

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.

Comparative analyses of immunoglobulin genes: surprises and portents.

The study of immunoglobulin genes in non-mouse and non-human models has shown that different vertebrate groups have evolved distinct methods of generating antibody diversity. By contrast, the development of T cells in the thymus is quite similar in all of the species that have been examined. The three mechanisms by which B cells uniquely modify their immunoglobulin genes -- somatic hypermutation, gene conversion and class switching -- are increasingly believed to share some fundamental mechanisms, which studies in different vertebrate groups have helped (and will continue to help) to resolve. When these mechanisms are better understood, we should be able to look to the constitutive pathways from which they have evolved and perhaps determine whether the rearrangement of variable, diversity and joining antibody gene segments -- V(D)J recombination -- was superimposed on an existing adaptive immune system.

Intraclass diversification of immunoglobulin heavy chain genes in the African lungfish.

Lungfish (Dipnoi) are the closest living relatives to tetrapods, and they represent the transition from water to land during vertebrate evolution. Lungfish are armed with immunoglobulins (Igs), one of the hallmarks of the adaptive immune system of jawed vertebrates, but only three Ig forms have been characterized in Dipnoi to date. We report here a new diversity of Ig molecules in two African lungfish species (Protopterus dolloi and Protopterus annectens). The African lungfish Igs consist of three IgMs, two IgWs, three IgNs, and an IgQ, where both IgN and IgQ originated evidently from the IgW lineage. Our data also suggest that the IgH genes in the lungfish are organized in a transiting form from clusters (IgH loci in cartilaginous fish) to a translocon configuration (IgH locus in tetrapods). We propose that the intraclass diversification of the two primordial gnathostome Ig classes (IgM and IgW) as well as acquisition of new isotypes (IgN and IgQ) has allowed lungfish to acquire a complex and functionally diverse Ig repertoire to fight a variety of microorganisms. Furthermore, our results support the idea that "tetrapod-specific" Ig classes did not evolve until the vertebrate adaptation to land was completed ~360 million years ago.

Detection of total and PRRSV-specific antibodies in oral fluids collected with different rope types from PRRSV-vaccinated and experimentally infected pigs.

BACKGROUND: Oral fluid collected by means of ropes has the potential to replace serum for monitoring and surveillance of important swine pathogens. Until now, the most commonly used method to collect oral fluid is by hanging a cotton rope in a pen. However, concerns about the influence of rope material on subsequent immunological assays have been raised. In this study, we evaluated six different rope materials for the collection of oral fluid and the subsequent detection of total and PRRSV-specific antibodies of different isotypes in oral fluid collected from PRRSV-vaccinated and infected pigs. RESULTS: An initial experiment showed that IgA is the predominant antibody isotype in porcine saliva. Moreover, it was found that synthetic ropes may yield higher amounts of IgA, whereas all rope types seemed to be equally suitable for IgG collection. Although IgA is the predominant antibody isotype in porcine oral fluid, the PRRSV-specific IgA-based IPMA and ELISA tests were clearly not ideal for sensitive detection of PRRSV-specific IgA antibodies. In contrast, PRRSV-specific IgG in oral fluids was readily detected in PRRSV-specific IgG-based IPMA and ELISA tests, indicating that IgG is a more reliable isotype for monitoring PRRSV-specific antibody immunity in vaccinated/infected animals via oral fluids with the currently available tests. CONCLUSIONS: Since PRRSV-specific IgG detection seems more reliable than PRRSV-specific IgA detection for monitoring PRRSV-specific antibody immunity via oral fluids, and since all rope types yield equal amounts of IgG, it seems that the currently used cotton ropes are an appropriate choice for sample collection in PRRSV monitoring.

Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing.

Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human κ and anti-His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs.

When binding is enough: nonactivating antibody formats.

Most therapeutic antibodies currently used in the clinic are based on the human IgG1 format, which is a bivalent molecule that efficiently interacts with the immune system's effector functions. In clinical applications where binding to the target alone is sufficient for therapeutic efficacy; however, engagement of the immune system is not required and may even cause unwanted side-effects. Likewise, bivalent binding to the target may negatively influence the therapeutic efficacy of an antibody. Here we discuss the state of the art for antibody-based therapeutics, designed to be nonactivating (i.e. do not engage the innate immune system's effector functions), in both monovalent and bivalent formats.

Expression of IgM, IgD, and IgY in a reptile, Anolis carolinensis.

The reptiles are the last major group of jawed vertebrates in which the organization of the IGH locus and its encoded Ig H chain isotypes have not been well characterized. In this study, we show that the green anole lizard (Anolis carolinensis) expresses three Ig H chain isotypes (IgM, IgD, and IgY) but no IgA. The presence of the delta gene in the lizard demonstrates an evolutionary continuity of IgD from fishes to mammals. Although the germline delta gene contains 11 C(H) exons, only the first 4 are used in the expressed IgD membrane-bound form. The mu chain lacks the cysteine in C(H)1 that forms a disulfide bond between H and L chains, suggesting that (as in IgM of some amphibians) the H and L polypeptide chains are not covalently associated. Although conventional IgM transcripts (four C(H) domains) encoding both secreted and membrane-bound forms were detected, alternatively spliced transcripts encoding a short membrane-bound form were also observed and shown to lack the first two C(H) domains (VDJ-C(H)3-C(H)4-transmembrane region). Similar to duck IgY, lizard IgY H chain (upsilon) transcripts encoding both full-length and truncated (IgYDeltaFc) forms (with two C(H) domains) were observed. The absence of an IgA-encoding gene in the lizard IGH locus suggests a complex evolutionary history for IgA in the saurian lineage leading to modern birds, lizards, and their relatives.