Design of miniprotein inhibitors targeting complement C9 to block membrane attack complex assembly

Domain: biology
Published: 2026-03-12
Editorial outcome: minor_revision

Abstract
The abnormal formation of the membrane attack complex (MAC) is intrinsically linked to a range of acute and chronic immune diseases. The insertion of complement C9 into the membrane is the final step and kinetic bottleneck of MAC formation. However, research on blocking the MAC formation of C9 is currently limited. Given its broad, flat, and polar functional interface, complement C9 is a challenging target for rational design. Here, we utilize deep learning-based methods for protein scaffold generation, sequence design, and complex structure prediction to de novo design mini-protein inhibitors that specifically block the membrane insertion of soluble complement C9. The binding affinity of the mini-protein inhibitor is further optimized to 700 pM via partial diffusion. Design accuracy and binding specificity are verified through X-ray crystallography and biochemical studies. An in vivo acute hemolysis inhibition assay reveals that the C9 mini-protein inhibitors remain effective against hemolysis even 8 minutes after complement activation, outperforming the complement C5 inhibitor eculizumab. The de novo designed C9 mini-protein inhibitors can offer an optional therapeutic approach for the prevention and treatment of acute or chronic immune diseases associated with abnormal complement activation.

Introduction
The complement system, ancient and evolutionarily conserved, is one of the most important components of the immune system. It defends against invading pathogens while preserving host homeostasis. Regardless of whether the initiating trigger is the classical, lectin, or alternative pathway, all three activation pathways converge on the formation of the membrane-attack complex (MAC). Aberrant MAC assembly on host cells underlies a spectrum of autoimmune disorders, including paroxysmal nocturnal hemoglobinuria (PNH)1, 2, atypical hemolytic uremic syndrome3, myasthenia gravis4, 5, and neuromyelitis optica spectrum disorders6, 7.

Complement C5 cleavage into C5a and C5b initiates MAC formation. C5b rapidly binds C6 to form the C5b6 complex, which sequentially recruits C7, C8, and multiple C9 molecules. C6, C7, C8\alpha, C8\beta and C9 undergo a conserved conformational switch that exposes a shared pore-forming domain, ultimately assembling into the membrane-attack complex (MAC) on the target membrane8, 9, 10, 11. Eculizumab, a monoclonal antibody that prevents cleavage of C5 into C5a and C5b, represents a landmark in complement therapeutics12, 13, 14, 15, 16, 17, 18, 19, 20. However, blockade of C5 also inhibits the release of C5a, which may increase infection risk, and some patients are insensitive to treatment with eculizumab due to the residual C5 activity21 and genetic polymorphisms of C522. Recent studies have therefore evaluated antibodies targeting C623 or C724, demonstrating that downstream inhibition can achieve equivalent or superior hemoprotection without compromising C5a-mediated immunity.

The activation of the complement system involves a series of enzymatic cascade reactions. Once initiated, the enzymatic cascade assembles the MAC in a relatively short period of time to attack the target and the inhibitors such as eculizumab are kinetically disadvantaged. In vitro, target cell lysis can be completed within 5 min23. Indeed, incorporation of the first C9 is the kinetic bottleneck of MAC formation, thereafter 18 soluble C9 molecules rapidly oligomerize to complete the pore25.

In fact, the widely expressed GPI-anchored glycoprotein CD59 could sterically block the insertion of the first C9 \beta-barrel hairpin into the membrane, preventing C9 polymerization and membrane-attack complex (MAC) formation11. Loss of the GPI anchor leaves CD59 unable to reach the membrane, depriving red cells of their final line of defense against complement and representing a central mechanism underlying the chronic hemolysis of PNH26. Besides, the constitutive expression of CD59 is insufficient to prevent massive MAC formation in hyperacute settings such as acute hemolytic transfusion reaction (AHTR)27, 28, paroxysmal cold hemoglobinuria (PCH)29 or cold agglutinin disease (CAD)30, 31, 32. Consequently, designing protein to direct blockade of C9 insertion may act as a “last-minute brake” even after cascade initiation, providing life-saving potential for both chronic and acute hemolysis. Despite this therapeutic rationale, C9-directed inhibitors are rarely reported. An early report showed that a commercially available anti-C9 monoclonal antibody X197 conferred inhibitory activity in a non-standard hemolysis-inhibition assay33. Separately, the O1-antigen of Klebsiella LPS was found to block C9 polymerization34. However, neither study evaluated the therapeutic potential of these inhibitors for hemolytic disorders. The key polymerization interface of C9 is highly conserved across mammals, which increases the difficulty in immunological screening of functional monoclonal antibodies. Moreover, the relatively high serum concentration of C9 requires C9 inhibitors have high doses and exquisite functional inhibition specificity.

With the rapid development of AI-based diffusion denoising technology for generating target binders, we can more accurately generate binding proteins for a broader range of target types35, 36. This technological advancement has to some extent compensated for the inherent shortcomings of antibody based screens that targets conserved function-critical sites. Additionally, it allows the production of high-affinity mini-proteins through low-throughput experimental workflows. Moreover, mini-protein binders can be produced in E. coli under simple culture conditions, with high expression levels and thermal stability37. Compared with the higher experimental barriers and greater production costs associated with antibody production, mini-protein binders hold vast potential for both research and pharmaceutical applications. Owing to strict structure-guided design, high affinity mini-protein binders have been reported for numerous ligands such as TNFR36, EGFR37, IL-1\beta38 and snake venom toxins39; nevertheless, the design of high-affinity and functional inhibitors against complement components, especially the structurally unique C9 protein remains challenging.

In this work, we utilize deep learning-based methods to de novo design mini-protein inhibitors that specifically block the membrane insertion of soluble complement C9. Screening of 103 candidates through an in vitro hemolysis inhibition assay identified the best-performing scaffold. The binding affinity of the mini-protein inhibitor is further optimized to 700 pM via partial diffusion. Design accuracy and binding specificity are verified through X-ray crystallography and biochemical studies. An in vivo acute hemolysis inhibition assay reveals that the C9 mini-protein inhibitors outperform the complement C5 inhibitor eculizumab.

Results
Design of mini-protein binder target C9 with RFdiffusion
As the monomeric structure of human complement C9 has not yet been experimentally determined, we employed the crystal structure of monomeric mouse C9 as our initial design template. C9 adopts a disc-shaped structure (Fig. 1a & b), and its transition from soluble monomer to membrane-inserted pore relies on an extensive 77 \times 50 \text{\AA} interface (Fig. 1b). This large interaction region is flat and contains few separated surface hydrophobic residues (Fig. 1a & b). Theoretically, binding to this region of soluble C9 can effectively inhibit its polymerization (Fig. 1a). Since binder design favors surface-exposed hydrophobic residues as hotspots35, 36, we carefully checked the C9 interaction region for spatially clustered, interface-critical hydrophobic residues and grouped them into several sets. Two independent hydrophobic hotspots were initially chosen for RFdiffusion scaffolding: Site-1 (V196, I199, V305) and Site-2 (L217, A233, F235) (Fig. 1b). For each site, 1,500 backbones (50-80 residues) were generated; three sequences per backbone were designed using ProteinAMPNN and scored initially with AlphaFold2 (Supplementary Fig. 1 & 2a). In the scoring scheme, pae_interaction represents the predicted aligned error that AlphaFold2 assigns to the binder–target interface. It is the key metric for assessing how confident the model is in the interface prediction: the lower the value, the more reliable the prediction and the better the spatial fit between binder and target. Designs with pae_interaction < 10 are generally considered acceptable. Complementarily, plddt_binder measures the per-residue confidence of the binder alone. A high value indicates that the sequence is likely to fold into a stable, well-defined monomeric structure; a threshold of > 90 is commonly required35, 36. After applying relaxed filters (pae_interaction < 15, plddt_binder > 88), Site-1 achieved a 1.20 % success rate, while Site-2 approached 0 % (Supplementary Fig. 2a). In the second round of calculation, we introduced a new site, Site-3 (F231, M476, P478, Y480, I505) for calculation (Fig. 1b), and generated 4,300 scaffolds. The initial success rate for Site-3 was 0.16% (Supplementary Fig. 2b). We further combined Site-1 and Site-3 to create a new hotspot combination, Site-1+3 (V196, I199, V305, P478, Y480, I505) (Fig. 1b), and generated 8000 scaffolds with lengths ranging from 80 to 130 residues to generate larger binders, achieving an initial success rate of 1.18% (Supplementary Fig. 2b). In a final round, the diffuser.T was set to 120, Site-3 residues were refined to P478, Y480, L485, I505, and a total of 14,000 backbones were sampled across Site-1, Site-3 and Site-1+3, yielding success rates of 1.57 %, 0.25 % and 2.72 %, respectively (Supplementary Fig. 2c). After filtering the combined ensemble (pae_interaction < 8.5, plddt_binder > 90), 103 top-ranking mini-protein binders that target three distinct binding sites were selected for experimental validation (Fig. 1c).

Initial screening of mini-protein inhibitors for complement C9
The predicted monomeric human C9 structure aligns closely with the mouse C9 crystal structure, suggesting the feasibility of screening for a mini-protein inhibitor that can inhibit C9 polymerization across mouse and human (Fig. 2a). 103 mini-protein binders were expressed in E. coli and purified by affinity chromatography. Unlike traditional in vitro protein–protein interaction screens, we directly evaluated C9 mini-protein inhibitors using a functional hemolysis inhibition assay with mouse serum and sheep red blood cells. The mini-protein inhibitors were tested at a fixed concentration of 40 \mu M for initial screening. The results showed that at a low serum concentration of 10%, five binders (18, 47, 50, 69, and 87) exhibited relatively good inhibitory effects (Fig. 2b). When the serum concentration was raised to 15 %, only Binder-47 and 87 retained potent inhibitory activity, with Binder-47 being more effective (Fig. 2c). Binder-47 revealed obvious dose-dependent hemolysis inhibition from 1.85% to 96.31% in a concentration range of 10 nM to 10 \mu M (Fig. 2d & e), suggesting a potential specific inhibition targeting complement C9 membrane insertion. Binder-47 was designed from the hotspot Site-1+3 scaffolding, which contain a relatively larger interface. This interface was highly conserved across mammals (Fig. 2a & Supplementary Fig. 3), only three contact residues were different between human and mouse C9 (Fig. 2a & Supplementary Fig. 3). Considering the potentially higher accuracy of AlphaFold3 in modeling protein–protein complexes, we used AlphaFold3 to predict the complex structure of Binder-47 with human C9. AlphaFold3 modeling predicted that Binder-47 also well interacts with human C9 (Fig. 2a), indicating that it may serve as a cross-species inhibitor. Consistently, Binder-47 also inhibited hemolysis mediated by human serum (Fig. 2d & e). Biolayer interferometry (BLI) analysis using recombinant Fc-tagged complement C9 proteins revealed that Binder-47 interacts more strongly with mouse C9, with Kd values of 3 and 22 nM (Fig. 2f), respectively. These results are consistent with the hemolysis inhibition assay (Fig. 2e).

Affinity maturation using partial diffusion
Hemolysis inhibition assays indicated that the scaffold of Binder-47 had strong optimization potential for complement C9 mediated MAC formation inhibition. We focused on optimizing the inhibitory effect of Binder-47 on human complement C9 function, which has greater pharmaceutical value compared with mouse derived C9. The complex structure of Binder-47 and human C9 was predicted by AlphaFold3 and further used as input for partial diffusion (Fig. 2a & Supplementary Fig. 1). In contrast to previous reports that fixed diffuser.partial_T at 15 or 2536 , we sampled a broader range (1-25, step = 1), generating 25,000 backbones. Generated backbones were applied to ProteinMPNN sequence design and ranked by AlphaFold2 prediction scores. Applying stricter cut-offs (pae_interaction < 6.2, plddt_binder > 90) yielded 60 top-ranking sequences for gene synthesis. We first fixed the mini-protein inhibitors concentration at 500 nM using human serum in hemolysis inhibition assay and identified 30 candidates that outperformed the parental Binder-47 (Fig. 3a & Supplementary Data 1). Dose responsive assay narrowed the list to six binders (P9, P14, P19, P39, P45 and P57), with P9 and P57 performing best. At the concentration of 12.5 nM, P9 and P57 achieved hemolysis inhibition rates of 98.21% and 55.92%, respectively (Fig. 3b), after setting a sufficiently low pae_interaction score for screening, the hemolysis inhibition rates obtained from experimental validation showed no strong correlation with the pae_interaction similar to initial screening, indicating the importance of experimental validation (Fig. 2b and 3a). Structural comparison revealed the C\alpha r.m.s.d values of P9 and P57 relative to Binder-47 were 1.01 and 0.875 \text{\AA}, suggesting a slight adjustment of scaffold during partial diffusion (Fig. 3c). The residues involved in interface interactions also exhibited diversity in P9 and P57 (Fig. 3c). Parallel screening using P1-P60 against mouse serum (Supplementary Fig. 4a & Supplementary Data 1) confirmed P57 as the most potent cross-reactive binder, reaching an 80% inhibition rate at a final concentration of 100 nM, exceeding the parental Binder-47 (Fig. 3d). BLI results indicated an obvious enhancement of affinity with a Kd of 0.7 nM between P9 and human complement C9 (Fig. 3e) and 1.3 nM for P57 and mouse complement C9 (Fig. 3f). Enzyme-Linked Immunosorbent Assay (ELISA) showed similar results with stronger human-complement binding for P9 and P57 versus Binder-47 (Supplementary Fig. 5a). These protein-protein interaction results are consistent with their enhanced in vitro hemolysis inhibition ability (Fig. 3b). Additionally, the P1–P60 group binders were screened against guinea-pig and rabbit sera, yielding 7 and 45 active binders, respectively (Supplementary Fig. 4b & Supplementary Data 1). At the same inhibitor concentration, the optimized P1–P60 group binders yielded more candidate binders that potently inhibited rabbit complement activity compared to guinea-pigs. In the future, optimizing partial diffusion using complement C9 structures from various species could yield better performance binders for more species.

Crystal structure and the binding specificity of the C9 mini-protein inhibitors
To further validate the accuracy of our designs, recombinant mouse C9 was mixed with the P57 mini-protein inhibitor and subjected to crystallization trials. SEC and SDS-PAGE confirmed stable complex formation (Supplementary Fig. 6). However, no diffraction-quality crystals of the complex were obtained. To verify the accuracy of our designs, we started screening the crystals of the P9 and P57 binders alone. Initial binder crystal screening obtained protein crystals, but diffraction was poor. Two to three surface-exposed hydrophilic residues distant from the binding interface were mutated to alanine to enhance crystal diffraction40, 41, 42, 43 (Supplementary Table 1). Two P57 variants, P57-M4 and P57-M5, yielded high-quality crystals that diffracted to 1.4 \text{\AA} and 1.8 \text{\AA}, respectively (Supplementary Table 2). The C\alpha r.m.s.d of two crystal structures was 0.49 \text{\AA} (Supplementary Fig. 7b) and superimposed onto the design model with C\alpha deviations of 0.85 \text{\AA} (P57-M4) and 1.50 \text{\AA} (P57-M5) (Fig. 4a & Supplementary Fig. 7a), demonstrating high consistency with the design model. Hemolysis inhibition assays showed that the crystal variants retained full inhibitory activity, indicating that surface alanine mutations do not impair P57 function (Fig. 4b). To further verify the interaction specificity between binders and C9, we performed C9 reconstitution experiments using C9-depleted human serum. C9-depleted human serum cannot cause hemolysis, reintroduction of purified human C9 to C9-depleted serum restored hemolytic activity, which was subsequently blocked by P9 and P57 (Fig. 4c), demonstrating that inhibition is strictly C9 dependent. Moreover, we performed BLI and ELISA assays using the well performed P9 and P57 mini-protein inhibitor against a panel of human complement proteins. BLI results showed that P9 specifically binds C9, but not C3, C4, C5, C6, C7, C8 complex (Fig. 4d). Since some complement proteins carry a polyhistidine tag, we performed direct ELISA using C-terminal horseradish-peroxidase (HLRP) labeled P57 (P57-HRP) (Supplementary Fig. 8). Similarly, ELISA data showed that P57-HRP interacts only with C9, not with other complement components (Fig. 4e). These results demonstrate that the mini-protein inhibitor is highly specific, binding specifically to C9. Finally, several key interface residues (positions 29, 36, 63) in P9 and P57 were mutated to arginine (Fig. 4a). Hemolysis inhibition assays revealed that, at a fixed binder concentration of 50 nM, mutations at these positions greatly attenuated the inhibition efficacy of both P9 and P57 against complement-mediated hemolysis (Fig. 4f). ELISA assays also showed that the binding of these mutants to human C9 was greatly reduced (Fig. 4g). These results indicate that P9 and P57 specifically inhibit soluble C9 to prevent the formation of MAC.

Comparison of C9 mini-protein inhibitor with antibodies
Having identified two well behaved C9 binders, we next compared their inhibitory capacity with the clinically approved anti-C5 monoclonal antibody eculizumab and two commercialized C9 monoclonal antibodies X197 and E-3. X197 previously showed inhibitory activity in a non-standard hemolysis assay33. E-3 is a widely used anti-human C9 antibody. In our classical gradient hemolysis inhibition assay, P9, P57 and eculizumab exhibited IC50 values of 7.49, 11.67 and 4.79 nM, respectively (Fig. 5a), suggesting that C9 mini-protein inhibitors could achieve hemolytic inhibition similar to the C5 inhibitor. On the other hand, both C9 monoclonal antibodies were markedly weaker and failed to reach the efficacy of P9 or P57 (Fig. 5a). Against mouse serum, P57 displayed an IC50 of 93.80 nM, whereas eculizumab showed no detectable inhibition (Fig. 5b). We also established an ABO-incompatible hemolysis assay in vitro, both P9 and P57 effectively suppressed hemolysis (Fig. 5c). Given that the first C9 insertion is the kinetic bottleneck of MAC formation25, to mimic inhibition of MAC formation during acute hemolysis, we adjusted the classic hemolysis inhibition assay. Different from mixing serum with MAC inhibitors including eculizumab or binders before being added to red blood cell, human serum was added directly to red blood cells. After 2 min of reaction, the MAC inhibitors were added respectively, and the results indicated that the hemolysis inhibition rate of the C5 inhibitor was markedly lower than C9 mini-protein inhibitors (Fig. 5d). Additionally, we tested the thermal stability and yield of the C9 mini-protein inhibitors, Circular dichroism (CD) data revealed that, Binder-47, P9 and P57 almost fully recovered their secondary structure after heating to 95 °C and cooling to 20 °C (Fig. 5e). Functionally, all three binders preserved their inhibitory activity following incubation at 95°C for 5 min, whereas eculizumab lost activity under the same conditions (Fig. 5f). Mass spectrometry confirmed the intact molecular weights and retention of N-terminal methionine (Supplementary Fig. 9). Without optimization, shake-flask expression in 100 mL yielded 49.98 mg/L for P9 and 126.85 mg/L for P57 (Supplementary Fig. 10) and both binders exhibited excellent solubility.

Inhibition of intravascular hemolysis in vivo
Furthermore, we established intravascular hemolysis assays in mice using human serum23. Firstly, P9, P57 or eculizumab was pre-incubated with human serum prior to tail vein injection (Fig. 6a). The results showed that all three inhibitors could completely suppress free hemoglobin release without significant differences (Fig. 6b). Additionally, consistent with the in vitro experiments, the serum was injected without prior incubation with the inhibitors. At different time intervals, P9 or eculizumab was then administered via the tail vein (Fig. 6a). The C9 mini-protein inhibitor showed greater hemolytic inhibition than the C5 inhibitor eculizumab at all time points, and could almost completely inhibit hemolysis even after complement activation had started for 8 min, compared to eculizumab which began to fail at 6 min (Fig 6c). These in vivo data demonstrated that C9 mini-protein inhibitors match eculizumab in potency under standard chronic conditions and provide a wider therapeutic window after acute complement activation.

Methods
Ethics statement
This study was approved by the Ethics Review Board of Shandong Second Medical University. The animal experiments were conducted in strict adherence to the guidelines and regulations set forth by the Laboratory Monitoring Committee of Shandong Province, China, and were approved by the Experimental Animal Ethics Committee of Shandong Second Medical University (NO.2025SDL798). BALB/c mice were bred and raised under specific pathogen-free conditions, and were housed in a 12-hour light/dark cycle environment and fed with standard feed. The temperature in the breeding room was maintained at 20-24℃, and the relative humidity was kept between 50% and 60%. The studies involving human samples were approved by Medical Research Ethics Committee of Shandong Second Medical University (NO.2025YX196). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Computational binder design
The binder design workflow basically followed previously published protocols35, 36. The initial target was the mouse C9 crystal structure (PDB: 6CXO)52 and its sequence was re-numbered continuously. Hotspots were selected manually based on structural features. Generated backbones were relaxed with FastRelax and redesigned with ProteinMPNN, generating three sequences per scaffold. The ProteinMPNN scripts were lightly modified to incorporate multi-threading, ensuring high-throughput processing. All ProteinMPNN generated designs were predicted using the AlphaFold2 prediction protocol. For partial diffusion, an AlphaFold3 model of the Binder-47 in complex with human C9 was first generated. Partial diffusion was executed with diffuser.partial_T spanning 1–25 (step = 1), producing 1,000 scaffolds per T value and further following similar ProteinMPNN and AlphaFold2 prediction protocols. AlphaFold3 predictions were performed using the online website (https://alphafoldserver.com/). The Supplement Figure 11 showed two view angles and the relevant scores with models presented in Figure 1c, 2a, 3c, 4a and Supplementary Figure 7a.

Protein expression and purification
Mini-protein inhibitor coding sequences were codon-optimized for expression in E. coli. Genes were synthesized and inserted between the NcoI and XhoI sites of pET-28a. For expression of human and mouse C9, mouse C9 (Q21-I548) and human C9 (Q22-K559) were codon-optimized for human cells, fused with an N-terminus mouse Ig kappa secretion signal (METDTLLLWVLLLWVPGSTGD) and a C-terminal (His)6 or human IgG1 Fc tag, inserted between the XbaI and BamHI sites of the pcDNA3.4. Mini-protein inhibitor mutant genes were directly synthesized and constructed. All genes were synthesized and constructed by Universe Gene Technology or GENCEFE Biotech. For mini-protein inhibitor expression and purification, plasmids were transformed into E. coli BL21(NDE3) and plated on LB agar plates overnight. A single colony was inoculated into 100 mL LB medium and grown at 37 °C. Expression was induced with 0.3 mM IPTG when OD600 reached 0.6–0.8, followed by 16 h culture at 24 °C and 220 rpm. Cells were harvested by centrifugation (10,000 g, 5 min), resuspended in 25 mL PBS, lysed by sonication and clarified by centrifugation (15,000 g, 20 min). The supernatant was applied to 1.5 mL Ni-NTA resin (QiaAgen), washed twice with 6× bead volumes of PBS containing 20–30 mM imidazole (pH 7.4), and eluted with PBS plus 300 mM imidazole (pH 7.4). Eluted proteins were analyzed on 4–20% SDS–PAGE (GenScript) and visualized with Coomassie Brilliant Blue. For complement C9 expression, plasmids were sterile-filtered and transiently expressed by Novoprotein using their mammalian expression system. After 5 days expression, supernatants were harvested. (His)6-tagged C9 was purified using Ni Smart beads (Smart-Lifesciences); Fc-tagged C9 was captured with rProtein A/G beads (Smart-Lifesciences), following the manufacturer’s protocols. Affinity-purified C9 was further purified by Size-exclusion chromatography (SEC). For C9 mini-protein complex formation, C9 and mini-protein were mixed at a 1:5 molar ratio, incubated at room temperature for 30 min, and subjected to SEC on a Superdex 75 Increase 10/300 GL column (GE Healthcare) using an FPLC system (Union-biotech, UEV25D). Protein concentrations were determined by measuring A280 on a multimode microplate reader (Tecan Spark).

In vitro hemolysis inhibition assay
For in vitro hemolysis inhibition assay, designed binders or antibodies of different concentrations were pre-incubated with mouse serum in a 96-well plate at 4°C for 45 min, antibody-sensitized sheep erythrocytes and GVB++ buffer (10 mM barbital, 145 mM NaCl, 0.15 mM CaCl2, 0.5 mM MgCl2 and 0.1% gelatin) were then added, and the mixture was incubated at 37°C for 30 min. Rabbit anti-sheep erythrocyte antibody (Beijing Bersee Biotechnology, BM351Y) was used to sensitize sheep erythrocytes (Zhengzhou Pingrui Biotechnology, ZPRMYHXB-100/4) at a dilution of 1:400 when using mouse serum. The blank control group was treated with an equal volume of GVB++ buffer. The positive control group obtained packed red blood cells by centrifuging the red blood cell suspension, and used an equal volume of ddH2O to completely lyse the red blood cells. The mouse serum concentration used in Figure 2c was 15%, all other mouse serum concentrations were 10%, and the normal human serum (NHS) concentration was 2%. After incubation, samples were centrifuged to pellet the remaining intact erythrocytes, and supernatants were collected. Hemoglobin release from erythrocyte lysis was detected by measuring the optical density of the supernatant at 414 nm using a multimode microplate reader (Tecan Spark). The sera of different species, including BALB/c mouse, guinea pig and rabbit, were all collected from 8-week-old male animals. For serum collection, a total of 50 BALB/c mice, 1 guinea pig, and 1 rabbit were used. All this animials were purchased from Shandong Pengyue Laboratory Animal Technology. Antibodies were purchased accordingly: eculizumab (AstraZeneca), C9 antibody (E-3; Santa Cruz Biotechnology, sc-390000), X197 antibody (Hycultbiotech, HM2111).

A complement C9 reconstitution hemolysis inhibition assay shown in Figure 4c was performed using 2% C9-depleted human serum (Complement Technology, A326) supplemented with 600 ng/well purified human C9 protein (Complement Technology, A126). For ABO-incompatible hemolysis assay, human red blood cells from blood group B+ donors, 1% were exposed to 50% incompatible human serum from blood group A+ donors in GVB++ buffer. For the adjusted acute hemolysis assay, human serum was used to induce hemolysis in sheep erythrocytes at 37°C for 2 min, followed by the addition of either eculizumab or designed binders, and the reaction was continued at 37°C for an additional 10 min. Samples were handled and detected as described in the above hemolysis assay. Ten healthy male volunteers aged 20–35 years, with no history of infectious or autoimmune diseases, were recruited for blood collection. After collection, the blood was first left to stand at room temperature for 30 min, and then centrifuged at 3000 g at 4°C for 10 min to obtain serum. For the hemolysis inhibition assay, animal blood was obtained by cardiac blood sampling, and serum was extracted through a similar process. To exclude the influence of sex on complement activity, only blood from male humans or animals was collected. Percentages of hemolysis = [(ODsample – ODblank)/(ODpositive – ODblank)] × 100; Percentage of inhibition = [1 – (ODsample – ODblank)/(ODpositive – ODblank)] × 100.

ELISA
Indirect ELISA was employed to detect interaction between native or mutant binders and soluble complement C9. Microtiter plates (Corning) were coated overnight with human C9 (Complement Technology, A126) at 0.5 \mu g per well. After discarding the coating solution, plates were washed three times with PBST (PBS with 0.05% Tween-20, pH 7.4). Wells were blocked with 5 % BSA at 37 °C for 2 h. Mini-protein inhibitors were added sequentially and incubated at 37 °C for 2 h, followed by three PBST washes. A Rabbit anti-(His)6 HRP-conjugated antibody (Proteintech, HRP-84814) was applied at 1:5000 dilution and incubated at 37 °C for 1 h. After washing, TMB (Thermo Fisher) was used as chromogenic substrate, and the reaction was stopped with 2 N H2SO4. Absorbance was measured at 450 nm using a multimode microplate reader (Tecan Spark).

Direct ELISA was used to assess interaction between P57-HRP and multiple complement components including complement C3 (Complement Technology, A113), C4 (Complement Technology, A105), C5 (Complement Technology, A120), C6 (Sino Biological, 12426-H08H), C7 (Sino Biological, 13848-H08H), C8 complex (Complement Technology), C8\alpha (Abcam, ab316549) and C9 (Complement Technology, A126). An extra cysteine was introduced before the C-terminal stop codon in the P57 expression plasmid to express the P57-C protein and enable HRP labeling. After expression and purification, P57-C was labeled with the HRP Maleimide Activator Labeling Kit (TCI Chemicals). Briefly, HRP and P57-C were mixed with the molar ratio of 1:5 with the reaction volume of 500 \mu L in PBS and incubated overnight. Free HRP was removed by Ni-NTA based affinity chromatography, and unlabeled P57-C was separated by size-exclusion chromatography, yielding pure P57-HRP (Supplementary Fig. 8).

BLI
The BLI experiments were conducted using an OCTET RED96E system (ForteBio) and the results were processed with the integrated software (ForteBio data analysis 12.0.1.2). For mini-protein binder gradient binding assay, Fc-tagged human or mouse C9 proteins were diluted to a final concentration of 5 \mu g/mL and loaded onto the Protein A sensor (ForteBio), with a loading time of 600 seconds. For multiple complement components binding assays, C3, C4, C5, C6, C7, C8 and C9 were biotinylated using chemical methods. Complement protein (100 \mu l, ~0.5 mg/mL in PBS) was biotinylated using a commercial kit (Genemore) following the manufacturer's protocol. Briefly, the protein was incubated with a predetermined molar excess of the biotinylation reagent for one hour at room temperature. The reaction mixture was purified using a desalting column to remove unconjugated biotin. Biotinylated complement proteins were diluted to a final concentration of 5 \mu g/mL and loaded onto the SA sensor (ForteBio), with a loading time of 600 seconds. Mini-protein inhibitors were serially diluted to achieve a range of concentrations from 128 to 2 nM, and the assay buffer was PBST. The concentration for P9 was 32 nM in multiple complement components binding assays (Fig. 4d). Except for the Binder-47 binding to human complement C9 in Figure 2f, where the association times were 180 seconds, the association and dissociation times for other experiments were all set to 240 seconds. The flow rate was 600 rpm for loading and 1000 rpm for other steps, all experiments were conducted at 25°C. Global fitting was applied for Figure 2f, 3e & 3f and Supplementary Figure 5b using a 1:1 fitting model. BLI analysis of mini-protein Binder-47 binding to mouse complement C9 yielded Rmax of 1.1505, R2 of 0.9821, X2 of 0.0052, and Kd of 3.3 nM; binding to human complement C9 yielded Rmax of 0.3737, R2 of 0.997281, X2 of 0.000364, and Kd of 22 nM (Fig. 2f). Mini-protein P9 with human complement C9 yielded Rmax of 0.1881, R2 of 0.976, X2 of 0.0118, and Kd of 0.7 nM (Fig. 3e) and P57 with mouse complement C9 yielded Rmax of 0.2211, R2 of 0.9713, X2 of 0.011, and Kd of 1.3 nM (Fig. 3f). P57 with human complement C9 resulted in Rmax of 0.1388, R2 of 0.9724, X2 of 0.0064, and Kd of 2.1 nM (Supplementary Fig. 5b). For Figure 4d, local fitting was applied using a 1:1 fitting model with Rmax of 0.1917, R2 of 0.9661, X2 of 0.0103. To subtract the background, each independent experiment included a reference well without mini-protein inhibitor added in association step. The buffer used for sensor activation, baseline stabilization, and protein dilution was PBST.

CD
Circular dichroism (CD) characterization was conducted using a Chirascan V100 instrument (Applied Photophysics). Briefly, 120 \mu L of 0.04 mM mini-protein inhibitor in PBS was placed into a quartz cuvette. Sequential wavelength scans were performed at 20°C, followed by heating to 95°C, and then a return to 20°C to observe recovery after heating. Far-ultraviolet CD spectra were recorded within the 180 - 280 nm range. The average protein spectrum was obtained by subtracting the buffer baseline spectrum and this measurement was replicated three times for accuracy. The CD signal was converted to mean residue ellipticity ([\theta]), dividing the corrected spectra by the product of the number of amino acid residues (N), the protein concentration (C), and the cuvette path length (L, which is 1 mm).

Crystallization and structural determination
Initial crystal screening was conducted using kits purchased from Hampton Research and Rigaku, which included Crystal Screen, PEGRx, and Wizard Classic 1-4. Initial screenings, along with subsequent optimizations, were carried out using a two-position deck mosquito LCP (SPT Labtech) within 96-well crystallization plates using the sitting-drop method. The crystals of the initially screened P9 and P57 binders had poor diffraction quality. To improve the crystal quality, 2-3 surface residue mutations were introduced into mini-protein inhibitors. Sequences are shown in Supplementary Table 1. (His)6 tag and design sequences were directly fused without GGS linker. The crystallization conditions for P57-form 1 were 0.1 M Citric acid pH 3.5, 34% v/v Polyethylene glycol 200. For P57-form 2 were 0.2 M Magnesium chloride hexahydrate, 0.1 M Tris pH 7.0 and 10% w/v Polyethylene glycol 8,000. The crystals were subjected to diffraction and indexing using an in-house X-ray diffraction system (Rigaku) utilizing Cu K\alpha radiation with a wavelength of 1.54 \text{\AA}. The structure was solved using the molecular replacement method, employing the designed models as search templates. The P57-form 2 was first solved by using P57 as the search model. The P57-form 1 structure was solved by using the crystal structure of P57-form 2 as the search model. Molecular replacement and structure refinement were executed using the PHENIX suite53 and COOT54. Visualization of the structures was performed using ChimeraX55 and PyMOL (The PyMOL Molecular Graphics System, 2002). The collection and refinement statistics are comprehensively presented in Supplementary Table 2.

In vivo intravascular hemolytic studies
To establish a complement-driven intravascular hemolysis assay, 8-week-old male BALB/c mice received NHS via the tail vein. Two protocols were employed: serum and inhibitors were pre-mixed and administered at a single 200 \mu l tail vein injection, doses were calculated based on mouse body weight: NHS 1.5 \mu l/g, eculizumab 6 \mu g/g, and anti-C9 mini-protein inhibitors (P9, P57) 6 \mu g/g, in sterile PBS. Negative controls received equal volume of PBS. For the classic intravascular hemolysis assays, serum was incubated with inhibitors (or PBS) and tail vein injected immediately. Mice were euthanized 10 mins after injection with inhibitors. Whole blood was collected by cardiac puncture, and plasma was separated via centrifugation at 3,000 g, 4 °C, 10 mins. The Percentage of hemolysis was quantified by measuring the absorbance at 414 nm, free hemoglobin was detected using a kit (Thermo Fisher) following manufacturer’s instructions.

For the adjusted intravascular hemolysis assays, 100 \mu l NHS was injected via the tail vein and allowed to circulate for defined intervals (2, 4, 6, 8, 10 or 12 mins); inhibitors (100 \mu l) were then delivered at the same dose as above. Mice were euthanized 10 mins after injection with inhibitors. NHS, inhibitor and blood samples were processed as described above.

Statistics
Data in the figures were presented as mean \pm SEM. Statistical analyses were performed using one-way analysis of variance for multiple-group comparisons. An unpaired, two-sided t-test was used for comparisons between two groups. Statistically significant P values were summarized in Supplementary Data 2. The gradient hemolysis assay used a standard four-parameter dose–response inhibition function to calculate IC50 (50% inhibition) values. All analyses were carried out using GraphPad Prism 8 for Windows (GraphPad Software, www.graphpad.com).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an exciting study that reports the development of a novel protein designed to bind C9. The protein was designed\nusing the murine structure as a template, and using ML-approaches made a series of potential binders by optimizing\ninteractions across 3 specified hot-spots.\nMinor comment: Figure 1a. Please adjust the color of C7, it is hard to see the distinction with C5 in some views.\nThey next leverage AF3 to prioritize binders and test functionality in hemolysis assays (both human and murine serum) and\nbinding assays (BLI). They show that key residues of the predicted interface are conserved across human/mouse and other\nmammals.\nMinor comment: Figure 2a. Needs to be clear in the legend this is an AF3 prediction, not an actual structure.\nThe authors then use partial diffusion to mature their designs and test for improved binding and functionality.\nThey then validate the designed C9 mini-binder with CD and by solving the crystal structure of 2 designs. They validate\ninteraction interfaces via ala mutagenesis and testing the functionality in a red blood cell lysis assay.\nMy main concern is regarding specificity. The authors clearly show that the newly designed protein can bind recombinant C9\nin vitro assays, but they need to show that it doesn’t bind other complement proteins that have a very similar MACPF\ndomain, for example C6, C7 or C8. In particular, the MACPF domain of C8a is very similar to C9.\nFinally they show that in a mouse model the mini-c9 binding protein has similar if not better potency than clinically used\nterminal pathway inhibitors.\nThe structural biology aspects of the manuscript are sound and the statistics for the refined model are good.\nOther minor corrections:\n“Given that C9 insertion is the kinetic bottleneck of MACformation”,\nBe careful, this paper showed that the 1st C9 addition is the kinetic bottleneck rather than polymerization of more C9s into 1\nmac, it didn’t give rates for C7, or C8 addition.\n“thereafter about 20 soluble C9 molecules rapidly oligomerize to complete the pore”.\nIt should be 18 copies of C9 (doi: 10.1038/ncomms10587)\n“E.coli” should be in italics throughout", "reviewer_2": "This paper describes high affinity mini-protein inhibitor targeting complement C9. Although AI-based technology for\ngenerating high-affinity mini-protein binders is not novel, it has not yet been applied for designing inhibitors of complement.\nSince the identified molecule targets a binding site in C9 that has no reported natural ligands, the work is significant for the\ncomplement field.\nMajor points:\n1. The Methodology description is very thorough (especially the parts on computational design, structure analyses). I see\nthis as a positive point, but also raises the questions whether the paper is more suited for a Methodology oriented journal\n(Nature Methods)\n2. The specificity of the mini-proteins for complement C9 is not accurately assessed. In Fig. 4C, the authors claim to\ndemonstrate specificity since the molecule only blocks lysis in the presence of C9. The interpretation is incorrect. Indeed\nthere are only functional MAC pores when C9 is present, but the inhibitor can still act on other complement components.\nEspecially binding to proteins homologous to C9 (C6, C7, C8) should be directly assessed by testing binding to purified\ncomplement proteins (for example ELISA). Other non-homologues (C3, C4, C5) should be included as control. Also in BLI,\nspecificity controls should be included!!\nMinor points:\n•A supplemental figure /schematic of binder design/selection process would be helpful.\n•Add the serum percentage to the relevant figures. This is important to interpret the results and to understand which\npathways may be contributing to response.\n[Introduction]\no C5 cleavage is the initiating event for MAC formation. Add to text.\no Reference 15 states that there are 18 copies of C9 (as well as other literature). Why do you state “about 20?”\noMention CD59, which binds C9 in the extended state. Makes a stronger case for needing a binder that prevents the\nconversion from closed to extended.\n[Results]\noIn the screening of the initial set of binders, they mention effect of the classical pathway but then use 10%/15% serum.\nAt this concentration AP should also be active.\noFig 1. Better description of the criteria used to choose the potential binder sites.\noDuring the binder generation, percentages are noted. Add a sentence explain what values are good.\noJumping between AF2/AF3. Provide an explanation for switching between the two versions.\noFig 1C not mentioned in text.\noFig 2A not mentioned in before B-D text.\noFig 2E Specify human/mouse in the 2E.\noSuppl 3 – nothing was mentioned about the potent effect of the inhibitors in rabbit serum.\n•Figure 4d – N29R does cause 50% inhibition whereas no binding is detected at all (fig. 4e). What would be the explanation\nfor this?\n•Fig 5E – adjust the x-axis to better read the scale\n•In the last results section, the final sentence is more appropriate for the discussion. “For emergency treatment of acute\nhemolysis, extending the therapeutic window by just a few minutes is crucial for saving patients’ lives and minimizing tissue\ndamage.\n•5D. why this approach of first allowing lysis and then add inhibitors. Repeat in the ‘classical way’\n[Discussion]\nFor discussion, consider expanding to include the following\noComputation resources required to pursue such a study. It’s only briefly mentioned and much of the emerging software is\nalso computationally expensive.\noImpact of depleting C9 on native state, chronic conditions, infection, etc.\noKon rates are discussed but what about Koff ?\noLongterm treatment of binder?", "reviewer_3": "Li et. al. present their design of mini-protein binders for complement C9, binders capable of inhibiting MAC formation. Using\nrecently established protein design methods to find C9 binders (RFDiffusion, ProtMPNN, AlphaFold2) they discover a\nnumber of binders to monomeric C9, some with impressively high affinity, and solve the structure of a bound complex, which\nis very similar to the predicted design. The in vitro tests reveal that these new inhibitors have potent activity, and in many\nrespects outform an approved antibody biologic. This is an experimentally solid, well-presented study that is an excellent\naddition to the growing number of studies that show the power diffusion methods in de novo protein design. Their argument\nabout the value of early experimental analysis, and some limits of computational metrics, will be valuable to the field. And\nthe new biologics found have clear biomedical potential.\nI would recommend accept, I only have minor changes, mostly the clarify some statements:\n“and the inhibitors are kinetically disadvantaged.” What are “the” inhibitors here? Existing monoclonal antibodies? Please\nclarify.\n\" Despite this therapeutic rationale, C9-directed inhibitors remain rarely reported.” If they have been reported at all,\nreferences are needed.\n“Moreover, the relatively high serum concentration of C9 requires that C9 inhibitors have high affinity” Is this right, I would\nexpect the opposite. If the concentrationof target is high, then any binder with a Kd below this concentration will bind. High\ndoses might be required, but not high affinity.\n“we can now generate binding proteins for any structural epitope” this could be too strong a statement. Indeed, in the same\nparagraphy it is claimed that “C9 remains challenging”", "reviewer_sheep_red_blood_cell_hemolysis_inhibition_assay": "he organism of the serum should be stated in the main text. As\nwritten, it could be mistaken for sheep serum.\n“In the future, optimizing partial diffusion using complement C9 structures from different species will result in better\nperformance binders.” What does “better” mean in this context? Able to bind more species, or bind human better?\n“were mutated to alanine to enhance diffraction”. Not obvious to me why this would work, and no reference given."}, "author_response": "Point-by-point response to reviewers’ comments on “De novo design of high\naffinity mini-protein inhibitor targeting complement C9 to block the final\nassembly step of membrane attack complexes”\nReviewer #1:\nThis is an exciting study that reports the development of a novel protein designed to bind C9. The\nprotein was designed using the murine structure as a template, and using ML-approaches made a\nseries of potential binders by optimizing interactions across 3 specified hot-spots:\n(1) Minor comment: Figure 1a. Please adjust the color of C7, it is hard to see the distinction\nwith C5 in some views.\nThanks, we change the color of C7 to yellow to better distinguish it from C5 in Figure 1a (p.\n25).\n(2) Minor comment: Figure 2a. Needs to be clear in the legend this is an AF3 prediction, not\nan actual structure.\nWe have provided a clear explanation in the figure legend (Fig. 2a) (p. 26, line 757-760).\n(3) The authors then use partial diffusion to mature their designs and test for improved binding\nand functionality. They then validate the designed C9 mini-binder with CD and by solving the\ncrystal structure of 2 designs. They validate interaction interfaces via ala mutagenesis and\ntesting the functionality in a red blood cell lysis assay. My main concern is regarding specificity.\nThe authors clearly show that the newly designed protein can bind recombinant C9 in vitro\nassays, but they need to show that it doesn’t bind other complement proteins that have a very\nsimilar MACPF domain, for example C6, C7 or C8. In particular, the MACPF domain of C8a\nis very similar to C9.\nThanks, this is indeed a critical issue. To determine whether the mini-protein inhibitor\ninteracts with any other complement components, we performed ELISA and BLI assays using the\nwell performed P9 and P57 mini-protein inhibitor against a panel of human complement proteins\nincluding C3, C4, C5, C6, C7, C8 complex, C8α, and C9. The results demonstrate that the mini-\nprotein inhibitor is highly specific, binding specifically to C9 but not other complement\ncomponents (Response Fig. 1) (p. 7-8, line 225-233). We added the Response Figure 1 Figure\n4d & 4e. The specific experimental details are described in the materials and methods section (p.\n15-16, line 435-460).\nResponse Figure 1. (a) The BLI experiment detects the interaction between P9 and complement components,\nincluding C3, C4, C5, C6, C7, C8, and C9. (b) Direct ELISA was employed to quantify the interaction between\ncomplement C3, C4, C5, C6, C7, C8 complex, C8α, C9 and P57-HRP.\n(4) Other minor corrections:\n“Given that C9 insertion is the kinetic bottleneck of MAC formation”. Be careful, this paper\nshowed that the 1st C9 addition is the kinetic bottleneck rather than polymerization of more C9s\ninto 1 mac, it didn’t give rates for C7, or C8 addition.\n“thereafter about 20 soluble C9 molecules rapidly oligomerize to complete the pore”.It should\nbe 18 copies of C9 (doi: 10.1038/ncomms10587)\n“E.coli” should be in italics throughout.\nThanks, we notice that these statements are indeed incorrect, and we have made\ncorresponding modifications (p. 8, line 253-254), and 18 copies of C9 has been changed correctly\n(p. 2, line 68-70).\nE.coli has been changed correctly.\nReviewer #2:\nThis paper describes high affinity mini-protein inhibitor targeting complement C9. Although AI-\nbased technology for generating high-affinity mini-protein binders is not novel, it has not yet been\napplied for designing inhibitors of complement. Since the identified molecule targets a binding site\nin C9 that has no reported natural ligands, the work is significant for the complement field:\n(1) The Methodology description is very thorough (especially the parts on computational design,\nstructure analyses). I see this as a positive point, but also raises the questions whether the paper\nis more suited for a Methodology oriented journal (Nature Methods).\nWe sincerely appreciate your positive appraisal. Since our study primarily integrates\npreviously published methods rather than introducing novel methodological advances, we\nconsidered Nature Communications to be the most suitable journal. We truly appreciate your\nencouragement.\n(2) The specificity of the mini-proteins for complement C9 is not accurately assessed. In Fig.\n4C, the authors claim to demonstrate specificity since the molecule only blocks lysis in the\npresence of C9. The interpretation is incorrect. Indeed there are only functional MAC pores\nwhen C9 is present, but the inhibitor can still act on other complement components. Especially\nbinding to proteins homologous to C9 (C6, C7, C8) should be directly assessed by testing binding\nto purified complement proteins (for example ELISA). Other non-homologues (C3, C4, C5) should be included as control. Also in BLI,\nspecificity controls should be included!!\nThanks, this is indeed a critical issue, also raised by Reviewer #1. To determine whether the\nmini-protein inhibitor interacts with any other complement components, we performed ELISA and\nBLI assays using the well performed P9 and P57 mini-protein inhibitor against a panel of human\ncomplement proteins including C3, C4, C5, C6, C7, C8 complex, C8α, and C9. The results\ndemonstrate that the mini-protein inhibitor is highly specific, binding specifically to C9 but not\nother complement components (Response Fig. 1) (p. 7-8, line 225-233). We added the Response\nFigure 1 to Figure 4d & 4e. The specific experimental details are described in the materials and\nmethods section (p. 15-16, line 435-460).\n(3) A supplemental figure /schematic of binder design/selection process would be helpful.\nThanks, we have added the Supplementary Fig. 1 to illustrate our binder design and selection\nprocess (Response Fig. 2).\nResponse Figure 2. Schematic representation of the complement C9 binder design and selection process.\nThe initial design and affinity maturation of the C9 mini-protein binder followed a workflow of hotspot\nselection, scaffold generation, sequence design, structure prediction, scoring, and screening. Detailed\ninformation is provided in the methods.\n(4) Add the serum percentage to the relevant figures. This is important to interpret the results\nand to understand which pathways may be contributing to response.\nThanks, the detailed serum concentration used was added on the y-axis of each hemolysis-\ninhibition assays (Fig. 2 & 3 & 4 & 5).\n(5) [Introduction]\n• C5 cleavage is the initiating event for MAC formation. Add to text.\nThanks, we have added this sentence to the introduction and provide relevant descriptions (p.\n1-2, line 53-57).\n• Reference 15 states that there are 18 copies of C9 (as well as other literature). Why do you\nstate “about 20?”\nThanks, we noticed this misstatement and made the corresponding correction (p. 2, line 68-\n70).\n• Mention CD59, which binds C9 in the extended state. Makes a stronger case for needing a\nbinder that prevents the conversion from closed to extended.\nThanks, we have added relevant descriptions about the working mechanism and the\ninspiration of CD59 in the introduction section (p. 2, line 70-80).\n(6) [Results]\n• In the screening of the initial set of binders, they mention effect of the classical pathway but\nthen use 10%/15% serum. At this concentration AP should also be active.\nThanks, we have corrected the relevant statements (p. 5, line 145-147).\n• Fig 1. Better description of the criteria used to choose the potential binder sites.\nThanks, we have added the relevant statements (p. 4, line 112 -114).\nTypically, binder design favour surface-exposed hydrophobic residues as hotspots. We\ncarefully checked the C9 interaction region for spatially clustered, interface-critical hydrophobic\nresidues and grouped them into several sets. In addition, our empirical experience indicated that\nexposed β-sheet region such as Site-1 yielded higher success rate. And we made a Supplement\nFigure 1 to introduce our screening and optimization process.\n• During the binder generation, percentages are noted. Add a sentence explain what values are\ngood.\nThanks, we have added relevant descriptions in the main text (p. 4, line 118-125).\nIn the scoring scheme, ‘pae_interaction’ is the predicted aligned error that AlphaFold2 assigns\nto the binder–target interface. It is the key metric for assessing how confident the model is in the\ninterface prediction: the lower the value, the more reliable the prediction and the better the spatial\nfit between binder and target. Designs with pae_interaction < 10 are generally considered\nacceptable. Complementarily, ‘plddt_binder’ measures the per-residue confidence of the binder\nalone. A high value indicates that the sequence is likely to fold into a stable, well-defined\nmonomeric structure; a threshold of > 90 is commonly required1, 2.\n• Jumping between AF2/AF3. Provide an explanation for switching between the two versions.\nThanks, we have added several sentences in the main text to explain why AlphaFold3 was\nemployed (p. 5, line 158-160).\nAfter large-scale MPNN sequence design, all candidates were scored with the early-released\nAlphaFold2-complex prediction pipeline that has been widely adopted in binder design. Owing to\nthe lack of a crystal structure for soluble monomeric human complement C9 and the potentially\nhigher accuracy of AlphaFold3 in modeling protein–protein complexes, we employed AlphaFold3\nto predict the single complex between human C9 and Binder-47 and subsequently used this model\nas the initial template for partial-diffusion calculations.\n• Fig 1C not mentioned in text.\nThanks, we have added the cition for Figure 1c (p. 4, line 136-138).\n• Fig 2A not mentioned in before B-D text.\nThanks, we have added the cition for Figure 2a before B-D (p. 5, line 142-144).\n• Fig 2E Specify human/mouse in the 2E.\nThanks, we have included species labels in Figure 2e (p. 26).\n• Suppl 3 – nothing was mentioned about the potent effect of the inhibitors in rabbit serum.\nThanks, we have added a description of the potent inhibition effect of rabbit complement\nactivity in the main text (p. 6-7, line 198-204).\n• Figure 4d – N29R does cause 50% inhibition whereas no binding is detected at all (fig. 4e).\nWhat would be the explanation for this?\nThank you for your question. Indeed, the results of the hemolysis assay and the ELISA\nexperiment do not fully align. Overall, mutations at these sites greatly weaken the binding ability\nof P9 and P57. Since the hemolysis-inhibition assay uses fresh serum, it may more accurately\nreflect the protein’s inhibitory effect. In contrast, our ELISA data primarily provide qualitative\nevidence of reduced binding affinity.\nHere are possible explanations for the N29R. First, the ELISA experiment used commercial\nC9 protein extracted from human serum through multi-step purification, the final preserved and\nused C9 protein may have undergone slight denaturation, leading to undetected binding between\nC9 and mutant binder. Second, the mutant binder may also interact nonspecifically with other\nserum complement proteins, resulting in partial inhibition effects in hemolysis.\n• Fig 5E – adjust the x-axis to better read the scale.\nThanks, we have adjusted the x-axis of Figure 5e to better read the scale (p. 30).\n• In the last results section, the final sentence is more appropriate for the discussion. “For\nemergency treatment of acute hemolysis, extending the therapeutic window by just a few\nminutes is crucial for saving patients’ lives and minimizing tissue damage.\nThanks, we have adjusted this sentence to the discussion section (p. 11, line 324-326).\n• 5D. why this approach of first allowing lysis and then add inhibitors. Repeat in the ‘classical\nway’.\nThanks, the in vitro experiment shown in Figure 5d was designed to compare the inhibitory\nefficacy of the miniprotein binder with the C5 inhibitor eculizumab after complement activation\nhad already been initiated for some time. The results of the classical inhibition assay, in which the\ninhibitors were pre-incubated with serum before being added to the erythrocyte-containing\nreaction system, are presented in Figure 5a, 5b and 5f (p. 30).\n(7) [Discussion]\nFor discussion, consider expanding to include the following\n•Computation resources required to pursue such a study. It’s only briefly mentioned and much\nof the emerging software is also computationally expensive.\nThanks, we have added discussion content on computational costs (p. 9-10, line 284-291).\n• Impact of depleting C9 on native state, chronic conditions, infection, etc.\nThanks, we have added relevant discussion on the possible impact of blocking C9 membrane\ninsertion (p. 10-11, line 311-318).\n• Kon rates are discussed but what about Koff ?\nThanks, we have added relevant description (p. 10, line 294-295).\n• Longterm treatment of binder?\nThanks, we have added discussion on longterm treatment of binder (p. 11, line 333-339).\nReviewer #3:\nLi et. al. present their design of mini-protein binders for complement C9, binders capable of\ninhibiting MAC formation. Using recently established protein design methods to find C9 binders\n(RFDiffusion, ProtMPNN, AlphaFold2) they discover a number of binders to monomeric C9, some\nwith impressively high affinity, and solve the structure of a bound complex, which is very similar\nto the predicted design. The in vitro tests reveal that these new inhibitors have potent activity, and\nin many respects outform an approved antibody biologic. This is an experimentally solid, well-\npresented study that is an excellent addition to the growing number of studies that show the power\ndiffusion methods in de novo protein design. Their argument about the value of early experimental\nanalysis, and some limits of computational metrics, will be valuable to the field. And the new\nbiologics found have clear biomedical potential.\nI would recommend accept, I only have minor changes, mostly the clarify some statements:\n• “and the inhibitors are kinetically disadvantaged.” What are “the” inhibitors here? Existing\nmonoclonal antibodies? Please clarify.\nThanks, we have clarified this statements (p. 2, line 67).\n• “Despite this therapeutic rationale, C9-directed inhibitors remain rarely reported.” If they\nhave been reported at all, references are needed.\nThanks, we have added reported inhibitors in the main text (p. 2-3, line 80-84).\nAn early report showed that a commercially available anti-C9 monoclonal antibody X197\nconferred inhibitory activity in a non-standard hemolysis-inhibition assay3. Separately, the O1-\nantigen of Klebsiella LPS was found to block C9 polymerization4. However, neither study\nevaluated the therapeutic potential of these inhibitors for hemolytic disorders.\n• “Moreover, the relatively high serum concentration of C9 requires that C9 inhibitors have\nhigh affinity” Is this right, I would expect the opposite. If the concentration of target is high,\nthen any binder with a Kd below this concentration will bind. High doses might be required, but\nnot high affinity.\nThanks, we have changed this statements (p. 3, line 86-88).\n• “we can now generate binding proteins for any structural epitope” this could be too strong\na statement. Indeed, in the same paragraphy it is claimed that “C9 remains challenging”\nThanks, this sentence is indeed inappropriate, we have revised the relevant wording (p. 3,\nline 90-92).\n• “The soluble, monomeric crystal structure of mouse C9” Why not the human, or the\npredicted human structure (that is used later)?\nThanks, we initially used murine complement C9 as the target, because the crystal structure\nof the soluble murine C9 monomer had been experimentally determined, whereas that of the\nhuman homolog had not. The experimentally resolved structure is more credible and, in principle,\nmore accurate than a predicted human soluble C9 monomer model. We added descriptions in the\nmain text (p. 3, line 106-108).\nIn addition, based on the membrane-inserted human C9 structure, it is inferred that the overall\narchitecture is structurally conserved between human and murine C9. Since the initial\ncomputational screen aimed to obtain the best mini-protein binder scaffolds capable of binding\nand inhibition, we performed the initial calculations with the crystal structure of murine C9 and\nswitched to human C9 for subsequent affinity maturation.\nFor the “sheep red blood cell hemolysis inhibition assay” the organism of the serum should be\nstated in the main text. As written, it could be mistaken for sheep serum.\nThanks, this sentence is indeed misleading, we have revised the relevant wording (p. 5, line\n145-147).\n• “In the future, optimizing partial diffusion using complement C9 structures from different\nspecies will result in better performance binders.” What does “better” mean in this context?\nAble to bind more species, or bind human better?\nThanks, this sentence is indeed misleading, we have revised the relevant wording (p. 7, line\n202-204).\n• “were mutated to alanine to enhance diffraction”. Not obvious to me why this would work,\nand no reference given.\nThanks, we have added relevant references in the main text (p. 7, line 212-214); alanine\nmutation of surface residues to promote crystal growth is a commonly used crystallographic\nstrategy5, 6, 7, 8. Alanine mutations enhance the probability of protein crystallization by reducing\nsurface entropy, optimizing crystal contact interfaces, and minimizing hydration interference8.\nReferences\n1. Watson JL, et al. De novo design of protein structure and function with RFdiffusion.\nNature 620, 1089-1100 (2023).\n2. Glögl M, et al. Target-conditioned diffusion generates potent TNFR superfamily\nantagonists and agonists. Science (New York, NY) 386, 1154-1161 (2024).\n3. Hatanaka M, Seya T, Yoden A, Fukamoto K, Semba T, Inai S. Analysis of C5b-8 binding\nsites in the C9 molecule using monoclonal antibodies: participation of two separate\nepitopes of C9 in C5b-8 binding. Molecular immunology 29, 911-916 (1992).\n4. Masson FM, et al. Klebsiella LPS O1-antigen prevents complement-mediated killing\nby inhibiting C9 polymerization. Scientific reports 14, 20701 (2024).\n5. Lu L, et al. De novo design of drug-binding proteins with predictable binding energy\nand specificity. Science (New York, NY) 384, 106-112 (2024).\n6. Yu B, et al. De novo design of light-responsive protein-protein interactions enables\nreversible formation of protein assemblies. Nature chemistry 17, 1910-1919 (2025).\n7. Yin W, et al. Crystal structure of the human 5-HT(1B) serotonin receptor bound to an\ninverse agonist. Cell discovery 4, 12 (2018).\n8. Derewenda ZS. The use of recombinant methods and molecular engineering in\nprotein crystallization. Methods (San Diego, Calif) 34, 354-363 (2004)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "all comments and issues have been addressed by the authors.", "reviewer_2": "The authors have addressed all concerns in an appropriate fashion", "reviewer_3": "All requested revisions are well addressed. I recommend publish, its an excellent study of its type."}, "author_response": ""}]

Decision letter