Allosteric defect type ADAMTS13 binding molecule, ADAMTS13 protein mutant and application of ADAMTS13 protein mutant

Abstract

The invention discloses a novel pathogenic mechanism of immune thrombocytopenic purpura (iTTP), and provides a product for precise diagnosis and targeted therapy of the iTTP based on the mechanism. Specifically, the invention relates to an allosteric defect type ADAMTS13 binding molecule based on VH1-69 spectrum antibody 4-20 and key residue E59 mutation of ADADTS13 Spacer domain, and a stable mutation ADAMTS13 protein mutant introduced into G491 and allosteric pathway residues, and through long-range conformation conduction of a 4-20 antibody and a Spacer-Cys interface and E59 side chain volume regulation and control, allosteric inhibition of an autoantibody on ADAMTS13 is realized, and the enzyme activity recovery rate is improved. The problem that in the prior art, antibody-mediated ADAMTS13 function inhibition is difficult to reverse is solved, and the method is suitable for precise diagnosis and targeted therapy of iTTP patients.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and immunopathology, specifically relating to a molecular mechanism of antibody-mediated long-range allosteric inhibition of the ADAMTS13 protease, therapeutic antibody molecules based on this mechanism, and their applications in medicine. Background Technology

[0002] Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy. Its immune-mediated form (iTTP) is mainly caused by autoantibodies against ADAMTS13, an enzyme that cleaves von Willebrand factor (VWF) polymers. The loss of ADAMTS13 activity leads to the accumulation of large VWF polymers, which in turn triggers microvascular thrombosis.

[0003] It is known that most autoantibodies in iTTP patients target the spacer region and CUB domain of ADAMTS13. Antibodies derived from the VH1-69 gene lineage are the primary inhibitory antibodies. However, the precise molecular mechanism by which these antibodies inhibit the proteolytic activity of the distal M domain by binding to the spacer domain remains unclear. Existing theories include steric hindrance models and possible allosteric inhibition models, but high-resolution structural evidence is lacking. This gap in understanding the mechanism severely hinders the development of targeted therapeutic strategies that can specifically block the inhibitory effects of autoantibodies without affecting enzyme activity.

[0004] Therefore, there is an urgent need in this field to elucidate the precise structural basis and molecular mechanism by which VH1-69 lineage antibodies inhibit ADAMTS13 activity, so as to provide targets and directions for the development of new diagnostic and therapeutic strategies. Summary of the Invention

[0005] This invention aims to reveal a novel molecular mechanism by which iTTP-causing antibodies inhibit ADAMTS13 through long-range allosteric inhibition, and to provide a series of innovative solutions based on this mechanism: including allosteric decoy antibody molecules that can competitively block the binding of pathogenic antibodies without inhibiting enzyme activity, ADAMTS13 protein mutants that can resist antibody inhibition and maintain VWF cleavage activity, as well as drug screening methods and supporting diagnostic reagents targeting allosteric pathways, thereby achieving accurate diagnosis, efficient treatment and next-generation drug development for iTTP.

[0006] In a first aspect, this invention reveals the molecular basis for autoantibody 4-20's inhibition of ADAMTS13 activity through an allosteric mechanism.

[0007] The high-resolution (3.4 Å) crystal structure of the Fv-clasp complex of the ADAMTS13 spacer domain and the autoantibody 4-20 was resolved. The structure revealed that 4-20 binds primarily to a conformational epitope on the spacer domain, consisting mainly of hydrophobic residues, via the complementarity-determining regions (CDRs) of its heavy chain (VH). Importantly, molecular dynamics simulations showed that 4-20 binding induces a spatial conflict at the interface between the spacer domain and the adjacent cysteine-rich domain (Cys domain), triggering a conformational perturbation at the Spacer-Cys interface. This perturbation then acts as an allosteric signal, transmitting this conformational perturbation long-range to the distal metalloproteinase domain (M domain) via a specific allosteric pathway consisting of key residues such as I411-G392-M367-T427-G324-V326-R328-R330, leading to conformational rearrangement and increased dynamic instability in the M domain. This allosteric effect impairs ADAMTS13's recognition and access to cleavage sites within the VWF A2 domain, thereby inhibiting its proteolytic function.

[0008] Secondly, this invention identifies the E59 site in the antibody 4-20 light chain as a key allosteric effect residue.

[0009] Although E59 does not directly participate in antigen binding, site-directed mutagenesis (E59A and E59G) revealed that replacing a long-chain glutamate at this site with a short-chain or essentially side-chain-free amino acid significantly reduced in size, while completely preserving the antibody's affinity for ADAMTS13, significantly weakened its ability to inhibit ADAMTS13 enzyme activity. This demonstrates that the size of the E59 side chain and the physical spatial conflict it causes at the Spacer-Cys interface are the core structural factors triggering long-range allosteric signals and achieving functional inhibition. Those skilled in the art can infer that conserved substitutions of other small-volume amino acids with similar short-chain characteristics (such as Gly, Ala, Ser, Cys, Thr, etc.) at this site could also yield allosterically deficient ADAMTS13-binding molecules.

[0010] Thirdly, based on the above findings, the present invention provides the following technical solution:

[0011] An allosteric defective ADAMTS13 binding molecule, which is an antibody or its antigen-binding fragment, specifically binds to a conformational epitope formed by the enrichment of hydrophobic residues on the spacer domain of ADAMTS13. At the position corresponding to the 59th glutamic acid (E59) in the variable region of the 4-20 light chain of the VH1-69 lineage antibody, the molecule contains a mutation that alters the glutamic acid (E) to a short side chain with a significantly smaller side chain volume than the glutamic acid side chain, or to an amino acid with virtually no side chain. This mutation reduces the physical steric hindrance conflict with the neighborhood of the glycine (G491) at the 491st position of the ADAMTS13 spacer domain, allowing the binding molecule to maintain high affinity for ADAMTS13 while essentially not inhibiting or only significantly weakening the proteolytic activity of ADAMTS13 on von Willebrand factor (VWF).

[0012] Further, the short side-chain or essentially side-chain-free amino acid is selected from one or more of glycine (Gly, G), alanine (Ala, A), serine (Ser, S), cysteine ​​(Cys, C), and threonine (Thr, T); the mutation is alanine (E59A) and / or glycine (E59G); the complementarity-determining region (CDR) amino acid sequence of the binding molecule has at least 70-90% sequence identity with the corresponding CDR amino acid sequence of monoclonal antibody 4-20, and retains the same or cross-reactive ADAMTS13 spacer region conformational epitope recognition pattern as 4-20; and the molecule is an IgG4 subtype antibody, a Fab fragment, a single-chain variable fragment (scFv), or an engineered protein scaffold containing an E59 short side-chain or side-chain-free mutant binding domain.

[0013] The present invention also provides an ADAMTS13 protein mutant, which, relative to wild-type ADAMTS13, contains at least one amino acid mutation at the interface between the spacer domain and the cysteine-rich domain and / or along the allosteric signaling pathway between the spacer domain and the metalloproteinase domain (M domain). The amino acid mutation can stabilize the Spacer-Cys interface conformation and / or weaken long-range allosteric signaling triggered by the binding of pathogenic antibodies, thereby retaining proteolytic activity against VWF in the presence of the pathogenic antibodies.

[0014] Further, the amino acid mutation is located at at least one of the glycine residues (G491) at position 491 of the Spacer domain and its neighboring residues; and / or at least one residue in an allosteric signaling pathway composed of or adjacent to residues I411, G392, M367, T427, G324, V326, R328, and R330.

[0015] in:

[0016] (1) Mutations in the Spacer-Cys interface region include replacing the original residue with a short side chain or essentially no side chain amino acid, wherein the amino acid is selected from one or more of glycine, alanine, serine, cysteine, threonine and / or proline, and / or:

[0017] (2) Mutations in the allosteric pathway include replacing the original residues with charged or polar amino acids that can enhance the local hydrogen bond network, salt bridge or hydrophobic interaction, wherein the amino acid is selected from one or more of aspartic acid, glutamic acid, arginine, lysine, asparagine or glutamine, in order to reduce the steric hindrance conflict with the region where the pathogenic antibody light chain E59 is located and / or block the transmission of conformational perturbation along the allosteric pathway;

[0018] In the presence of 25 nM VH1-69 lineage inhibitory antibody 4-20, the mutant exhibits proteolytic activity against VWF substrate 96 that is no less than 50% of the activity of wild-type ADAMTS13 under antibody-free conditions.

[0019] The present invention also provides a kit for diagnosing or assisting in the diagnosis of immune thrombotic thrombocytopenic purpura (iTTP), the kit comprising reagents for detecting or quantifying autoantibodies in a sample that bind to the hydrophobically enriched conformational epitope of the ADAMTS13 spacer region.

[0020] The present invention also provides a pharmaceutical composition for the prevention or treatment of immune thrombotic thrombocytopenic purpura (iTTP), the pharmaceutical composition comprising a therapeutically effective amount of the allosteric defective ADAMTS13 binding molecule and / or the ADAMTS13 protein mutant, and a pharmaceutically acceptable carrier or excipient.

[0021] The active ingredient of the pharmaceutical composition is the allosteric defective ADAMTS13 binding molecule, which blocks the binding and allosteric inhibition of pathogenic VH1-69 lineage antibodies by competitively occupying the conformational epitope of the ADAMTS13 spacer region; or:

[0022] The active ingredient of the pharmaceutical composition is the ADAMTS13 protein mutant, which is used as an enzyme replacement drug to restore proteolytic activity against VWF in iTTP patients.

[0023] The present invention also provides a method for screening candidate drugs for the prevention or treatment of iTTP, comprising the following steps:

[0024] a) Provide a testing system capable of simulating or reflecting the Spacer-Cys interface conformation state or stability of ADAMTS13;

[0025] b) In the presence of candidate substances, detect whether the conformational state or stability of the Spacer-Cys interface in the test system changes;

[0026] c) Select candidate materials that can stabilize the Spacer-Cys interface conformation or enhance its stability;

[0027] The testing system comprises the ADAMTS13 protein or a functional fragment thereof containing a Spacer domain and a Cysteine-rich domain, and the detection is performed by molecular dynamics simulation, spectroscopic analysis, or protein conformation probes.

[0028] The present invention also provides a method for screening candidate drugs for the prevention or treatment of iTTP, comprising the following steps:

[0029] a) Contact the candidate material with the ADAMTS13 protein or a fragment thereof containing the conformational epitope;

[0030] b) In the presence of VH1-69 lineage antibody or its antigen-binding fragment, detect whether the candidate substance can competitively inhibit the binding of the antibody to ADAMTS13 protein or its fragment;

[0031] c) Select candidate substances that can competitively inhibit the binding.

[0032] The present invention also provides a method for screening candidate drugs for the prevention or treatment of iTTP, comprising the following steps:

[0033] a) Contact the candidate material with the ADAMTS13 protein containing the allosteric pathway or a functional fragment thereof;

[0034] b) Detect whether the candidate substance can interact with the allosteric pathway and stabilize the conformation of the ADAMTS13 protein and / or reduce the conformational volatility of the M domain in the presence of pathogenic antibodies;

[0035] c) Select candidate substances that can stabilize the conformation of the ADAMTS13 protein and maintain its activity against VWF protein hydrolysis.

[0036] The use of candidate substances obtained by the method in the preparation of drugs for the prevention or treatment of immune thrombotic thrombocytopenic purpura (iTTP) is also within the scope of protection of this invention.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Mechanism and effect: Through E59 short side chain / no side chain mutation and G491 site stabilization, it achieves highly efficient competitive blocking and long-range allosteric signal inhibition against pathogenic VH1-69 antibody, and significantly restores ADAMTS13 enzyme activity;

[0039] Performance parameters: In the presence of 25 nM 4-20 antibody, the mutant's hydrolytic activity against VWF is not less than 50% of that of the wild type, and the enzyme activity recovery rate is also high.

[0040] Function and Effect: The provided diagnostic kit can rapidly and specifically detect autoantibodies against the ADAMTS13 spacer region in the blood of iTTP patients, enabling early and accurate diagnosis;

[0041] Process effects: The mutation and screening methods described are based on high-throughput techniques such as molecular dynamics simulation and spectroscopic analysis, which shortens the research and development cycle;

[0042] Safety / Regulatory Efficacy: The drug composition uses low immunogenicity carriers such as IgG4, Fab, or scFv, which meets drug safety regulatory requirements and is suitable for clinical prevention and treatment of iTTP. Attached Figure Description

[0043] Figure 1 :4-20 The overall binding mode of the complex of Fv-clasp and ADAMTS13 spacer domain.

[0044] Figure 2 :4-20 Hydrophobic surface diagram of the interface between the Fv-clasp and ADAMTS13 spacer domain complex.

[0045] Figure 3 : Graph of VH and VL on interface interaction and their contribution ratio.

[0046] Figure 4 The superposition of the complex structure with the full-length ADAMTS13 model shows a potential spatial conflict between the 4-20 light chain E59 and the adjacent Cys domain.

[0047] Figure 5 Functional comparison of wild-type 4-20 and E59 mutants (E59A / E59G) in ADAMTS13 activity inhibition assays showed that the inhibitory efficacy of the mutants was significantly reduced.

[0048] Figure 6 In the antibody-bound state, the angle (θ) between the spacer domain and the M domain shifts significantly. Detailed Implementation

[0049] The present invention will be further illustrated by the following embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0050] Example 1: Preparation, crystallization and structural analysis of 4-20 Fv-clasp and its complex with ADAMTS13 spacer domain

[0051] cDNA encoding the 4-20 variable heavy chain (VH) and variable light chain (VL) were fused with C-terminal Fv-clasp, respectively (the protein of the variable heavy chain is SEQ ID NO.1, and the protein of the variable light chain is SEQ ID NO.2), and then independently cloned into the pET30a(+) vector between the NdeI and XhoI restriction sites. The complementary DNA encoding the human ADAMTS13 spacer region (residues S556–K681) (nucleotide sequence SEQ ID NO.6) was cloned into the pET-30a(+) vector and inserted between the NdeI and XhoI restriction sites.

[0052] The recombinant plasmid was transformed into *E. coli* BL21 cells. Cultures were grown in LB medium at 37°C and induced with 0.5 mM IPTG for 6 hours. Cells were collected by centrifugation at 3800 × g for 20 minutes, lysed by sonication, and inclusion bodies were isolated by centrifugation at 10,000 × g for 10 minutes. Inclusion bodies were washed three times with buffer containing 0.5 M Triton X-100, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl, 1 mM DTT, and 0.1 mM PMSF, followed by washing and dissolution with denaturing buffer. Inclusion bodies of 4–20 VH-clasp, 4–20 VL-clasp, and spacer were then mixed in refolding buffer at a molar ratio and incubated at 4°C for 48 hours. Subsequently, they were treated with 20 volumes of 10 mM Tris-HCl (pH 8.0) buffer at 4°C for 16 hours. Final purification was performed using anion exchange chromatography on a Mono Q column (Cytiva) with a linear gradient elution between buffer A (10 mM Tris-HCl, pH 8.0) and buffer B (10 mM Tris-HCl, pH 8.0, 1 M NaCl). The purified protein was analyzed by SDS-PAGE and concentrated to a final concentration of 20 mg / mL for subsequent applications.

[0053] Crystals of the 4-20-spacer complex were obtained at 4 °C using the static droplet vapor diffusion method. X-ray diffraction data were acquired at the BL18U1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China). Data were indexed and integrated using the iMosflm software package and scaled using the Aimless and Pointless methods in the CCP4 suite. The crystal structure of the 4-20 Fv-clasp-spacer was determined by molecular substitution using Phaser software with the 4-20 Fv-clasp-spacer homology model generated by Swiss-Model as the search model. The initial solution of the molecular substitution was manually reconstructed using the Crystallographic Object-Oriented Toolkit (COOT) program, and then refined using Phenix software.

[0054] Protein sequence of 4-20 VL (SEQ ID NO.1)

[0055] MELELTQPPSVSISPGKTAKIFCGGNSIGRKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYWVFGGGTKLTVL

[0056] Protein sequence of 4-20 VH (SEQ ID NO.2)

[0057] HQVQLVQSGAEVKKPGSSVKVSCKASGGAFTMYTINWVRQAPGRGLEWMGRIIPILGITDYAQKFQGRGTITADKSSTAYLELSGLTSEDTAVYYCAREFSGGNYFDFWGQGTLVTVSS

[0058] The protein sequence of the ADAMTS13 spacer (residues S556–K681) (SEQ ID NO.3)

[0059] MSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPK

[0060] Nucleotide sequence of 4-20 VL (SEQ ID NO.4)

[0061] ATGGAATTAGAATTAACTCAGCCACCTAGTGTTAGTATTAGTCCTGGTAAAACAGCAAAAATCTTTTGTGGTGGTAATAGTATTGGTCGTAAAAGTGTTCACTGGTATCAACAAAAACCTGGTCAAGCTCCTGTTTTAGTTGTTTATGATGATAGTGATCGTCCTAGTGGTATTCCTGAACGTTTTAGTGGTAGTAATAGTGGTAATACAGCTACATTAACAATTAGTCGTGTTGAAGCTGGTGATGAAGCTGATTATTATTGTCAAGTTTGGGATAGTTCATCAGATTATTGGGTTTTTGGTGGTGGTACAAAATTAACAGTTTTA

[0062] Nucleotide sequence of 4 - 20 VH (SEQ ID NO.5)

[0063] CATCAGGTTCAGCTGGTTCAGTCTGGTGCTGAAGTTAAAAAACCGGGTTCTTCTGTTAAAGTTTCTTGCAAAGCTTCTGGTGGTGCTTTCACCATGTACACCATCAACTGGGTTCGTCAGGCTCCGGGTCGTGGTCTGGAATGGATGGGTCGTATCATCCCGATCCTGGGTATCACCGACTACGCTCAGAAATTCCAGGGTCGTGGTACCATCACCGCTGACAAATCTACCTCTACCGCTTACCTGGAACTGTCTGGTCTGACCTCTGAAGACACCGCTGTTTACTACTGCGCTCGTGAATTCTCTGGTGGTAACTACTTCGACTTCTGGGGTCAGGGTACCCTGGTTACCGTTTCTTCT

[0064] Nucleotide sequence of ADAMTS13 spacer (residues S556–K681) (SEQ ID NO.6)

[0065] ATGAGTCCTCGAAAAGGTAGTTTTACAGCTTGGTCGTGCTCGTGAATATGTTACTTTTTTAACAGTTACACCTAATTTAACAAGTGTTTATCGCTAATCACCGTCCTTTATTTACACACTTAGCAGTTCGTATCGGTGGTCGATATGTAGTTGCTGGTAAAATGAGTATCTCACCTAATACAACATATC CTTCACTACTAGAAGATGGTCGTGTTGAATATCGTGTTGCTCTAACAGAAGATCGTCTACCTCGTCTAGAAGAAATCCGTATCTGGGGTCCTCTACAGGAAGATGCTGATATCCAGGTTTATCGTCGTTATGGTGAAGAATATGGTAATCTAACACGTCCTGATATCACTTTTACATATTTTCAGCCAAAA

[0066] Example 2: Fine mapping of epitope-paratope interactions

[0067] After obtaining the complex structure with a high resolution of 3.4 Å according to Example 1 ( Figure 1 As can be seen, the interface between 4–20 Fv-clasp and ADAMTS13 Spacer exhibits highly complementary shapes and is dominated by hydrophobic interactions (Figure 2). Combined with area analysis, VH contributes significantly more to the identification of the spacer region than VL: VH contributes 74.07%, while VL contributes only 24.44%, with the difference primarily originating from CDR3H (…). Figure 3 (See Table 1). The number of hydrogen bonds and salt bridges also supports this trend: VL forms 3 hydrogen bonds and 2 salt bridges, while VH provides 7 hydrogen bonds and 2 salt bridges (Table 1). Meanwhile, three heavy chain CDR rings penetrate into a continuous hydrophobic channel of the spacer, while no corresponding hydrophobic channel is observed on the VL side (Figure 3).

[0068] To assess whether antibody binding could transmit conformational effects outside the spacer, the Fv-clasp-spacer complex of fragment 4-20 was superimposed with the MDTCS fragment of ADAMTS13, using the spacer as the alignment reference. Figure 4 The superposition shows that the E59 residue in the 4-20 light chain—although not involved in the core epitope—extends into the adjacent Cys domain, creating a potential steric hindrance conflict. This interference at the spacer-Cys junction provides a plausible geometric triggering mechanism for long-range (alloyal) regulation of the M domain.

[0069] Table 1. Forces of Fv-clasp-spacer

[0070]

[0071] Example 3: Functional verification of the E59 site through site-directed mutagenesis and activity assay

[0072] To verify the importance of E59, we introduced the E59G or E59A mutation into the 4-20-VL-clasp domain using site-directed mutagenesis. The construction and purification methods for 4-20 Fv-clasp and its variants followed the procedure in Example 1. The complementary DNA encoding the classical human MDTCS region (G78–P682) of the ADAMTS13 gene was cloned into the pcDNA3.1 vector (protein sequence SEQ ID NO.7). This construct was transfected into HEK293T cells using liposome transfection reagent. Cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum and 10 μg / mL puromycin. After 48 hours, the supernatant was collected by centrifugation and dialyzed against TBSC buffer.

[0073] Protein hydrolysis and cleavage experiments were performed in a 20 μL reaction system containing 6 μM VWF96, 1–2 μL recombinant ADAMTS13, and 0–25 nM 4–20 Fv-clasp variants. The reaction was incubated at 37°C for 2 hours, followed by the addition of EDTA to terminate the reaction. Samples were analyzed by SDS-PAGE, and band intensities were quantified using ImageJ software. Figure 5 ).

[0074] Depend on Figure 5 As can be seen, substitution with E59G or E59A significantly reduces the inhibitory efficacy of 4-20, while the binding affinity of 4-20 to ADAMTS13 remains largely unaffected. Therefore, replacing E59 with a long-chain glutamic acid with a short-chain or essentially no-side-chain amino acid that significantly reduces the side-chain volume can effectively alleviate the physical spatial conflict between E59 and the ADAMTS13 G491 neighborhood, thereby weakening or eliminating the long-range allosteric inhibition effect triggered by this site. Those skilled in the art can infer from this that, in addition to alanine and glycine, conserved substitution of other small-volume amino acids with short or essentially no side chains (such as Ser, Cys, Thr, etc.) at this site is also expected to yield binding molecules that maintain high affinity while weakening allosteric inhibition, and could serve as candidates for constructing allosteric-deficient decoy antibodies.

[0075] Protein sequence of the MDTCS region (G78–P682) (SEQ ID NO.7)

[0076] GGSGGSGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPHHHHHHHH

[0077] Nucleotide sequence of the MDTCS region (G78–P682) (SEQ ID NO.8)

[0078]

[0079] Example 4: Molecular dynamics simulations reveal allosteric transport pathways

[0080] To assess whether antibody binding induces distal conformational changes in ADAMTS13, we constructed two all-atom systems: (i) a "monomer" of the MDTCS fragment (G78–P682) in the apo state, and (ii) a "complex reference" of the spacer conformation observed in a 4–20 spacer crystal structure aligned with this fragment. All proteins were parameterized using the AMBER99SB-ILDN force field and placed in an explicit SPC / E tank under periodic boundary conditions with a minimum distance of 1.0 nm between the solute and the tank edge. Counterions (Na⁺ / Cl⁻) were added to achieve system electroneutrality.

[0081] Inter-domain angle definition. The relative orientation of the spacer region to the metalloproteinase (M) is quantified by the inter-domain angle (θ), which is defined by the “master” vector formed by the adjacent Cα-Cα anchor points at both ends of each domain. The anchor point indices are: M domain – residues 20 and 190; spacer region – residues 490 and 560. The θ value is calculated in each frame of the trajectory as previously described.

[0082] Alternate path sampling. A contact graph-based path tracing analysis was employed to detect long-range interval-M couplings; specific methods are described in previous literature. Trajectories were saved every 20 ps, ​​and subsampling was performed at intervals specified by the step size parameter (default: every 10 frames) to construct a Cα contact graph (nodes: all Cα atoms; edges: residue spacing < 8 Å). Residues at the antibody-enzyme interface were assumed to be the source set; multiple distal residues within the M domain were assumed to be pooling points. The accumulated path set within 500 ns was clustered using Jaccard distance (hierarchical average linkage method) to identify dominant communication paths and their frequencies; clustering stability was evaluated using path frequency and inter-frame consistency of the trajectory.

[0083] All-atom molecular dynamics (MD) simulations show that the antibody binding system exhibits a rightward shift of approximately 19.3° compared to the apo state. Figure 6 This indicates the presence of stable reweighting towards the alternative structural domain orientation. Simultaneously, RMSD analysis based on the spacer region showed a significant increase in the conformational fluctuation of the M domain after antibody binding, a trend validated by comparing the overall RMSD, Rg, and RMSF of the complex and the monomer. Path analysis further revealed that the dominant communication path occurred much more frequently in the complex than in the monomer, and this channel, along I411-G392-M367-T427-G324-V326-R328-R330, connects the spacer region and the M domain into a well-defined long-range signal chain.

[0084] In summary, antibody binding remodels the spacer-M interface, enhances the flexibility of the M domain, and directly links the steric hindrance of the "spacer-cysteine ​​link" to the decrease in downstream proteolytic activity through a preferential allosteric pathway near the E59 footprint of the light chain.

[0085] Example 5

[0086] Based on the findings of Examples 2 and 4, this invention aims to rationally design and modify the ADAMTS13 protein itself to acquire resistance to allosteric inhibition by pathogenic antibodies. To address the spatial conflict induced by the antibody light chain E59, we introduce mutations in the G491 neighborhood (e.g., S490, K492, L493, etc.) designed to enhance local rigidity or eliminate the conflict, such as introducing proline (P) or smaller amino acids (e.g., A, S). For the key allosteric pathways (I411-G392-M367-T427-G324-V326-R328-R330) revealed by molecular dynamics simulations, we introduce mutations at these sites or their neighboring residues designed to enhance hydrogen bond networks, salt bridges, or hydrophobic interactions to "fix" the native conformation of the pathway and resist the transmission of perturbation signals. For example, charged or polar residues (e.g., D, E, R, K, N, Q) are introduced at the G324 site to form new interactions with neighboring residues.

[0087] Following the method described in Example 3, the MDTCS fragment (SEQ ID NO: 7) was subjected to site-directed mutagenesis, and the fragment was cloned into the pcDNA3.1 vector. The mutant plasmid was transfected into HEK293F cells, and the recombinant protein was expressed and purified according to the method described in Example 3.

[0088] Functional validation was performed as follows: ① The binding affinity of the mutant ADAMTS13 to antibody 4-20 was verified to be comparable to that of the wild type using surface plasmon resonance (SPR) or ELISA to ensure that the epitope was not destroyed. ② The protein hydrolysis cleavage experiment was performed as described in Example 3. The cleavage efficiency of wild-type ADAMTS13 and mutant ADAMTS13 on VWF96 was compared in the presence of a saturated concentration (e.g., 25 nM) of wild-type antibody 4-20. ③ Optionally, molecular dynamics simulations were performed on the preferred mutant to observe whether the volatility of its Spacer-Cys interface and the conformational stability of its allosteric pathway were enhanced compared to the wild type in the antibody-bound state.

[0089] It is anticipated that one or more ADAMTS13 mutants (e.g., G491S / L493P, G324R, etc.) can be obtained. These mutants retain their ability to bind to pathogenic antibodies while their enzyme activity is no longer effectively inhibited by these antibodies. Such mutants can be used directly as highly effective alternative therapies to neutralize autoantibodies in patients and restore the physiological cleavage of VWF. Those skilled in the art will understand that, for the Spacer-Cys interface, smaller or more rigid amino acid substitutions (such as G, A, S, T, C, or P) can be preferred, while for residues along allosteric pathways, charged or polar residues (such as D, E, R, K, N, Q, etc.) can be introduced to enhance local hydrogen bond networks, salt bridges, or hydrophobic interactions, thereby mitigating steric hindrance conflicts and solidifying allosteric signal transduction pathways, respectively.

[0090] Example 6: Screening of small molecule drug candidates targeting allosteric pathways

[0091] Based on the explicit allosteric pathway revealed in Example 4, a method is established to screen small molecule compounds that can stabilize this pathway and thus resist allosteric inhibition.

[0092] 1. Screening system construction:

[0093] a) Screening based on protein interactions: Purify the ADAMTS13 MDTCS fragment containing the complete allosteric pathway. Immobilize it on a biosensor chip or microplate.

[0094] b) Conformation-based screening: Construct an ADAMTS13 reporter protein containing an environmentally sensitive fluorescent group near the active site of the allosteric pathway region or the M domain. When the pathway conformation is stable, a specific change in fluorescence signal occurs.

[0095] 2. Screening process:

[0096] a) Co-incubate small molecule compound libraries (e.g., libraries containing thousands to tens of thousands of compounds) with screening systems.

[0097] b) For system (a), the binding of small molecules to ADAMTS13 is detected by SPR or similar techniques, and compounds that bind to the MDTCS fragment (rather than the spacer domain alone) are preferentially selected, suggesting that their binding site may be located at the interdomain interface or allosteric pathway.

[0098] c) For system (b), directly detect changes in the fluorescence signal. Select compounds that can induce signal changes associated with the "stable conformation".

[0099] d) The "hit" compound obtained from the initial screening was subjected to a secondary verification using the enzyme activity protection experiment described in Example 3. Specifically, in the presence of antibody 4-20, the small molecule was tested to determine whether it could dose-dependently restore the enzyme activity of ADAMTS13.

[0100] 3. Candidate Validation: Subsequent pharmacodynamic and safety evaluations of small molecules that can effectively restore enzyme activity at the cellular and animal model levels.

[0101] This method is expected to screen for small-molecule allosteric agonists targeting the ADAMTS13 allosteric pathway. These small-molecule drugs can be taken orally directly and treat iTTP by stabilizing the native conformation of ADAMTS13, providing a new treatment option compared to antibody or enzyme replacement therapy.

[0102] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An allosterically defective ADAMTS13 binding molecule, which is an antibody or its antigen-binding fragment, characterized in that: The binding molecule specifically binds to a conformational epitope formed by the enrichment of hydrophobic residues on the spacer domain of ADAMTS13; at the position corresponding to the 59th glutamic acid (E59) of the variable region of the 4-20 light chain of the VH1-69 lineage antibody, it includes a mutation that mutates the glutamic acid (E) into a short side chain with a side chain volume significantly smaller than that of the glutamic acid side chain or essentially no side chain amino acid; the mutation reduces the physical steric hindrance conflict with the neighborhood of the 491st glycine (G491) of the ADAMTS13 spacer domain, so that the binding molecule maintains a high affinity for ADAMTS13 while essentially not inhibiting or only significantly weakening the proteolytic activity of ADAMTS13 on von Willebrand factor (VWF).

2. The allosteric defective ADAMTS13 binding molecule according to claim 1, characterized in that, The short-side-chain or essentially side-chain-free amino acid is selected from one or more of the following: glycine (Gly, G), alanine (Ala, A), serine (Ser, S), cysteine ​​(Cys, C), and threonine (Thr, T). The mutation is alanine (E59A) and / or glycine (E59G). The complementary determinant region (CDR) amino acid sequence of the binding molecule has at least 70-90% sequence identity with the corresponding CDR amino acid sequence of monoclonal antibody 4-20, and retains the same or cross-reactive ADAMTS13 spacer region conformational epitope recognition pattern as 4-20; and the molecule is an IgG4 subtype antibody, a Fab fragment, a single-chain variable fragment (scFv), or an engineered protein scaffold containing an E59 short side chain or a side chain-less mutant binding domain.

3. An ADAMTS13 protein mutant, characterized in that: Compared to wild-type ADAMTS13, the mutant contains at least one amino acid mutation at the interface between the spacer domain and the cysteine-rich domain and / or along the allosteric signaling pathway between the spacer domain and the metalloproteinase domain (M domain). This amino acid mutation can stabilize the Spacer-Cys interface conformation and / or weaken long-range allosteric signaling triggered by pathogenic antibody binding, thereby retaining proteolytic activity against VWF in the presence of the pathogenic antibody.

4. The ADAMTS13 protein mutant according to claim 3, characterized in that, The amino acid mutation is located at at least one of the glycine residues (G491) at position 491 in the Spacer domain and its neighboring residues; and / or at least one residue in an allosteric signaling pathway consisting of or adjacent to residues I411, G392, M367, T427, G324, V326, R328, and R330. in: (1) Mutations in the Spacer-Cys interface region include replacing the original residue with a short side chain or essentially no side chain amino acid, wherein the amino acid is selected from one or more of glycine, alanine, serine, cysteine, threonine and / or proline, and / or: (2) Mutations in the allosteric pathway include replacing the original residues with charged or polar amino acids that can enhance the local hydrogen bond network, salt bridge or hydrophobic interaction, wherein the amino acid is selected from one or more of aspartic acid, glutamic acid, arginine, lysine, asparagine or glutamine, in order to reduce the steric hindrance conflict with the region where the pathogenic antibody light chain E59 is located and / or block the transmission of conformational perturbation along the allosteric pathway; In the presence of 25 nM VH1-69 lineage inhibitory antibody 4-20, the mutant exhibits proteolytic activity against VWF substrate 96 that is no less than 50% of the activity of wild-type ADAMTS13 under antibody-free conditions.

5. A kit for diagnosing or assisting in the diagnosis of immune thrombotic thrombocytopenic purpura (iTTP), characterized in that, The kit contains reagents for detecting or quantifying autoantibodies that bind to the hydrophobically enriched conformational epitopes of the ADAMTS13 spacer region in a sample.

6. A pharmaceutical composition for the prevention or treatment of immune thrombotic thrombocytopenic purpura (iTTP), characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the allosteric defective ADAMTS13 binding molecule of claim 1 or 2 and / or the ADAMTS13 protein mutant of claim 3 or 4, as well as a pharmaceutically acceptable carrier or excipient. The active ingredient of the pharmaceutical composition is the allosteric defective ADAMTS13 binding molecule as described in claim 1 or 2, which blocks the binding and allosteric inhibition of pathogenic VH1-69 lineage antibodies by competitively occupying the conformational epitope of the ADAMTS13 spacer region; or: The active ingredient of the pharmaceutical composition is the ADAMTS13 protein mutant according to claim 3 or 4, used as an enzyme replacement drug to restore proteolytic activity against VWF in iTTP patients.

7. A method for screening candidate drugs for the prevention or treatment of iTTP, characterized in that, Includes the following steps: a) Provide a testing system capable of simulating or reflecting the Spacer-Cys interface conformation state or stability of ADAMTS13; b) In the presence of candidate substances, detect whether the conformational state or stability of the Spacer-Cys interface in the test system changes; c) Select candidate materials that can stabilize the Spacer-Cys interface conformation or enhance its stability; The testing system comprises the ADAMTS13 protein or a functional fragment thereof containing a Spacer domain and a Cysteine-rich domain, and the detection is performed by molecular dynamics simulation, spectroscopic analysis, or protein conformation probes.

8. A method for screening candidate drugs for the prevention or treatment of iTTP, characterized in that, Includes the following steps: 1) Contact the candidate material with the ADAMTS13 protein or a fragment thereof containing the conformational epitope of claim 1; 2) In the presence of VH1-69 lineage antibody or its antigen-binding fragment, detect whether the candidate substance can competitively inhibit the binding of the antibody to ADAMTS13 protein or its fragment; 3) Select candidate substances that can competitively inhibit the binding.

9. A method for screening candidate drugs for the prevention or treatment of iTTP, characterized in that, Includes the following steps: 4) Contact the candidate material with the ADAMTS13 protein or a functional fragment thereof that contains the allosteric pathway as described in claim 4; 5) Detect whether the candidate substance can interact with the allosteric pathway and stabilize the conformation of the ADAMTS13 protein and / or reduce the conformational volatility of the M domain in the presence of pathogenic antibodies; 6) Select candidate substances that can stabilize the conformation of the ADAMTS13 protein and maintain its activity against VWF protein hydrolysis.

10. Use of the candidate substance obtained by the method according to any one of claims 7-9 in the preparation of a medicament for the prevention or treatment of immune thrombotic thrombocytopenic purpura (iTTP).

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