Importance and Monitoring of Therapeutic Immunoglobulin G Glycosylation.

The complex diantennary-type oligosaccharides at Asn297 residues of the IgG heavy chains have a profound impact on the safety and efficacy of therapeutic IgG monoclonal antibodies (mAbs). Fc glycosylation of a mAb is an established critical quality attribute (CQA), and its oligosaccharide profile is required to be thoroughly characterized by state-of-the-art analytical methods. The Fc oligosaccharides are highly heterogeneous, and the differentially glycosylated species (glycoforms) of IgG express unique biological activities. Glycoengineering is a promising approach for the production of selected mAb glycoforms with improved effector functions, and non- and low-fucosylated mAbs exhibiting enhanced antibody-dependent cellular cytotoxicity activity have been approved or are under clinical evaluation for treatment of cancers, autoimmune/chronic inflammatory diseases, and infection. Recently, the chemoenzymatic glycoengineering method that allows for the transfer of structurally defined oligosaccharides to Asn-linked GlcNAc residues with glycosynthase has been developed for remodeling of IgG-Fc oligosaccharides with high efficiency and flexibility. Additionally, various glycoengineering methods have been developed that utilize the Fc oligosaccharides of IgG as reaction handles to conjugate cytotoxic agents by "click chemistry", providing new routes to the design of antibody-drug conjugates (ADCs) with tightly controlled drug-antibody ratios (DARs) and homogeneity. This review focuses on current understanding of the biological relevance of individual IgG glycoforms and advances in the development of next-generation antibody therapeutics with improved efficacy and safety through glycoengineering.

Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Structural Determination of the Broadly Reactive Anti-IGHV1-69 Anti-idiotypic Antibody G6 and Its Idiotope.

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine.

Development of a rapid and quantitative lateral flow assay for the simultaneous measurement of serum κ and λ immunoglobulin free light chains (FLC): inception of a new near-patient FLC screening tool.

BACKGROUND: Serum free light chains (FLC) are sensitive biomarkers used for the diagnosis and management of plasma cell dyscrasias, such as multiple myeloma (MM), and are central to clinical screening algorithms and therapy response criteria. We have developed a portable, near-patient, lateral-flow test (Seralite®) that quantitates serum FLC in 10 min, and is designed to eliminate sample processing delays and accelerate decision-making in the clinic. METHODS: Assay interference, imprecision, lot-to-lot variability, linearity, and the utility of a competitive-inhibition design for the elimination of antigen-excess ('hook effect') were assessed. Reference ranges were calculated from 91 healthy donor sera. Preliminary clinical validation was conducted by retrospective analysis of sera from 329 patients. Quantitative and diagnostic results were compared to Freelite®. RESULTS: Seralite® gave a broad competitive-inhibition calibration curve from below 2.5 mg/L to above 200 mg/L, provided good assay linearity (between 1.6 and 208.7 mg/L for κ FLC and between 3.5 and 249.7 mg/L for λ FLC) and sensitivity (1.4 mg/L for κ FLC and 1.7 mg/L for λ FLC), and eliminated anomalous results from antigen-excess. Seralite® gave good diagnostic concordance with Freelite® (Roche Hitachi Cobas C501) identifying an abnormal FLC ratio and FLC difference in 209 patients with newly diagnosed MM and differentiating these patients from normal healthy donors with polyclonal FLC. CONCLUSIONS: Seralite® sensitively quantitates FLC and rapidly identifies clinical conditions where FLC are abnormal, including MM.

Humanized mouse G6 anti-idiotypic monoclonal antibody has therapeutic potential against IGHV1-69 germline gene-based B-CLL.

In 10-20% of the cases of chronic lymphocytic leukemia of B-cell phenotype (B-CLL), the IGHV1-69 germline is utilized as VH gene of the B cell receptor (BCR). Mouse G6 (MuG6) is an anti-idiotypic monoclonal antibody discovered in a screen against rheumatoid factors (RFs) that binds with high affinity to an idiotope expressed on the 51p1 alleles of IGHV1-69 germline gene encoded antibodies (G6-id(+)). The finding that unmutated IGHV1-69 encoded BCRs are frequently expressed on B-CLL cells provides an opportunity for anti-idiotype monoclonal antibody immunotherapy. In this study, we first showed that MuG6 can deplete B cells encoding IGHV1-69 BCRs using a novel humanized GTL mouse model. Next, we humanized MuG6 and demonstrated that the humanized antibodies (HuG6s), especially HuG6.3, displayed ∼2-fold higher binding affinity for G6-id(+) antibody compared to the parental MuG6. Additional studies showed that HuG6.3 was able to kill G6-id(+) BCR expressing cells and patient B-CLL cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Finally, both MuG6 and HuG6.3 mediate in vivo depletion of B-CLL cells in NSG mice. These data suggest that HuG6.3 may provide a new precision medicine to selectively kill IGHV1-69-encoding G6-id(+) B-CLL cells.

Enhanced sialylation of a human chimeric IgG1 variant produced in human and rodent cell lines.

Glycosylation of the IgG-Fc is essential for optimal binding and activation of Fcγ receptors and the C1q component of complement. However, it has been reported that the effector functions are down-regulated when the Fc glycans terminate in sialic acid residues and that sialylated IgG mediates anti-inflammatory effects of intravenous immunoglobulin (IVIG). Although recombinant IgG is hypo-sialylated, Fc sialylation is shown to be markedly increased when a mouse/human chimeric IgG3 Phe243Ala (F243A) variant is expressed in Chinese hamster ovary (CHO)-K1 cells. Here we investigate whether sialylation is increased in IgG1 F243A when expressed in CHO-K1, mouse myeloma J558L and human embryonic kidney (HEK) 293. Although the sialylation level was 2-5% for IgG1 wild type (WT), it was increased to 31%, 10% and 33% for the variant from CHO-K1, J558L and HEK293 cells, respectively. Interestingly, an increased addition of bisecting GlcNAc and α(1-3)-galactose residues to the Fc glycan was observed for HEK293-derived and J558L-derived IgG1 F243A, respectively. Fucosylation of HEK293-derived IgG1 F243A was maintained despite increased bisecting GlcNAc content. Although sialic acid and bisecting GlcNAc residues are reported to have an opposing effect on antibody-dependent cellular cytotoxicity (ADCC), IgG1 F243A showed 7 times lower ADCC activities than IgG1 WT, irrespective of bisecting GlcNAc residue. Thus, highly sialylated, human cell-derived IgG1 F243A with lowered ADCC activity may be of interest for the development of therapeutic antibodies with anti-inflammatory properties as an alternative to IVIG.

Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection, galactosylation, and sialylation.

Granulomatosis with polyangiitis (GPA) is associated with circulating immunoglobulin (Ig) G anti-proteinase 3 specific (anti-PR3) anti-neutrophil cytoplasm antibodies (ANCA), which activate cytokine primed neutrophils via Fcgamma receptors. ANCA are class switched IgG antibodies implying T cell help in their production. Glycosylation of IgG Fc, under the control of T cell cytokines, determines the interaction between IgG and its receptors. Previous studies have reported aberrant glycosylation of Ig Fc in GPA patients. We investigated whether aberrant Fc glycosylation was present on anti-PR3 ANCA as well as whole IgG subclass preparations compared to healthy controls and whether this correlated with Birmingham vasculitis activity scores (BVAS), serum cytokines, and time to remission. Here, IgG Fc glycosylation of GPA patients and controls and anti-PR3 ANCA Fc glycosylation were determined by mass spectrometry of glycopeptides. IgG1 and IgG2 subclasses from GPA patients showed reduced galactosylation, sialylation, and bisection compared to healthy controls. Anti-PR3 IgG1 ANCA Fc galactosylation, sialylation, and bisection were reduced compared to total IgG1 in GPA. Galactosylation of anti-PR3 ANCA Fc correlated with inflammatory cytokines and time to remission but not BVAS. Bisection of anti-PR3 ANCA Fc correlated with BVAS. Total IgG1 and anti-PR3 IgG1 Fc galactosylation were weakly correlated, while bisection of IgG1 and anti-PR3 showed no correlation. Our data indicate that aberrant ANCA galactosylation may be driven in an antigen-specific manner.

Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine.

Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1 mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5 mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated.

Aggregation, immune complexes and immunogenicity.

The development of an immune response to a protein therapeutic may nullify its beneficial activity or result in adverse events. Immunogenicity is, therefore, a major concern for clinicians, regulatory authorities and the biopharmaceutical industry. These concerns are particularly acute for the treatment of chronic diseases, as opposed to cancer, that may require repeated exposure to therapeutic over extended cycles of remission/relapse. There are many parameters that may be contributory to immunogenicity; however, the "bête noire," for the past decade has been aggregation. ( 1-3).

IgG1 heavy chain-coding gene polymorphism (G1m allotypes) and development of antibodies-to-infliximab.

OBJECTIVE: The chimeric anti-tumor necrosis factor-alpha antibody infliximab is known to induce antibodies-to-infliximab (ATI) in some treated patients. Immunogenicity in murine variable domains is expected; however, constant domains of its human heavy gamma1 chain may also be implicated as it expresses G1m1 and G1m17 allotypes. This allelic form may be immunogenic in patients that are homozygous for the G1m3 allotype commonly expressed in Caucasoid populations. METHODS: As G1m allotypic divergence may explain the presence of ATI or may influence their concentration, a genotyping method was developed and validated to determine antithetical (i.e. mutually exclusive) G1m3 and G1m17 allotypes (amino acid 120 of CH1 according to the international ImMunoGeneTics information system unique numbering) at the IGHG1 gene level (CH1 359g/a nucleotide polymorphism). Two hundred forty-five blood donors and 118 previously described patients suffering from Crohn's disease, treated with infliximab, and having developed ATI in 73 of them, were genotyped. RESULTS: The IGHG1 CH1 359g/a polymorphism does not depart from the Hardy-Weinberg equilibrium in the control population, and allele frequencies were similar in controls and patients. No association was found between the patient G1m allotypes and the presence of ATI or their concentration. It remains possible that anti-Gm1 antibodies are not well detected by the enzyme-linked immunosorbent assays used for ATI detection and/or that the G1m allotypes are minor antigens on IgG1. CONCLUSION: The IGHG1 polymorphism does not seem to play a major role in the induction of ATI. Further analyses will be required to determine whether it is also the case for humanized or fully human antibodies bearing the same G1m allotypes.

Glycosylation as a strategy to improve antibody-based therapeutics.

To date, more than 20 recombinant immunoglobulin G (IgG) antibody therapeutics are licensed for the treatment of various diseases. The mechanism of action of recombinant monoclonal antibodies (rMAbs) has been extensively investigated and several distinct pathways have been defined; selective activation of specific pathways may optimize clinical outcomes for different diseases, such as cancer and chronic inflammation. Human IgG is a glycoprotein with oligosaccharides attached at a single site. These are essential to the mode of action of rMAbs, and the antibody efficacy can vary depending on the particular oligosaccharide that is attached. Methods are now becoming available that allow the production of rMAbs bearing pre-selected oligosaccharides - glycoforms - to provide maximum efficacy for a given disease indication. This Review summarizes current knowledge of these methods and avenues for their exploitation in the clinic.

Antibody therapeutics: isotype and glycoform selection.

Recombinant monoclonal antibody (rMAb) therapy may be instituted to achieve one of two broad outcomes: i) killing of cells or organisms (e.g., cancer cells, bacteria); and ii) neutralisation of soluble molecules (e.g., cytokines in chronic disease or toxins in infection). The choice of rMAb isotype is a critical decision in the development of a therapeutic antibody as it will determine the biological activities triggered in vivo. It is not possible, however, to accurately predict the in vivo activity because multiple parameters impact on the functional outcome, for example, IgG subclass, IgG-Fc glycoform, epitope density, cellular Fc receptors polymorphisms and so on. The present understanding of the molecular interactions between IgG-Fc and effector ligands in vitro has allowed the generation of new antibody structures with altered/improved effector function profiles that may prove optimal for given disease indications. Thus, when maximal antibody-dependent cell-mediated cytotoxicity activity is indicated a non-fucosylated IgG1 format may be optimal; when minimal activity is indicated an aglycosylated IgG2 may be the form of choice.

Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.

A conserved structural feature of human IgG molecules is the presence of an oligosaccharide moiety within the Fc region at Asn297. In addition, 15-20% of normal polyclonal IgG molecules bear N-linked oligosaccharides in the variable (V) regions of the light (L) and/or heavy (H) chains. Electrospray ionization mass spectrometry (ESI-MS) has been applied to the glycan analysis of two IgG1 myeloma proteins (Wid and Cri) after mild reduction and acidification. Heterogeneous ion peaks were observed for both the H and L chains of Wid in contrast to Cri whose L chain peak was homogeneous. Site-specific deglycosylation of the H and L chains of IgG Wid was achieved under native conditions with peptide-N-glycosidase F and endoglycosidase F2, respectively. The Fc glycoforms differed between the two proteins in that Cri-Fc bears diantennary complex-type glycans that are fully core-fucosylated and partially sialylated while Wid-Fc glycans are non-fucosylated, partially galactosylated and non-sialylated. In contrast to the Fc glycans, the L chain glycans of Wid were shown to be fucosylated, fully galactosylated and sialylated, indicating that the glycosylation machinery of the Wid-producing myeloma cells is intact. Thus, combination of the two endoglycosidases can provide a simple means of glycan analysis of both Fab and Fc by ESI-MS, which may contribute to the development of therapeutic IgG with customized glycan profiles.

Structure-function relationships of the variable domains of monoclonal antibodies approved for cancer treatment.

Due to their exquisite specificity for a given epitope on the target antigen, recombinant monoclonal antibodies (rmAb) can deliver "targeted therapy" in oncology. This review focuses on the structural bases of "antigen specificity" to aid clinical researchers and pharmacologists in managing these new drugs. The fine structure of the Fv (Fragment variable) module (combination of VH and VL domains) from the five unconjugated antibodies currently approved for cancer treatment, namely rituximab, cetuximab, alemtuzumab, trastuzumab and bevacizumab, is presented and analysed. Co-crystal and functional studies are reviewed to define rmAb residues contributing to antigen binding site (paratope)-epitope interfaces. The genetic origin of these recombinant monoclonal antibodies, determined through the IMGT/3Dstructure-DB database and IMGT/V-QUEST (http://imgt.cines.fr), is presented, allowing the evaluation of homologies between antibodies and their closest germline human counterparts and hence their possible immunogenicity. Overall, the IMGT standards appear as a first and crucial step in the evaluation of recombinant antibodies.