Molecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and Antigen.

The classical complement pathway contributes to the natural immune defense against pathogens and tumors. IgG antibodies can assemble at the cell surface into hexamers via Fc:Fc interactions, which recruit complement component C1q and induce complement activation. Biophysical characterization of the C1:IgG complex has remained elusive primarily due to the low affinity of IgG-C1q binding. Using IgG variants that dynamically form hexamers efficient in C1q binding and complement activation, we could assess C1q binding in solution by native mass spectrometry and size-exclusion chromatography. Fc-domain deglycosylation, described to abrogate complement activation, affected IgG hexamerization and C1q binding. Strikingly, antigen binding by IgG hexamers or deletion of the Fab arms substantially potentiated complement initiation, suggesting that Fab-mediated effects impact downstream Fc-mediated events. Finally, we characterized a reconstituted 2,045.3 ± 0.4-kDa complex of intact C1 bound to antigen-saturated IgG hexamer by native mass spectrometry, providing a clear visualization of a complete complement initiation complex.

Staphylococcal protein A inhibits complement activation by interfering with IgG hexamer formation.

Immunoglobulin (Ig) G molecules are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of Staphylococcus aureus, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)6:C1q complexes and prevents downstream complement activation on the surface of S. aureus. Finally, we demonstrate that IgG3 antibodies against S. aureus can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.

Minimizing population health loss due to scarcity in OR capacity: validation of quality of life input.

OBJECTIVES: A previously developed decision model to prioritize surgical procedures in times of scarce surgical capacity used quality of life (QoL) primarily derived from experts in one center. These estimates are key input of the model, and might be more context-dependent than the other input parameters (age, survival). The aim of this study was to validate our model by replicating these QoL estimates. METHODS: The original study estimated QoL of patients in need of commonly performed procedures in live expert-panel meetings. This study replicated this procedure using a web-based Delphi approach in a different hospital. The new QoL scores were compared with the original scores using mixed effects linear regression. The ranking of surgical procedures based on combined QoL values from the validation and original study was compared to the ranking based solely on the original QoL values. RESULTS: The overall mean difference in QoL estimates between the validation study and the original study was - 0.11 (95% CI: -0.12 - -0.10). The model output (DALY/month delay) based on QoL data from both studies was similar to the model output based on the original data only: The Spearman's correlation coefficient between the ranking of all procedures before and after including the new QoL estimates was 0.988. DISCUSSION: Even though the new QoL estimates were systematically lower than the values from the original study, the ranking for urgency based on health loss per unit of time delay of procedures was consistent. This underscores the robustness and generalizability of the decision model for prioritization of surgical procedures.

Intraoperative assessment of resection margins by Raman spectroscopy to guide oral cancer surgery.

Patients with oral cavity cancer are almost always treated with surgery. The goal is to remove the tumor with a margin of more than 5 mm of surrounding healthy tissue. Unfortunately, this is only achieved in about 15% to 26% of cases. Intraoperative assessment of tumor resection margins (IOARM) can dramatically improve surgical results. However, current methods are laborious, subjective, and logistically demanding. This hinders broad adoption of IOARM, to the detriment of patients. Here we present the development and validation of a high-wavenumber Raman spectroscopic technology, for quick and objective intraoperative measurement of resection margins on fresh specimens. It employs a thin fiber-optic needle probe, which is inserted into the tissue, to measure the distance between a resection surface and the tumor. A tissue classification model was developed to discriminate oral cavity squamous cell carcinoma (OCSCC) from healthy oral tissue, with a sensitivity of 0.85 and a specificity of 0.92. The tissue classification model was then used to develop a margin length prediction model, showing a mean difference between margin length predicted by Raman spectroscopy and histopathology of -0.17 mm.

The course of health-related quality of life in the first 2 years after a diagnosis of head and neck cancer: the role of personal, clinical, psychological, physical, social, lifestyle, disease-related, and biological factors.

PURPOSE: The aim of this prospective cohort study was to estimate the relationship between the course of HRQOL in the first 2 years after diagnosis and treatment of head and neck cancer (HNC) and personal, clinical, psychological, physical, social, lifestyle, HNC-related, and biological factors. METHODS: Data were used from 638 HNC patients of the NETherlands QUality of life and BIomedical Cohort study (NET-QUBIC). Linear mixed models were used to investigate factors associated with the course of HRQOL (EORTC QLQ-C30 global quality of life (QL) and summary score (SumSc)) from baseline to 3, 6, 12, and 24 months after treatment. RESULTS: Baseline depressive symptoms, social contacts, and oral pain were significantly associated with the course of QL from baseline to 24 months. Tumor subsite and baseline social eating, stress (hyperarousal), coughing, feeling ill, and IL-10 were associated with the course of SumSc. Post-treatment social contacts and stress (avoidance) were significantly associated with the course of QL from 6 to 24 months, and social contacts and weight loss with the course of SumSc. The course of SumSc from 6 to 24 months was also significantly associated with a change in financial problems, speech problems, weight loss, and shoulder problems between baseline and 6 months. CONCLUSION: Baseline clinical, psychological, social, lifestyle, HNC-related, and biological factors are associated with the course of HRQOL from baseline to 24 months after treatment. Post-treatment social, lifestyle, and HNC-related factors are associated with the course of HRQOL from 6 to 24 months after treatment.

Raman spectroscopy to discriminate laryngeal squamous cell carcinoma from non-cancerous surrounding tissue.

As for many solid cancers, laryngeal cancer is treated surgically, and adequate resection margins are critical for survival. Raman spectroscopy has the capacity to accurately differentiate between cancer and non-cancerous tissue based on their molecular composition, which has been proven in previous work. The aim of this study is to investigate whether Raman spectroscopy can be used to discriminate laryngeal cancer from surrounding non-cancerous tissue. Patients surgically treated for laryngeal cancer were included. Raman mapping experiments were performed ex vivo on resection specimens and correlated to histopathology. Water concentration analysis and CH-stretching region analysis were performed in the high wavenumber range of 2500-4000 cm-1. Thirty-four mapping experiments on 22 resection specimens were used for analysis. Both laryngeal cancer and all non-cancerous tissue structures showed high water concentrations of around 75%. Discriminative information was only found to be present in the CH-stretching region of the Raman spectra of the larynx (discriminative power of 0.87). High wavenumber region Raman spectroscopy can discriminate laryngeal cancer from non-cancerous tissue structures. Contrary to the findings for oral cavity cancer, water concentration is not a discriminating factor for laryngeal cancer.

Identification of Functional HLA-A*01:01-Restricted Epstein-Barr Latent Membrane Protein 2-Specific T-Cell Receptors.

BACKGROUND: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated malignancies of latency type II/III that express EBV antigens (LMP1/2). Patients who are HLA-A*01:01 positive could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far. METHODS: HLA-A*01:01-restricted EBV-LMP2-specific T cells were isolated using peptide major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-γ) and cytotoxicity when stimulated with EBV-LMP2-expressing cell lines. Functionality of primary T cells transduced with HLA-A*01:01-restricted EBV-LMP2-specific TCRs was optimized by knocking out the endogenous TCRs of primary T cells (∆TCR) using CRISPR-Cas9 technology. RESULTS: EBV-LMP2-specific T cells were successfully isolated and their TCRs were characterized. TCR gene transfer in primary T cells resulted in specific pMHC tetramer binding and reactivity against EBV-LMP2-expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5-2 fold when the endogenous TCRs of CD8+ T cells was knocked out. CD8+/∆TCR T cells modified to express EBV-LMP2-specific TCRs showed IFN-γ secretion and cytotoxicity toward EBV-LMP2-expressing malignant cell lines. CONCLUSIONS: We isolated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can potentially be used in future TCR gene therapy to treat EBV-associated latency type II/III malignancies.

Study retention and attrition in a longitudinal cohort study including patient-reported outcomes, fieldwork and biobank samples: results of the Netherlands quality of life and Biomedical cohort study (NET-QUBIC) among 739 head and neck cancer patients and 262 informal caregivers.

BACKGROUND: Longitudinal observational cohort studies in cancer patients are important to move research and clinical practice forward. Continued study participation (study retention) is of importance to maintain the statistical power of research and facilitate representativeness of study findings. This study aimed to investigate study retention and attrition (drop-out) and its associated sociodemographic and clinical factors among head and neck cancer (HNC) patients and informal caregivers included in the Netherlands Quality of Life and Biomedical Cohort Study (NET-QUBIC). METHODS: NET-QUBIC is a longitudinal cohort study among 739 HNC patients and 262 informal caregivers with collection of patient-reported outcome measures (PROMs), fieldwork data (interview, objective tests and medical examination) and biobank materials. Study retention and attrition was described from baseline (before treatment) up to 2-years follow-up (after treatment). Sociodemographic and clinical characteristics associated with retention in NET-QUBIC components at baseline (PROMs, fieldwork and biobank samples) and retention in general (participation in at least one component) were investigated using Chi-square, Fisher exact or independent t-tests (p< 0.05). RESULTS: Study retention at 2-years follow-up was 80% among patients alive (66% among all patients) and 70% among caregivers of patients who were alive and participating (52% among all caregivers). Attrition was most often caused by mortality, and logistic, physical, or psychological-related reasons. Tumor stage I/II, better physical performance and better (lower) comorbidity score were associated with participation in the PROMs component among patients. No factors associated with participation in the fieldwork component (patients), overall sample collection (patients and caregivers) or PROMs component (caregivers) were identified. A better performance and comorbidity score (among patients) and higher age (among caregivers) were associated with study retention at 2-years follow-up. CONCLUSIONS: Retention rates were high at two years follow-up (i.e. 80% among HNC patients alive and 70% among informal caregivers with an active patient). Nevertheless, some selection was shown in terms of tumor stage, physical performance, comorbidity and age, which might limit representativeness of NET-QUBIC data and samples. To facilitate representativeness of study findings future cohort studies might benefit from oversampling specific subgroups, such as patients with poor clinical outcomes or higher comorbidity and younger caregivers.

Phosphorylcholine Monoclonal Antibody Therapy Decreases Intraplaque Angiogenesis and Intraplaque Hemorrhage in Murine Vein Grafts.

Phosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies and low levels of these antibodies have been associated with human vein graft (VG) failure. Although PC antibodies are recognized for their anti-inflammatory properties, their effect on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH)-interdependent processes contributing to plaque rupture-are unknown. We hypothesized that new IgG phosphorylcholine antibodies (PC-mAb) could decrease vulnerable lesions in murine VGs.Therefore, hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery and weekly IP injections of (5 mg/kg) PCmAb (n = 11) or vehicle (n = 12) until sacrifice at day 28. We found that PCmAb significantly decreased vein graft media (13%), intima lesion (25%), and increased lumen with 32% compared to controls. PCmAb increased collagen content (18%) and decreased macrophages presence (31%). PCmAb resulted in 23% decreased CD163+ macrophages content in vein grafts whereas CD163 expression was decreased in Hb:Hp macrophages. PCmAb significantly lowered neovessel density (34%), EC proliferation and migration with/out oxLDL stimulation. Moreover, PCmAb enhanced intraplaque angiogenic vessels maturation by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a 62% decrease in IPH. PCmAb effectively inhibits murine atherosclerotic lesion formation in vein grafts by reducing IPA and IPH via decreased neovessel density and macrophages influx and increased neovessel maturation. PC-mAb therefore holds promise as a new therapeutic approach to prevent vein graft disease.

Phosphorylcholine antibodies restrict infarct size and left ventricular remodelling by attenuating the unreperfused post-ischaemic inflammatory response.

Phosphorylcholine is a pro-inflammatory epitope exposed on apoptotic cells, and phosphorylcholine monoclonal immunoglobulin (Ig)G antibodies (PC-mAb) have anti-inflammatory properties. In this study, we hypothesize that PC-mAb treatment reduces adverse cardiac remodelling and infarct size (IS) following unreperfused transmural myocardial infarction (MI). Unreperfused MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery in hypercholesterolaemic APOE*3-Leiden mice. Three weeks following MI, cardiac magnetic resonance (CMR) imaging showed a reduced LV end-diastolic volume (EDV) by 21% and IS by 31% upon PC-mAb treatment as compared to the vehicle control group. In addition, the LV fibrous content was decreased by 27% and LV wall thickness was better preserved by 47% as determined by histological analysis. Two days following MI, CCL2 concentrations, assessed by use of ELISA, were decreased by 81% and circulating monocytes by 64% as assessed by use of FACS analysis. Additionally, local leucocyte infiltration determined by immunohistological analysis showed a 62% decrease after three weeks. In conclusion, the local and systemic inflammatory responses are limited by PC-mAb treatment resulting in restricted adverse cardiac remodelling and IS following unreperfused MI. This indicates that PC-mAb holds promise as a therapeutic agent following MI limiting adverse cardiac remodelling.

Atorvastatin pleiotropically decreases intraplaque angiogenesis and intraplaque haemorrhage by inhibiting ANGPT2 release and VE-Cadherin internalization.

OBJECTIVE: Statins pleiotropically provide additional benefits in reducing atherosclerosis, but their effects on intraplaque angiogenesis (IPA) and hemorrhage (IPH) remain unclear. Therefore, we discriminated statin's lipid-lowering dependent and independent effects on IPA and IPH. APPROACH AND RESULTS: ApoE3*Leiden mice are statin-responsive due to ApoE and LDLR presence, but also allow to titrate plasma cholesterol levels by diet. Therefore, ApoE3*Leiden mice were fed a high-cholesterol-inducing-diet (HCD) with or without atorvastatin (A) or a moderate-cholesterol-inducing-diet (MCD). Mice underwent vein graft surgery to induce lesions with IPA and IPH. Cholesterol levels were significantly reduced in MCD (56%) and HCD + A (39%) compared to HCD with no significant differences between MCD and HCD + A. Both MCD and HCD + A have a similar reduction in vessel remodeling and inflammation comparing to HCD. IPA was significantly decreased by 30% in HCD + A compared to HCD or MCD. Atorvastatin treatment reduced the presence of immature vessels by 34% vs. HCD and by 25% vs. MCD, resulting in a significant reduction of IPH. Atorvastatin's anti-angiogenic capacity was further illustrated by a dose-dependent reduction of ECs proliferation and migration. Cultured mouse aortic-segments lost sprouting capacity upon atorvastatin treatment and became 30% richer in VE-Cadherin expression and pericyte coverage. Moreover, Atorvastatin inhibited ANGPT2 release and decreased VE-Cadherin(Y685)-phosphorylation in ECs. CONCLUSIONS: Atorvastatin has beneficial effects on vessel remodeling due to its lipid-lowering capacity. Atorvastatin has strong pleiotropic effects on IPA by decreasing the number of neovessels and on IPH by increasing vessel maturation. Atorvastatin improves vessel maturation by inhibiting ANGPT2 release and phospho(Y658)-mediated VE-Cadherin internalization.

Minimizing Population Health Loss in Times of Scarce Surgical Capacity During the Coronavirus Disease 2019 Crisis and Beyond: A Modeling Study.

OBJECTIVES: Coronavirus disease 2019 has put unprecedented pressure on healthcare systems worldwide, leading to a reduction of the available healthcare capacity. Our objective was to develop a decision model to estimate the impact of postponing semielective surgical procedures on health, to support prioritization of care from a utilitarian perspective. METHODS: A cohort state-transition model was developed and applied to 43 semielective nonpediatric surgical procedures commonly performed in academic hospitals. Scenarios of delaying surgery from 2 weeks were compared with delaying up to 1 year and no surgery at all. Model parameters were based on registries, scientific literature, and the World Health Organization Global Burden of Disease study. For each surgical procedure, the model estimated the average expected disability-adjusted life-years (DALYs) per month of delay. RESULTS: Given the best available evidence, the 2 surgical procedures associated with most DALYs owing to delay were bypass surgery for Fontaine III/IV peripheral arterial disease (0.23 DALY/month, 95% confidence interval [CI]: 0.13-0.36) and transaortic valve implantation (0.15 DALY/month, 95% CI: 0.09-0.24). The 2 surgical procedures with the least DALYs were placing a shunt for dialysis (0.01, 95% CI: 0.005-0.01) and thyroid carcinoma resection (0.01, 95% CI: 0.01-0.02). CONCLUSION: Expected health loss owing to surgical delay can be objectively calculated with our decision model based on best available evidence, which can guide prioritization of surgical procedures to minimize population health loss in times of scarcity. The model results should be placed in the context of different ethical perspectives and combined with capacity management tools to facilitate large-scale implementation.

Releasing Nonperipheral Subunits from Protein Complexes in the Gas Phase.

The quaternary structure is an important feature regulating protein function. Native mass spectrometry contributes to untangling quaternary structures by preserving the integrity of protein complexes in the gas phase. Tandem mass spectrometry by collision-induced dissociation (CID) can then be used to release subunits from these intact complexes, thereby providing structural information on the stoichiometry and topology. Cumulatively, such studies have revealed the preferred release of peripheral subunits during CID. In contrast, here we describe and focus on dissociation pathways that release nonperipheral subunits from hetero-complexes in CID at high collision energies. We find that nonperipheral subunits are ejected with a high propensity, as a consequence of sequential dissociation events, upon initial removal of peripheral subunits. Alternatively, nonperipheral subunits can be released directly from a charge-reduced or an elongated intact complex. As demonstrated here for a range of protein assemblies, releasing nonperipheral subunits under controlled conditions may provide unique structural information on the stoichiometry and topology of protein complexes.

Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces.

IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.

IRF3 and IRF7 mediate neovascularization via inflammatory cytokines.

OBJECTIVE: To elucidate the role of interferon regulatory factor (IRF)3 and IRF7 in neovascularization. METHODS: Unilateral hind limb ischaemia was induced in Irf3-/- , Irf7-/- and C57BL/6 mice by ligation of the left common femoral artery. Post-ischaemic blood flow recovery in the paw was measured with laser Doppler perfusion imaging. Soleus, adductor and gastrocnemius muscles were harvested to investigate angiogenesis and arteriogenesis and inflammation. RESULTS: Post-ischaemic blood flow recovery was decreased in Irf3-/- and Irf7-/- mice compared to C57BL/6 mice at all time points up to and including sacrifice, 28 days after surgery (t28). This was supported by a decrease in angiogenesis and arteriogenesis in soleus and adductor muscles of Irf3-/- and Irf7-/- mice at t28. Furthermore, the number of macrophages around arterioles in adductor muscles was decreased in Irf3-/- and Irf7-/- mice at t28. In addition, mRNA expression levels of pro-inflammatory cytokines (tnfα, il6, ccl2) and growth factor receptor (vegfr2), were decreased in gastrocnemius muscles of Irf3-/- and Irf7-/- mice compared to C57BL/6 mice. CONCLUSION: Deficiency of IRF3 and IRF7 results in impaired post-ischaemic blood flow recovery caused by attenuated angiogenesis and arteriogenesis linked to a lack of inflammatory components in ischaemic tissue. Therefore, IRF3 and IRF7 are essential regulators of neovascularization.

Kinetic mechanism of controlled Fab-arm exchange for the formation of bispecific immunoglobulin G1 antibodies.

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Förster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the CH3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reaction.

CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering.

CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.

Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry.

The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.

Cysteine-SILAC Mass Spectrometry Enabling the Identification and Quantitation of Scrambled Interchain Disulfide Bonds: Preservation of Native Heavy-Light Chain Pairing in Bispecific IgGs Generated by Controlled Fab-arm Exchange.

Bispecific antibodies (bsAbs) are one of the most versatile and promising pharmaceutical innovations for countering heterogeneous and refractory disease by virtue of their ability to bind two distinct antigens. One critical quality attribute of bsAb formation requiring investigation is the potential randomization of cognate heavy (H) chain/light (L) chain pairing, which could occur to a varying extent dependent on bsAb format and the production platform. To assess the content of such HL-chain swapped reaction products with high sensitivity, we developed cysteine-stable isotope labeling using amino acids in cell culture (SILAC), a method that facilitates the detailed characterization of disulfide-bridged peptides by mass spectrometry. For this analysis, an antibody was metabolically labeled with 13C3,15N-cysteine and incorporated into a comprehensive panel of distinct bispecific molecules by controlled Fab-arm exchange (DuoBody technology). This technology is a postproduction method for the generation of bispecific therapeutic IgGs of which several have progressed into the clinic. Herein, two parental antibodies, each containing a single heavy chain domain mutation, are mixed and subjected to controlled reducing conditions during which they exchange heavy-light (HL) chain pairs to form bsAbs. Subsequently, reductant is removed and all disulfide bridges are reoxidized to reform covalent inter- and intrachain bonds. We conducted a multilevel (Top-Middle-Bottom-Up) approach focusing on the characterization of both "left-arm" and "right-arm" HL interchain disulfide peptides and observed that native HL pairing was preserved in the whole panel of bsAbs produced by controlled Fab-arm exchange.