C1q antibody and application thereof, medicament for inhibiting classical pathway activation of complement and application of medicament

Abstract

The invention relates to the technical field of biomedicine, in particular to a C1q antibody and application thereof, and a medicament for inhibiting classical pathway activation of complement and application thereof. The C1q antibody comprises a VHCDR1, a VHCDR2 and a VHCDR3 of which the amino acid sequences are shown as SEQ ID NO: 1 to SEQ ID NO: 3, and a VLCDR1, a VLCDR2 and a VLCDR3 of which the amino acid sequences are shown as SEQ ID NO: 4 to SEQ ID NO: 6. The antibody has good affinity with C1q antigen, and can effectively inhibit classical pathway activation of complement.

Description

Technical Field

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

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

[0003] However, excessive complement activation is associated with many disease conditions, including a variety of inflammatory and autoimmune diseases. Therefore, an antibody that inhibits complement activation is urgently needed to solve the problem of excessive complement activation. Summary of the invention

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a C1q antibody, which comprises 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.

[0007] Preferably, the nucleotide sequence encoding VHCDR1 is shown as SEQ ID NO:7; the nucleotide sequence encoding VHCDR2 is shown as SEQ ID NO:8; the nucleotide sequence encoding VHCDR3 is shown as SEQ ID NO:9; the nucleotide sequence encoding VLCDR1 is shown as SEQ ID NO:10; the nucleotide sequence encoding VLCDR2 is shown as SEQ ID NO:11; and the nucleotide sequence encoding VLCDR3 is shown as 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 a medicament for inhibiting activation of the classical complement pathway, comprising the C1q antibody.

[0010] The present invention also provides the use of the C1q antibody in preparing a medicament for treating diseases related to activation of the classical complement pathway.

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

[0012] The present invention also provides the use of the C1q antibody in preparing a medicament for inhibiting rejection reaction after organ transplantation.

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

[0014] Beneficial effects of the present invention:

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

[0016] C1q is the initiator of activation of the classical complement pathway. Effective inhibition of activation of the classical complement pathway can be used to treat diseases related to activation of the classical complement pathway, such as Guillain-Barré syndrome, amyotrophic lateral sclerosis, Huntington's disease, warm autoimmune hemolytic anemia or autoimmune thrombocytopenic purpura; in addition, it can also effectively inhibit the rejection reaction after organ transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 It is the inhibitory effect of C1q antibody on activation of the classical complement pathway. DETAILED DESCRIPTION

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

[0020] The SEQ ID NO: 1 is DYNMD;

[0021] The SEQ ID NO: 2 is DINPDYDGTIYNQKFKG;

[0022] The SEQ ID NO: 3 is RVYDSDVWYFDV;

[0023] The SEQ ID NO:4 is TASSSVSSSYLH;

[0024] The SEQ ID NO:5 is STSNLAS;

[0025] The SEQ ID NO:6 is HQYHRSPFT;

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

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

[0028] Preferably, 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; the nucleotide sequence encoding VLCDR3 is 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] Nucleotide sequence encoding the heavy chain region of C1q antibody such as SEQ ID NO:15:GAGGTTCAGTTGCAGCAGTTTGGAACAGAGTTGGTGAAGCCAGGAGCAAGCGTGAAGATTAGTTGTAAGGCAAGCGGGTATACATTCACAGACTATAACATGGACTGGGTGAAGCAGAGCCACGGAAAGAGCCTGGAGTGGATCGGAGACATTAACCCCGATTACGACGGCACAATCTAC AACCAGAAATTCAAGGGCAAGGCCACCCTGACAGTGGACAAGTCTAGCAGCACAGCATATATGGAACTGAGGAGTCTGACCAGCGAAGATACTGCTGTGTACTACTGTGCAAGGAGGGTGTACGACTCCGACGTTTGGTACTTCGACGTGTGGGGAGCAGGCACAACCGTTACAGTGAGCTCC;

[0036] Nucleotide sequence encoding the C1q antibody light chain region such as SEQ ID NO:16:CAGATCGTCCTCACCCAGAGTCCAGCTATCATGTCCGCCTCCCTCGGAGAAAGAGTGACCATGACCTGCACCGCCTCCTCTTCAGTCTCCTCATCCTACTCCACTGGTACCAGCAGAAAAGCGGCTCCTCCCCTAAACTCTGGATCTACAGCACCTCCA ACCTCGCCAGCGGAGTTCCTACAAGGTTCTCCGGTTCCGGCTCCGGAACTTCATTCTCCCTCACTATCTCCCGGATGGAAGCTGAGGACGCCGCTACCTATTTCTGCCACCAGTACCACCGCTCCCCCTTTACATTCGGCGGAGGAACTAAGCTGGAGATGAAA is shown below:

[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 a medicament for inhibiting activation of the classical complement pathway, comprising the C1q antibody.

[0039] The present invention also provides the use of the C1q antibody in preparing a medicament for treating diseases related to activation of the classical complement pathway.

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

[0041] The present invention also provides the use of the C1q antibody in preparing a medicament for inhibiting rejection reaction after organ transplantation.

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

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

[0044] Example 1 Synthesis of C1q Antibody

[0045] Obtain C1q antibody through mammalian system expression: construct the heavy and light chain sequences of C1q antibody into pcDNA3.4 expression vector respectively, extract the plasmid, and then co-transfect the heavy and light chain plasmids into CHOK1 cell line. After 4 days of expression, collect the cell supernatant, purify the supernatant with protein A, and the obtained antibody is C1q antibody.

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

[0047] The nucleotide sequence encoding the C1q antibody heavy chain variable region is as SEQ ID NO:15 shows: GAGGTTCAGTTGCAGCAGTTTGGAACAGAGTTGGTGAAGCCAGGAGCAAGCGTGAAGATTAGTTGTAAGGCAAGCGGGTATACATTCACAGACTATAACATGGACTGGGTGAAGCAGAGCCACGGAAAGAGCCTGGAGTGGATCGGAGACATTAACCCCGATTACGACGGCACAATCT ACAACCAGAAATTCAAGGGCAAGGCCACCCTGACAGTGGACAAGTCTAGCAGCACAGCATATATGGAACTGAGGAGTCTGACCAGCGAAGATACTGCTGTGTACTACTGTGCAAGGAGGGTGTACGACTCCGACGTTTGGTACTTCGACGTGTGGGGAGCAGGCACAACCGTTACAGTGAGCTCC;

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

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

[0050] Example 2 C1q antibody performance verification

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

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

[0053] (3) Binding: The C1q antibody (anti-C1q) described in Example 1 was added to the solid-phase antigen. The initial concentration of the C1q antibody added to the first well was 400 nM. The antibody was diluted 3-fold with casein blocking solution, and 11 gradients were diluted in sequence. The last well was left blank as a blank control. At the same time, the C1q antibody was added to the solid-phase antigen at a concentration of 400 nM. The C1q antibody was diluted 3-fold with casein blocking solution, and 11 gradients were diluted in sequence. The last well was left blank as a blank control. ,Jonathan Wickel, Nina Hahn, et al. Microgliamediate neurocognitive deficits by eliminating C1q-tagged synapses in sepsis-associated encephalopathy. Sci Adv. 2023, 9(21): eabq7806.) reported C1q antibody as positive control (PC), Mouse IgG as negative control (NC), the specific binding steps are the same as the steps of adding C1q; binding at 25°C for 1 hour, washing the plate 6 times, patting dry, and obtaining a solid phase antigen-antibody complex;

[0054] (4) Horseradish peroxidase labeling: dilute the horseradish peroxidase-labeled anti-mouse Fc specific antibody (Anti-MouseFc specific HRP) with casein blocking solution at a ratio of 1:65000 to obtain the diluted horseradish peroxidase-labeled anti-mouse Fc specific antibody; add the diluted horseradish peroxidase-labeled anti-mouse Fc specific antibody to the solid phase antigen-antibody complex, adding 100 μL to each well; label at 25°C for 1 hour, wash the plate 6 times, and pat dry to obtain the label;

[0055] (5) Color development: Add color development solution (Biyuntian, NO: P0209) to the marker, add 100 μL to each well; perform color development reaction at 25°C in the dark;

[0056] (6) Termination: After 4 min of color development, add 50 μL of stop solution (Biyuntian, NO: P0215) to each well to terminate the color development reaction;

[0057] (7) Reading: Read the OD of each well using an ELISA reader 450 Value, the result is Figure 1 shown.

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

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

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

[0061] (2) adding the target cell suspension to a 96-well cell culture plate, adding 100 μL to each well to obtain a cell culture plate containing the target cell suspension;

[0062] (3) Adding 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, wherein the final concentration of the C1q antibody solution in human serum is 20 μg / mL, mixing to obtain a mixed solution, and then adding 50 μL of the mixed solution to each well of the cell culture plate containing the target cell suspension and incubating for 30 min;

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

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

[0065] from Figure 2 It can be seen 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, and a medicament for inhibiting activation of the classical complement pathway and its application. The antibody has good affinity with the C1q antigen and can effectively inhibit activation of the classical complement pathway.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection 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. Use of the C1q antibody according to claim 1 or 2 in inhibiting activation of the classical complement pathway.

4. 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.

5. Use of the C1q antibody according to claim 1 or 2 in the preparation of a medicament for treating diseases associated with activation of the classical complement pathway.

6. The use according to claim 5, characterized in that: The diseases associated with activation of the classical complement pathway include Guillain-Barré syndrome, amyotrophic lateral sclerosis, Huntington's disease, warm autoimmune hemolytic anemia or autoimmune thrombocytopenic purpura.

7. Use of the C1q antibody according to claim 1 or 2 in the preparation of a medicament for inhibiting rejection reaction after organ transplantation.

8. 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.

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