Complement C9 mini-binding protein or its mutants and their applications

Abstract

This application relates to complement C9 mini-binding protein or its mutants and their applications. The complement C9 mini-binding protein of this application comprises an amino acid sequence selected from the following: SEQ ID No. 6-SEQ ID No. 65. Experimental results have demonstrated that the complement C9 mini-binding protein of this application exhibits excellent hemolysis inhibition effects, significantly superior to eculizumab in terms of protein thermostability and inhibition of acute hemolysis. Therefore, the series of complement C9 mini-binding proteins developed in this application have broad application prospects in the development of drugs for the treatment of diseases related to complement C9-mediated intravascular hemolysis.

Description

Technical Field

[0001] This invention belongs to the field of proteins, specifically relating to a complement C9 mini-binding protein or its mutants and their applications. Background Technology

[0002] The complement system is one of the most important components of the immune system. Recent studies have explored the potential value of C6 and C7 monoclonal antibodies in the treatment of complement-related diseases, demonstrating that inhibition of proteins downstream of C5 can have equivalent or superior biological efficacy.

[0003] With the rapid development of AI-based diffusion-denoised target binder generation technology, it is now possible to directly generate binding proteins targeting any structural epitope with relatively high precision. This technological advancement has, to some extent, compensated for the inherent limitations of immune screening in identifying antibodies targeting key conserved sites. Although numerous high-affinity mini-binding proteins for ligands such as Il2ra, Il7ra, EGFR, and TNFR have been reported, the design of mini-binding proteins targeting complement components, especially structurally unique proteins like C9, remains challenging and has not yet been reported. Summary of the Invention

[0004] To address the shortcomings of the prior art, the present invention provides a complement C9 mini-binding protein or a mutant thereof, comprising an amino acid sequence selected from the following: SEQ ID No. 6-SEQ ID No. 65.

[0005] In a specific embodiment, the complement C9 mini-binding protein comprises an amino acid sequence selected from SEQ ID No. 14, SEQ ID No. 19, SEQ ID No. 24, SEQ ID No. 44, SEQ ID No. 50, and SEQ ID No. 62.

[0006] In a specific embodiment, the amino acid sequence of the complement C9 mini-binding protein is selected from SEQ ID No. 66-SEQ ID No. 125.

[0007] In a specific embodiment, the amino acid sequence of the complement C9 mini-binding protein is selected from one of SEQ ID No. 74, SEQ ID No. 79, SEQ ID No. 84, SEQ ID No. 104, SEQ ID No. 110, and SEQ ID No. 122. Preferably, the amino acid sequence of the complement C9 mini-binding protein is SEQ ID No. 74 or SEQ ID No. 122.

[0008] In a specific embodiment, the amino acid sequence of the mutant complement C9 mini-binding protein is selected from one of SEQ ID No. 127, SEQ ID No. 128, and SEQ ID No. 126.

[0009] In another aspect, the present invention provides a fusion protein comprising the above-mentioned complement C9 mini-binding protein or a mutant thereof.

[0010] On the other hand, the present invention provides a polynucleotide that encodes the complement C9 mini-binding protein or a mutant thereof as described above.

[0011] On the other hand, the present invention provides an expression vector comprising the polynucleotides described above.

[0012] In a specific embodiment, the expression vector is selected from pET28a.

[0013] On the other hand, the present invention provides a host cell comprising the expression vector described above.

[0014] In a specific embodiment, the host cell is selected from BL21(DE3).

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising the complement C9 mini-binding protein or a mutant thereof as described above, the fusion protein as described above, the expression vector as described above, or the host cell as described above, and optionally, a pharmaceutically acceptable vector.

[0016] In another aspect, the present invention provides the use of the complement C9 mini-binding protein or mutant thereof as described above, the fusion protein as described above, the expression vector as described above, the host cell as described above, or the pharmaceutical composition as described above in the preparation of C9 complement inhibitors.

[0017] In another aspect, the present invention provides the use of the complement C9 mini-binding protein or mutant thereof as described above, the fusion protein as described above, the expression vector as described above, the host cell as described above, or the pharmaceutical composition as described above in the preparation of a C9 complement detection agent.

[0018] In another aspect, the present invention provides the use of the complement C9 mini-binding protein or mutant thereof as described above, the fusion protein as described above, the expression vector as described above, the host cell as described above, or the pharmaceutical composition as described above in the preparation of a medicament for treating or preventing diseases related to intravascular hemolysis associated with complement C9-mediated membrane attack complex.

[0019] In specific embodiments, the diseases associated with complement C9-mediated intravascular hemolysis include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), cold agglutinin disease (CAD), paroxysmal cold hemoglobinuria (PCH), clinically erroneous transfusion, myasthenia gravis (MG) associated with abnormal formation of membrane attack complex attacking its own tissues, and neuromyelitis optica spectrum disorder (NMOSD).

[0020] In another aspect, the present invention provides a kit for detecting soluble C9 protein, which contains at least the complement C9 mini-binding protein or its mutant or the fusion protein as described above.

[0021] Beneficial effects

[0022] (1) This application utilizes diffusion-based denoising backbone generation technology, protein sequence design technology MPNN based on deep learning neural network, and structure prediction technology Alphafold2 and Alphafold3 based on deep learning to obtain a series of mini binding proteins through cluster computing and computational screening of a certain scale, in order to antagonize the complement C9-mediated membrane attack complex formation function in mammals, especially humans and mice.

[0023] (2) The affinity of the mini-binding proteins obtained in the preliminary screening was optimized by partial diffusion noise reduction to obtain complement C9 mini-binding proteins with high affinity.

[0024] (3) The screened mini-binding proteins can be highly expressed in E. coli and can be purified to high purity in one step.

[0025] (4) In vitro and mouse models, complement C9 mini-binding proteins P9 and P57 exhibited hemolytic inhibition effects close to or exceeding those of eculizumab. Furthermore, in in vitro and in vivo hemolytic inhibition experiments with complement activation, P9 and P57 showed significantly better hemolytic inhibition effects than the C5 inhibitor eculizumab. Therefore, the complement C9 mini-binding proteins developed in this application have broad application prospects in the development of drugs for the treatment of diseases related to complement C9-mediated intravascular hemolysis.

[0026] (5) The complement C9 mini-binding proteins P9 and P57 can maintain their inhibitory activity against complement C9 after being heated to room temperature to a certain extent, which is significantly better than eculizumab and has high thermal stability.

[0027] (6) Complement C9 mini-binding proteins P9 and P57 can be detected by ELISA. Attached Figure Description

[0028] Figure 1This diagram illustrates the design, synthesis, and screening of the complement C9 mini-binding protein in this application, and its application in a hemolytic disease model. As shown in the figure, the mouse C9 structure was consulted based on the PDB database. The mini-binding protein was designed based on the contact surface of the MAC formed by C9 polymerization. After preliminary screening of the protein through in vitro hemolysis inhibition experiments, partial diffusion technology was used to enhance the affinity of the mini-binding protein for human complement C9. Functional and specific analyses were performed using hemolysis inhibition experiments, protein structure crystallography, ELISA, and other experiments. Its inhibitory activity was further compared with that of known monoclonal antibodies. Finally, the results were validated in a mouse hemolytic disease model.

[0029] Figure 2 The diagram illustrates the synthesis of the membrane attack complex and the process by which the complement C9 mini-binding protein inhibits its assembly. The formation of the membrane attack complex depends on the sequential binding and assembly of complement C5, C6, C7, C8, and C9. The complement C9 mini-binding protein can prevent the assembly of complement C9 with the complement C5678 complex. Furthermore, since C9 is the final step in the formation of the membrane attack complex, inhibiting C9 insertion can act as a brake, preventing the membrane attack complex from fully forming even after the complement system has been initiated.

[0030] Figure 3 This diagram shows the designed binding site structure of the complement C9 mini-binding protein for mouse complement C9. a) Designed binding site of the complement C9 mini-binding protein for mouse complement C9; b) Model structure of the complement C9 mini-binding protein for mouse complement C9. The left side (purple) represents the first designed site, Site 1; the middle (pink) represents the second introduced site, Site 3; and the right side (yellow) represents the third fusion site, Site 1+3.

[0031] Figure 4 This diagram illustrates the structural differences in the binding site epitopes of mouse complement C9, human complement C9, and mini-binding protein. Gray: mouse complement C9; red: human complement C9; orange: initially designed mini-binding protein I47.

[0032] Figure 5The results of the in vitro hemolysis inhibition experiments of the initially designed mini-binding proteins are shown. Specifically: a. A graph showing the inhibition of in vitro hemolysis mediated by mouse complement C9 and the pae_interaction score of the initially designed mini-binding proteins. A lower pae_interaction score indicates a better binding effect between the mini-protein and the target. Mini-binding proteins with inhibition rates >90% are marked (orange), and red represents the best-performing mini-binding protein I47 in this screening; b. Graphs showing the results of in vitro hemolysis inhibition experiments of mini-binding proteins I18, I47, I50, I69, and I87 at different concentrations mediated by mouse complement C9; c. Graphs showing the inhibition of in vitro hemolysis mediated by mouse complement C9 (top) and human complement C9 (bottom) by mini-binding protein I47 at different concentration gradients; d. Statistical graphs showing the results of in vitro hemolysis inhibition by mini-binding protein I47 at different concentration gradients mediated by mouse complement C9 (top) and human complement C9 (bottom).

[0033] Figure 6 The results of BLI for I47 mini-binding protein with mouse complement C9 (top) and human complement C9 (bottom) are shown.

[0034] Figure 7 The results of the in vitro hemolysis inhibition assay for the optimized mini-binding protein are shown, along with the structural differences between the I47 mini-binding protein and P9 and P57 models, and the BLI results for P9, P57, and complement C9. Specifically, a. Graph showing the in vitro hemolysis inhibition mediated by human complement C9 and the calculated pae-interaction score of the optimized mini-binding protein. a. Orange represents mini-binding proteins with an inhibition rate >80%; b. Results of in vitro hemolysis inhibition experiments mediated by human complement C9 for mini-binding proteins I47, P9, P14, P19, P39, P45, and P57 at different concentrations; c. Differences in model structures of mini-binding protein I47 (gray), P9 (orange), and P57 (red); d. Results of in vitro hemolysis inhibition experiments mediated by mouse complement C9 for mini-binding proteins I47, P9, P31, P45, P51, and P57 at different concentrations; e. BLI results of P9 and human complement C9; f. BLI results of P57 and mouse complement C9.

[0035] Figure 8The diagram illustrates the binding specificity of P57 crystalloid P57-M4, P57-M5, P57, and P9 to human complement C9. Specifically: a. Structural differences between the P57-M4 crystalloid (red) and computer simulation (blue), with the mutation sites involved in d and e highlighted on the right; b. In vitro hemolysis inhibition results of P57, P57-M4, and P57-M5 mediated by human complement C9; c. In vitro hemolysis inhibition results of P9 and P57 mediated by human complement C9 after complement C9 recombinant. NHS represents normal human serum; C9-dpl represents human complement C9 depleted serum. d. Results of in vitro hemolysis inhibition mediated by human complement C9 in P9, P57 and its mutants P9-N29R, P9-L36R, P9-Q63R, P57-H29R, P57-D36R, and P57-Q63R; e. ELISA results of P9, P57 and its mutants P9-N29R, P9-L36R, P9-Q63R, P57-H29R, P57-D36R, and P57-Q63R.

[0036] Figure 9 The comparison results of P9 and P57 with eculizumab, E-3 antibody, and X197 antibody are shown. Specifically: a. IC50 calculation of P9, P57, eculizumab, E-3 antibody, and X197 antibody in human complement C9-mediated in vitro hemolysis; b. IC50 calculation of P57 and eculizumab in murine complement C9-mediated in vitro hemolysis; c. Experimental results of P9 and P57 inhibiting the occurrence of ABO hemolysis (mistransfusion) in vitro; d. Inhibition results of P9 and P57 in human complement C9-mediated in vitro hemolysis after constructing acute hemolysis; e. Circular dichroism chromatograms validating the thermal stability of I47, P9, and P57; f. Inhibition results of P9 and P57 in human complement C9-mediated in vitro hemolysis after heating denaturation and recovery to room temperature.

[0037] Figure 10 This diagram illustrates the construction of acute intravascular hemolysis in a mouse model and the comparison of P9, P57, and eculizumab. a. Schematic diagram of the construction of acute intravascular hemolysis in mice. Two methods were used to validate the function of the mini-binding protein: one involved mixing normal human serum (NHS) with the mini-binding protein and then infusing it via the mouse tail vein to observe the inhibitor's functional effect; the other involved injecting NHS for a period of time (2, 4, 6, 8, 10, 12 min) before infusing the inhibitor to observe its functional effect; b. Measurement of free hemoglobin (top) and plasma OD414 absorbance (bottom) after simultaneous injection of P9, P57, and eculizumab with normal human serum (NHS); c. Measurement of free hemoglobin (top) and plasma OD414 absorbance (bottom) after different time points following the construction of acute intravascular hemolysis in vivo.

[0038] Figure 11 The success rates of Alphafold2 complex structure predictions for three batches of mini-binding protein designs are shown. Red dots represent successful designs. a. Initial design success rate; b. Second design success rate; c. Third design success rate.

[0039] Figure 12 The results of the screening experiments for the optimized mini-binding protein in Example 3, mediated by complement in different species, are shown. a) Screening results of the mini-binding protein in mouse serum complement-mediated in vitro hemolysis inhibition experiments, where orange represents mini-binding proteins with an inhibition rate >90%, and the dashed line indicates the inhibition rate of the initially designed mini-binding protein I47 under these screening conditions; b) Screening results of the mini-binding protein in guinea pig serum complement (left) and rabbit serum complement (right) mediated in vitro hemolysis inhibition experiments.

[0040] Figure 13 The results of gel chromatography and SDS-PAGE are shown. a. Gel chromatography results for P57; b. SDS-PAGE results for P57.

[0041] Figure 14 This diagram illustrates the binding specificity of P57-M5 protein crystals to human complement C9 and the structural characteristics of P57-M4 and P57-M5. Specifically: a. Structural comparison between the P57-M5 crystal structure (yellow) and the computer model of the C9-P57 complex (blue); b. Structural deviation comparison between the P57-M4 (purple) and P57-M5 (green) crystal structures.

[0042] Figure 15 The mass spectrometry analysis results for P9, P14, P39, P45, and P57 are shown.

[0043] Figure 16 The following table shows the protein yields of P9, P14, P39, P45, and P57, as well as the ELISA results of the I47 mini-binding protein, P9, and P57. Specifically: a. SDS-PAGE results of P9, P14, P39, P45, and P57; b. Table of protein yields of P9, P14, P39, P45, and P57; c. ELISA results of the interaction between the I47 mini-binding protein, P9, P57, and human C9.

[0044] Figure 17 The image shows a purified P57-C protein labeled with HRP enzyme, along with SDS-PAGE and ELISA data. a. SDS-PAGE indicates successful P57-labeling of HRP and purification to a high purity via gel chromatography. b. ELISA data shows that P57-HRP can directly detect soluble human C9 protein. Detailed Implementation

[0045] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0046] the term:

[0047] In this document, the term "mutant" refers to a sequence that shares more than 96% sequence identity with the complement C9 mini-binding protein of this application and possesses the same or similar complement C9 binding specificity. Specifically, the two alanine mutants of the P57 mini-binding protein, P57-M4 (SEQ ID No. 127) and P57-M5 (SEQ ID No. 128), are derived by selecting inactive amino acid sites in the amino acid sequence of the P57 mini-binding protein and performing alanine mutations to enhance the crystallinity of the mini-binding hemoglobin; P57-C (SEQ ID No. 126) is derived by adding a cysteine ​​residue to the carboxyl terminus of the P57 mini-binding protein, and the P57-C expression plasmid is obtained through gene synthesis. These mutants can also recognize the complement C9 protein.

[0048] In the following section, several polymerization-binding regions of the C9 protein that are most critical for the formation of the membrane attack complex were selected as targets for the design of mini-binding proteins. Based on the screening of mini-binding proteins with excellent performance, further affinity optimization was carried out to obtain mini-binding proteins that outperformed conventional antibody drugs in in vitro hemolysis and routine and acute hemolysis experiments in mice.

[0049] Example 1: Complement C9 mini-binding protein was generated using RFdiffusion through computational protein design.

[0050] Currently, the structure of the mouse-derived free soluble C9 monomer protein has been resolved, and it was selected as the design target in this embodiment. The C9 protein has a rather unique structure, exhibiting a "disc-like" shape, with dimensions of approximately 77 × 50 × 13 Å. Figure 3 The process by which the C9 protein further polymerizes and stacks from soluble monomeric proteins to form a membrane-attacking complex depends on a broad 77 × 50 Å protein contact surface. In this embodiment, this binding contact surface was selected to inhibit C9 intercalation. Figure 2 , Figure 3 First, two locations on the entire contact surface were selected as hotspots for RFdiffusion to generate the mini binding protein backbone: Site 1 (V176, I179, V285) and Site 2 (L197, A213, F215). Figure 3(a) In the initial calculation, 1500 backbones were generated for each site, and the generated mini-binding proteins were 50-80 amino acids in length. In the subsequent MPNN sequence design, three sequence designs were generated for each backbone, and finally scored using Alphafold2 initial guess. Analysis of the results showed that the feasibility of successful designs was evaluated by setting relatively lenient screening conditions: pae_interaction < 15, plddt_binder > 88. The results indicated that the success rate for Site 1 was 1.20%, while the success rate for Site 2 was almost 0%. Figure 11 (a). In the second calculation, a new site, Site3 (F211, M456, P458, Y460, I485), was introduced. Figure 3 (a) 4300 skeletons were generated, with an initial success rate of 0.16% for site 3. Figure 11 (b) Further fusing site 1 and site 3 to generate a new hotpot combination Site1+3 (V176, I179, V285, P458, Y460, I485), generating a backbone of 8000 amino acids with a length of 80-130 amino acids, the preliminary success rate was calculated to be 1.18%. Figure 11 (b). In the final calculation, diffuser.T was set to 120, the hotspots at Site3 were adjusted to P458, Y460, L465, and I485, and a total of 14,000 skeletons were generated for Site1, Site3, and Site1+3. The design success rates obtained were 1.57%, 0.25%, and 2.72%, respectively. Figure 11 (c) The above results were organized, and the cut-off was set to pae_interaction < 8.5 and plddt_binder > 90. From the screened mini-binding proteins, 100 were selected for subsequent experimental screening and verification. The computational design method in this invention mainly uses (PMID: 39636970, 37433327), in which the computational programs of FastRelax and ProteinMPNN were slightly modified to add concurrency mechanisms and retry loops to meet the needs of generating a large number of sequences.

[0051] Example 2: Initial screening for complement C9 mini-binding protein

[0052] In this embodiment, all gene synthesis was performed by Jiutian Gene Technology (Tianjin) Co., Ltd. All animals involved were purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd. The 100 designed mini-binding protein genes were optimized according to the *E. coli* codons and synthesized between NcoI and XhoI in the pET28a vector. Except for I69 and I87, which have an HHHHHHggs sequence at the amino terminus, the other proteins were constructed without an additional 6×His tag at the amino terminus. Except for I69 and I87, the remaining sequences retained the 6×His tag on the vector at the carboxyl terminus, and were expressed and purified in *E. coli*.

[0053] Specific procedures: The synthesized mini-binding protein-binding particle gene was transformed into *E. coli* BL21(DE3) competent cells (Shanghai Weidi Biotechnology). After a series of ice bath, 42℃ heat shock, and ice bath operations, the cells were added to LB liquid medium and thawed at 37℃ for 1 h. The cells were then plated on kanamycin-resistant plates and cultured for 12-16 h. Single colonies were picked and inoculated into LB liquid medium containing kanamycin. The cells were shaken at 37℃ until the OD600 reached 0.6-0.8, then IPTG inducer (Sangon Biotech) was added at a ratio of 1:3000 and induced overnight at 24℃. The cells were collected by centrifugation at 1000g for 5 min at 4℃, resuspended in PBS, and sonicated on ice (Ningbo Xinzhi Biotechnology). The supernatant was collected by centrifugation at 12000g for 20 min. The supernatant was passed through a nickel column and impurities were removed using 20 mM, 30 mM, and 40 mM imidazole buffer. Finally, the target protein was collected using 300 mM imidazole buffer. Protein purity was determined by SDS-PAGE electrophoresis, and the target band was observed by Coomassie brilliant blue staining. After ultrafiltration to remove imidazole, the target protein concentration was detected at 280 nm using a multi-functional microplate reader (TECAN).

[0054] Unlike traditional protein-protein interaction screening, this embodiment directly uses the classic in vitro hemolysis inhibition experiment for verification.

[0055] Materials: Rabbit anti-sheep erythrocyte antibody (Beijing Bosi Technology Co., Ltd.), sheep erythrocytes (Zhengzhou Pingrui Biotechnology Co., Ltd.), and serum from Balb / c mice.

[0056] The specific procedure is as follows: First, pre-incubate 10% Balb / c mouse serum with a final concentration of 40 μM mini-binding protein at 4°C for 45 min. Then, add sensitized sheep red blood cells at a final concentration of 1% and react at 37°C for 30 min to induce hemolysis. The total reaction volume is 200 μL. GVB is used. ++The reaction volume was brought up with buffer (10 mM barbital, 145 mM NaCl, 0.15 mM CaCl2, 0.5 mM MgCl2, and 0.1% gelatin). After the reaction, the mixture was centrifuged at 2000 rpm for 10 min, and 150 μL of the supernatant was measured at OD414 nm using a multi-mode microplate reader. Sheep red blood cells were first processed using GVB. ++ After washing three times with buffer, the cells were sensitized with rabbit anti-sheep erythrocyte antibody at a ratio of 1:400 before the experiment was conducted (subsequent in vitro hemolysis inhibition experiments were performed according to the above procedure).

[0057] Experimental results showed that at a serum concentration of 10%, the five mini-binding proteins numbered I18, I47, I50, I69, and I87 exhibited good inhibitory effects. Further increasing the serum concentration to 15%, I47 and I87 showed stronger inhibitory effects. Figure 5 (See Table 1 below and section b), among which the I47 mini-binding protein showed the best inhibitory effect, achieving a hemolysis inhibition rate of ~70% at 5 μM ( Figure 5 (b) In this embodiment, the I47 mini-binding protein was further tested, showing significant dose-dependent inhibition ( Figure 5 The I47 mini-binding protein originates from the backbone formation of Site 1+3. In fact, this site exhibits strong overall structural conservation in mammalian C9. Compared to human C9, the binding interface residues of the I47 mini-binding protein are almost identical to those of the mouse C9 protein. Figure 4 Alphafold3 predictions also indicate that the I47 mini-binding protein can bind well to the human C9 protein. Figure 4 This suggests that the I47 mini-binding protein and its backbone may act as a pan-mammal C9 protein inhibitor. Hemolysis inhibition experiments also showed that the I47 mini-binding protein could inhibit human serum-mediated hemolysis against sheep erythrocytes. Figure 5 (C in the middle, D in the middle of 5).

[0058] Table 1

[0059]

[0060] Furthermore, human and mouse complement C9 protein-encoding genes (UniProt ID: P02748; P06683) were synthesized, and the encoding nucleic acid sequences were codon-optimized according to human standards. Overexpression plasmids were synthesized in human HEK293F cells (Thermo Fisher Scientific), specifically EcoRI and BamHI into the pCDNA3.4 plasmid. Two constructions were developed: mouse C9 containing either (His)6 or the Fc segment of human IgG1 at the C-terminus, and human C9 tagged with the Fc segment of human IgG1 at the C-terminus. Transfection was performed using PEI (Polyscience) at a plasmid mass ratio of 3:1, with a transfection cell density of 2*103. 6 Five days after expression, the supernatant was collected and then subjected to affinity purification. The (His)6-tagged protein was purified using Ni Smart gel media, and the Fc-tagged protein was purified using rProtein A / G gel media. The gel media were purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd., and the affinity purification process followed the supplier's standard procedure. Further purification was performed by gel chromatography after affinity purification. The gel chromatography instrument used in this invention was a UEV25D (Yonglian Biotechnology), and the chromatography column was a Superdex 75 Increase 10 / 300GL column (GE Healthcare). Subsequently, the purified Fc-tagged C9 (C9-Fc) was used for BLI experiments. The experimental instrument was an OCTET RED96E (ForteBio), and the sensor was a Protein A sensor (ForteBio). Mouse and human C9-Fc proteins were diluted to 10 μg / ml with PBST (PBS with a final concentration of 0.05% Tween-20, pH 7.4) as the sensor-binding protein. Protein I47 was serially diluted 2-fold to a concentration range of 128-2 nM.

[0061] Experimental results show that the KD of the I47 mini-binding compound with mouse C9 is ~3 nM ( Figure 6 (above), with an affinity of approximately ~22 nM for human C9 ( Figure 6 (Below), the I47 mini-binding protein has a stronger affinity for mouse C9, consistent with the inhibitory effect on mouse serum in the hemolysis experiment.

[0062] Example 3: Partial diffusion technology enhances the affinity between complement C9 mini-binding protein and complement C9.

[0063] Hemolytic inhibition experiments of the I47 mini-binding protein against human C9 demonstrate that it is a mini-binding protein backbone with strong potential for C9 function inhibition. This application focuses more on the inhibition of human C9 protein by the mini-binding protein; therefore, the complex structure prediction model obtained by combining the I47 mini-binding protein with human C9 is applied to the calculation of partial diffusion.

[0064] First, the diffuser.partial_T parameter was set to a wider range, with a minimum of 1 and a maximum of 26, and a step size of 1. A total of 25,000 skeletons were generated, and MPNN was used to generate 3 sequences for each skeleton. Then, Alphafold2's initial guess was used for scoring, and models with pae_interaction < 6.2 and plddt_binder > 90 were selected. The 60 mini-binding protein model sequences with the highest scores were subjected to gene synthesis and experimental screening in the same manner as above.

[0065] The experiment used human serum (from healthy volunteers) and sheep red blood cells to conduct a hemolysis inhibition test.

[0066] First, the concentration of 60 mini-binding proteins was fixed at 500 nM, with a serum proportion of 2%. Real-time in vitro hemolysis inhibition experiments showed that 30 proteins exhibited inhibitory effects exceeding those of the original I47 protein. Figure 7 (a) and Table 2 below).

[0067] Table 2. Sequence of complement C9 mini-binding protein (binder) and its inhibition rate (%) in different sera.

[0068]

[0069] In the sequences P1-P60 (SEQ ID No. 6-65) above, the starting amino acid M is added at the N-terminus and ggsHHHHHHH is added at the C-terminus, forming sequences SEQ ID No. 66-125 respectively.

[0070] Furthermore, the inhibitory effects of proteins that performed well in the initial screening were tested. P9, P14, P39, P45, and P57 proteins were identified as exhibiting superior inhibitory effects. Among them, P9 and P57 proteins showed the best inhibition against human serum, achieving inhibition rates of 98.21% and 55.92%, respectively, at 12.5 nM. Figure 7 (b). Structurally, P9 and P57 show small overall structural differences from the original I47 mini-binding protein: Cα biases of 1.01 and 0.875, respectively.

[0071] Further screening of 60 proteins for their inhibitory effects on mouse serum complement was conducted using a similar method. The mini-binding protein concentration was fixed at 2.5 μM, using 10% mouse serum concentration. It was found that 21 mini-binding proteins showed better inhibitory effects than the initially designed I47, with 5 of them exhibiting inhibition rates exceeding 95%. Figure 12(See Table 2, a). Further gradient experiments showed that the P57 protein had the best inhibitory effect, achieving an inhibition rate of 80% at 100 nM, significantly surpassing the original I47 mini-binding protein (…). Figure 7 (d). P57 protein has hemolytic inhibitory effects in both humans and mice. Figure 7 (d).

[0072] Further BLI assays were performed to determine the dissociation constant. The results showed that the P9 mini-binding protein and human C9 have a Kd of 700 pM ( Figure 7 In the middle e), the dissociation constant of P57 protein with mouse C9 reaches 1.3 nM ( Figure 7 (f). ELISA experiments also showed that P9 and P57 had stronger complement-binding activity compared to the original I47 mini-binding protein. Figure 16 (c)

[0073] The specific procedure for the indirect ELISA experiment is as follows: Human C9 protein (Complement Technology) 0.5 μg per well is coated overnight. After removing the coating solution, the plate is washed three times with PBST. Blocked with 5% BSA at 37°C for 2 h, then the mini-binding proteins are added sequentially and incubated at 37°C for 2 h. 6*His-HRP-labeled secondary antibody (Proteintech) is used at a 1:5000 ratio, and incubated at 37°C for 1 h. Three washes with PBST are required during this period. Subsequently, TMB (Thermo Fisher) is used for color development, and the color development is stopped with 2N H2SO4. Absorbance is measured at OD450 nm using a multi-mode microplate reader (subsequent ELISA experiments are performed according to the above procedure). This is consistent with the excellent hemolytic inhibition effect of these two mini-binding proteins. Figure 7 (b)

[0074] In addition, this embodiment also screened 60 candidate proteins for their inhibitory effects on complement function in guinea pig and rabbit serum. The final concentration of the mini-binding protein was fixed at 40 μM for screening, with guinea pig serum accounting for 2% and rabbit serum accounting for 3%. The results showed that 7 proteins had good inhibitory effects on guinea pig serum, with inhibition rates >90%. Figure 12 (See Table 2, left side of b), 45 proteins showed good inhibitory effects on rabbit complement, with an inhibition rate >90% ( Figure 12 (See Table 2, right side of b). These findings further demonstrate that binding to this site of action of soluble complement C9 can inhibit complement C9 function across species.

[0075] Example 4: Crystal structure and binding specificity analysis of complement C9 mini-binding protein

[0076] To further confirm the accuracy of the design, in this embodiment, the recombinant expressed mouse C9 protein and P57 mini-binding protein were subjected to protein crystallization and structural analysis. The results of gel chromatography and SDS-PAGE (using 4-20% gel purchased from GenScript) showed that the C9 protein and P57 formed a protein complex. Figure 13 (a & b), but crystal screening experiments of the complex have not yet yielded diffractable protein crystals.

[0077] In this embodiment, crystallization screening of the mini-binding protein was further performed. Two to three hydrophilic residues at the non-binding interface were mutated to alanine to enhance crystallization and protein crystal quality. The synthesis of the mutant plasmid was completed by Jiutian Gene. Crystallization screening kits were purchased from Hampton Research and Rigaku, including Crystal Screen, PEGRx, and Wizard Classic 1-4 crystallization screening kits. Crystallization screening was performed using 96-well drop plates (Xunjing Biotechnology) and a two-position deck mosquito LCP (SPT Labtech) machine. Two alanine mutants of the P57 mini-binding protein, P57-M4 (SEQ ID No. 127) and P57-M5 (SEQ ID No. 128, specific mutation sites are as follows). Figure 14 (As shown in Figure b) High-quality protein crystals were obtained, and the structure was resolved to a resolution of 1.46 and 1.8 Å. Figure 8 a& Figure 14 In section a), the structures of the two analytical crystal structures are consistent, with Cα deviating by 0.49 Å ( Figure 14 (b) Diffraction and data processing of the protein crystals were performed using an indoor X-ray diffraction system (Rigaku) ​​with Cu Kα as the diffraction source. Structural analysis was performed using a designed mini-binding protein model as the initial replacement model, and the replacement and analysis were performed using Phenix software. The Cα deviations of P57-M4 and P57-M5 from the designed mini-binding protein model were 0.85 Å and 1.50 Å, respectively. Figure 8 a, Figure 14 (a) indicates that the design model is highly consistent with the actual structure.

[0078] Hemolysis inhibition experiments showed that the two surface mutations had no effect on the function of the mini-binding protein, and both proteins effectively inhibited hemolysis. Figure 8 (b)

[0079] To further verify the specific binding and functional inhibition of C9 by the mini-binding protein, a complement C9 protein reintroduction experiment was first performed. Hemolysis inhibition was then conducted using human serum (purchased from Complement Tech) with complement C9 protein removed. The results showed that the reintroduction of human complement C9 protein (purchased from Complement Tech) induced hemolysis of sheep erythrocytes, and that P9 and P57 proteins inhibited this hemolysis. Figure 8 (c) suggests that P9 and P57 proteins specifically inhibit human complement C9 protein.

[0080] Furthermore, key residues at the binding interface of P9 (SEQ ID No. 14) and P57 (SEQ ID No. 62) were mutated: amino acids at positions 29, 36, and 63 were mutated to arginine, respectively. Figure 8 (a) The mutations P9-N29R (SEQ ID No. 129), P9-L36R (SEQ ID No. 130), P9-Q63R (SEQ ID No. 131), P57-H29R (SEQ ID No. 132), P57-D36R (SEQ ID No. 133), and P57-Q63R (SEQ ID No. 134) were obtained. Hemolysis inhibition experiments showed that mutations at these sites significantly reduced the hemolytic inhibition effect of P9 and P57. Figure 8 (d) ELISA experiments also showed that the interaction between these interface site mutant proteins and C9 was significantly reduced ( Figure 8 (e), which further confirms the specificity of the binding interface.

[0081] Example 5: Comparison of complement C9 mini-binding protein and monoclonal antibody

[0082] Building upon the discovery of two excellent complement C9 mini-binding proteins—P9 and P57—this study aims to compare their inhibitory functions with those of the existing C5 monoclonal antibody Eculizumab (purchased from AstraZeneca), and two C9 monoclonal antibodies: E-3 (purchased from Santa Cruz, sc-390000) and X197 (purchased from Hycultbiotech, HM2111). X197 has been reported in earlier literature to inhibit hemolysis to some extent in unconventional hemolysis inhibition experiments (PMID: 1378934).

[0083] Gradient hemolysis inhibition assay: The procedure is the same as described in Example 2, wherein... Figure 9 In the study, 2% human serum concentration was used. Except for the E-3 antibody and X197 antibody, which were at concentrations of 0.2 nM - 100 nM, the concentrations of other inhibitors were 0.2 nM - 200 nM, using a 2-fold serial dilution. Figure 9 In the b-drug assay, 10% mouse serum concentration was used, and the inhibitor concentration was 2 nM - 1 μM, also using a 2-fold serial dilution.

[0084] Experimental results showed that the IC50 values ​​of P9 and P57 were 7.49 nM and 11.67 nM, respectively, with P9 exhibiting a very similar inhibitory effect to Eculizumab. Figure 9 (a). The two monoclonal antibodies against C9 are very weak, and their inhibitory activity against C9 is not comparable to that of P9 and P57. Figure 9 (a). The IC50 of P57 in inhibiting complement activity in mouse serum was 93.80 nM, while eculizumab could not inhibit complement activity in mouse serum (a). Figure 9 (b)

[0085] Since C9 insertion is the rate-limiting step in the formation of the membrane attack complex (PMID: 31061395), in addition to the conventional hemolysis inhibition experiment, this embodiment constructed an in vitro ABO blood group incompatibility hemolysis experiment. Specifically, P9 and P57 at concentrations of 1 μM and 200 nM, respectively, were pre-incubated with 50% type A serum for 45 min, followed by the addition of 1% type B erythrocytes for 30 min to induce hemolysis. The experimental results showed that both P9 and P57 could inhibit hemolysis. Figure 9 (c)

[0086] In addition, this embodiment also conducted an inhibition experiment simulating acute hemolysis. Specifically, after the complement system had been activated, inhibitors were added to inhibit hemolysis. Serum was added to lyse sheep erythrocytes for 2 minutes, followed by the addition of P9, P57, and eculizumab at final concentrations of 200 nM and 100 nM, respectively, and the reaction was carried out at 37°C for 10 minutes. The experimental results showed that, after complement had been activated for some time, inhibition of C9 was significantly better than inhibition of C5. Figure 9 (d).

[0087] In addition, the secondary structure thermal stability of mini-binding proteins such as I47, P9, and P57 was tested in this embodiment. Circular dichroism spectroscopy results showed that after heating at 95°C and then restoring to 20°C, P9 could completely recover its secondary structure, while mini-binding protein I47 and P57 could largely recover their original structures. Figure 9(e). Circular dichroism spectroscopy was performed using a Chirascan V100 instrument (Applied Photophysics), specifically by adding 130 μL of 0.04 mM mini-binding protein to a quartz tube for analysis. Functional experiments also showed that the protein retained its biological activity to a large extent after heating at 95°C for 5 min, while eculizumab completely lost its activity. Figure 9 (f)

[0088] Mass spectrometry results showed that the N-terminal methionine residues of several mini-binding proteins (P9, P14, P39, P45, P57) were retained, and the complete molecular weights of the obtained proteins were consistent with the predictions. Figure 15 Mass spectrometry experiments were performed using the mass spectrometry and metabolism platform at Westlake University. Furthermore, without optimization, using a 100 ml expression system, the yields of P9 and P57 reached 29.98 mg / L and 126.85 mg / L, respectively. Figure 16 (b), and can be concentrated to at least 200 mg / ml without precipitation, indicating extremely strong protein stability. Figure 16 (a) Protein concentration was determined using a TECAN multi-functional microplate reader to measure the absorbance at 280 nm.

[0089] Example 6: Validation of the role of complement C9 mini-binding protein in a mouse hemolytic model

[0090] In this embodiment, an intravascular hemolytic mouse model was constructed using 8-week-old BALB / c male mice and human serum. First, P9, P57, and eculizumab were mixed with human serum and injected into the mice via the tail vein to induce hemolysis. Figure 10 In step a), the dosage of human serum and inhibitors was calculated based on mouse body weight, i.e., the human serum dosage was 1.5 μl / g, and the mini-binding protein and eculizumab were administered at a dosage of 6 μg / g. After 10 min, mice were sacrificed, and blood was collected from their hearts. The free hemoglobin content in plasma was detected using a free hemoglobin assay kit (Thermo Fisher Scientific), and the plasma absorbance was detected at OD414 nm using a multi-functional microplate reader. The experimental results showed that all three proteins could effectively inhibit the production of free hemoglobin. Figure 10 (b)

[0091] In this embodiment, an acute hemolytic model was subsequently constructed, in which serum was injected into mice via the tail vein for a period of time, followed by the injection of P9 or Eculizumab into mice via the tail vein. Figure 10(c) After a 10-minute reaction, mice were sacrificed and their hearts were blooded. The results showed that injection of the inhibitory protein at 2 and 4 minutes post-injection effectively suppressed hemolysis. At 6 and 8 minutes post-injection, the P9 protein showed significantly stronger inhibitory effects on hemolysis than the C5 inhibitor Eculizumab. Injection at 8 minutes post-injection rendered all three inhibitors ineffective. Figure 10 (c)

[0092] These animal experiments demonstrate that the complement C9 mini-binding protein of this application can inhibit complement function to the same level as Eculizumab, and can inhibit hemolysis for a longer period after acute hemolysis occurs. Given the rapid onset of acute hemolysis, gaining even a few minutes of resuscitation time is crucial for saving the patient's life and minimizing damage to the patient's body.

[0093] Example 7: Horseradish peroxidase-labeled P57 mini-binding protein for ELISA detection of C9 protein

[0094] In Examples 3 and 4 above, soluble C9 protein was detected using an indirect ELISA assay. In this example, a cysteine ​​residue was added to the carboxyl terminus of the P57 mini-binding protein, and the P57-C expression plasmid was synthesized. After purification, this mini-binding protein was labeled using the TGI Horseradish Peroxidase Maleimide Activator Labeling Kit (TGI, H1621). The specific labeling method is as follows: the molar ratio of HRP to mini-binding protein was 1:5, and the total reaction volume was supplemented with PBS at pH 7.0 to 500 μL for overnight labeling. After labeling, affinity purification was performed using a nickel column. First, the total reaction volume was diluted to 10 mL with PBS, and then His-tag and Ni were purified using a nickel column. 2+ The protein was bound to the target mini-binding protein, and then washed with 10 mL of PBS to remove impurities. The target mini-binding protein was then eluted with a mixture of 5 mL of 300 mM imidazole (pH 7.4) and PBS. The eluent was concentrated to 500 μL using a 3000 Da ultrafiltration tube at room temperature. Finally, gel chromatography was used to further purify the P57-HRP protein, which can be directly used to detect soluble C9 protein.

[0095] Experimental materials: HRP-labeled mini protein inhibitor, C9 protein purified from serum (specifically PMID: 26841934), ELISA plate, washing buffer PBST, chromogenic solution TMB (Thermo Fisher), and stop solution H2SO4.

[0096] Specific procedures: C9 protein purified from serum was coated overnight at a concentration of 0.5 μg / well. After discarding the coating solution, the plate was washed three times with PBST. Blocking was performed with 5% BSA at 37°C for 2 h, followed by the addition of 20 μg / ml P57-HRP protein. Incubation was carried out at 37°C for 1 h, followed by color development using TMB (Thermo Fisher) and termination of the color development with 2N H2SO4. Absorbance was measured at OD450 nm using a multi-mode microplate reader.

[0097] The results are as follows Figure 17 As shown, this protein can be used directly to detect soluble C9 protein without the use of enzyme-labeled secondary antibody. The sequence number of P57-C is SEQ ID No. 126. Although P57 is used here, those skilled in the art will understand that it can also be replaced by P9, P14, P39 or P45.

Claims

1. A complement C9 mini-binding protein, characterized in that, Its amino acid sequences are selected from: SEQ ID No. 14, 62, 74 and 122.

2. A polynucleotide, characterized in that, Encodes the complement C9 mini-binding protein as described in claim 1.

3. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 2.

4. A pharmaceutical composition, characterized in that, It contains the complement C9 mini-binding protein as described in claim 1 or the expression vector as described in claim 3, and optionally, a pharmaceutically acceptable vector.

5. Use of the complement C9 mini-binding protein of claim 1, the expression vector of claim 3, or the pharmaceutical composition of claim 4 in the preparation of a C9 complement detection agent.

6. Use of the complement C9 mini-binding protein of claim 1, the expression vector of claim 3, or the pharmaceutical composition of claim 4 in the preparation of a medicament for the treatment or prevention of intravascular hemolysis associated with complement C9-mediated membrane attack complex.

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