A C1q antibody and its application, an agent for inhibiting the activation of the classical complement pathway and its application

Abstract

The present invention relates to the field of biomedical technologies, and in particular, to a C1q antibody and its applications, an agent for inhibiting the activation of the classical complement pathway and its applications. A C1q antibody, wherein the C1q antibody comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown in SEQ ID NO: 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown in SEQ ID NO: 4-6. This antibody has a good affinity with the C1q antigen and can effectively inhibit the activation of the classical complement pathway.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a C1q antibody and its application, and an agent for inhibiting the activation of the classical complement pathway and its application. Background Art

[0002] Complement is an immune molecular system composed of soluble proteins and membrane proteins. After cascade activation, it participates in innate and adaptive immune responses and plays an important role in the occurrence and development of diseases such as infections, autoimmune diseases, and tumors. The activation process of the classical complement pathway is as follows: after an activator binds to C1q, C1r and C1s are further activated. C1s has esterase activity and cleaves C4 into two fragments, C4b and free C4a. In the presence of Mg 2+ When present, C1 and C4b together cleave C2 into a large fragment C2b and a free small fragment C2a; C2b and C4b can combine to form C4b2b (C3 convertase), which cleaves C3 into a large fragment C3b and a free small fragment C3a; then C3b binds to the adjacent cell membrane attached to C4b2b, forming a trimolecular complex C4b2b3b, that is, C5 convertase; C5 convertase cleaves C5 into C5b and free small molecule C5a. C5b binds to the cell membrane, then binds C6, C7, and C8 to form C5b678, and then binds to 1 to 18 C9 molecules and catalyzes C9 to polymerize into a transmembrane channel with a hydrophilic inner wall, resulting in cell rupture.

[0003] However, excessive complement activation is associated with many disease conditions, including various inflammatory and autoimmune diseases. Therefore, there is an urgent need for an antibody that inhibits complement activation to solve the problem of excessive complement activation. Summary of the Invention

[0004] The purpose of the present invention is to provide a C1q antibody that can effectively inhibit the activation of the classical complement pathway.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a C1q antibody, wherein the C1q antibody comprises VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1 to 3, and VLCDR1, VLCDR2, and VLCDR3 with amino acid sequences as shown in SEQ ID NO: 4 to 6.

[0007] Preferably, the nucleotide sequence encoding VHCDR1 is as shown in SEQ ID NO:7; the nucleotide sequence encoding VHCDR2 is as shown in SEQ ID NO:8; the nucleotide sequence encoding VHCDR3 is as shown in SEQ ID NO:9; the nucleotide sequence encoding VLCDR1 is as shown in SEQ ID NO:10; the nucleotide sequence encoding VLCDR2 is as shown in SEQ ID NO:11; the nucleotide sequence encoding VLCDR3 is as shown in SEQ ID NO:12.

[0008] The present invention also provides the use of the C1q antibody in inhibiting the activation of the classical complement pathway.

[0009] The present invention also provides an agent for inhibiting the activation of the classical complement pathway, comprising the C1q antibody.

[0010] The present invention also provides the use of the C1q antibody in the preparation of an agent for treating diseases associated with the activation of the classical complement pathway.

[0011] Preferably, the diseases associated with the activation of the classical complement pathway include Guillain-Barré syndrome, amyotrophic lateral sclerosis, Huntington's chorea, warm autoimmune hemolytic anemia or autoimmune thrombocytopenic purpura.

[0012] The present invention also provides the use of the C1q antibody in the preparation of an agent for inhibiting rejection occurring after organ transplantation.

[0013] The present invention also provides a kit for inhibiting the activation of the classical complement pathway, comprising the C1q antibody.

[0014] Advantages of the present invention:

[0015] The present invention provides a C1q antibody, which has a good affinity with C1q and can effectively inhibit the activation of the classical complement pathway.

[0016] C1q is the starting molecule for activating the classical complement pathway. Effectively inhibiting the activation of the classical complement pathway can be used to treat diseases associated with the activation of the classical complement pathway, such as Guillain-Barré syndrome, amyotrophic lateral sclerosis, Huntington's chorea, warm autoimmune hemolytic anemia or autoimmune thrombocytopenic purpura; in addition, it can also effectively inhibit rejection occurring after organ transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a diagram showing the binding effect between the C1q antibody and the C1q antigen;

[0018] Figure 2 It is the inhibitory effect of the C1q antibody on the activation of the classical complement pathway. Detailed implementation mode

[0019] The present invention provides a C1q antibody, which comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown in SEQ ID NOs: 1 to 3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown in SEQ ID NOs: 4 to 6;

[0020] SEQ ID NO: 1 is DYNMD;

[0021] SEQ ID NO: 2 is DINPDYDGTIYNQKFKG;

[0022] SEQ ID NO: 3 is RVYDSDVWYFDV;

[0023] SEQ ID NO: 4 is TASSSVSSSYLH;

[0024] SEQ ID NO: 5 is STSNLAS;

[0025] SEQ ID NO: 6 is HQYHRSPFT;

[0026] The amino acid sequence of the heavy chain region of the C1q antibody is as shown in SEQ ID NO: 13: EVQLQQFGTELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWI GDINPDYDGTIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARRVYDSDVWYFDVWGAGTTVTVSS;

[0027] The amino acid sequence of the light chain region of the C1q antibody is as shown in SEQ ID NO: 14: QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKSGSSPKLWIY STSNLASGVPTRFSGSGSGTSFSLTISRMEAEDAATYFCHQYHRSPFTFGGGTKLEMK.

[0028] Preferably, the nucleotide sequence encoding VHCDR1 is as shown in SEQ ID NO: 7; the nucleotide sequence encoding VHCDR2 is as shown in SEQ ID NO: 8; the nucleotide sequence encoding VHCDR3 is as shown in SEQ ID NO: 9; the nucleotide sequence encoding VLCDR1 is as shown in SEQ ID NO: 10; the nucleotide sequence encoding VLCDR2 is as shown in SEQ ID NO: 11; the nucleotide sequence encoding VLCDR3 is as shown in SEQ ID NO: 12;

[0029] The SEQ ID NO:7 is GACTATAACATGGAC;

[0030] The SEQ ID NO:8 is GACATTAACCCCGATTACGACGGCACAATCTACAACCAGAAATTCAAGGGC;

[0031] The SEQ ID NO:9 is AGGGTGTACGACTCCGACGTTTGGTACTTCGACGTG;

[0032] The SEQ ID NO:10 is ACCGCCTCCTCTTCAGTCTCCTCATCCTACCTCCAC;

[0033] The SEQ ID NO:11 is AGCACCTCCAACCTCGCCAGC;

[0034] The SEQ ID NO:12 is CACCAGTACCACCGCTCCCCCTTTACA;

[0035] The nucleotide sequence encoding the heavy chain region of the C1q antibody is as shown in SEQ ID NO:15: GAGGTTCAGTTGCAGCAGTTTGGAACAGAGTTGGTGAAGCCAGGAGCAAGCGTGAAGATTAGTTGTAAGGCAAGCGGGTATACATTCACAGACTATAACATGGACTGGGTGAAGCAGAGCCACGGAAAGAGCCTGGAGTGGATCGGAGACATTAACCCCGATTACGACGGCACAATCTACAACCAGAAATTCAAGGGCAAGGCCACCCTGACAGTGGACAAGTCTAGCAGCACAGCATATATGGAACTGAGGAGTCTGACCAGCGAAGATACTGCTGTGTACTACTGTGCAAGGAGGGTGTACGACTCCGACGTTTGGTACTTCGACGTGTGGGGAGCAGGCACAACCGTTACAGTGAGCTCC;

[0036] The nucleotide sequence encoding the light chain region of the C1q antibody is as shown in SEQ ID NO:16: CAGATCGTCCTCACCCAGAGTCCAGCTATCATGTCCGCCTCCCTCGGAGAAAGAGTGACCATGACCTGCACCGCCTCCTCTTCAGTCTCCTCATCCTACCTCCACTGGTACCAGCAGAAAAGCGGCTCCTCCCCTAAACTCTGGATCTACAGCACCTCCAACCTCGCCAGCGGAGTTCCTACAAGGTTCTCCGGTTCCGGCTCCGGAACTTCATTCTCCCTCACTATCTCCCGGATGGAAGCTGAGGACGCCGCTACCTATTTCTGCCACCAGTACCACCGCTCCCCCTTTACATTCGGCGGAGGAACTAAGCTGGAGATGAAA

[0037] The present invention also provides the use of the C1q antibody in inhibiting the activation of the classical complement pathway.

[0038] The present invention also provides an agent for inhibiting the activation of the classical complement pathway, comprising the C1q antibody.

[0039] The present invention also provides the use of the C1q antibody in the preparation of an agent for treating diseases associated with the activation of the classical complement pathway.

[0040] In the present invention, the diseases associated with the activation of the classical complement pathway include Guillain-Barré syndrome, amyotrophic lateral sclerosis, Huntington's chorea, warm autoimmune hemolytic anemia, or autoimmune thrombocytopenic purpura.

[0041] The present invention also provides the use of the C1q antibody in the preparation of an agent for inhibiting rejection occurring after organ transplantation.

[0042] The present invention also provides a kit for inhibiting the activation of the classical complement pathway, comprising the C1q antibody.

[0043] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1 Synthesis of the C1q antibody

[0045] Obtaining C1q antibody through expression in the mammalian system: The heavy and light chain sequences of the C1q antibody were respectively constructed into the pcDNA3.4 expression vector. After plasmid extraction, the heavy and light chain plasmids were co-transfected into the CHO K1 cell line. After 4 days of expression, the cell supernatant was collected and purified using protein A, and the obtained antibody was the C1q antibody.

[0046] The amino acid sequence of the variable region of the heavy chain of the C1q antibody is shown in SEQ ID NO:13: EVQLQQFGTELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWI GDINPDYDGTIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCA RRVYDSDVWYFDVWGAGTTVTVSS;

[0047] The nucleotide sequence encoding the variable region of the heavy chain of the C1q antibody is shown in SEQ ID NO:15: GAGGTTCAGTTGCAGCAGTTTGGAACAGAGTTGGTGAAGCCAGGAGCAAGCGTGAAGATTAGTTGTAAGGCAAGCGGGTATACATTCACAGACTATAACATGGACTGGGTGAAGCAGAGCCACGGAAAGAGCCTGGAGTGGATCGGAGACATTAACCCCGATTACGACGGCACAATCTACAACCAGAAATTCAAGGGCAAGGCCACCCTGACAGTGGACAAGTCTAGCAGCACAGCATATATGGAACTGAGGAGTCTGACCAGCGAAGATACTGCTGTGTACTACTGTGCAAGGAGGGTGTACGACTCCGACGTTTGGTACTTCGACGTGTGGGGAGCAGGCACAACCGTTACAGTGAGCTCC;

[0048] The amino acid sequence of the variable region of the light chain of the C1q antibody is shown in SEQ ID NO:14: QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKSGSSPKLWIY STSNLASGVPTRFSGSGSGTSFSLTISRMEAEDAATYFCHQYHRSPFTFGG GTKLEMK;

[0049] The nucleotide sequence encoding the light chain variable region of the C1q antibody is shown in SEQ ID NO: 16: CAGATCGTCCTCACCCAGAGTCCAGCTATCATGTCCGCCTCCCTCGGAGAAAGAGTGACCATGACCTGCACCGCCTCCTCTTCAGTCTCCTCATCCTACCTCCACTGGTACCAGCAGAAAAGCGGCTCCTCCCCTAAACTCTGGATCTACAGCACCTCCAACCTCGCCAGCGGAGTTCCTACAAGGTTCTCC GGTTCCGGCTCCGGAACTTCATTCTCCCTCACTATCTCCCGGATGGAAGCTGAGGACGCCGCTACCTATTTCTGCCACCAGTACCACCGCTCCCCCTTTACATTCGGCGGAGGAACTAAGCTGGAGATGAAA。

[0050] Example 2 Performance Verification of C1q Antibody

[0051] (1) Coating: Use 100 μL of coating solution PBS pH 7.3 to coat 2 μg of C1q antigen (abcam, NO: ab282858 / 20230815), coat at 37 °C for 1 h, wash the plate 6 times, pat dry, and obtain the coated C1q antigen;

[0052] (2) Blocking: Add 200 μL / well of casein blocking solution to the coated C1q antigen, block at 25 °C for 1 h, wash the plate 6 times, pat dry, and obtain the solid-phase antigen;

[0053] (3) Binding: Add the C1q antibody (anti-C1q) described in Example 1 to the solid-phase antigen. The initial concentration of the C1q antibody added to the first well is 400 nM, and it is diluted 3-fold with casein blocking solution, successively diluted in 11 gradients; the last well is blank, serving as a blank control; at the same time, with reference to the literature (Ha-Yeun Chung ,The C1q antibody reported by Jonathan Wickel, Nina Hahn, et al. in "Microglia mediate neurocognitive deficits by eliminating C1q-tagged synapses in sepsis-associated encephalopathy. Sci Adv. 2023, 9(21): eabq7806." was used as the positive control (PC), and Mouse IgG was used as the negative control (NC). The specific binding steps were the same as those for adding C1q; binding was carried out at 25°C for 1 h, the plate was washed 6 times, and patted dry to obtain the solid-phase antigen-antibody complex;

[0054] (4) Horseradish peroxidase labeling: The anti-mouse Fc specific HRP labeled with horseradish peroxidase was diluted at a ratio of 1:65000 with casein blocking solution to obtain the diluted anti-mouse Fc specific HRP labeled with horseradish peroxidase; the diluted anti-mouse Fc specific HRP labeled with horseradish peroxidase was added to the solid-phase antigen-antibody complex, 100 μL was added to each well; labeling was carried out at 25°C for 1 h, the plate was washed 6 times, and patted dry to obtain the labeled product;

[0055] (5) Color development: Color development solution (Beyotime, NO: P0209) was added to the labeled product, 100 μL was added to each well; the color development reaction was carried out in the dark at 25°C;

[0056] (6) Termination: After 4 min of color development, 50 μL of termination solution (Beyotime, NO: P0215) was added to each well to terminate the color development reaction;

[0057] (7) Reading: The OD 450 value of each well was read with an enzyme-linked immunosorbent assay reader, and the results were as Figure 1 shown.

[0058] It can be seen from Figure 1 that the C1q antibody and the C1q antigen have good affinity.

[0059] Example 3 Effect of C1q antibody on inhibiting the activation of the classical complement pathway

[0060] (1) The target cells Raji cells (purchased from Shanghai Rubai Biotechnology Co., Ltd.) in the logarithmic growth phase were resuspended and counted to obtain the target cell suspension;

[0061] (2) The target cell suspension was added to a 96-well cell culture plate, 100 μL was added to each well, and a cell culture plate containing the target cell suspension was obtained;

[0062] (3) Add the C1q antibody (anti-C1q) described in Example 1 and the positive control antibody Sutimlimab (PC) to human serum with a concentration of 25% respectively. The final concentration of the C1q antibody solution in human serum is 20 μg / mL. After mixing, a mixture is obtained. Then, add 50 μL of the mixture to each well of the cell culture plate containing the target cell suspension and incubate for 30 min;

[0063] (4) Add 50 μL of rituximab (RTX) and the negative control human IgG1 control (NC) to the corresponding wells respectively, and culture at 37 °C and 5% CO2 for 2 h;

[0064] (5) Collect the cells, then add 100 μL of propidium iodide buffer (PI buffer, staining buffer: propidium iodide = 1000:1) to each well, and incubate in the dark for 10 min; Finally, collect data using FACS and perform data analysis using GraphPad Prism software. The analysis results are as Figure 2 shown.

[0065] It can be seen from Figure 2 that the C1q antibody described in Example 1 has a good effect of inhibiting the activation of the classical complement pathway.

[0066] As can be seen from the above examples, the present invention provides a C1q antibody and its application, an agent for inhibiting the activation of the classical complement pathway and its application. The antibody has a good affinity for the C1q antigen and can effectively inhibit the activation of the classical complement pathway.

[0067] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A C1q antibody, characterized in that The C1q antibody includes VHCDR1, VHCDR2 and VHCDR3 whose amino acid sequences are shown in SEQ ID NOs: 1 to 3, and VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4 to 6.

2. The C1q antibody according to claim 1, characterized in that The nucleotide sequence encoding VHCDR1 is shown in SEQ ID NO:7; the nucleotide sequence encoding VHCDR2 is shown in SEQ ID NO:8; the nucleotide sequence encoding VHCDR3 is shown in SEQ ID NO:9; the nucleotide sequence encoding VLCDR1 is shown in SEQ ID NO:10; the nucleotide sequence encoding VLCDR2 is shown in SEQ ID NO:11; and the nucleotide sequence encoding VLCDR3 is shown in SEQ ID NO:

12.

3. A drug for inhibiting activation of the classical complement pathway, characterized in that: The invention comprises the C1q antibody according to claim 1 or 2.

4. Use of the C1q antibody according to claim 1 or 2 in the preparation of a medicament for treating a disease associated with activation of the classical complement pathway; the disease associated with activation of the classical complement pathway is Guillain-Barré syndrome, amyotrophic lateral sclerosis or Huntington's disease.

5. A kit for inhibiting activation of the classical complement pathway, characterized in that: The invention comprises the C1q antibody according to claim 1 or 2.

Images