Inhibitor Of Alternative Pathway Research Articles

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

Complement inhibition in paroxysmal nocturnal hemoglobinuria: From biology to therapy.

Complement inhibitors are the mainstay of paroxysmal nocturnal hemoglobinuria (PNH) treatment. The anti-C5 monoclonal antibody eculizumab was the first treatment to improve hemolysis, thrombotic risk, and survival in PNH although at the price of a life-long intravenous fortnightly drug. Additionally, suboptimal response may occur in up to 2/3 of patients with persistent anemia due to incomplete control of intravascular hemolysis, development of upstream C3-mediated extravascular hemolysis (EVH), or concomitant bone marrow failure. Ravulizumab, a longer half-life anti-C5 developed from eculizumab, administered every 8 weeks, improved patient convenience, and reduced pharmacokinetic breakthrough hemolysis (BTH) by establishing more stable anti-C5 concentrations. More recently, several other anti-C5 compounds (crovalimab, pozelimab, tesidolumab, cemdisiran, zilucoplan, and coversin) are on study in clinical trials. Upstream inhibition of complement cascade was also explored with the anti-C3 pegcetacoplan, and with the alternative pathway inhibitors iptacopan (anti-factor B) and danicopan (anti-factor D). These drugs efficiently target EVH and are able to improve anemia and transfusion need in suboptimal responders to anti-C5. The route and schedule of administration (twice weekly subcutaneously for pegcetacoplan and twice or thrice oral daily dosing for iptacopan and danicopan, respectively) are very convenient but pose novel issues regarding adherence. Additionally, both anti-C5 and upstream inhibitors do not resolve the unmet need of pharmacodynamic BTH events due to complement amplifying conditions such as infections, traumas, and surgery. In this review, we will recapitulate PNH physiopathology, clinical presentation, and diagnosis and describe available and developing drugs that will lead to a precision medicine approach for this rare though heterogenous disease.

Abstract B083: Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer

Abstract KRAS is the most frequently mutated oncogene, with high prevalence in tumor types with a significant unmet clinical need, such as colorectal cancer (CRC). Activating mutations in the KRAS gene leading to amino acid exchange at position 12 from Glycine to Asparagine, G12D, occur in approximately 12.5% of CRC. The approval of mutant specific KRASG12C inhibitors MRTX849 and AMG510 by the FDA raised the question if KRAS targeted therapy approaches could provide benefit to cancer patients with other mutant alleles. Several KRASG12D inhibitors have recently been described and MRTX1133, a selective KRASG12D inhibitor from Mirati Therapeutics, has now entered clinical trial, recruiting patients with advanced solid tumors harboring a KRAS G12D mutation. Alongside the great hopes placed in KRAS targeting therapy, resistance is likely to occur, as it was observed in patients relapsing in response to KRASG12C inhibitors. We aimed to use this pre-clinical study to predict and understand potential resistance mechanisms that may arise during targeted treatment against KRASG12D in the CRC setting. To this aim, the colorectal cancer cell line GP2d was continuously treated with MRTX1133 to generate resistance and individual outgrowing clones were subsequently profiled in a series of assays to identify potential mechanism(s). These assays included analysis of gene expression changes, mutation calling, as well as qPCR and protein-based assays. Interestingly, we identified two individual secondary on-target mutations in the KRAS gene, which could affect binding of MRTX1133 to the pocket in KRAS. These findings were confirmed in an orthogonal assay, by screening for resistance in Ba/F3 cells transduced with the corresponding KRAS double mutant. Combination assays were then performed in parental and resistant cells to study whether additional vertical or alternative pathway inhibition could address the resistance. Collectively our pre-clinical study identified secondary KRAS mutations in the compound binding pocket as a mechanism of resistance to KRAS G12D inhibition by MRTX1133 in colorectal cancer cells. Citation Format: Simone Lieb, Sabine Jurado, Sergej Nowoshilow, Krzysztof M. Zak, Marco H. Hofmann. Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B083.

Regulatable Complement Inhibition of the Alternative Pathway Mitigates Wet Age-Related Macular Degeneration Pathology in a Mouse Model.

Risk for developing age-related macular degeneration (AMD) is linked to an overactive complement system. In the mouse model of laser-induced choroidal neovascularization (CNV), elevated levels of complement effector molecules, including complement C3, have been identified, and the alternative pathway (AP) is required for pathology. The main soluble AP regular is complement factor H (fH). We have previously shown that AP inhibition via subretinal AAV-mediated delivery of CR2-fH using a constitutive promoter is efficacious in reducing CNV. Here we ask whether the C3 promoter (pC3) effectively drives CR2-fH bioavailability for gene therapy. Truncated pC3 was used to generate plasmids pC3-mCherry/CR2-fH followed by production of corresponding AAV5 vectors. pC3 activation was determined in transiently transfected ARPE-19 cells stimulated with H2O2 or normal human serum (+/- antioxidant or humanized CR2-fH, respectively). CNV was analyzed in C57BL/6J mice treated subretinally with AAV5-pC3-mCherry/CR2-fH using imaging (optical coherence tomography [OCT] and fundus imaging), functional (electroretinography [ERG]), and molecular (protein expression) readouts. Modulation of pC3 in vitro is complement and oxidative stress dependent, as shown by mCherry fluorescence. AAV5-pC3-CR2-fH were identified as safe and effective using OCT and ERG. CR2-fH expression significantly reduced CNV compared to mCherry and was correlated with reduced levels of C3dg/C3d in the retinal pigment epithelium/choroid fraction. We conclude that complement-dependent regulation of AP inhibition ameliorates AMD pathology as effectively as using a constitutive promoter. The goal of anticomplement therapy is to restore homeostatic levels of complement activation, which might be more easily achievable using a self-regulating system.

In vaccinated individuals serum bactericidal activity against B meningococci is abrogated by C5 inhibition but not by inhibition of the alternative complement pathway.

Several diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against encapsulated Gram-negative bacteria. Therefore, C3 and C5 inhibition increases the risk of invasive disease, in particular by Neisseria meningitidis. As inhibitors against complement components other than C3 and C5 may carry a reduced risk of infection, we compared the effect of inhibitors targeting the terminal pathway (C5), the central complement component C3, the alternative pathway (FB and FD), and the lectin pathway (MASP-2) on SBA against serogroup B meningococci. Serum from adults was collected before and after vaccination with the meningococcal serogroup B vaccine 4CMenB and tested for meningococcal killing. Since the B capsular polysaccharide is structurally similar to certain human polysaccharides, 4CMenB was designed to elicit antibodies against meningococcal outer membrane proteins. While only a few pre-vaccination sera showed SBA against the tested B meningococcal isolates, 4CMenB vaccination induced potent complement-activating IgG titers against isolates expressing a matching allele of the bacterial cell surface-exposed factor H-binding protein (fHbp). SBA triggered by these cell surface protein-specific antibodies was blocked by C5 and reduced by C3 inhibition, whereas alternative (factor B and D) and lectin (MASP-2) pathway inhibitors had no effect on the SBA of post-4CMenB vaccination sera. Compared to the SBA triggered by A,C,W,Y capsule polysaccharide conjugate vaccination, SBA against B meningococci expressing a matching fHbp allele was remarkably resilient against the alternative pathway inhibition.

Trial in Progress: An Open-Label, Phase 2 Study to Evaluate the Safety, Tolerability, Pharmacokinetics, Pharmacodynamics, and Efficacy of KP104 in Complement Inhibitor-Naïve Subjects with Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a life-threatening disease characterized by destruction of red blood cells by the complement system, leading to pathologies such as anemia and thrombosis. Currently approved complement inhibitors target C5 which addresses primarily intravascular hemolysis (IVH). However, many treated patients are still anemic and transfusion dependent since C5 inhibitors cannot treat extravascular hemolysis (EVH), which is driven by the alternative pathway (AP). C3 inhibitors need frequent administration, high dosage, and are associated with breakthrough hemolysis (BTH) (Hillmen, 2021; Rother, 2007; Stern, 2019; Rosario, 2022). Thus, the treatment of PNH still remains a challenge with significant unmet medical needs to develop more efficacious and convenient complement drugs. KP104 is a bi-functional complement inhibitor that can inhibit both C5 and C3 convertase activities and thereby achieve high potency in treating complement mediated pathologies. KP104 is an engineered anti-C5 immunoglobulin G4 monoclonal antibody (mAb) fused with the N-terminal 5 short consensus repeats of human factor H (a potent AP inhibitor) and engineered to extend serum half-life. KP104 was generally well tolerated and safe in the First-in-Human (FIH), healthy volunteer Phase 1 trial (n=66). PD data from this FIH study also support the dual mechanisms of KP104 inhibiting both AP and terminal pathway (TP). In addition, an extended half-life for KP104 has been observed in healthy volunteers, compared to other approved bispecific/fusion proteins on the market. Details of the FIH study will be published at another major conference (Wabinitz, 2022). KP104 is being developed for multiple diseases with complement cascade dysregulation, with PNH being the first indication to be investigated. Study Design and Methods: This is an open-label, 2-part, Phase 2 study to evaluate safety, tolerability, PK, PD, and efficacy of KP104 in complement inhibitor-naïve subjects with PNH (NCT05476887). Approximately 35 subjects with PNH are planned to be enrolled, including up to 18 in the dose-ranging Part 1, and up to 17 in the proof-of-concept (POC) Part 2. The primary objectives of Part 1 are to assess safety and tolerability and estimate optimal biologic dose (OBD) and regimen of KP104 in complement inhibitor-naïve subjects with PNH. Part 1 will include up to 3 cohorts of 3 to 6 subjects/cohort dosed for 12 weeks (Figure 1). When the third subject in the last cohort of Part 1 completes 4 weeks of treatment, a KP104 OBD will be estimated for evaluation in Part 2. Part 2 is to confirm POC by studying the KP104 OBD in 1 cohort of complement inhibitor-naïve subjects with PNH (Figure 1). The primary efficacy endpoint is the proportion of subjects with a ≥2 g/dL increase in hemoglobin from baseline to the end of the treatment period, with the absence of transfusion through the treatment period. The percent of subjects showing an increase from baseline in hemoglobin of ≥2 g/dL at the end of the treatment period will be compared to a 15% reference rate using an exact binomial one-sample test. The sample size was calculated to achieve approximately 90% power with 0.025 type 1 error rate (one-sided). In summary, KP104, through effective inhibition of both AP and TP complement pathways, may achieve high potency and address IVH, EVH, and BTH for treating subjects with PNH. Hillmen P, Szer J, et al. 2021. Pegcetacoplan versus eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med 384:1028-1037. Rosario N, Lucio L. 2022. Breakthrough Hemolysis in PNH with Proximal or Terminal Complement Inhibition. N Engl J Med 387(2):160-166. Rother, R.P., et al. 2007. Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. Nat Biotechnol 25:1256-1264. Stern, R.M., and Connell, N.T. 2019. Ravulizumab: a novel C5 inhibitor for the treatment of paroxysmal nocturnal hemoglobinuria. Ther Adv Hematol 10:2040620719874728. Wabinitz, P., et al. 2022. SYNERGY-1: A Phase 1, first-in-human, randomized, double-blind, placebo-controlled safety, tolerability, immunogenicity, PK and PD study of KP104 in escalating single and multiple doses. ASN abstract. Under review. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of the Alternative Pathway Masp-3 Inhibitor OMS906 in a Phase 1 Study of Healthy Subjects

Introduction: The alternative pathway of the complement system is implicated in a wide variety of diseases. Complement Factor D (CFD) drives formation of alternative pathway C3 convertase, generating C3b and creating a positive feedback loop of protease complexes. Left unchecked, this amplification cycle leads to inflammation, tissue destruction, and the intravascular and extravascular hemolysis observed in paroxysmal nocturnal hemoglobinuria (PNH). Mannan-binding lectin-associated serine protease-3 (MASP-3) is the activator of CFD, responsible for conversion of its zymogen form (pro-CFD) to mature CFD. OMS906 is a humanized IgG4 monoclonal antibody (mAb) that binds to and inhibits MASP-3. Preclinical studies of OMS906 in mice and cynomolgus monkeys have demonstrated inhibition of alternative pathway activity via MASP-3 inhibition, suggesting therapeutic potential of OMS906 in the treatment of diseases associated with alternative pathway dysregulation. Here we report the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of OMS906 in healthy subjects. Methods: This was a Phase 1, first-in-human, randomized, double-blind, single-center, placebo-controlled study of OMS906 in healthy subjects. Males or females aged 18-64 years with a body mass index (BMI) of 20-32 kg/m2 were enrolled. Subjects were grouped into 9 single-ascending dose cohorts from 0.1-5 mg/kg intravenous (IV) OMS906 and 3-8 mg/kg subcutaneous (SC) OMS906. Cohorts comprised 8 subjects each (6:2 active:placebo). Study drug was delivered by 30-min infusion (50 mL volume for IV and no dilution for SC). The primary objective was to assess safety and tolerability of OMS906 in healthy subjects. The secondary objectives were to characterize PK and PD of single-ascending IV and SC doses of OMS906, and to assess the presence of anti-drug antibodies (ADAs) against OMS906 in healthy subjects. Results: Seventy-two healthy subjects were studied; 54 received OMS906 (30 IV and 24 SC) and 18 received placebo. Mean age was 43 years (range, 20-63), mean BMI 26.7 kg/m2 (range, 21.0-31.4) and mean weight 77.7 kg (range, 50.8-105.7). Males (n=35, 48.6%) and females (n=37, 51.4%) were represented in nearly equal numbers. Subjects were White (n=40, 55.6%), Black or African American (n=22, 30.6%), Asian (n=3, 4.2%), American Indian (n=2, 2.8%) or designated as Multiple Ethnicity (n=5, 6.9%). There were no serious adverse events (AEs) or discontinuations due to AEs. Most treatment-emergent AEs (TEAEs) were mild in severity; there were no severe TEAEs. Most TEAEs were determined unlikely due to OMS906 with exception of injection site reactions (ISRs), which were the most frequently reported AEs. Reported ISRs comprised: 27 ISRs in 17 of 24 subjects receiving SC OMS906 (70.8%); 1 ISR in a subject receiving IV OMS906 (3.3%); 3 ISRs in 2 of 8 subjects receiving SC placebo (25.0%); and none in subjects receiving IV placebo. There were no hypersensitivity reactions. ADAs were detected in subjects receiving OMS906 and the degree of reactivity increased to Day 85. The overall confirmed positive rate of ADAs was 20.4% (n=11/54 receiving OMS906), consistent with the reported frequency of other mAbs in development (Vaisman-Mentesh A, et al. Front. Immunol. 2020;11:1951). Among subjects with ADAs, there was no obvious dose response or difference in ADA frequencies between IV and SC OMS906 administration and no evidence of neutralizing activity. OMS906 displayed consistent PK properties with dose proportionality and non-linearity for both IV and SC administration (Table). Tmax for SC cohorts was 96-239 hours (median). A long half-life (103-423 hours) was observed, with measurable drug concentrations detected at Day 85 for the 5 mg/kg IV and 8 mg/kg SC OMS906 cohorts. The key pharmacodynamic marker, mature CFD, showed a dose-proportional response with rapid suppression and substantial degree of suppression of long duration. Conclusion: In this Phase 1 study of healthy subjects, MASP-3 inhibitor OMS906 was well tolerated with no safety concerns. There were no serious AEs. The most common AEs were ISRs, predominantly observed in the SC OMS906 cohorts. The observed PK and PD profiles were consistent with predictions from preclinical assessments, showing predictable systemic exposure, evidence of alternative pathway inhibition, and a long duration of action. Further clinical study of OMS906 is warranted, including in patients with PNH. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Vemircopan (ALXN2050) Monotherapy in Paroxysmal Nocturnal Hemoglobinuria: Interim Data from a Phase 2 Open-Label Proof-of-Concept Study

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a deficit of complement inhibitory proteins on the blood cell surface, causing intravascular hemolysis (IVH) and risk of thrombosis owing to terminal complement activation. Treatment of patients with PNH with complement C5 inhibitors has greatly improved clinical outcomes and survival; however, some patients taking C5 inhibitors develop clinically significant extravascular hemolysis (EVH) that may result in symptomatic anemia and require blood transfusions. Thus, when treating PNH, complete and sustained terminal complement inhibition is needed. Pharmacologic inhibition of factor D, the rate-limiting enzyme of the complement alternative pathway (AP), even as a monotherapy, may control IVH while preventing EVH, eventually improving anemia and the need for transfusions. The oral factor D inhibitor ALXN2040 (danicopan) showed efficacy as a monotherapy and an add-on treatment to the C5 inhibitor eculizumab, but there was suboptimal control of IVH in some patients when used as a monotherapy. A latter oral factor D inhibitor, ALXN2050 (vemircopan), has the same mechanism of action as danicopan but demonstrates increased potency and binding affinity for factor D. It also achieves immediate, complete, and sustained AP inhibition with twice-daily administration (BID). Vemircopan is being investigated in a phase 2 proof-of-concept study as a monotherapy to treat patients with PNH. Methods: This is an ongoing, phase 2, open-label study (NCT04170023) to assess the efficacy, safety, and PK/PD of vemircopan monotherapy in patients with PNH. The study comprises a 60-day screening period, a 12-week treatment period, and a 96-week long-term extension. Three patient groups are included: treatment-naïve, eculizumab switch, and danicopan monotherapy rollover. This interim analysis focuses on efficacy and safety data from the treatment-naïve group to establish proof of concept in PNH. Key inclusion criteria for the treatment-naïve group include lactate dehydrogenase (LDH) ≥1.5 × upper limit of normal (ULN), absolute reticulocyte count ≥100x109/L, and anemia (hemoglobin [Hgb] <10.5 g/dL). Patients receive vemircopan 120 mg BID, which can be escalated to 180 mg BID per investigator discretion based on guidelines in the protocol (Hgb has not increased by ≥1 g/dL by week 4 relative to baseline; blood transfusion needed; or LDH >1.5 × ULN by day 14 in 2 consecutive assessments). Primary endpoint is change from baseline to week 12 in Hgb. Key secondary endpoints include change from baseline to week 12 in LDH and Functional Assessment of Chronic Illness Therapy - Fatigue (FACIT-Fatigue) score. Safety is reported as treatment-emergent adverse events (TEAEs). Results: As of a data cutoff date of April 30, 2022, 11 treatment-naïve patients were enrolled (mean [SD] age, 44.4 [17.8] y; 63.6% male; 72.7% Asian, 54.5% received transfusions during screening period). Nine patients completed 12 weeks in the study and were included in this analysis. From baseline to week 12, mean (SD) Hgb increased by 3.9 (1.11) g/dL from 7.9 (1.29) g/dL to 11.8 (1.25) g/dL (Fig. 1A), LDH rapidly decreased by 81% from 7 × ULN to 1.4 × ULN (Fig. 1B), absolute reticulocyte count decreased from 212.4 (86.47) x 103/uL to 120.0 (51.44) x 103/uL, and a 13-point improvement in FACIT-Fatigue score was observed. No patients needed transfusions except 1 case on day 2 owing to low Hgb (5.1 g/dL). As of the data cutoff date, 31 TEAEs were reported (n=9 patients); most (25/31; 80.6%) were considered unrelated to study drug. No serious TEAEs, grade ≥3 TEAEs, discontinuations, or deaths were reported. The most common TEAE was headache (n=4; 36.4%). There were no thrombotic events, seizures, or meningococcal infections. Thus far, 7 of 11 patients received dose escalation from 120 mg BID to 180 mg BID, which was associated with increased Hgb levels. Conclusions: This interim analysis of treatment-naïve patients with PNH suggests that monotherapy with vemircopan controlled IVH (reduction in LDH to almost normal levels) and prevented EVH (clinically meaningful increases in Hgb values, reticulocyte count reductions). No new safety signals were identified during the 12-week evaluation period. This interim efficacy and safety analysis provides proof of concept in PNH and suggests that phase 3 trials of vemircopan in patients with PNH are warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alternative pathway amplification and infections.

The alternative pathway (AP) is the phylogenetically oldest arm of the complement system and may have evolved to mark pathogens for elimination by phagocytes. Studies using purified AP proteins or AP-specific serum showed that C3b amplification on bacteria commenced following a lag phase of about 5 min and was highly dependent on the concentration of complement. Most pathogens have evolved several elegant mechanisms to evade complement, including expressing proteases that degrade AP proteins and secreting proteins that block function of C3 convertases. In an example of convergent evolution, many microbes recruit the AP inhibitor factor H (FH) using molecular mechanisms that mimic FH interactions with host cells. In most instances, the AP serves to amplify C3b deposited on microbes by the classical pathway (CP). The role of properdin on microbes appears to be restricted to stabilization of C3 convertases; scant evidence exists for its role as an initiator of the AP on pathogens in the context of serum. Therapeutic complement inhibition carries with it an increased risk of infection. Antibody (Ab)-dependent AP activation may be critical for complement activation by vaccine-elicited Ab when the CP is blocked, and its molecular mechanism is discussed.

Complement killing of clinical Klebsiella pneumoniae isolates is serum concentration dependent

Klebsiella pneumoniae is an opportunistic gram-negative pathogen causing serious infections, including sepsis. In plasma, activation of the complement cascades is important for killing bacteria. Thirty clinical Klebsiella spp. blood isolates were analyzed for serum susceptibility in 75% normal human serum (NHS).Twenty-two were serum resistant and eight were serum sensitive, and subsequently tested in 5–75% NHS. Two isolates were killed in 5% and the remaining six in 50%–75% NHS. The two 5% sensitive isolates showed binding of complement (C)4 and C3 in 5% NHS with formation of membrane attack complex (MAC). Inhibition of the classical/lectin mediated pathways (CP/LP) using a C4 specific nanobody, hC4Nb8, led to survival of both isolates in 5% NHS. Using nanobody hC3Nb1, inhibiting the alternative pathway (AP), the isolates were killed in 5% NHS, and amplification of the CP/LP by AP was not necessary for killing. Sole AP killing of these isolates when inhibiting CP/LP with hC4Nb8 was observed in 50% NHS, stressing the concentration dependent functionality of AP.For the less sensitive isolates, killing required activation of CP/LP and AP demonstrated by inhibition with nanobodies. AP inhibition resulted in no C3 deposition on the serum resistant isolate, supporting that AP was the sole activation pathway.

Minimal role for the alternative pathway in complement activation by HIT immune complexes

BackgroundAnti‐platelet factor 4 (PF4)/heparin immune complexes that cause heparin‐induced thrombocytopenia (HIT) activate complement via the classical pathway. Previous studies have shown that the alternative pathway of complement substantially amplifies the classical pathway of complement activation through the C3b feedback cycle. ObjectivesThese studies sought to examine the contributions of the alternative pathway to complement activation by HIT antibodies. MethodsUsing IgG monoclonal (KKO) and/or patient‐derived HIT antibodies, we compared the effects of classical pathway (BBK32 and C1‐esterase inhibitor [C1‐INH]), alternative pathway (anti‐factor B [fB] or factor D [fD] inhibitor) or combined classical and alternative pathway inhibition (soluble complement receptor 1 [sCR1]) in whole blood or plasma. ResultsClassical pathway inhibitors BBK32 and C1‐INH and the combined classical/alternative pathway inhibitor sCR1 prevented KKO/HIT immune complex–induced complement activation, including release of C3 and C5 activation products, binding of immune complexes to B cells, and neutrophil activation. The alternative pathway inhibitors fB and fD, however, did not affect complement activation by KKO/HIT immune complexes. Similarly, alternative pathway inhibition had no effect on complement activation by unrelated immune complexes consisting of anti‐dinitrophenyl (DNP) antibody and the multivalent DNP‐‐keyhole limpet hemocyanin antigen. ConclusionsCollectively, these findings suggest the alternative pathway contributes little in support of complement activation by HIT immune complexes. Additional in vitro and in vivo studies are required to examine if this property is shared by most IgG‐containing immune complexes or if predominance of the classic pathway is limited to immune complexes composed of multivalent antigens.

Low-molecular weight inhibitors of the alternative complement pathway.

Dysregulation of the alternative complement pathway predisposes individuals to a number of diseases. It can either be evoked by genetic alterations in or by stabilizing antibodies to important pathway components and typically leads to severe diseases such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. In addition, the alternative pathway may also be involved in many other diseases where its amplifying function for all complement pathways might play a role. To identify specific alternative pathway inhibitors that qualify as therapeutics for these diseases, drug discovery efforts have focused on the two central proteases of the pathway, factor B and factor D. Although drug discovery has been challenging for a number of reasons, potent and selective low-molecular weight (LMW) oral inhibitors have now been discovered for both proteases and several molecules are in clinical development for multiple complement-mediated diseases. While the clinical development of these inhibitors initially focuses on diseases with systemic and/or peripheral tissue complement activation, the availability of LMW inhibitors may also open up the prospect of inhibiting complement in the central nervous system where its activation may also play an important role in several neurodegenerative diseases.

How we (will) treat PNH?

Clinical signs arising from intravascular hemolysis, hemolysis-related transfusions and thrombosis are indications for treatment initiation in paroxysmal nocturnal hemoglobinuria (PNH), whereas clone size per se is not. Eculizumab prevents intravascular hemolysis and reduces significantly thromboembolic risk resulting in a five-year overall survival of >90%. Hemoglobin value, LDH and reticulocyte count are used to define treatment response. Residual intravascular hemolysis is mainly caused by an incomplete C5 blockage and can lead to continuous low‐grade hemolysis or transient breakthrough hemolysis episodes in 10-15% of PNH patients. Additional complement‐amplifying conditions such as infections, surgery or pregnancy may overcome efficient therapeutic levels of Eculizumab and therefore require dose adjustments. C3‐mediated extra‐vascular hemolysis represents the main reason for residual anemia during anti‐C5 treatment. Patients with an inherited C5-variant lack response to Eculizumab and have been directed (in past) towards allogeneic HSCT. Transplantation has an overall mortality of up to 30%, with a higher risk in patients with previous thrombosis. A plethora of novel therapeutic agents are reported to impact on both; residual intravascular hemolysis and C3‐mediated extra‐vascular hemolysis. The new C5 inhibitor Ravalizumab with an eight‐week i.v. dosing interval showed non-inferiority to Eculizumab. Crovalimab, binding on the single missense C5 heterozygous mutation is injected s.c. monthly; two large phase III trials are ongoing as add on- and mono-therapy. Others, such as Pozelimab, injected subcutaneously on a weekly basis after an initial IV loading dose or Tesidolumab are still under current investigation. Currently investigated proximal inhibitors are acting towards: (i) the C3 complement; (ii) complement factor D or (iii) the complement factor B. They are aiming in particular to prevent C3‐mediated extra‐vascular hemolysis. Pegcetacoplan is a PEGylated version of compstatin which binds to C3 and is injected s.c. in monotherapy 4 weeks after initial concomitant therapy with Eculizumab. In a recent phase III trial, pegcetacoplan showed superiority to eculizumab in hemoglobin change from baseline and is now approved by the FDA for patients with PNH who are either treatment‐naive or switching from anti‐C5 monoclonal antibodies. Danicopan is an oral first‐in‐class factor D complement alternative pathway inhibitor and decreased significantly transfusion requirement, as shown in a phase II trial (phase III ongoing). BCX9930, another FD inhibitor in early development is given orally and demonstrated initial clinical efficacy both as add‐on therapy in patients with inadequate response to eculizumab as well as in monotherapy in treatment‐naive patients. In conclusion, novel proximal and distal complement inhibitors with different application modalities, in part as add-on or monotherapy seem to improve significantly intra- and extra‐vascular hemolysis in PNH, resulting in a better hematological benefit. Before choosing specific treatment, hematologists have to assess hemolysis, thrombosis and patients‘ bone marrow function. Future studies will help to explore long-term efficacy and safety of these novel agents.

Alternative Complement Pathway Inhibition With Iptacopan for the Treatment of C3 Glomerulopathy-Study Design of the APPEAR-C3G Trial

IntroductionComplement 3 glomerulopathy (C3G) is a rare kidney disease characterized by dysregulation of the alternative pathway (AP) of the complement system. About 50% of patients with C3G progress to kidney failure within 10 years of diagnosis. Currently, there are no approved therapeutic agents for C3G. Iptacopan is an oral, first-in-class, potent, and selective inhibitor of factor B, a key component of the AP. In a Phase II study, treatment with iptacopan was associated with a reduction in proteinuria and C3 deposit scores in C3G patients with native and transplanted kidneys, respectively.MethodsAPPEAR-C3G (NCT04817618) is a randomized, double-blind, and placebo-controlled Phase III study to evaluate the efficacy and safety of iptacopan in C3G patients, enrolling 68 adults with biopsy-confirmed C3G, reduced C3 (<77 mg/dl), proteinuria ≥1.0 g/g, and estimated glomerular filtration rate (eGFR) ≥30 ml/min per 1.73 m2. All patients will receive maximally tolerated angiotensin-converting enzyme inhibitor/angiotensin receptor blocker and vaccination against encapsulated bacteria. Patients with any organ transplantation, progressive crescentic glomerulonephritis (GN), monoclonal gammopathy of undetermined significance, or kidney biopsy with >50% interstitial fibrosis/tubular atrophy, will be excluded. Patients will be randomized 1:1 to receive either iptacopan 200 mg twice daily or placebo for 6 months, followed by open-label treatment with iptacopan 200 mg twice daily for all patients for 6 months. The primary objective is to evaluate the efficacy of iptacopan versus placebo on proteinuria reduction urine protein:creatinine ratio (UPCR) (24 h urine). Key secondary endpoints will assess kidney function measured by eGFR, histological disease total activity score, and fatigue.ConclusionThis study aims to demonstrate the clinical benefits of AP inhibition with iptacopan in C3G.

Discovery of APL-1030, a Novel, High-Affinity Nanofitin Inhibitor of C3-Mediated Complement Activation.

Uncontrolled complement activation contributes to multiple immune pathologies. Although synthetic compstatin derivatives targeting C3 and C3b are robust inhibitors of complement activation, their physicochemical and molecular properties may limit access to specific organs, development of bifunctional moieties, and therapeutic applications requiring transgenic expression. Complement-targeting therapeutics containing only natural amino acids could enable multifunctional pharmacology, gene therapies, and targeted delivery for underserved diseases. A Nanofitin library of hyperthermophilic protein scaffolds was screened using ribosome display for C3/C3b-targeting clones mimicking compstatin pharmacology. APL-1030, a recombinant 64-residue Nanofitin, emerged as the lead candidate. APL-1030 is thermostable, binds C3 (KD, 1.59 nM) and C3b (KD, 1.11 nM), and inhibits complement activation via classical (IC50 = 110.8 nM) and alternative (IC50 = 291.3 nM) pathways in Wieslab assays. Pharmacologic activity (determined by alternative pathway inhibition) was limited to primate species of tested sera. C3b-binding sites of APL-1030 and compstatin were shown to overlap by X-ray crystallography of C3b-bound APL-1030. APL-1030 is a novel, high-affinity inhibitor of primate C3-mediated complement activation developed from natural amino acids on the hyperthermophilic Nanofitin platform. Its properties may support novel drug candidates, enabling bifunctional moieties, gene therapy, and tissue-targeted C3 pharmacologics for diseases with high unmet need.

Minimal Role for the Alternative Pathway in Complement Activation By HIT Immune Complexes

Background: Recent studies show that ultra-large immune complexes consisting of IgG and platelet factor 4 and heparin (P+H) potently activate complement and facilitate complement dependent activation of cellular FcgRIIA (PMID 34189574). In whole blood assays using KKO, a monoclonal anti-PF4/heparin antibody, or antibodies from patients with heparin induced thrombocytopenia (HIT), we showed that classical pathway (CP) inhibition reduced immune complex-mediated complement activation (C3c and soluble C5b-9 generation), cell surface deposition of immune complexes and cellular activation.Aims: As previous studies suggest that the alternative pathway (AP) provides significant amplification (>80%) of the CP pathway, (PMID: 15544620) we compared the effects of AP, CP, and CP/AP inhibitors by KKO and HIT immune complexes in whole blood.Methods: Inhibitors of the CP (BBK32, a borrelia protein inhibitor to C1r), AP (anti-factor B antibody (α-fB), or Factor D (fD inhibitor or fD-INH) Alexion Pharmaceuticals, Boston, MA) or combined AP/CP (C1-esterase inhibitor, C1-INH, Berinert, CSL Behring; or soluble complement receptor 1, sCR1, Alexion) were tested in hemolytic assays of CP or AP to confirm pathway specificity. To examine effects of CP or AP inhibition on complement activation by immune complexes consisting of KKO or HIT IgG, whole blood was pre-incubated with CP, AP or CP/AP inhibitors prior to addition of P+H ± KKO/HIT IgG or isotype controls. WB was incubated for 45 minutes at 37ºC followed by addition of 10mM EDTA to quench further complement activation. Complement activation products (C3c and sC5b-9) and neutrophil degranulation (MMP9) markers were measured using commercial immunoassays. Effects of complement inhibitors on cellular deposition of immune complexes was examined by flow, using previously described methods (PMID 34189574) using fluorescently labeled anti-C3c antibody (Quidel, San Diego, CA) and anti-mouse or human IgG (Biolegend, San Diego, CA).Results: Consistent with prior publications (PMID: 26808924), BBK32 showed marked reduction CP, but not AP-dependent hemolytic assays. The converse was true of AP inhibitors: α-fB and fD-INH prevented AP-dependent, but not CP-dependent hemolysis (data not shown). C1-INH and sCR1 showed activity in both CP- and AP-dependent assays. The CP or CP/AP inhibitors showed potent inhibition of C3c and sC5b-9 generation by KKO and HIT immune complexes, while AP inhibitors had no effect (Figure A for KKO C3c generation; and Table 1 for KKO/HIT C3c generation; sC5b-9 data not shown). For a given CP or CP/AP inhibitor, the concentrations leading to 50% inhibition (IC 50) were generally comparable for KKO and HIT immune complexes for C3c (Figure A and Table 1) and sC5b-9 generation (data not shown), with potency as follows: C1-INH>>BBK32>sCR1 (Table 1). On the other hand, the AP inhibitors, α-fB and fD-INH, showed no inhibitory activity in C3c (Figure A and Table 1)/sC5b-9 (data not shown) generation by KKO or HIT ULICs. As our recent studies indicate that complement activation is critical to cell surface deposition of immune complexes and cellular activation via FcgRIIA, we examined effects of complement inhibitors on IC deposition on B-cells and MMP9 release from neutrophils. CP or CP/AP inhibitors, but not AP inhibitors, reduced cell surface binding of immune complexes (Figure B) as well as MMP9 release (Figure C and Table 1).Conclusion: Together, these studies demonstrate that the AP has a minimal role in supporting complement activation by KKO/HIT ULICs. Future studies should examine CP inhibition as a therapeutic strategy for modulating the cellular activating effects of HIT antibodies. To what extent these findings apply to other immune complexes and/or CP activators requires further study.Funding Agency: NIH HL151730; α-fB antibody, fD inhibitor and sCR1 was provided by Alexion Pharmaceuticals, Boston, MA. BBK32 was provided by Dr. Brandon Garcia, East Carolina University, Greenville, NC. [Display omitted] DisclosuresCines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board. OffLabel Disclosure:C1-esterase inhibitor off label for HIT

12-Month Analysis of a Phase 2 Study of Iptacopan (LNP023) Monotherapy for Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hematological disorder caused by somatic mutations in the phosphatidylinositol glycan A (PIGA) gene in hematopoietic stem cells, resulting in complement alternative pathway (AP)-mediated severe hemolysis, life-threatening thrombosis, and impaired bone marrow function. The current standard-of-care for PNH consists of anti-C5 blockade with either eculizumab or ravulizumab. While both monoclonal antibodies effectively control intravascular hemolysis (IVH), reduce thrombosis and improve long-term survival, a significant proportion of patients remains anemic and continues to require transfusions, largely due to persistent extravascular hemolysis (EVH). Conversely, pegcetacoplan, a recently FDA-approved anti-C3 inhibitor, prevents both IVH and EVH and showed superiority to eculizumab in improving hemoglobin (Hb) levels in PNH. Nevertheless, the need for effective oral treatment options in PNH remains unmet. Iptacopan (LNP023) is a new, oral, selective and potent first-in-class inhibitor of factor B, a key component of the AP. Recent phase 2 data showed that iptacopan effectively controls both IVH and EVH and leads to rapid, transfusion-free improvements in Hb levels in the majority of PNH patients.Methods: CLNP023X2204 (NCT03896152) is a multicenter, randomized, open-label phase 2 study in adult PNH patients with active hemolysis and no complement inhibitor treatment within 3 months prior to study entry. Patients are randomized to receive twice daily (BID) iptacopan in one of two dose-sequences, either 25 mg for 4 weeks followed by 100 mg for up to 2 years (Cohort 1) or 50 mg followed by 200 mg (Cohort 2). The key objectives are to assess the effect of iptacopan on markers of IVH/EVH (incl. lactate dehydrogenase (LDH)) and on Hb levels, as well as safety. 12-month interim results are summarized below.Results: The study has completed enrolment. A total of 13 patients (mean age 38.2 years, 7 female) were randomized to either Cohort 1 (n=7) or 2 (n=6). Mean (SD) lab values at baseline were LDH 2097.8 (911.2) U/L, reticulocytes 203.3 (82.9) x10E9/L, bilirubin 32.4 (10.1) µmol/L, and Hb 85.8 (13.2) g/L, and most patients required red blood cell transfusions in the prior 12 months (median 3.0, range 0-19). At the time of the interim analysis, 11 patients had been treated with iptacopan for at least 52 weeks, with a maximum treatment duration of 81 weeks; 2 patients discontinued treatment early, one after 2 days due to a non-serious adverse event of headache (hence not evaluable for the primary endpoint), the other after 13 weeks due to physician decision. Amongst the 12 evaluable patients, all of whom were anti-C5 naive, all reached the primary endpoint of lowering LDH by at least 60% within the first 12 weeks. The LDH response was rapid and durable, with all patients treated with ≥50 mg BID reaching this threshold after only one week of treatment and all ongoing patients except one maintaining the threshold up until the data cutoff, i.e., for at least 52 weeks. Equivalent improvements were also observed for other markers of IVH and EVH. Similarly, Hb levels improved significantly and durably in most patients, and all except one of the ongoing patients have remained transfusion-free since the start of iptacopan treatment. Moreover, no thromboembolic events occurred during the study, and the FACIT fatigue score improved significantly in most patients. Iptacopan monotherapy was safe, with no severe or serious adverse reported up to the data cutoff.Conclusion: Iptacopan is a new, well tolerated oral complement AP inhibitor that blocks both IVH and EVH in adult PNH patients with hemolytic PNH. 12-month results from this non-pivotal study demonstrate that iptacopan monotherapy leads to rapid and durable improvements in various hemolytic markers and meaningful and sustained clinical benefit as seen in improvements in Hb levels, transfusion requirement and FACIT fatigue score. These results suggest that proximal inhibition of the complement cascade parallels and further improves the hematological benefit seen with anti-C5 therapies, paving the way for the phase 3 evaluation of iptacopan as potentially new oral first-line therapy for patients with PNH. [Display omitted] DisclosuresWong: Astellas Pharma, INc.: Research Funding. Li: Novartis: Current Employment, Current equity holder in publicly-traded company. Rozenberg: Novartis: Current Employment, Current equity holder in publicly-traded company. Nidamarthy: Novartis: Current Employment, Current equity holder in publicly-traded company. Chawla: Novartis: Current Employment, Current equity holder in publicly-traded company. Junge: Novartis: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure:Iptacopan (LNP023) is a new, oral, selective and potent first-in-class inhibitor of factor B, a key component of the complement alternative pathway. It is currently being investigated in paroxysmal nocturnal hemoglobinuria (PNH) as well as in several renal indications.

Alternative Complement Pathway Inhibition Does Not Abrogate Meningococcal Killing by Serum of Vaccinated Individuals.

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors.

Nitric oxide-mediated alternative pathway alleviates aluminum-induced programmed cell death in soybean root tips

Alternative pathway (AP) plays essential roles in plant adaptation to environmental stress. However, the exact role of AP in response to aluminum (Al) toxicity remains elusive. We here provide solid evidences that the activated AP capacity in root tips of soybean alleviated Al toxicity. Furthermore, inhibition of AP by pharmacological or transgenic approach aggravated Al-induced programmed cell death (PCD) occurrence mediated through reactive oxygen species (ROS)-dependent mitochondrial pathway. Our results also demonstrated that nitric oxide (NO) plays a negative role in PCD occurrence caused by Al in soybean root tips. Interestingly, the alleviating effect of NO on Al-induced PCD could be blocked by AP inhibition. Further investigation showed that NO mediates the induction of AP resulting from the upregulation of AOX expression and pyruvate content in Al-treated root tips of soybean. Taken together, our results clearly suggest that AP participates in the alleviation of Al toxicity and also plays a critical role in the alleviating effect of NO on Al-induced PCD occurrence, which will open up new avenues for the improvement of plant growth in acidic soils.

In Vitro Combination Studies of ACH-4471 with Eculizumab to Assess a Potential “Switch” Treatment Approach for Paroxysmal Nocturnal Hemoglobinuria

Introduction: ACH-4471 is an oral complement factor D (fD) inhibitor that has demonstrated clinical efficacy in an ongoing phase 2 clinical study for naïve patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). PNH is a rare clonal blood disorder in which erythrocytes lacking the GPI-anchored complement regulators CD55 and CD59 are susceptible to destruction by the terminal complement complex (also known as membrane attack complex, MAC) following the normal low-level activation of the complement alternative pathway (AP) in the fluid phase. The therapeutic agent eculizumab, a monoclonal antibody directed against the terminal complement component C5, blocks MAC assembly on erythrocyte membranes and so prevents most intravascular hemolytic destruction. Eculizumab treatment however leads to increased deposition of complement C3 fragments on PNH membranes, which can result in extravascular phagocytic elimination of opsonized erythrocytes (A. M. Risitano et al, Blood, 2009, 113:4094) and incomplete inhibition of intravascular hemolysis (M. J. Harder et al, Blood, 2017, 129:970), and consequently to the continued anemia and transfusion dependence observed in a significant subset of patients. As an AP inhibitor, in contrast to eculizumab, ACH-4471 prevents both MAC assembly and C3 fragment deposition on PNH cell surfaces and therefore is expected to address the medical needs of these suboptimal responders to eculizumab. To assess this notion directly and also to support a future switch treatment approach, a further clinical study has been planned to evaluate ACH-4471 co-administration in PNH patients who respond suboptimally to eculizumab. In the present study, we conducted in vitro combination experiments to explore the potential pharmacological consequences when ACH-4471 is added on to eculizumab or another anti-C5 monoclonal antibody, using functional AP assays that include the hemolysis of erythrocytes from a PNH patient.Methods: Concentration series of ACH-4471 were assessed in pairwise combinations with eculizumab and a second monoclonal antibody inhibitor of complement C5. Functional assays for human serum AP activity included a single-plate ELISA-based functional assay (Wieslab) and the hemolysis of red blood cells from a PNH patient under AP-restricted conditions but at neutral pH. Analyses of interactions were performed using a three-dimensional surface graphing method and, where applicable, a median-effect plot method.Results: We assessed pairwise interactions using two assays for AP function. In the AP Wieslab assay, interactions between ACH-4471 and an anti-C5 monoclonal antibody ranged from additive to moderately synergistic. Next, to better approximate the physiological setting, we assessed inhibitor interactions against the AP-mediated hemolysis of red blood cells obtained from a PNH patient. ACH-4471 in this assay showed strongly synergistic interactions when tested with eculizumab. In light of a recent report on incomplete inhibition by eculizumab following strong complement activation, we propose a possible mechanism for synergy between ACH-4471 and eculizumab wherein ACH-4471 reduces the density of deposited C3b clusters, to which C5 binding is thereby more readily inhibited by eculizumab.Conclusions: ACH-4471 showed strong synergy with eculizumab. These results suggest the potential clinical utility of ACH-4471 as a PNH therapeutic agent that may be used in suboptimal eculizumab responders in support of a “switch” treatment strategy. DisclosuresPatel:Achillion: Employment. Thanassi:Achillion: Employment. Yang:Achillion: Employment. Brodsky:Alexion Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Grant Funding. Huang:Achillion: Employment. Podos:Achillion: Employment.