APL-2 Prevents Both C3 and C5 Convertase Formation and Activity: A Potential Therapeutic for Renal Diseases

American Society of Nephrology (ASN) Kidney Week 2019 | November 5-10, 2019 | Washington, DC

Introduction • C3 glomerulopathy (C3G) is a group of rare inflammatory renal

diseases classified into 2 major subgroups: C3 glomerulonephritis (C3GN) and dense deposit disease (DDD),1,2 which cause end-stage renal disease in up to 50% of affected patients within 5-10 years of diagnosis.1-4

• A key feature of C3G is uncontrolled sustained activation of the alternative pathway of the complement system. This is caused by the presence of autoantibodies, C3 and C5 nephritic factors (NeFs), which bind and stabilize the respective convertases responsible for C3/C5 activation, or by genetic abnormalities in complement regulatory proteins.4,5

• Overactivation of complement leads to excessive deposition of C3 and C5 breakdown products in the glomeruli, and subsequent renal damage.3,4 In C3G, overactivation of convertases exceeds the capabilities of complement regulatory proteins factor H, CR1, or CD55.6

• Currently, there are no treatments approved for C3G.7

• APL-2 (pegcetacoplan) is a 15–amino acid cyclic peptide conjugated to each end of a linear polyethylene glycol (PEG) molecule that binds to C3 and inhibits C3 activation. Additionally, APL-2 binds to and prevents the activity of C3b, a breakdown product of C3 (Figure 1).

• Based on its mechanism of action, APL-2 has the potential to address the underlying disease process of C3 overactivation in C3G, and to reduce renal damage.

MethodsC3 and C5 Convertase Hemolytic Assays• An established in vitro model of hemolysis (C3b-bearing sheep

erythrocytes) was used to analyze the effects of APL-2 on the activities of C3 convertase (Figure 2A) and C5 convertase (Figure 2B). Serial dilutions of APL-2 (final concentration 0.07-36.5 µM) or PEG (mol to mol of APL-2) were tested to study the impact on the hemolytic activities of the C3 and C5 convertases.

Cell-Bound C3bBb and Properdin-C3bBb Stabilization Assays• Cell-bound C3bBb and properdin-C3bBb stabilization assays were

performed to study the overactivation of C3 and C5 convertases by C3 NeF and C5 NeF, respectively, isolated from 14 patients with C3GN or DDD (Figure 3; Table).1,2

Figure 5. APL-2 Prevented (A) Formation and (B) Activity of the Alternative Pathway C5 Convertase

SA-PO609

References: 1. Fakhouri F, et al. C3 glomerulopathy: a new classification. Nat Rev Nephrol. 2010;6(8):494-499. 2. Sethi S, et al. Membranoproliferative glomerulonephritis and C3 glomerulopathy: resolving the confusion. Kidney Int. 2012;81(5):434-441. 3. Bomback AS, et al. Eculizumab for dense deposit disease and C3 glomerulonephritis. Clin J Am Soc Nephrol. 2012;7(5):748-756. 4. Servais A, et al. Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies. Kidney Int. 2012;82(4):454-464. 5. Marinozzi MC, et al. C5 nephritic factors drive the biological phenotype of C3 glomerulopathies. Kidney Int. 2017;92(5):1232-1241. 6. Paixao-Cavalcante D, et al. Sensitive and specific assays for C3 nephritic factors clarify mechanisms underlying complement dysregulation. Kidney Int. 2012;82(10):1084-1092. 7. Smith RJH, et al. C3 glomerulopathy - understanding a rare complement-driven renal disease. Nat Rev Nephrol. 2019;15(3):129-143. 8. Lu DF, et al. Clinical features and outcomes of 98 children and adults with dense deposit disease. Pediatr Nephrol. 2012;27(5):773-781. Acknowledgments: This study was funded by Apellis Pharmaceuticals, Inc. Medical writing support was provided by Malcolm Darkes, PhD, MPS, of Excel Medical Affairs, Horsham, UK, and funded by Apellis Pharmaceuticals, Inc.

Noémie Simon-Tillaux,1 Sophie Chauvet,1 Delphine El Mehdi,2 Pascal Deschatelets,2 Veronique Fremeaux-Bacchi1 1Laboratoire d’Immunologie, Hôpital Européen Georges Pompidou, Paris, France; 2Apellis Pharmaceuticals, Inc., Waltham, MA

Conclusions• APL-2 inhibits the activities of the

preformed C3 and C5 convertases. Due to its capacity to bind C3b, APL-2 may abolish the subsequent attachment of additional C3b molecules near the C3 convertase that are essential for C5 convertase formation.

• APL-2 significantly inhibits the cleavage of C3 and C5 by their respective convertases stabilized by patients’ plasma IgG positive for C3 and C5 NeFs, which decreases the subsequent activation of complement.

• These data support the ability of APL-2 to significantly decrease the NeF-mediated overactivation of complement. As such, APL-2 may have the potential to mitigate or prevent kidney impairment in patients with C3G.

Patient Diagnosis C3 NeFa C5 NeF C3, mg/Lb C4, mg/Lb sC5b-9, μg/mLb

1 C3GN + − 254 183 267

2 C3GN + + 240 103 1017

3 C3GN + + 137 174 353

4 C3GN + − 58 151 1700

5 C3GN − + 221 136 912

6 C3GN + + 129 234 1578

7 C3GN + + <45 236 1675

8 C3GN + + 461 155 565

9 DDD + + 435 266 227

10 C3GN + − 1020 157 186

11 C3GN + + 326 262 861

12 DDD + − 89 168 352

13 C3GN + + 853 134 204

14 C3GN + − 302 320 1780

C3GN, C3 glomerulonephritis; DDD, dense deposit disease; IgG, immunoglobulin G; NeF, nephritic factor; sC5b-9, soluble C5b-9.aPatient IgG was isolated from the plasma of patients with C3GN or DDD using DE 52 resin (Sigma-Aldrich).bNormal reference ranges: C3 = 660-1250 mg/L; C4 = 93-380 mg/L; sC5b-9 <440 µg/mL.

Table. Baseline Characteristics of Patients With C3GN and DDD With IgG Positive for C3 and C5 NeFs

ResultsImpact on C3 Convertase Activity• APL-2 inhibited alternative pathway C3 convertase activity (Figure 4). • PEG had no impact on C3 convertase activity.

C3

APL-2

Bb

C3bX

Prevent docking with the convertase complex

B

C3

APL-2

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C3bX

Interfere with the binding of C3

C

C3b

A

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C5C5

C5b

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ALTERNATIVE PATHWAY

C3 convertase

C3 binding to convertase

C3a cleavage Convertasedecay

Amplification loop

Bb

C3b C3b C3b C3b C3b

MAC

C5a

C3

Bb Bb

Bb

C3bC3bC3bC3b

C3 convertase

C3a

C3

C3

Bb

C5 convertaseC3 convertase

Bb

Figure 1. Overview of the Complement Cascade and Putative Mechanism of Action of APL-2

Figure 2. Functional Hemolytic Assays8

AP C3 convertase (C3bBb) was generated by incubating SRs bearing C3b (SR-C3b, 107/mL) with an appropriate concentration of FB (Complement Technology) and FD (Sigma, 400 ng/mL) in order to obtain a Z (lytic sites per cell) between 1 and 1.5. Fresh-frozen rat serum diluted in GVB-EDTA was used as a source of C3 to C9 to develop C3bBb convertase sites. In this assay, APL-2 was added at the same time as rat serum. APL-2 binds to human but not rodent C3, a property that allowed us to study the impact of APL-2 exclusively on the human convertase.

A. C3 Convertase Hemolytic Assay Using SRs Bearing C3bBb

B. C5 Convertase Hemolytic Assay Using SRs Bearing Properdin-C3bBb

Objective• The objective of this proof-of-concept in vitro study was to show

that APL-2 prevents C3 and C5 cleavage by their respective convertases in the absence and presence of C3 NeF and C5 NeF.

The cleavage of C5 by a preformed membrane-bound C5 convertase was measured using SR-C3bBb generated by incubating SR-C3b with FD, human C3, P, and appropriate concentrations of FB to obtain a Z (lytic sites per cell) between 1 and 1.5. Human purified C5, C6, C7, C8, and C9 were used to form the human lytic complex C5b-9. In this assay, APL-2 was added either at the step of C5 convertase formation or at the step of MAC formation.

AP, alternative pathway; C3bBb, C3 convertase; C3b(n)Bb, C3 or C5 convertase; C3bBbC3b, C5 convertase; EDTA, ethylenediaminetetraacetic acid; FB, factor B; FD, factor D; MAC, membrane attack complex (C5b-9); P, properdin; SR, sheep erythrocyte.

Figure 3. In Vitro Model of Hemolysis5 Induced by C3- and C5-Stabilized Convertases in the Presence and Absence of APL-2

% stabilization = % of the residual convertase compared with the reference.

APL-2 and patient IgG positive for C3 and/or C5 NeFs were added after the formation of the C3 convertase (C3bBb) or the C5 convertase (C3bBbC3b) during the dissociation step. The residual C3 or C5 convertase activity was revealed using rat sera 20 or 30 minutes after the addition of patient IgG with or without APL-2.

C3b(n)Bb, C3 or C5 convertase; EDTA, ethylenediaminetetraacetic acid; FB, factor B; FD, factor D; IgG, human purified immunoglobulin G from patients or healthy controls; NeF, nephritic factor; P, properdin; SR, sheep erythrocyte.

Figure 4. APL-2 Inhibits C3 Cleavage by C3 Convertase

Purified FB and FD were added to the media as required to generate the C3 convertase to obtain a Z (lytic sites per cell) between 1 and 1.5 on the C3b SR, and rat C5 to C9 were added to trigger the lysis step. Results are expressed as percentage ± SD of convertase lysis activity in the absence of APL-2. N = 3 separate experiments.

C3bBb, C3 convertase; FB, factor B; FD, factor D; PEG, polyethylene glycol (control); SR, sheep erythrocyte.

Impact on C5 Convertase Formation and Activity• APL-2 induced a decreased density of solid-phase C3b and thus

inhibited the formation of C3bBbC3b complexes, resulting in less C5 convertase at the cell surface (Figure 5A).

• The binding of APL-2 to C3b of the C5 convertase interfered with the binding of C5 to C5 convertase (Figure 5B).

APL-2 Affects C3 and C5 Cleavage by C3 and C5 Convertases That Are Stabilized by C3 and C5 NeFs, Respectively• APL-2 decreased C3 and C5 convertase activity despite the presence of

C3 NeF (Figure 6) and C5 NeF (Figure 7), respectively. The difference in convertase activity was significant between the 2 groups (with and without APL-2) per the Wilcoxon matched-pairs rank test (P < 0.0001 for C3 convertase; P < 0.002 for C5 convertase).

A. Addition of APL-2 at the Time of Formation of the Alternative Pathway C5 Convertase C3bBbC3b

B. Addition of APL-2 After the Formation of C5 Convertase

Figure 6. APL-2 Prevents C3 Cleavage by C3 NeF-Stabilized C3 Convertase

APL-2 and patient plasma IgG positive for NeFs were added at the same time in the in vitro model. APL-2 was used at a concentration of 0.6 µM, as indicated in the square. At this concentration, APL-2 inhibited 70% of C3 convertase activity relative to the absence of APL-2.

IgG, immunoglobulin G; NeF, nephritic factor.

*P < 0.0001 for C3 convertase.

A. APL-2 Restrains C3 Convertase Activity in the Presence of the C3 NeF (Example: Patient 7)

B. Residual C3 Convertase Activity of Patients IgG positive for NeFs With or Without APL-2*

Figure 7. APL-2 Prevents C5 Cleavage by C5 NeF-Stabilized C5 Convertase

APL-2 and patient plasma IgG positive for NeFs were used at a concentration of 0.6 µM (30% remaining activity relative to the absence of APL-2), as indicated in the square. Patients 1, 4, 5, 13, and 14 were negative for C5 NeFs and thus were not tested.

IgG, immunoglobulin G; NeF, nephritic factor.

*P < 0.002 for C5 convertase.

Residual C5 Convertase Activity of Patients IgG Positive for NeFs With or Without APL-2*

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Human purified C3, FD, FB, and properdin were added to generate C5 convertases, and human C5-C9 were added to trigger the lysis step. Results expressed as percentage ± SD of convertase lysis activity in the absence of APL-2. N = 2 separate experiments for the first 2 time points; N = 1 experiment for the third time point. No differences were observed when APL-2 was added during the formation of convertase (panel A) or after formation, at the lysis step (panel B).

C3bBbC3b, C5 convertase; FB, factor B; FD, factor D; SR, sheep erythrocyte.

PEG

APL-2

APL-2 (µM)

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0.0 0.5 1.0 1.5 2.0 2.5 2826 303.00

40

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APL-2 (µM)

% ±

SD

of

Co

nve

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ctiv

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0.0 0.5 1.5 2.0 2.5 3.0 P1 P20

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WithAPL-2

0

320280240

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P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14

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260120

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0

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Without APL-2With APL-2

MAC, membrane attack complex (C5b-9).

C3b SR C3bBb SR Lysis

FB

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APL-2

APL-2

APL-2

APL-2

IgG

(EDTA) Convertase stabilization

Residual convertase

45’ 37°C (EDTA)

45’ 37°C

C3b(n)Bb

FB+FD (+P)30’ 30°C

1 lytic site/cell

Z = 1

Decay 20’ (30’)

30°C

No decay Reference

Rat serum (C5-C9)

Rat serum

FB

FD

FD

C3

P

C3bBb

Step 1: addition of FB and FD

Step 1: addition of C3, FB, FD, and P

Step 2: addition of rat serum

Step 2: addition of rat serum

Step 2: addition of C5 to C9 purified human proteins

Formation of the AP C3 convertase C3bBb

Formation of the AP C5 convertase C3bBbC3b

Amplification of the C3b deposits and MAC formation

MAC formation

C3b(n)Bb

C3bBb

C3bBbC3b

C3, C5, C6, C7, C8, and C9

C3, C5, C6, C7, C8, and C9

C5, C6, C7, C8, and C9

C5, C6, C7, C8, and C9

C5, C6, C7, C8, and C9

C3b SRC3b(n)Bb

SR Lysis

SR SR

SR

Lysis

Lysis

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C3bBbC3b