Human Complement Component Research Articles

Multifunctional interaction of CihC/FbpC orthologs of relapsing fever spirochetes with host-derived proteins involved in adhesion, fibrinolysis, and complement evasion.

Relapsing fever (RF) remains a neglected human disease that is caused by a number of diverse pathogenic Borrelia (B.) species. Characterized by high cell densities in human blood, relapsing fever spirochetes have developed plentiful strategies to avoid recognition by the host defense mechanisms. In this scenario, spirochetal lipoproteins exhibiting multifunctional binding properties in the interaction with host-derived molecules are known to play a key role in adhesion, fibrinolysis and complement activation. Binding of CihC/FbpC orthologs to different human proteins and conversion of protein-bound plasminogen to proteolytic active plasmin were examined by ELISA. To analyze the inhibitory capacity of CihC/FbpC orthologs on complement activation, a microtiter-based approach was performed. Finally, AlphaFold predictions were utilized to identified the complement-interacting residues. Here, we elucidate the binding properties of CihC/FbpC-orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to human fibronectin, plasminogen, and complement component C1r. All CihC/FbpC-orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by tranexamic acid, indicating that lysine residues are involved in the interaction with this serine protease. In addition, a strong inactivation capacity toward the classical pathway could be demonstrated for the wild-type CihC/FbpC-orthologs as well as for the C-terminal CihC fragment of B. recurrentis. Pre-incubation of human serum with borrelial molecules except CihC/FbpC variants lacking the C-terminal region protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing AlphaFold2 predictions and existing crystal structures, we mapped the putative key residues involved in C1r binding on the CihC/FbpC orthologs attempting to explain the relatively small differences in C1r binding affinity despite the substitutions of key residues. Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of relapsing fever spirochetes proposed to be involved in pathogenesis and virulence.

Binding profiles of human and mouse complement component 8γ to trisubstituted organometallic compounds

Complement component 8gamma (C8γ), a member of the lipocalin protein family, is suggested to act as a carrier protein for various chemicals. Although C8γ has been identified in both humans and rodents for some time, our understanding of the species differences in its chemical binding properties remains limited. In the present study, with the aim to elucidate the potential role of C8γ as a carrier protein in both humans and mice, we conducted a radioligand binding assay to examine the chemical binding properties of human C8γ (hC8γ) and mouse C8γ (mC8γ). Scatchard analysis revealed that [14C]TPT bound to hC8γ with an equilibrium dissociation constant (Kd) of 64.2 ± 32.4 nM, comparable to that of [14C]TPT to mC8γ. Competitive ligand-binding assays demonstrated binding of TPT and TBT to hC8γ, while diphenyltin, dibutyltin, monophenyltin, monobutyltin, and tetrabutyltin did not exhibit binding. These results suggest that for effective binding to C8γ, chemicals must possess substituents of appropriate bulkiness. Further analyses with other group 14 compounds with triphenyl substituents revealed that a central metal atom, rather than a central non-metal or semi-metal atom, is crucial for specific binding to both hC8γ and mC8γ. Overall our findings imply that C8γ may play a role in the physiological or toxicological actions of group 14 metal compounds with tributyl or triphenyl substituents by binding to these chemicals in both humans and mice.

Molecular recognition and binding between human plasminogen Kringle 5 and α-chain of human complement component C3b by frontal chromatography and dynamics simulation

The binding and molecular recognition between α-chain of human complement C3b (α-chain of C3b) and human plasminogen Kringle 5 (Kringle 5) were studied and explored by frontal chromatography and dynamics simulation in the combination of bio-specific technologies. The specific interaction between the α-chain of C3b and Kringle 5 was initially confirmed by ligand blot and ELISA (Kd = 4.243×10−6 L/mol). Furthermore, the binding determination conducted via frontal chromatography showed that the presence of a single binding site between them, with the binding constant of 2.98 × 105 L/mol. Then the molecular recognition by dynamics simulation and molecular docking showed that there were 9 and 13 amino acid residues respective in the Kringle 5 and α-chain of C3b directly implicated in the binding and the main stabilizing forces were electrostatic force (-55.99 ± 11.82 kcal/mol) and Van der Waals forces (-42.70 ± 3.45 kcal/mol). Additionally, a loop structure (65–71) in Kringle 5 underwent a conformational change from a random structure to an α-helix and a loop structure (417–425) in α-chain of C3b was closer to the molecular center, both of them were more conducive to the binding between them. Meanwhile, the involvement of the lysine binding site of Kringle 5 played an important role in the binding process. In addition, the erythrocyte-antibody complement rosette assay substantiated that the presence of Kringle 5 hindered the transportation of α-chain of C3b to antigen-antibody complex in a dose-dependent manner. These findings collectively indicated that the α-chain of C3b is very likely a receptor protein for Kringle 5, which provides a methodology for other similar investigations and valuable insights into expansion of the pharmacological effects and potential application of Kringle 5 in immune-related diseases.

ZGGS15, a Novel IgG4 Bispecific Monoclonal Antibody Targeting Both LAG-3 and Tigit, Demonstrates Outstanding Anti-Tumor Activity and Can be Used As Monotherapy or in Combination with Anti-PD-1 Antibodies for Cancer Immunotherapy

Background This study aims to evaluate the anti-tumor efficacy of ZGGS15, a novel IgG4 bispecific monoclonal antibody (BsAb) targeting TIGIT and LAG-3, for cancer immunotherapy. Methods: ZGGS15 was generated in-house with the standard recombinant BsAb technology. ZGGS15 proteins were purified with protein A affinity column chromatography and evaluated for purity by size-exclusion high-performance liquid chromatography. The ZGGS15 affinity was assessed using a biolayer interferometry (BLI) assay. The EC50s of ZGGS15 binding to TIGIT and LAG-3 were determined using the ELISA technique. The EC50s of ZGGS15 binding to REH, Hela, MCF-7, and HCC1954 cells were measured by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities were also evaluated by flow cytometry. BALB/c-hPD-1/hLAG-3 and BALB/c-hPD-1/hTIGIT subcutaneously transplanted with CT26.WT tumor mouse models, and peripheral blood mononuclear cells (PBMC) -humanized NOG bearing A375 tumor mouse model were used for in vivo anti-tumor efficacy evaluation. Results ZGGS15 binds specifically to activated CD4 + and CD8 + T cells and human LAG-3 and TIGIT proteins expressed by CHO-K1 cells identically with a dose-dependent nanomolar affinity of 0.69 nM and 1.87 nM for human LAG-3 and TIGIT, respectively. In vitro kinetic analysis BLI results revealed ZGGS15 has high affinity for human LAG-3 and human TIGIT proteins, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 binds to human LAG-3 and human TIGIT simultaneously with similar affinity. Furthermore, ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50=0.24 nM). ZGGS15 induced better T cell activation than single anti-LAG3 or anti-TIGIT mAb agent or combined with different stimuli with or without cancer cells. When combined with nivolumab, ZGGS15 can further enhance the T cell activation in a dose-dependent manner. ADCC/CDC assay results showed that ZGGS15 binding to target cells does not induce ADCC or CDC. ZGGS15 interacts with the human neonatal Fc receptor in a pH-dependent manner, which can promote recirculation of ZGGS15 with a prolonged half-life in plasma. Besides, ZGGS15 interacts with human Fc-γ receptors and the human complement component, C1q, with low binding affinities. However, ZGGS15 does not induce obvious cytokine production from PBMCs, including IFN-γ, IL-2, IL-6, IL-10, and TNF-α. In vivo anti-tumor efficacy for ZGG15 in PBMC humanized NOG mice bearing A375 tumors showed that ZGGS15 had significant anti-tumor effects in the subcutaneous A375 xenograft model. ZGGS15 showed a better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agent and a synergetic effect when combined with nivolumab. On day 18 post-treatment, the ZSAB015 plus nivolumab treatment demonstrated a significantly tumor growth inhibition (TGI) of 95.80% ( p =0.001), compared with the vehicle group. In vivo efficacy tests in BALB/c-hPD-1/hLAG-3 mice subcutaneously transplanted with CT26.WT tumor model showed that both high and low doses of ZGGS15 could inhibit the growth of CT26.WT colon cancer. The combination of high-dose ZGGS15 plus nivolumab has better efficacy than high-dose ZGGS15 and is slightly better than nivolumab alone. The tumor volume inhibition rate (TGITV) for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% ( p < 0.001). During the experiment, the tumor-bearing mice tolerated it well, and no adverse reactions were observed. In vivo efficacy tests of ZGG15 in BALB/c-hPD-1/hTIGIT mice subcutaneously transplanted with CT26.WT tumor model showed that ZGGS15 at a low dose (1.5 mg/kg) demonstrated a significant tumor inhibitory effect, with the TGITV of 51.42% ( p < 0.01). The combination of ZGGS15 high-dose with nivolumab (5 mg/kg + 1 mg/kg) showed a better tumor inhibitory effect compared with ZGGS15 high-dose (5 mg/kg), with the TGITV of 81.15% and 41.54%, respectively ( p < 0.05). Conclusions ZGGS15 has excellent anti-tumor activity by inhibiting LAG-3 and TIGIT as a new IgG4 BsAb. ZGGS15 also exhibits potent anti-tumor efficacy in transgenic mouse models combined with nivolumab. Additionally, ZGGS15 does not elicit ADCC or CDC, therefore avoiding the cross-reaction of non-target binding. Finally, ZGGS15 does not induce cytokine production, mitigating any potential adverse effects of CRS.

Efficacy of Parallel and Crossover Analysis As Well As Pharmacokinetic Similarity Were Confirmed between ABP 959 and Eculizumab Reference Product in Patients with PNH

Introduction: ABP 959, a biosimilar to eculizumab reference product (RP), binds to the human complement component 5 protein to inhibit terminal complement activation. Eculizumab RP is approved for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, generalized myasthenia gravis, and neuromyelitis optical spectrum disorder. Regulatory approval of biosimilars is based on a totality of evidence approach which includes demonstration of similarity in analytical and non-clinical characteristics, and in comparative clinical studies. Analytical and non-clinical similarities of ABP 959 with eculizumab RP and clinical pharmacokinetics (PK)/pharmacodynamics (PD) and safety similarities in healthy adults have been previously reported. Similarity of the primary efficacy and safety results from the parallel and crossover comparisons in patients with PNH have also been previously reported. Here, we report together the primary efficacy results from both the parallel and the cross-over comparisons along with additional PK analyses in patients with PNH to further support the demonstration of similarity of ABP 959 with eculizumab RP. Methods: A multicenter, randomized, double-blind, active-controlled, 2-period crossover study evaluated the clinical similarity of ABP 959 compared with eculizumab RP in adult patients with PNH. Patients were randomized 1:1 to receive each investigational product (900 mg of ABP 959 or eculizumab RP IV q14d) in 1 of 2 treatment sequences (ABP 959/eculizumab RP or eculizumab RP/ABP 959). Primary efficacy determined by hemolysis, measured by lactate dehydrogenase (LDH), was analyzed for both the parallel and crossover comparisons. The parallel comparison endpoint was measured at week 27 and clinical similarity was assessed by comparing the 1-sided 97.5% upper confidence interval (CI) limit for the geometric least squares (LS) mean ratio of LDH between ABP 959 treatment group and eculizumab RP treatment group with a non-inferiority (NI) margin of 2.873. The crossover comparison endpoint was measured by the time-adjusted area under the effect curve (AUEC) of LDH from weeks 13 to 27, 39 to 53, and 65 to 79. The similarity of efficacy between treatment groups was assessed by comparing the 2-sided 90% CI for the geometric mean ratio (GMR) of the time-adjusted AUEC of LDH with a similarity margin of (0.77, 1.30). The total and unbound PK area under the curve (AUC) of ABP 959 and eculizumab RP from week 13 to week 15 and trough PK were identified as secondary endpoints in this study. Results: Forty-two patients (20 in ABP 959/eculizumab RP group; 22 in eculizumab RP/ABP 959 group) were randomized across 25 centers. Similarity of efficacy was established in both the parallel and crossover comparisons. In the parallel comparison, the ratio of the geometric LS means of LDH at week 27 (ABP 959 vs eculizumab RP) was 1.0628, with a 1-sided 97.5% upper CI of 1.1576 was contained within the NI margin of 2.873. In the crossover comparison, a point estimate of the GMR of time-adjusted AUEC of LDH (ABP 959 vs eculizumab RP) of 0.9812 with a 2-sided 90% CI of (0.9403-1.0239) was contained within the prespecified margin of similarity. The GMR (90% CI) for the total and unbound PK AUC from week 13 to week 15 was 0.9122 (0.7586, 1.0968) and 0.9508 (0.7454, 1.2130), respectively. Geometric mean values for trough total and unbound concentrations of ABP 959 and eculizumab RP were similar between the treatment groups at all time points tested over the entire study. Conclusions: Similarity in clinical efficacy in both parallel and crossover comparisons as determined by hemolysis in patients with PNH was established between ABP 959 and eculizumab RP. Additionally, analyses of serum total and unbound PK concentrations for patients with PNH further demonstrated PK similarity between ABP 959 and eculizumab RP. The results of this study, along with previously demonstrated analytical, non-clinical, clinical PK/PD in healthy adults, and efficacy and safety evaluations in PNH patients, further support a demonstration of no clinically meaningful differences between ABP 959 and eculizumab RP.

Iberdomide Enhances Dara Mediated Cytotoxicity through Upregulation of CDC Activity and Elevated NK Cell Mediated ADCC

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with current treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent anti-tumor activity and immunomodulatory effects. Clinical studies utilizing the combination of IMiD agents and daratumumab (DARA), an anti-CD38 monoclonal antibody, have shown improved patient outcomes for MM. However, patients ultimately relapse demonstrating the need for novel therapies. Iberdomide (IBER) is a novel cereblon E3 ligase modulator. It promotes faster and deeper degradation of the substrate targets compared to IMiDs, resulting in enhanced anti-proliferative, pro-apoptotic and immune stimulatory activity. In early clinical studies, IBER has been shown to overcome resistance to IMiDs and in the ongoing Phase 3 EXCALIBER study (NCT04975997) is being explored for treatment of newly diagnosed (ND) and relapsed/refractory (RR) MM in combination with DARA and dexamethasone. Here, we investigate the potential mechanisms of synergy between IBER and DARA in preclinical models. Methods: Complement dependent cytotoxicity (CDC) of DARA was assessed with 15% human serum for human complement components and assays were performed by titrating either single agent or combinations in a tritiated thymidine assay. Antibody dependent cellular cytotoxicity (ADCC) was performed by co-culturing MM cells with varying expression levels of CD38 and effector cells and assessing target cell viability with annexin-v/topro-3 flow. A humanized PBMC adoptive transfer mouse model, utilizing human PBMC engraftment 14 days prior to implantation, was used to assess in vivo activity of IBER, DARA and IBER + DARA. Results: In CDC assays, we observed variable sensitivity of MM cells to complement-specific CDC in the presence of DARA (0.1-1 µg/ml), regardless of cell surface CD38 expression. Notably, IBER treatment resulted in upregulation of CD38 surface expression in 60% of MM cell lines tested. LP1 and MOLP8 displayed susceptibility to DARA-mediated CDC. DARA showed a dose-dependent increase in CDC activity in both cell lines. Notably, IBER+DARA also showed ~35% more synergistic CDC activity in both cell lines, than the single agents. In ADCC assays, we observed pretreatment of PBMC with IBER (vs. DMSO) significantly increased DARA mediated ADCC in H929 and LP-1 cells. To confirm that this effect was directed by NK cells, we cultured NK cells for 24 hr in supernatants from stimulated PBMC in presence of IBER prior to co-culture with MM cells. We noted that IBER-treated PBMC supernatant significantly enhanced the NK ADCC activity of DARA suggesting that IBER enhancement of cytokine secretion from PBMC increases NK cell mediated ADCC. Orthogonally, we pre-treated MM cells with IBER or DMSO prior to co-culture in ADCC assay. Notably, IBER treatment of MM cells, without treating immune cells, also increased DARA-mediated ADCC, indicating that IBER directly increases sensitivity to DARA-directed ADCC. To investigate the MOA of IBER-DARA synergy in ADCC, we analyzed gene expression and cell surface changes of a panel of NK-specific receptor ligands on MM cells following treatment with IBER. We observed IBER-induced upregulation of MICA mRNA levels (~2x) and surface expression (~40%) in MM target cells. We used RNAi to knock down Ikaros, an IBER substrate, which resulted in increased MICA expression (~3x) in MM cells. Furthermore, we observed that RNAi knock down of MICA in MM cells resulted in a significant decrease (60%, p<0.0001) in MM cell killing by NK cells, suggesting IBER-enhanced sensitivity of MM cells to NK-cell killing was in part, due to the upregulation of MICA through degradation of Ikaros. Lastly, we examined the anti-MM effects of IBER in a humanized xenograft model with a lenalidomide resistant cell line. Longitudinal analysis identified persistence of human PBMC engraftment and activation of immune subsets (NK cells and T-cells) that were increased upon IBER treatments, consistent with clinical observations. The combination of IBER/DARA resulted in ~22% tumor growth inhibition (TGI) in comparison to single agents, DARA and IBER and ~37% TGI in comparison to vehicle. Conclusion: Taken together, these data demonstrate that IBER and DARA work synergistically in preclinical models primarily through enhancement of both ADCC and CDC. This combination has the potential to improve outcomes in MM.

Surface antibody changes protein corona both in human and mouse serum but not final opsonization and elimination of targeted polymeric nanoparticles

BackgroundNanoparticles represent one of the most important innovations in the medical field. Among nanocarriers, polymeric nanoparticles (PNPs) attracted much attention due to their biodegradability, biocompatibility, and capacity to increase efficacy and safety of encapsulated drugs. Another important improvement in the use of nanoparticles as delivery systems is the conjugation of a targeting agent that enables the nanoparticles to accumulate in a specific tissue. Despite these advantages, the clinical translation of therapeutic approaches based on nanoparticles is prevented by their interactions with blood proteins. In fact, the so-formed protein corona (PC) drastically alters the biological identity of the particles. Adsorbed activated proteins of the complement cascade play a pivotal role in the clearance of nanoparticles, making them more easily recognized by macrophages, leading to their rapid elimination from the bloodstream and limiting their efficacy. Since the mouse is the most used preclinical model for human disease, this work compared human and mouse PC formed on untargeted PNPs (uPNPs) and targeted PNPs (tPNPs), paying particular attention to complement activation.ResultsMouse and human serum proteins adsorbed differently to PNPs. The differences in the binding of mouse complement proteins are minimal, whereas human complement components strongly distinguish the two particles. This is probably due to the human origin of the Fc portion of the antibody used as targeting agent on tPNPs. tPNPs and uPNPs mainly activate complement via the classical and alternative pathways, respectively, but this pattern did not affect their binding and internalization in macrophages and only a limited consumption of the activity of the human complement system was documented.ConclusionsThe results clearly indicate the presence of complement proteins on PNPs surface but partially derived from an unspecific deposition rather than an effective complement activation. The presence of a targeting antibody favors the activation of the classical pathway, but its absence allows an increased activation of the alternative pathway. This results in similar opsonization of both PNPs and similar phagocytosis by macrophages, without an impairment of the activity of circulating complement system and, consequently, not enhancing the susceptibility to infection.Graphical abstract

Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.

The complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885. The interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers. Zilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies. Our findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.

Optimizing the use of vilobelimab for the treatment of COVID-19

ABSTRACT Introduction On 4 April 2023i4 April 2023, the United States Food and Drug Administration issued an emergency use authorization for the use of vilobelimab (GohibicTM) for the treatment of COVID-19 in hospitalized adults when initiated within 48 hours of receiving invasive mechanical ventilation or extracorporeal membrane oxygenation. Areas Covered Vilobelimab is a human-mouse chimeric IgG4 kappa antibody that targets human complement component 5a, a part of the immune system that is thought to play an important role in the systemic inflammation due to SARS-CoV-2 infection that leads to COVID-19 disease progression. Expert Opinion A pragmatic, adaptive, randomized, multicenter phase II/III study evaluating vilobelimab for the treatment of severe COVID-19 found that patients receiving invasive mechanical ventilation and usual care who were treated with vilobelimab had a lower risk of death by day 28 and day 60 compared to those receiving placebo. This manuscript explores what is known about vilobelimab and explores how this treatment may be used in the future to treat severe COVID-19.

Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.

Human complement component C3 N-glycome changes in type 1 diabetes complications.

Changes in N-glycosylation have been described in numerous diseases and are being considered as biomarkers of ongoing pathological condition. Previous studies demonstrated the interrelation of N-glycosylation and type 1 diabetes (T1D), particularly linking serum N-glycan changes with complications accompanying the disease. Moreover, the role of complement component C3 in diabetic nephropathy and retinopathy has been implicated, and C3 N-glycome was found to be altered in young T1D patients. Therefore, we investigated associations between C3 N-glycan profiles and albuminuria and retinopathy accompanying T1D, as well as glycosylation connection with other known T1D complication risk factors. Complement component C3 N-glycosylation profiles have been analyzed from 189 serum samples of T1D patients (median age 46) recruited at a Croatian hospital centre. Using our recently developed high-throughput method, relative abundances of all six of the C3 glycopeptides have been determined. Assessment of C3 N-glycome interconnection with T1D complications, hypertension, smoking status, estimated glomerular filtration rate (eGFR), glycaemic control and duration of the disease was done using linear modelling. Significant changes of C3 N-glycome in severe albuminuria accompanying type 1 diabetes were observed, as well as in T1D subjects with hypertension. All except one of the C3 glycopeptides proved to be associated with measured HbA1c levels. One of the glycoforms was shown to be changed in non-proliferative T1D retinopathy. Smoking and eGFR showed no effect on C3 N-glycome. Furthermore, C3 N-glycosylation profile was shown to be independent of disease duration. This study empowered the role of C3 N-glycosylation in T1D, showing value in distinguishing subjects with different diabetic complications. Being independent of the disease duration, these changes may be associated with the disease onset, making C3 N-glycome a potential novel marker of the disease progression and severity.

Killer to cure: Expression and production costs calculation of tobacco plant-made cancer-immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have achieved huge clinical success. However, many still have limited response rates, and are prohibitively costly. There is a need for effective and affordable ICIs, as well as local manufacturing capacity to improve accessibility, especially to low-to-middle income countries (LMICs). Here, we have successfully expressed three key ICIs (anti-PD-1 Nivolumab, anti-NKG2A Monalizumab, and anti-LAG-3 Relatimab) transiently in Nicotiana benthamiana and Nicotiana tabacum plants. The ICIs were expressed with a combination of different Fc regions and glycosylation profiles. They were characterized in terms of protein accumulation levels, target cell binding, binding to human neonatal Fc receptors (hFcRn), human complement component C1q (hC1q) and various Fcγ receptors, as well as protein recovery during purification at 100 mg- and kg-scale. It was found that all ICIs bound to the expected target cells. Furthermore, the recovery during purification, as well as Fcγ receptor binding, can be altered depending on the Fc region used and the glycosylation profiles. This opens the possibility of using these two parameters to fine-tune the ICIs for desired effector functions. A scenario-based production cost model was also generated based on two production scenarios in hypothetical high- and low-income countries. We have shown that the product accumulation and recovery of plant production platforms were as competitive as mammalian cell-based platforms. This highlights the potential of plants to deliver ICIs that are more affordable and accessible to a widespread market, including LMICs.

Design, Synthesis of Dioxopiperidinamide Derivatives by Amide Coupling Reaction and Study of Their Biological Activity

AbstractA series of fifteen dioxopiperidinamide derivatives synthesized and their spectra chemical data were reported. An alternative amide coupling reaction has been followed by using 3‐aminopiperidine‐2,6‐dione through amide bond functionalization between N‐Ethyl‐N′‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and Hydroxybenzotriazole (HOBt). All the designed compounds were docked in to the active sites of 2 A73: Human Complement Component C3. All the docked compounds is favorable to make both hydrogen and hydrophobic interaction with amino acids located at the binding site. The most of potential compounds (E)‐N‐(2,6‐dioxopiperidin‐3‐yl)‐3‐(m‐tolyl)acrylamide (4 g), (E)‐3‐(4‐bromophenyl)‐N‐(2,6‐dioxopiperidin‐3‐yl)acrylamide (4 h) and N‐(2,6‐dioxopiperidin‐3‐yl)‐3‐methyl‐4‐oxo‐4,6,7,8,9,10‐hexahydrothieno [2′,3′:4,5]pyrimido[1,2‐a]azepine‐2‐carboxamide (7) studied for their anticancer activity on the THP‐1 cancer cells line. It was also further established their Anti‐inflammatory activity and Antioxidant activity with the comparable efficiency against those of clinically used drugs.

Efficacy and Safety of Biosimilar Candidate ABP 959 As Compared with Eculizumab Reference Product in Paroxysmal Nocturnal Hemoglobinuria

Introduction: ABP 959 is a biosimilar candidate to eculizumab reference product (RP). Eculizumab is a humanized recombinant immunoglobulin antibody that binds to the human complement component 5 protein to inhibit progression of complement cascades and is approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, generalized myasthenia gravis, and neuromyelitis optica spectrum disorder. Methods: This multicenter, randomized, double-blind, active-controlled, 2-period crossover study compared the efficacy and safety of ABP 959 and eculizumab RP in adult patients with PNH who were stable on eculizumab 900 mg IV every 14 ± 2 days. The primary objective of this study was to evaluate the efficacy of ABP 959 compared to eculizumab RP based on control of intravascular hemolysis, with a secondary objective to compare their safety, pharmacokinetics (PK), and immunogenicity. Results from Period 1 of the crossover study are presented here. Patients were randomized 1:1 to receive either ABP 959 at 900 mg IV for 52 weeks in Period 1 followed by eculizumab RP 900 mg IV for 26 weeks in Period 2 (ABP 959/RP) or eculizumab RP 900 mg IV for 52 weeks followed by ABP 959 at 900 mg IV for 26 weeks (RP/ABP 959) (Figure). The primary efficacy endpoint for the parallel comparisons was hemolysis, measured by lactate dehydrogenase (LDH) at week 27; clinical similarity between treatments was assessed by comparing the 1-sided 97.5% upper confidence interval (CI) limit for the geometric least squares (LS) mean ratio between ABP 959 and eculizumab RP, with a non-inferiority (NI) margin of 2.873. To assess the robustness of the primary analysis result, a sensitivity analysis was conducted using a subset of randomized patients who did not experience any important protocol deviation affecting their primary efficacy evaluation. Secondary endpoints included total complement (50% total hemolytic complement activity [CH50]), LDH-time profile, number of units of packed red blood cells (pRBCs) transfused, safety, immunogenicity, and PK area under the serum concentration-time curve (PK AUC) of ABP 959 and eculizumab RP. Results: Forty-two patients (20 in the ABP 959/RP group; 22 in the RP/ABP 959 group) were randomized and treated in Period 1 across 25 centers. Forty-one (97.6%) patients (20 [100%] in ABP 959/RP and 21 [95.5%] in RP/ABP 959) completed Period 1 dosing. Baseline demographic and disease characteristics were comparable between the 2 treatment groups - mean (SD) age: 50.2 (16.6) years; time since original PNH diagnosis: 128.3 (116.1) months; duration of pre-study treatment: 68.4 (39.8) months. Results for week 27 LDH geometric LS means are presented below (Table). The ratio of the geometric LS means of LDH (ABP 959/RP) was 1.0628, with a 1-sided 97.5% upper CI of 1.1576 contained within the prespecified NI margin of 2.873, establishing similarity in clinical efficacy between ABP 959 and eculizumab RP in the parallel comparison; 95% CI was 0.9758 to 1.1576. Results from the sensitivity analysis were identical with results from the primary efficacy analysis. Total complement (CH50), total hemoglobin, serum-free hemoglobin, haptoglobin, bilirubin, and type III erythrocytes values were generally stable over time and results were comparable between the 2 treatment groups. The overall LDH profile and the mean number of units of pRBCs transfused during the study period were also comparable. Total PK AUC from week 13 to week 15 were similar between the 2 treatment groups. A comparable safety profile was observed in Period 1 between treatment groups, with only a single patient (4.5%) in the eculizumab RP group discontinuing the study due to any adverse event (grade 2 non-serious asthenia and grade 2 non-serious fatigue). The nominal difference in serious adverse event incidence was determined to be not clinically meaningful. Additionally, immunogenicity results through Period 1 were similar between ABP 959 and eculizumab RP groups. No patients in either treatment group tested positive for binding antidrug antibodies (ADAs), neutralizing ADAs, or treatment-boosted ADAs. Conclusions: Similarity in clinical efficacy was established between ABP 959 and eculizumab RP in the primary endpoint of hemolysis, measured by LDH at week 27. Additionally, comparable safety, PK, and immunogenicity results support the conclusion of clinical similarity between ABP 959 and eculizumab RP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analysis of the relationship between human complement components C3 & C4 deficiency and C– reactive protein levels as an indicator of the increase of microbial infections

The complement system is an important component of the immune system that consists of a group of plasma proteins produced by the liver and serving a defense function. As a result, the deficiency in any of the complement system components leads to the loss of the function of the deficient protein causing a specific clinical problem or disease. For that reason, this study was designed to investigate C3 and C4 deficiencies in young patients and to inspect the possibility of using C-Reactive Protein (CRP) test and the variation of C3 and C4 concentrations in children`s blood as an indicator of the onset of infection. This study was conducted in the children hospital in Benghazi city, Libya. The blood samples were collected from 446 children in all hospital units to examine the proportion of complement deficiency among those children and C3, C4 deficiency was set as an indicator of infection occurrence. The results of this study have shown a deficiency in complement proteins C3 and C4 were in 44 (10%) samples and 2 (0.4%) samples respectively, the female candidates had a higher rate of C3 and C4 deficiency than males. In addition, the CRP levels were elevated in 167 (55.9%) cases. Furthermore, the association between the increase of CRP levels and low levels of C3 and C4 was observed in 26 (8.7%) cases and 2 (0.6%) cases. These results suggest a strong association between complement proteins C3 and C4 deficiency and an elevation of CRP levels (P-Value=0.00), which supports the concept of using such association as an indicator of microbial infection

Quantification of the pro-form of human complement component factor D (adipsin)

Factor D (also known as adipsin) is a serine protease and part of the complement system, involved in innate immune responses and effector functions of antibodies. Factor D cleaves factor B complexed with C3b, leading to the C3 convertase C3bBb. This C3 convertase is central in the alternative activation pathway and the amplification loop, which amplifies the two other complement activation pathways: the classical pathway and the lectin pathway. Adipocytes synthesize factor D as a pro-form comprising 6 additional residues that must be cleaved off to generate a mature form. The MBL-associated serine protease 3 (MASP-3), found in complex with the pattern recognition molecules of lectin activation pathway, converts the pro-form to mature factor D, which reportedly is the most abundant form found in the circulation at concentrations of 1–2 μg/ml among healthy individuals.The mature factor D is rate-limiting for complement activation, but little is known about the distribution of pro vs. mature factor D in the circulation, the regulation hereof and the potential activation stimuli of the lectin pathway, responsible for activation of MASP-3 and subsequent conversion of pro-form of factor D.In this light we established and validated an ELISA specific for measuring the pro-form of complement factor D. With a working range of 0.82–25 ng/ml, acceptable intra and inter assay CVs, and a relative recovery rate above 90%, we found that the median plasma concentration in Danish blood donors was 134 ng/ml; corresponding to that 8–15% factor D circulates as pro-form. We also found that blood sampling procedures affect conversion and hence the levels measured in serum and plasma.

Intrinsic chemical reactivity of activated human complement component C3: A historical glimpse into research during 1979–1980 on the covalent binding properties of C3, C4 and alpha-2 macroglobulin

A new type of intrinsic activity was revealed in the complement protein C3 during investigations of nucleophilic complement inhibitors. Nascent C3b molecules produced by cleavage-activation of C3 have highly reactive acyl groups which form short-lived sites for covalent binding to biological materials. Alpha-2 macroglobulin and C4 may utilise similar “autoligation” mechanisms.

The BS variant of C4 protects against age-related loss of white matter microstructural integrity.

Age-related loss of white matter microstructural integrity is a major determinant of cognitive decline, dementia and gait disorders. However, the mechanisms and molecular pathways that contribute to this loss of integrity remain elusive. We performed a genome-wide association study of white matter microstructural integrity as quantified by diffusion MRI metrics (mean diffusivity and fractional anisotropy) in up to 31 128 individuals from UK Biobank (age 45-81 years) based on a two degrees of freedom (2df) test of single nucleotide polymorphism (SNP) and SNP × Age effects. We identified 18 loci that were associated at genome-wide significance with either mean diffusivity (n = 16) or fractional anisotropy (n = 6). Among the top loci was a region on chromosome 6 encoding the human major histocompatibility complex (MHC). Variants in the MHC region were strongly associated with both mean diffusivity [best SNP: 6:28866209_TTTTG_T, beta (standard error, SE) = -0.069 (0.009); 2df P = 6.5 × 10-15] and fractional anisotropy [best SNP: rs3129787, beta (SE) = -0.056 (0.008); 2df P = 3.5 × 10-12]. Of the imputed human leukocyte antigen (HLA) alleles and complement component 4 (C4) structural haplotype variants in the human MHC, the strongest association was with the C4-BS variant [for mean diffusivity: beta (SE) = -0.070 (0.010); P = 2.7 × 10-11; for fractional anisotropy: beta (SE) = -0.054 (0.011); P = 1.6 × 10-7]. After conditioning on C4-BS no associations with HLA alleles remained significant. The protective influence of C4-BS was stronger in older participants [age ≥ 65; interaction P = 0.0019 (mean diffusivity), P = 0.015 (fractional anisotropy)] and in participants without a history of smoking [interaction P = 0.00093 (mean diffusivity), P = 0.021 (fractional anisotropy)]. Taken together, our findings demonstrate a role of the complement system and of gene-environment interactions in age-related loss of white matter microstructural integrity.

Affinity Purification–Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency

The vast majority of plant viruses are transmitted by insect vectors, with many crucial aspects of the transmission process being mediated by key protein-protein interactions. Still, very few vector proteins interacting with viruses have been identified and functionally characterized. Potato leafroll virus (PLRV) is transmitted most efficiently by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using affinity purification coupled to high-resolution mass spectrometry (AP-MS), we identified 11 proteins from M. persicaedisplaying a high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Three of these aphid proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV in cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Artificial diet delivery to aphids of a chemical inhibitor of C1QBP leads to increased PLRV acquisition by aphids and subsequently increased titer in inoculated plants, supporting a role for C1QBP in the acquisition and transmission efficiency of PLRV by M. persicae. This study presents the first use of AP-MS for the in vivo isolation of a functionally relevant insect vector-virus protein complex. MS data are available from ProteomeXchange.org using the project identifier PXD022167.

Genomic Variation Within the C4 Locus and Cerebrospinal Fluid Levels of C4 Isotype Proteins in Acute and Chronic Schizophrenia

The human complement component 4 (C4) locus includes the genes C4A and C4B. Copy numbers (CNs) of C4A increase risk of schizophrenia and in patient-derived models of synaptic pruning we have shown that C4A-CNs contribute to increased synapse elimination in schizophrenia. However, no data exist regarding C4A/C4B protein levels in the CNS of living humans, and the extent to which they may be related to variation within the C4 locus as well as to schizophrenia.