Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1 Domain Deletion

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component, C1q. To provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for quantitative visualization of the interaction between IgG and the C1 complex composed of C1q, C1r, and C1s. Results showed that C1q in the C1 complex is restricted regarding internal motion and has a stronger binding affinity for on-membrane IgG assemblages than C1q alone, presumably because of smaller conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG variant that lacks the entire CH1 domain in the absence of antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

Herbal NF-κB Inhibitors Sensitize Rituximab-Resistant B Lymphoma Cells to Complement-Mediated Cytolysis

BackgroundDrug resistance remains a serious challenge to rituximab therapy in B-NHL (B cell non-Hodgkin’s lymphoma). CDC (complement-dependent cytotoxicity) has been proposed as a major antitumor mechanism of rituximab, and direct abrogation of CD59 function partially restores rituximab sensitivity with high efficacy. However, universal blockade of CD59 may have deleterious effects on normal cells. Sp1 regulates constitutive CD59 expression, whereas NF-κB and CREB regulate inducible CD59 expression.MethodsImmunohistochemistry (IHC) assay was used to detect the expression levels of CD59 and other related molecules. Quantitative Real-time PCR (RT-PCR) analysis was used to explore the levels of transcripts in the original and resistant cells. We chose LY8 cells to test the effects of NF-κB and CBP/p300 inhibition on CD59 expression using flow cytometry (FACS). Immunoblotting analysis was employed to detect the effects of curcumin and POH. The in vitro and in vivo experiments were used to evaluate the toxicity and combined inhibitory effect on tumor cells of curcumin and POH.ResultsWe demonstrated that herbal (curcumin and perillyl alcohol) blockade of NF-κB specifically suppresses the expression of inducible CD59 but not CD20, thus sensitizing resistant cells to rituximab-mediated CDC. Moreover, activation of NF-κB and CREB is highly correlated with CD59 expression in B-NHL tissues.ConclusionsOur findings suggest the potential of CD59 expression as a predictor of therapeutic efficacy of NF-κB inhibitors in clinical application as well as the rationality of a NF-κB inhibitor-rituximab regimen in B-NHL therapy.

Gain-of-Function Mutations R249C and S250C in Complement C2 Protein Increase C3 Deposition in the Presence of C-Reactive Protein

The impairment of the alternative complement pathway contributes to rare kidney diseases such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). We recently described an aHUS patient carrying an exceptional gain-of-function (GoF) mutation (S250C) in the classical complement pathway component C2 leading to the formation of hyperactive classical convertases. We now report the identification of the same mutation and another C2 GoF mutation R249C in two other patients with a glomerulopathy of uncertain etiology. Both mutations stabilize the classical C3 convertases by a similar mechanism. The presence of R249C and S250C variants in serum increases complement-dependent cytotoxicity (CDC) in antibody-sensitized human cells and elevates deposition of C3 on ELISA plates coated with C-reactive protein (CRP), as well as on the surface of glomerular endothelial cells. Our data justify the inclusion of classical pathway genes in the genetic analysis of patients suspected of complement-driven renal disorders. Also, we point out CRP as a potential antibody-independent trigger capable of driving excessive complement activation in carriers of the GoF mutations in complement C2.

Mechanisms, Characteristics, and Treatment of Neuropathic Pain and Peripheral Neuropathy Associated with Dinutuximab in Neuroblastoma Patients

Prognosis of metastatic neuroblastoma is very poor. Its treatment includes induction chemotherapy, surgery, high-dose chemotherapy, radiotherapy, and maintenance with retinoic acid, associated with the anti-GD2 monoclonal antibody (ch14.18) dinutuximab. Immunotherapy determined a significant improvement in survival rate and is also utilized in relapsed and resistant neuroblastoma patients. Five courses of dinutuximab 100 mg/m2 are usually administered as a 10-day continuous infusion or over 5 consecutive days every 5 weeks. Dinutuximab targets the disialoganglioside GD2, which is highly expressed on neuroblastoma cells and minimally present on the surface of normal human neurons, peripheral pain fibers, and skin melanocytes. Anti GD2 antibodies bind to surface GD2 and determine the lysis of neuroblastoma cells induced by immune response via the antibody-dependent cellular cytotoxicity and the complement-dependent cytotoxicity. Dinutuximab has significant side effects, including neuropathic pain, peripheral neuropathy, hypersensitivity reactions, capillary leak syndrome, photophobia, and hypotension. The most important side effect is neuropathic pain, which is triggered by the same antibody–antigen immune response, but generates ectopic activity in axons, which results in hyperalgesia and spontaneous pain. Pain can be severe especially in the first courses of dinutuximab infusion, and requires the administration of gabapentin and continuous morphine infusion. This paper will focus on the incidence, mechanisms, characteristics, and treatment of neuropathic pain and peripheral neuropathy due to dinutuximab administration in neuroblastoma patients.

Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system. Most NMOSD patients are seropositive for immunoglobulin G (IgG) autoantibodies against astrocyte water channel aquaporin-4 (AQP4), called AQP4-IgG. AQP4-IgG binding to aquaporin-4 causes complement-dependent cytotoxicity (CDC), leading to inflammation and demyelination. Here, CDC was measured in AQP4-expressing cells exposed to human complement and heat-inactivated sera from 108 AQP4-IgG seropositive NMOSD subjects and 25 non-NMOSD controls. AQP4-IgG positive sera produced a wide range of CDC, with 50% maximum cytotoxicity produced by as low as 0.2% serum concentration. Unexpectedly, 58 samples produced no cytotoxicity, and of those, four sera were cytoprotective against cytotoxic AQP4-IgG. Cytoprotection was found against different cytotoxic monoclonal AQP4-IgGs and NMOSD patient sera, and in primary astrocyte cultures. Mechanistic studies revealed that the protective factor is an IgG antibody that did not inhibit complement directly, but interfered with binding of cytotoxic AQP4-IgG to AQP4 and consequent C1q binding and complement activation. Further studies suggested that non-pathogenic AQP4-IgG, perhaps with altered glycosylation, may contribute to reduced or ineffectual binding of cytotoxic AQP4-IgG, as well as reduced cell-surface AQP4. The presence of natural cytoprotective antibodies in AQP4-IgG seropositive sera reveals an added level of complexity in NMOSD disease pathogenesis, and suggests the potential therapeutic utility of ‘convalescent’ serum or engineered protective antibody to interfere with pathogenic antibody in AQP4-IgG seropositive NMOSD.

On-target IgG hexamerisation driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation

The majority of depleting monoclonal antibody (mAb) drugs elicit responses via Fc-FcγR and Fc-C1q interactions. Optimal C1q interaction is achieved through hexameric Fc:Fc interactions at the target cell surface. Herein is described an approach to exploit the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Fusion of the C-terminal tailpiece of IgM promoted spontaneous hIgG hexamer formation, resulting in enhanced C1q recruitment and complement-dependent cytotoxicity (CDC) but with off-target complement activation and reduced in-vivo efficacy. Mutation of the penultimate tailpiece cysteine to serine (C575S) ablated spontaneous hexamer formation, but facilitated reversible hexamer formation after concentration in solution. C575S mutant tailpiece antibodies displayed increased complement activity only after target binding, in-line with the concept of ‘on-target hexamerisation’, whilst retaining efficient in-vivo efficacy and augmented target cell killing in the lymph node. Hence, C575S-tailpiece technology represents an alternative format for promoting on-target hexamerisation and enhanced CDC.

Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity

The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats.

Establishment of HLA class I and MICA/B null HEK-293T panel expressing single MICA alleles to detect anti-MICA antibodies

Pre- and post-transplantation anti-MICA antibody detection development are associated with an increased rejection risk and low graft survival. We previously generated HLA class I null HEK-293T using CRISPR/Cas9, while MICA and MICB genes were removed in this study. A panel of 11 cell lines expressing single MICA alleles was established. Anti-MICA antibody in the sera of kidney transplant patients was determined using flow cytometric method (FCM) and the Luminex method. In the 44 positive sera, the maximum FCM value was 2879 MFI compared to 28,135 MFI of Luminex method. Eleven sera (25%) were determined as positive by FCM and 32 sera (72%) were positive by the Luminex method. The sum of total MICA antigens, MICA*002, *004, *009, *019, and *027 correlation showed a statistically significant between the two methods (P = 0.0412, P = 0.0476, P = 0.0019, P = 0.0098, P = 0.0467, and P = 0.0049). These results demonstrated that HEK-293T-based engineered cell lines expressing single MICA alleles were suitable for measuring specific antibodies against MICA antigens in the sera of transplant patients. Studies of antibodies to MICA antigens may help to understand responses in vivo and increase clinical relevance at the cellular level such as complement-dependent cytotoxicity.

Mechanisms of Action of the New Antibodies in Use in Multiple Myeloma

Monoclonal antibodies (mAbs) directed against antigen-specific of multiple myeloma (MM) cells have Fc-dependent immune effector mechanisms, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP), but the choice of the antigen is crucial for the development of effective immuno-therapy in MM. Recently new immunotherapeutic options in MM patients have been developed against different myeloma-related antigens as drug conjugate-antibody, bispecific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR)-T cells. In this review, we will highlight the mechanism of action of immuno-therapy currently available in clinical practice to target CD38, SLAMF7, and BCMA, focusing on the biological role of the targets and on mechanisms of actions of the different immunotherapeutic approaches underlying their advantages and disadvantages with critical review of the literature data.

The influence of the antithymocyte globulin dose on clinical outcomes of patients undergoing kidney retransplantation

Optimizing antithymocyte globulin (rATG) dosage is critical for high immunological risk patients undergoing a repeat kidney transplant. This natural retrospective cohort study compared clinical outcomes of two successive cohorts of consecutive recipients of retransplants receiving 5 x 1 mg/kg (rATG-5, n = 100) or a single 3 mg/kg (rATG-3, n = 110) dose of rATG induction therapy. All patients had negative complement-dependent cytotoxicity crossmatch and no anti-HLA A, B, DR donor-specific antibodies (DSA). The primary endpoint was efficacy failure (first biopsy-proven acute rejection, graft loss, or death) at 12 months. There was no difference in the cumulative incidence of efficacy failure (18.0% vs. 21.8%, HR = 1.22, 95% CI 0.66–2.25), respectively. There were no differences in 3-years freedom from biopsy proven acute rejection, and patient, graft, and death-censored graft survivals. There were no differences in the incidence of surgical complications (25.0% vs. 18.2%; p 0.151), early hospital readmission (27.8% vs. 29.5%; p = 0.877) and CMV infections (49% vs. 40%; p = 0.190). There were also no differences in the incidence (59.6% vs. 58.7%, p = 0.897) and duration of delayed graft function but a stable difference in estimate glomerular filtration rate was observed from month 1 (54.7±28.8 vs. 44.1±25.3 ml/min/1.73 m2, p = 0.005) to month 36 (51.1±27.7 vs. 42.5±24.5, p = 0.019). Mean urinary protein concentration (month 36: 0.38±0.81 vs. 0.70±2.40 g/ml, p = 0.008) and mean chronic glomerular Banff score in for cause biopsies (months 4–36: 0.0±0.0 vs. 0.04±0.26, p = 0.044) were higher in the rATG-3 group. This cohort analysis did not detect differences in the incidence of efficacy failure and in safety outcomes at 12 months among recipients of kidney retransplants without A, B, and DR DSA, receiving induction therapy with a single 3 mg/kg rATG dose or the traditional 5 mg/kg rATG.