Activation Of Classical Complement Pathway Research Articles

#2314 Circulating immune complexes concentration and complement activation in sensitized kidney transplant recipients

Abstract Background and Aims Chronic antibody mediated rejection of the kidney transplant is the most common cause of graft loss in the late posttransplant period. Crucial in this process seems to be the role of classical pathway complement activation due to the formation of immune complexes. The formation of immune complexes is a benign process of a normally functioning immune system. In kidney transplant recipients (KTRs), the production of donor specific IgG/IgM antibodies (DSAs) against donor antigens (MHC molecules, ABO blood group antigens) leads to the attachment of immune complexes to cell membranes and subsequent tissue damage through complement activation. The attachment of C1q complement fragment to the immune complexes holds an important part in this process. This study aims to determine the levels of immune complexes as well as classical pathway complement activation in sensitized kidney transplant recipients. Method In this cross-sectional study 3 groups of KTRs were compared as to the levels of immunocomplexes concentration and classical pathway complement activation. Fourteen KTRs with preformed or de novo donor specific antibodies were compared to 28 kidney transplant recipients with no DSAs and to 22 subjects with no history of chronic kidney disease (controls). We used MicroVue Complement CIC-C1q EIA that is designed to detect immunocomplexes that bind to C1q in human serum or plasma. In order to estimate total classical pathway complement activation in serum, we used MicroVue Complement CH50 Eq EIA. Results Kidney function (eGFR) of KTRs with and without DSAs was similar (53.1 ± vs. 60.8 ± ml/min/1.73 m2, p = 0.54) but lower in comparison to controls (106.4 ml/min/1.73 m2, p < 0.001). We found a significantly higher concentration of C1q-immunocomplexes in the serum of KTRs with donor specific antibodies, in comparison to controls, as well as to the group of kidney transplant recipients with no DSAs (6.78 ± 2.7 vs. 4.83 ± 1.9 vs. 5.04 ± 1.2 μgEq/ml respectively, p = 0.0078). Furthermore, there was no significant difference of C1q-immunocomplexes between kidney transplant recipients with no DSAs and controls. Finally, a significant reduction of CH50 levels was observed in KTRs with DSAs as compared to controls and KTRs with no DSAs (39.2 ± 14.9 vs. 70.6 ± 34.3 vs. 67.9 ± 39.9 U/ml respectively, p = 0.003), which suggests higher levels of classical pathway complement activation. CH50 levels in KTRs with DSAs, who were further subcategorized in those with preformed and those with de novo DSAs were similar (39.68 ± 15.6 vs 39.08 ± 16.04 U/ml). Conclusion Kidney transplant recipients with donor specific antibodies have increased serum levels of immune complexes and increased classical pathway complement activation.

Targeted complement inhibition using bispecific antibodies that bind local antigens and endogenous complement regulators

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases.

Tumid lupus erythematosus in C1-inhibitor deficiency.

A case of a 28-year-old woman with known C1-inhibitor deficiency (functional, Type II) with persistent bilateral non-pruritic, mildly photosensitive facial rash for 10 months following delivery of her second child is presented. Histology of the skin was suggestive of tumid lupus erythematosus (LE), but no other features of systemic LE (ANA, dsDNA negative) were evident. She had stopped danazol which was controlling the underlying disease, and once this was restarted and treatment for tumid lupus was started, she improved. More rigorous control preventing all C1-inhibitor deficiency-related attacks proved successful. The hypothesis that uncontrolled classical pathway complement activation that led to the lupus-like skin lesions is being presented as a clinical case, highlighting the complex interrelationships between immunodeficiency and autoimmunity in inborn errors in immunity.

Anticardiolipin Antibody Plays a More Important Role Than Anti-β2-Glycoprotein I Antibody in Activating Complement in Patients with Lupus Nephritis.

This research aimed to explore the correlation between antiphospholipid antibodies (aPLs) and complement activation in lupus nephritis (LN) patients. A retrospective analysis was carried out on patients diagnosed with LN based on renal biopsy from June 2019 to June 2022. The study assessed levels of IgM, IgA, and IgG subtypes of anticardiolipin antibodies (aCLs) and anti-β2-glycoprotein I (anti-β2-GPI) antibodies. Pathological and clinical data were collected concurrently with the renal biopsy. The analysis included 76 LN patients, with 44.7% testing positive for aPLs. LN patients with positive aPLs exhibited increased hematuria, higher SLEDAI scores, reduced serum C3 and C4 levels, and more C1q deposits in the glomerulus compared to those with negative aPLs (P<0.05). Correlation analysis demonstrated the inverse relationships between IgG-aCL levels and serum C3 and C4 levels (r=-0.29, P=0.005; r=-0.24, P=0.016, respectively), as well as a positive correlation with C4 deposits in the glomerulus (r=0.20, P=0.041). This investigation suggests that aPLs, particularly IgG-aCLs, may be associated with the severity of LN and could contribute to the activation of classical complement pathways.

Complement System Deficiencies in Elite Athletes.

Although regular physical activity improves immune competency and reduces the prevalence of inflammatory diseases, strenuous training in elite athletes is associated with an increased susceptibility to infectious complications. Therefore, the objective of our study was to assess the routinely examined parameters of the complement system in elite athletes. The study was carried out in a cohort of elite athletes (n = 134) and healthy control subjects (n = 110). In all subjects, besides a routine laboratory check-up, serum concentrations of the C3 and C4 complement components, mannose-binding lectin (MBL), as well as activation of all three complement pathways were determined. Compared to healthy controls, lower C3 and C4 complement component concentrations were observed in elite athletes (0.96 ± 0.1 vs. 1.08 ± 0.2mg/L, and 0.18 ± 0.1 vs. 0.25 ± 0.1mg/L, respectively, p < 0.05); with much higher frequency rates of C3 and C4 deficiencies in athletes (31.3 vs. 14.5%, and 6 vs. 0%, p < 0.05). Simultaneously, athletes had much higher frequency rates of deficiencies of activation of classical and alternative complement pathways; while, deficiency of activation of the lectin pathway was similar in both cohorts. We confirmed a high frequency of defects in the complement system in elite athletes. Lower concentrations of C3 and C4 complement components, with high frequencies of deficiencies of the classical and alternative complement activation pathways were the most prevalent disorder of the complement system in elite athletes. Further studies are needed to uncover the functional impacts of these observations upon the susceptibility to infectious diseases.

Thrombotic microangiopathy following systemic AAV administration is dependent on anti-capsid antibodies.

BACKGROUNDSystemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome-like complement activation, immune-mediated myocardial inflammation, and hepatic toxicity.METHODSWe describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 individuals following 2 distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation.RESULTSGroup 1 participants had a rapid increase in immunoglobulin M (IgM) and IgG. Increase in D-dimer, decline in platelet count, and complement activation are indicative of TMA. All Group 1 participants demonstrated activation of both classical and alternative complement pathways, as indicated by depleted C4 and elevated soluble C5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation.CONCLUSIONSThis study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune-mediated events that impact the safety and efficacy of systemic gene therapy.

Prevalence and Incidence of Primary Autoimmune Hemolytic Anemia and Cold Agglutinin Disease in the United States, 2016-2022: A Retrospective Study in Administrative Claims

Background Autoimmune hemolytic anemia (AIHA) is a decompensated acquired hemolysis caused by autoantibodies that act against red blood cells leading to anemia. Cold agglutinin disease (CAD) is a rare subtype of AIHA mediated by classical complement pathway activation, characterized by chronic hemolysis, severe anemia, fatigue, weakness, dizziness, and circulatory problems. The aim of this study was to update the epidemiology of AIHA and CAD in the United States using administrative claims data that includes commercially insured patients, as well as those with Medicare Advantage, Medicare Fee for Service (FFS) and Managed Medicaid plans. Methods Administrative closed claims from Optum CDM database (commercially insured single-payer and Medicare Advantage), Inovalon MORE 2 Registry (commercially insured multi-payer, Medicare Advantage, and Managed Medicaid), and 100% Medicare FFS (Part A/B services) data were used for this analysis. AIHA cases were defined as patients with at least 2 claims with ICD-9/10 diagnosis codes for AIHA (283.0, D59.1, D59.10, D59.11, D59.12, D59.13, D59.19) at least 30 days apart. CAD cases were defined as patients with at least 2 claims with ICD-10 diagnosis codes for CAD (D59.12) or at least 1 claim with diagnosis code for AIHA followed by 1 claim with diagnosis code for CAD (D59.12) at least 30 days apart. Index date was assigned to the first observed claims with the AIHA/CAD diagnosis. Analyses included patients 18 years of age and above with at least 180 days of continuous enrollment in their health plan prior to index for the assessment of the exclusion criteria. Patients with secondary causes of AIHA/CAD (e.g., lymphoma, leukemia, myeloma, cytomegalovirus, or Epstein-Barr virus) were excluded. US census data from years 2016-2021 was used to standardize the incidence and prevalence estimates by age and sex. Point prevalence was calculated as of January 1 st of each year using an all-data lookback. Period prevalence was calculated using a one calendar year assessment period. Incidence estimated new cases in a given year among patients with no prior evidence of AIHA in all data lookback. For CAD, estimates were presented for years 2022/23 due to the recency of the ICD-10 code specific to CAD (active as of 1 October 2020). Results Age and sex standardized point prevalence of AIHA per 1,000,000 persons was approximately 210 in the Medicare FFS across years 2016-2021, 160 in the Optum claims across years 2016-2022, and 50 in the MORE 2 database across years 2016-2022. One-year prevalence per 1,000,000 persons was approximately 190 in the Medicare FFS, 70 in the Optum claims, and 50 in the MORE 2 database across study years. Incidence per 1,000,000 was 50-60 in the Medicare FFS, 20-30 in the Optum claims, and 15 in the MORE 2 database. While overall standardized estimates differed across databases, incidence and prevalence estimates were similar when stratified by age group, indicating that standardization did not fully account for the underlying differences in the composition and representativeness of the overall US population in the three databases. Incidence and prevalence were higher among females than males and increased with age as expected. Incidence per 1,000,000 for year 2021 ranged from roughly 10-80 among those under the age of 45 years to 30-80 among those over the age of 65 years across the three databases. Point prevalence per 1,000,000 for year 2021 had a broader range from 30 to 220 among those under the age of 45 years and from 120 to 360 among those over the age of 65 years. The standardized point prevalence per 1,000,000 for CAD was 31 in Optum claims (year 2023), 33 in Medicare FFS (year 2021), and 14 in MORE 2 (year 2021). Prevalence of CAD was higher among females and increased with age. Conclusion This multi-database analysis provides updated epidemiology estimates for AIHA and CAD in the US. Age and sex standardized point prevalence for AIHA ranged from 60 to 210 per 1,000,000 across the 3 databases, with higher prevalence in the Medicare FFS database which primarily captures population over the age of 65 and was lower in the two databases that capture younger age groups, such as Managed Medicaid and commercially insured populations. Similarly, incidence of AIHA ranged from 15 to 60 per 1,000,000. Point prevalence of CAD ranged from 14 to 33 per 1,000,000. These estimates are in line with those previously reported for North America and Western Europe.

Efficacy of Sutimlimab in Improving FACIT-Fatigue Scores in Patients with Cold Agglutinin Disease: A Post-Hoc Analysis of the Phase 3 Cadenza (Part A) Study

Background Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia characterized by chronic hemolysis mediated by classical complement pathway activation. Chronic inflammation and anemia associated with CAD can lead to profound fatigue and general weakness, impacting the patients' quality of life. Sutimlimab is a first-in-class humanized monoclonal antibody approved for the treatment of CAD. To assess the effect of sutimlimab on patients' self-reported fatigue (Functional Assessment of Chronic Illness Therapy-Fatigue [FACIT-Fatigue] scores) in CAD, a post-hoc analysis of the CADENZA Part A study was carried out. Objective To assess the effect of sutimlimab vs placebo on FACIT-Fatigue total score, and individual items of the FACIT-fatigue scale in patients with CAD. Methods CADENZA (NCT03347422) Part A was a phase 3, double-blind, 26-Week study where patients were randomized (1:1) to receive intravenous sutimlimab or placebo. The 13-item FACIT-Fatigue questionnaire comprising 11 fatigue items and 2 energy items (‘I have energy’ or 'I am able to do my usual activities') was scored on a 0 (“not at all”) to 4 (“very much”) response scale, and the total scores ranged from 0 to 52; higher scores indicated less fatigue. Least square (LS) mean difference (sutimlimab vs placebo) in change from the baseline (BL) in FACIT-Fatigue total score were estimated at all visits using mixed model for repeated measures. Treatment assessment timepoint (TAT) was the average of values from Week 23, 25, and 26 visits. Patients were considered responders if they achieved a change from BL in FACIT-Fatigue total score of ≥5, did not receive blood transfusions from Week 5 through TAT, and did not take any protocol prohibited CAD medications. Patients with missing data at a given visit were considered non-responders. Time to first response to treatment was assessed using the Kaplan-Meier product limit method. For the item level analysis of the FACIT-Fatigue scale, the proportion of patients reporting the two worst response options 'Quite a bit' or ‘Very much’ for fatigue items and ‘Not at all’ or ‘A little bit’ for energy items was calculated at BL and Week 26. Results A total of 42 patients were included in the study (sutimlimab group: n=22; placebo group: n=20). The median (range) age of patients was 66.0 (46-88) years and 78.6% (n=33) were female. The mean (SD) FACIT-Fatigue total score for the pooled population at BL was 32.30 (11.83). About 79% of patients (n=33) had a total score below the general healthy population mean of 43. The LS mean (95% CI) difference (sutimlimab vs placebo) in change from BL in the FACIT-Fatigue total score was 4.97 (0.35─9.60) at Week 1 and 8.93 (4.00─13.85) at TAT. The nominal significant difference that started at Week 1 (rapid treatment effect) was sustained throughout Part A ( Figure 1). Overall, the fatigue response rate was higher in the sutimlimab group than placebo group throughout the study. The median time (95% CI) to first FACIT-Fatigue response in the sutimlimab and placebo groups were 3.1 (1.1─3.1) and 9.1 (3.3─22.4) weeks (log-rank test p&amp;lt;0.05), respectively. At BL, ~25-30% of the patients had reported the two worst response options (‘Quite a bit’ or 'Very much') for most of the FACIT-fatigue items. Around 40.5% (17/42) of the patients responded with one of the two worst options for item 'I feel tired', and more than half of the patients (22/42; 52.4%) reported one of the two worst options (‘Not at all’ or 'A little bit') for item 'I have energy'. At Week 26, lower proportion of patients in sutimlimab group reported one of the two worst options for fatigue and energy items compared with the placebo group ( Figure 2). Conclusion Sutimlimab treatment led to rapid and sustainable improvement in FACIT-Fatigue scores and higher response rates as compared to placebo. Number needed to treat to observe a sutimlimab-related FACIT-Fatigue benefit was 4. The proportion of severe FACIT-Fatigue answers for fatigue and energy items was noticeably lower in sutimlimab-treated patients at Week 26 compared to placebo, highlighting the effect of sutimlimab in improving almost all dimensions of FACIT-Fatigue scale.

Classical Complement Inhibition By SAR445088 (BIVV020) in Adults with Cold Agglutinin Disease: Safety, Tolerability and Activity Results from the Open-Label, Non-Randomized, Single-Dose Phase 1b Study

Background:Cold agglutinin disease (CAD) is a rare, chronic autoimmune hemolytic anemia characterized by complement-mediated hemolysis. The classical complement inhibitor sutimlimab, is the first approved pharmacotherapy for treating patients with CAD. The second-generation complement inhibitor SAR445088 (BIVV020) is a humanized monoclonal antibody that selectively inhibits the activated form of C1s (sutimlimab inhibits total C1s). Furthermore, SAR445088 contains mutations that increase FcRn binding, preventing its degradation and allowing it to be recycled back into the system, resulting in reduced drug clearance and a prolonged half-life, which may allow a longer dosing interval than that of sutimlimab. Aims:This Phase 1b open-label study evaluated the safety, tolerability, effect on hemolysis, and pharmacodynamics (PD)/pharmacokinetics (PK) of a single intravenous (IV) dose of SAR445088. The study aimed to assess the impact of SAR445088 on complement-mediated hemolysis to support proof of concept for SAR445088 as a treatment for patients with CAD. Methods:The study design included up to three single-dose cohorts with a maximum of six patients per cohort. SAR445088 was administered on Day 1 with follow-up visits from Day 2 to Day 71; end of study (EOS) visit was on Day 106. The first cohort received a dose of 30 mg/kg IV with decisions about the selection of the dose for the next cohort being based on safety, biomarker activity, and PD. In the second cohort, patients received a lower dose of 15 mg/kg IV; a third, higher-dose cohort was not enrolled. To be enrolled in the study, participants needed to be aged ≥18 years with a confirmed diagnosis of CAD and a hemoglobin level ≤11.0 g/dL at time of screening. The primary endpoint was assessment of safety, with secondary endpoints of hematologic biomarkers and PD/PK. Results:Twelve patients received a single IV dose of SAR445088 (n=6, 30 mg/kg; n=6, 15 mg/kg). Patients enrolled in the study were mainly female (91.7%, n=11) with a median age (range) of 69 (54-80) years, and median (range) hemoglobin at baseline was 9.60 (4.8-10.9) g/dL. There were no treatment-emergent serious adverse events (AEs), discontinuations or deaths reported in the study. Fifty-eight treatment-emergent AEs (TEAEs) were reported in 11 patients (91.7%). There was one reported pre-treatment severe AE (Grade 3) of hemolytic anemia in a patient in the 15 mg/kg IV cohort which began prior to treatment and continued during the treatment period; the remainder of TEAEs were mild or moderate in severity (Grade 1 or 2). The most frequently reported AE was hematuria. No serious infections, meningococcal infections, hypersensitivity, or thromboembolic events were reported. Nine patients (n=4, 30 mg/kg IV cohort; n=5, 15 mg/kg IV cohort) reached an increase of hemoglobin ≥1.5 g/dL from baseline to Day 106 (EOS visit); eight patients (n=4 for both cohorts) reached an increase in hemoglobin &amp;gt;1.0 g/dL from baseline to Day 15 (Figure A). Total mean bilirubin decreased by Day 1 and this was sustained to Day 106 (EOS visit; Figure B).The decrease was more pronounced in the 30 mg/kg IV cohort. Improvements in clinical markers correlated with sustained reductions in CH50 in both treatment cohorts. Reductions in mean percent classical complement pathway (CP) activity were noted 1 hour after a single dose of 30 or 15 mg/kg IV, followed by gradual rebound of CP activity over the 15-week study period. Conclusion:SAR445088 was generally well tolerated; no safety concerns were identified in patients with CAD. A single IV dose of SAR445088 led to classical complement inhibition, control of hemolysis, and improvement in anemia, which was sustained for 15 weeks.

Combined Safety Data for Sutimlimab in Cold Agglutinin Disease: A Post-Hoc Analysis of the Phase 3 Cardinal and Cadenza Studies

Background: Cold agglutinin disease (CAD) is a rare, chronic, autoimmune hemolytic anemia mediated by the classical complement pathway (CP).Sutimlimab is a first-in-class, humanized, monoclonal antibody that selectively inhibits C1s of the C1 complex, preventing CP activation and CP-mediated hemolysis. CARDINAL (NCT03347396) was an open-label, single-arm, Phase 3 study of sutimlimab in patients with CAD and recent history of transfusion; CADENZA (NCT03347422) was a randomized, double-blind, placebo-controlled, Phase 3 study of sutimlimab in patients with CAD and no recent history of transfusion. Both studies had a 26-week treatment period (Part A) followed by a long-term extension (Part B), reporting data up to 2 years after the last patient finished Part A of CARDINAL and up to 1 year after the last patient finished Part A of CADENZA. With long-term treatment in both studies, sutimlimab demonstrated sustained efficacy with improvements in hemolysis and anemia, sustained clinically meaningful improvements in quality of life, and it had a favorable safety profile. Aims: To report combined safety data from Part A and B of the Phase 3 CARDINAL and CADENZA studies in sutimlimab-treated patients with CAD. Methods: In CARDINAL Part A, patients received sutimlimab on Days 0 &amp; 7, then biweekly until the end of Part B. In CADENZA Part A, patients received sutimlimab or placebo on Days 0 &amp; 7, then biweekly; in Part B, patients continued to receive biweekly sutimlimab or switched from placebo to receive sutimlimab on Days 0 &amp; 7, then biweekly. Data fromall enrolled patients who received at least 1 dose of sutimlimab in CARDINAL (N=24) and CADENZA (N=42), including a post-treatment follow-up 9 weeks after the last dose, were combined into the Safety Analysis Set. Endpoints for this analysis included the incidence of treatment-emergent adverse events (TEAEs), treatment-emergent serious adverse events (TESAEs), and adverse events of special interest (AESIs). AESIs were selected based on a list of important identified risks and important potential risks for sutimlimab. Results: The Safety Analysis Set included 66 patients. At baseline, the median age was 69.5 years (range 46-88) and the majority (72.7%) were female, with a median duration since CAD diagnosis of 5.7 years (range 0-33). The median patient follow-up was 129.1 weeks (range 5-175). Sixty-four (97.0%) patients experienced ≥1 TEAE ( Table 1). Eighty-six TEAEs assessed as related to sutimlimab by the investigator occurred in 30 (45.5%) patients. Fifty-three TESAEs were reported in 22 (33.3%) patients. Three (4.6%) patients each experienced one TESAE assessed as related or possibly related to sutimlimab by the investigator (related vitreous hemorrhage, related viral infection, and possibly related severe cerebral venous thrombosis). Seven patients discontinued sutimlimab treatment and/or the studies due to ≥1 TEAE, 4 in CARDINAL and 3 in CADENZA. Four patients died during the studies: 1 in CADENZA with a TESAE of lung squamous cell carcinoma and sutimlimab was withdrawn due to this TESAE prior to the patient's death; 3 in CARDINAL, 1 patient died after premature discontinuation of sutimlimab due to AEs (fatal infection of Klebsiella pneumoniae, acrocyanosis), 1 patient died due to a newly diagnosed hepatic cancer in the first month of the study, and 1 patient died due to exacerbation of CAD during the 9-week washout period approximately 1.5 months after the last dose of sutimlimab. No deaths were assessed as related to sutimlimab by the investigator. Treatment-emergent AESIs (selected based on a list of important identified risks and important potential risks for sutimlimab) are presented in Table 1. Fourteen TEAEs of serious infection were reported in 9 patients (13.6%; one assessed as related to sutimlimab by the investigator), 31 TEAEs of hypertension were reported in 17 patients (25.8%), 28 TEAEs of acrocyanosis and/or Raynaud's phenomenon were reported in 13 patients (19.7%), and 4 TEAEs of thromboembolic events were reported in 4 patients (6.1%). No TEAEs of serious hypersensitivity reaction and/or anaphylaxis, meningococcal infection or development of systemic lupus erythematosus were reported. Conclusion: The combined safety analysis of the Phase 3 CARDINAL and CADENZA studies (Part A and B) demonstrated that sutimlimab was generally well tolerated, with the type and frequency of TEAE consistent with an older and medically complex population.

PSIX-9 Activation of Multiple Inflammatory Signaling Pathways May Contribute to the Pathogenesis of Idiopathic Cystitis in Felines

Abstract Feline idiopathic cystitis (FIC) is a common urinary disorder in domestic cats. Clinical signs include straining or failing to urinate, frequent urination, and discolored urine. Inflammation is a key characteristic in the pathogenesis of cystitis. We assessed circulating inflammatory markers in a retrospective study in cats housed in the Hill’s colony diagnosed with cystitis (n = 4; 15.2 to 18.6yr) and in control cats (n = 8; 5.9 to 16.5 yr). Blood samples were collected at end-of-life immediately before necropsy after they had lived their full lives. End-of-life pathology reports indicated inflammation and presence of inflammatory infiltrates in the bladder with some cats also showing renal inflammation. RNA was extracted from blood collected in PAXgene tubes, and gene expression investigated using NanoString nCounter platform. Analysis was performed using the nSolver software. There was a significant increase in IRAK4, a kinase that activates the NFκB pro-inflammatory signaling pathway, in FIC when compared with controls (1.56, P &amp;lt; 0.05). Small molecule inhibitors of IRAK4 are being developed as targets of inflammation. Signaling molecules in the NFκB pathway were also significantly increased including Rel A (p65 subunit; 1.3 fold), NFκB1 (p100 subunit; 1.3 fold), IKBKB (1.29 fold), and TANK (1.46 fold) in FIC cats (all P &amp;lt; 0.05). Another signaling pathway contributing to pro-inflammatory signaling is the JAK/STAT pathway. There was a significant increase in JAK2 (1.65) and STAT3 (1.48). Blockade of JAK2/STAT3 pathway can attenuate cytokine synthesis and reduce inflammation. STAT6 was also significantly increased in FIC cats (1.35). Activation of STAT6, by IL-4 and IL-13, can lead to systemic inflammation. Activation of the complement immune pathway was also seen in FIC. While the complement system is important for an innate immune response, its sustained activation leads to chronic pathological inflammation. There was a significant increase in complement C1s, an initiator of classical complement activation pathway, in FIC cats (1.43; P &amp;lt; 0.05). A late component, complement C7, was also upregulated in FIC (2.01 fold; P &amp;lt; 0.05). C7 binds to the C5b-6 complex which eventually becomes part of the terminal complement complex. Increased abundance of C7 and eventual terminal complement complex are associated with increased inflammation and may be important in the progression of FIC. There was also an increase in PECAM1 in FIC cats (1.87; P &amp;lt; 0.05). Signaling through PECAM1 leads to the activation of neutrophils and monocytes. DUSP1 was also elevated in FIC (2.24; P &amp;lt; 0.05). Increased abundance of DUSP1 is hypothesized to be a predictor of chronic subclinical inflammation in humans with cardiovascular disease. Our results identify activation of pro-inflammatory signaling pathways that may be potential targets for alleviating inflammation in cystitis. Pathogenesis of FIC is likely multi-factorial, and nutritional intervention to attenuate the activation of inflammatory pathways may be an important strategy in the management of cystitis.

Changes in expression levels of erythrocyte and immune-related genes are associated with high altitude polycythemia

BackgroundAs a chronic mountain sickness(CMS) with the highest incidence and the greatest harm, the pathogenesis of high altitude polycythemia (HAPC) is still not fully understood.Methods37 HAPC patients and 42 healthy subjects were selected from plateau, and peripheral venous blood samples were collected for transcriptome sequencing on Illumina NovaSeq platform. The sequenced data were analyzed by bioinformatics and phenotypic association analysis.ResultsThe results showed significant differences in multiple clinical indicators including RBC and HGB et al. existed between HAPC and control. Based on the RNA-seq data, 550 genes with significant differential expression were identified in HAPC patients. GO and KEGG pathway enrichment analysis showed that the up-regulated genes were mainly enriched in processes such as erythrocyte differentiation and development and homeostasis of number of cells, while the down-regulated genes were mainly enriched in categories such as immunoglobulin production, classical pathway of complement activation and other biological processes. The coupling analysis of differential expression genes(DEGs) and pathological phenotypes revealed that 91 DEGs were in close correlation with in the phenotype of red blood cell volume distribution (width-CV and width-SD), and they were all up-regulated in HAPC and involved in the process of erythrocyte metabolism. Combined with the functional annotation of DEGs and literature survey, we found that the expression of several potential genes might be responsible for pathogenesis of HAPC. Besides, cell type deconvolution analysis result suggested that the changes in the number of some immune cell types was significantly lower in HAPC patients than control, implying the autoimmune level of HAPC patients was affected to a certain extent.ConclusionThis study provides an important data source for understanding the pathogenesis and screening pathogenic genes of HAPC. We found for the first time that there was a significant correlation between HAPC and the pathological phenotype of width-CV and width-SD, wherein the enriched genes were all up-regulated expressed and involved in the process of erythrocyte metabolism. Although the role of these genes needs to be further studied, the candidate genes can provide a starting point for functionally pinning down the underlying mechanism of HAPC.

Complement Components in Peripheral Blood from Adult Patients with IDH Wild-Type Glioblastoma

The complement system seems to influence cancer pathophysiology. The primary aim of this study was to explore complement components associated with the classical pathway (CP) of the complement system in peripheral blood from patients with IDH-wild-type (IDH-wt) glioblastoma. Patients undergoing primary surgery due to glioblastoma in the years 2019-2021 were prospectively included in the present study. Blood samples were collected prior to surgery, and analyzed with regard to CP complement components, as well as standard coagulation tests. In total, 40 patients with IDH-wt glioblastomas were included. C1q was reduced in 44% of the cases compared to the reference interval. C1r was reduced in 61% of the analyzed samples. Both C1q and C1r are parts of the initial steps of the classical complement activation pathway, which, however, was not correspondingly altered. Activated pro-thromboplastin time (APTT) was shorter in 82% of the analyzed samples compared to the reference interval. APTT was shorter in those with reduced C1q and C1r levels. C1q is an important link between the innate and acquired immunity, and C1q and C1r also interact with the coagulation system. Patients who displayed reduced levels of both C1q and C1r preoperatively had a significantly shorter overall survival compared with the rest of the cohort. Our findings demonstrate that there are alterations in C1q and C1r concentrations in peripheral blood from patients with IDH1-wt glioblastoma compared with the normal population. Patients who displayed reduced C1q and C1r levels had a significantly shorter survival.

#4015 RECOMBINANT HIGH-DENSITY LIPOPROTEIN MODULATES INFLAMMATORY RESPONSE AND RENAL DYSFUNCTION IN A SWINE MODEL OF SEPSIS-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Sepsis is a severe and dysregulated inflammatory disease that often precedes the development of acute kidney injury (AKI) with consequent worsening outcome. Although clinical data demonstrate that high-density lipoprotein (HDL) levels drop in septic patients with a poor prognosis, little is known about the molecular basis of HDL's role in systemic inflammation and renal function. Here we investigate the possible effects of a novel engineered HDL-mimetic (CER-001) in a swine model of lipopolysaccharide (LPS)-induced AKI. Method Sepsis was induced by intravenous infusion of a saline solution containing 300 μg/kg of LPS in a porcine model. The animals were randomized into three groups: LPS (endotoxemic pigs, n = 6), CER20 (endotoxemic pigs treated with a single dose of CER-001 20mg/kg; n = 6), and CER20 × 2 (endotoxemic pigs treated with two doses of CER001 20mg/kg; n = 6). Animals were sacrificed after 24h from the start of experimental procedure. Renal histologic and biochemical changes were analyzed. Endothelial dysfunction biomarkers, circulating pro-inflammatory mediators, LPS and Apolipoprotein A-I (Apo A-I) levels were quantified with ELISA assay. Systemic complement activation was evaluated by Wieslab kit. Results Untreated animals were highly susceptible to LPS challenge and usually succumbed before completing the study protocol (LPS group 16.7%). CER-001 treatment increased the survival rate of endotoxemic pigs, compared to the LPS group (CER20, 50%; CER20 × 2, 66.7%). Furthermore, as shown in Figure 1A, LPS injection led to a time-dependent increase of IL-6 in endotoxemic animals respect to basal condition (T0). CER-001 treatment was able to reverse LPS effects. In particular, the second infusion of CER-001 three hours after the first dose (T3) strongly reduced IL-6 serum levels back to basal level (LPS p &amp;lt;0.05). Similarly, we found high levels of TNF-α and MCP-1 in endotoxemic pigs that were significantly decreased in both CER-001 treatment arms (T24, CER20 × 2 vs LPS group, IL-6, p = 0.0086; TNF-α, p&amp;lt;0.0001; MCP-1, p = 0.0009). In addition, CER-001 treatment ameliorated systemic endothelial dysfunction by reducing VCAM and ICAM serum levels. A significant activation of classical and alternative complement pathway (vs T0 p &amp;lt;0.05) was observed at 1h, 3h and 24h after LPS infusion. CER001 treatment significantly prevented systemic complement activation in both treated groups (vs LPS p &amp;lt;0.05). We also investigated whether CER001 infusions significantly prevented renal tissue damage. Endotoxemic pigs presented oliguric AKI with increased tubulo-interstitial infiltrate, extensive collagen deposition, and glomerular thrombi; CER-001 treatment preserved renal parenchyma, recovered urine output, decreased creatine levels, and reduced the biomarkers of tubular damage, Cystatin C and KIM-1, both in serum and urine samples. Considering that HDL has a very high affinity for LPS, we evaluated the circulating LPS concentration in treated animals. We observed that LPS levels were greatly reduced in treated animals and the effects are more evident after the second infusion of CER-001(Figure 1B). Therefore, we also examined LPS levels in bile samples and we observed a dose-dependent increasing amount of endotoxin in the CER001 treated septic pigs. Conclusion This preclinical data indicates that CER001 treatment prevents systemic inflammation thereby limiting renal damage. The mechanism of action is two-fold consisting of both the scavenging of endotoxin and a direct anti-inflammatory effect of CER-001.

A nanobody-based complement inhibitor targeting complement component 2 reduces hemolysis in a complement humanized mouse model of autoimmune hemolytic anemia

C2 is an attractive therapeutic target for many complement-mediated diseases. We developed Nab1B10, a new anti-C2 nanobody that potently and selectively inhibits both the classical and lectin pathways of complement activation. Mechanistically, Nab1B10 binds to the C2a portion of C2 and inhibits the assembly of C3 convertase C4b2a. Nab1B10 cross-reacts with monkey but not rodent C2 and inhibits classical pathway-mediated hemolysis. Using a new complement humanized mouse model of autoimmune hemolytic anemia (AIHA), we demonstrated that Nab1B10 abolished classical pathway complement activation-mediated hemolysis in vivo. We also developed C2-neutralizing bi- and tetra-valent antibodies based on Nab1B10 and found these antibodies significantly more potent than the other anti-C2 monoclonal antibody that is already in clinical trials. These data suggest that these novel C2-neutralizing nanobodies could be further developed as new therapeutics for many complement-mediated diseases, in which pathogenesis is dependent on the classical and/or lectin pathway of complement activation.

Complement-directed therapy for cold agglutinin disease: sutimlimab

ABSTRACT Introduction Cold agglutinin disease (CAD) is a rare subtype of autoimmune hemolytic anemia defined as a distinct, low-grade lymphoproliferative disorder and characterized by the presence of immunoglobulin M (IgM) antibodies that recognize the ‘I’ antigen on red blood cell membranes. Hemolysis in CAD is mediated by activation of the classical complement pathway by IgM–antigen complexes. Sutimlimab directly targets classical complement pathway activation and has been shown to be generally well tolerated with rapid and sustained effects on hemoglobin levels, hemolytic markers, and fatigue in patients with CAD. Areas covered This review will outline the drug profile of sutimlimab and summarize the key efficacy and safety data focusing on the Phase 3 studies that formed the basis of the approval of sutimlimab in patients with CAD in the US, the EU, and Japan. Expert opinion Sutimlimab provides patients with an approved therapeutic option that can be used as part of a holistic approach to CAD management. The beneficial effects of sutimlimab go beyond rapid inhibition of hemolysis and include sustained meaningful improvements in fatigue and quality-of-life measures. Further, real-world evidence of the effectiveness and safety of sutimlimab in CAD and cold agglutinin syndrome will be assessed via the CADENCE registry.

Sustained inhibition of complement C1s with sutimlimab over 2 years in patients with cold agglutinin disease.

Cold agglutinin disease (CAD) is a rare, autoimmune, classical complement pathway (CP)-mediated hemolytic anemia. Sutimlimab selectively inhibits C1s of the C1 complex, preventing CP activation while leaving the alternative and lectin pathways intact. In Part A (26 weeks) of the open-label, single-arm, Phase 3 CARDINAL study in patients with CAD and a recent history of transfusion, sutimlimab demonstrated rapid effects on hemolysis and anemia. Results of the CARDINAL study Part B (2-year extension) study, described herein, demonstrated that sutimlimab sustains improvements in hemolysis, anemia, and quality of life over a median of 144 weeks of treatment. Mean last-available on-treatment values in Part B were improved from baseline for hemoglobin (12.2 g/dL on-treatment versus 8.6 g/dL at baseline), bilirubin (16.5 μmol/L on-treatment versus 52.1 μmol/L at baseline), and FACIT-Fatigue scores (40.5 on-treatment versus 32.4 at baseline). In the 9-week follow-up period after sutimlimab cessation, CP inhibition was reversed, and hemolytic markers and fatigue scores approached pre-sutimlimab values. Overall, sutimlimab was generally well tolerated in Part B. All 22 patients experienced ≥1 treatment-emergent adverse event (TEAE); 12 (54.5%) patients experienced ≥1 serious TEAE, including seven (31.8%) with ≥1 serious infection. Three patients discontinued due to a TEAE. No patients developed systemic lupus erythematosus or meningococcal infections. After cessation of sutimlimab, most patients reported adverse events consistent with recurrence of CAD. In conclusion, the CARDINAL 2-year results provide evidence of sustained sutimlimab effects for CAD management, but that disease activity reoccurs after treatment cessation. NCT03347396. Registered November 20, 2017.

Abstract 154: Crossroads Of Immunity And Coagulation: Protease Activity Of Complement Component C1s Inhibits Protein C Activation In Covid-19

Introduction: There is an abundance of crosstalk between innate immunity and coagulation in thrombo-inflammatory diseases such as COVID-19. Several potential procoagulant effects of complement activation have been described, but whether complement proteases affect antithrombotic pathways in is not known. Aims: We tested the hypothesis that complement component 1 (C1) impairs the protein C antithrombotic pathway. Methods: Plasma levels of the C1 complex were measured in plasma collected from healthy individuals and COVID-19 patients hospitalized early in the COVID-19 pandemic (NCT04360824). Activation of protein C by recombinant thrombomodulin was measured in a two-stage assay. In the first stage, protein C, thrombin, and thrombomodulin were incubated to generate activated protein C. The reaction mix was incubated with either healthy or COVID-19 plasma in the presence or absence of C1 inhibitor (which binds to C1 to inhibit its activity) or sutimlimab (a specific C1s inhibitor). During the second stage, protein C activation was measured using spectrophotometry with the chromogenic substrate S-2366. Results: In the inflammatory condition COVID-19, plasma levels of the C1 complex were increased compared to healthy controls ( p = 0.004). Generation of activated protein C by recombinant thrombomodulin was decreased in the presence of COVID-19 plasma ( p = 0.001), whereas the addition of healthy plasma had no significant effect ( p = 0.469). Addition of C1 inhibitor or sutimlimab normalized protein C activation in the presence of COVID-19 plasma ( p = 0.022 and 0.039, respectively). Addition of recombinant C1s protease, but not C1r protease or C1q, impaired the generation of activated protein C in a dose dependent fashion. Conclusions: We have uncovered a novel role of the protease C1s in regulating the generation of anticoagulant activated protein C. These findings suggest that the classical pathway of complement activation may impair anticoagulant responses in COVID-19.

The choice of new treatments in autoimmune hemolytic anemia: how to pick from the basket?

Autoimmune hemolytic anemia (AIHA) is defined by increased erythrocyte turnover mediated by autoimmune mechanisms. While corticosteroids remain first-line therapy in most cases of warm-antibody AIHA, cold agglutinin disease is treated by targeting the underlying clonal B-cell proliferation or the classical complement activation pathway. Several new established or investigational drugs and treatment regimens have appeared during the last 1-2 decades, resulting in an improvement of therapy options but also raising challenges on how to select the best treatment in individual patients. In severe warm-antibody AIHA, there is evidence for the upfront addition of rituximab to prednisolone in the first line. Novel agents targeting B-cells, extravascular hemolysis, or removing IgG will offer further options in the acute and relapsed/refractory settings. In cold agglutinin disease, the development of complement inhibitors and B-cell targeting agents makes it possible to individualize therapy, based on the disease profile and patient characteristics. For most AIHAs, the optimal treatment remains to be found, and there is still a need for more evidence-based therapies. Therefore, prospective clinical trials should be encouraged.

Functional Analysis of Two Divergent C4 Isotypes in the Classical and Lectin Pathways of Complement Activation in the Common Carp (Cyprinus carpio)

In the evolution of the complement system, a major humoral innate immune factor, the existence of multiple isotypes of the complement components is considered as a key strategy to enhance innate immune defense. Complement C4 is also diversified in a wide range of vertebrate species including teleost fish, possibly supporting the robust activation mechanism of the complement. To better understand the functional diversity of C4 isotypes in the teleost complement system, two C4 isotypes, C4-1 and C4-2, sharing only 32% amino acid sequence identity, were examined for binding specificities towards model target molecules representing microbe antigens and towards Gram-positive and -negative bacteria. The results suggest that C4-1 and C4-2 behave similarly in binding to the tested targets, despite the predicted difference in binding specificity based on the thioester catalytic site. The participation of C4-1 in the classical and lectin pathways of complement activation was also explored using pathway-specific activating enzyme complexes, C1r/s and MBL-MASP2. As a result, C4-1 can be activated in both the classical and the lectin pathways, at higher efficiency in the classical pathway. Taken together, the present results imply that both C4-1 and C4-2 isotypes are fully functional in the complement activation cascades, probably playing comparable roles in innate immunity.