CN114149501B - anti-C5 antibodies and uses thereof - Google Patents
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Abstract
The invention provides an anti-C5 antibody and application thereof, wherein the variable region of the anti-C5 antibody comprises the amino acid sequences shown in SEQ ID NO 19-20, SEQ ID NO 21-22 or SEQ ID NO 23-24. The anti-C5 antibodies CR3#24, CR3#30 and CR3#75, especially CR3#75, have remarkable binding capacity with human recombinant C5 protein, show remarkable CDC inhibition effect, have better binding activity compared with Eculizumab and C5, have long half-life, good solubility and good thermal stability, and have potential application value in the aspect of pharmaceutical development.
Description
The present application claims priority from patent application No. 202011459619.0 (the date of application of the previous application is 12/11 in 2020, the name of the invention is anti-C5 antibody and its use).
Technical Field
The invention belongs to the technical field of biological medicines, and relates to an anti-C5 antibody and application thereof.
Background
The complement system consists of more than 30 plasma proteins and membrane proteins, is widely present on blood, tissue fluid and cell surfaces, promotes phagocytosis of phagocytes and the ability of dissolving target cells, and is an important component of the immune defense mechanism of the organism. Of these, C5 is an important complement, C5 convertase breaks down C5 into C5a and C5b during the inflammatory reaction, C5a is free in the liquid phase and is an important inflammatory mediator, and C5b forms a Membrane Attack Complex (MAC) through a series of complement reactions, leading to cell lysis and death.
Normally complement activation and inhibition mechanisms work together to maintain a healthy body state, while abnormal autoimmunity is produced when C5 is overactivated due to certain conditions. For example, paroxysmal Nocturnal Hemoglobinuria (PNH) is caused by impaired C5 inhibition due to CD59 deficiency of PNH erythrocytes, and atypical hemolytic uremic syndrome (aHUS) is caused by excessive activation of the alternative complement pathway resulting in the formation of a membrane attack complex and damage of microvascular endothelial cells, all of which share the mechanism of action of excessive activation of C5.
Disclosure of Invention
The invention provides an anti-C5 antibody and application thereof, wherein the anti-C5 antibody can be combined with C5 with high affinity, prevents the anti-C5 antibody from being cracked by C5 invertase, is beneficial to blocking the formation of a membrane attack complex, and finally achieves the aim of relieving and improving clinical symptoms.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an anti-C5 antibody comprising a heavy chain variable region and a light chain variable region;
the heavy chain variable region comprises CDR3 shown in SEQ ID NO. 3, SEQ ID NO. 9 or SEQ ID NO. 15;
the light chain variable region comprises CDR3 as shown in SEQ ID NO. 6, SEQ ID NO. 12 or SEQ ID NO. 18.
Preferably, the heavy chain variable region further comprises CDR1 as shown in SEQ ID NO. 1, SEQ ID NO. 7 or SEQ ID NO. 13.
Preferably, the heavy chain variable region further comprises CDR2 as shown in SEQ ID NO. 2, SEQ ID NO. 8 or SEQ ID NO. 14.
Preferably, the light chain variable region further comprises CDR1 as shown in SEQ ID NO. 4, SEQ ID NO. 10 or SEQ ID NO. 16.
Preferably, the light chain variable region further comprises CDR2 as shown in SEQ ID NO. 5, SEQ ID NO. 11 or SEQ ID NO. 17.
In the invention, the CDRs 1-3 of the heavy chain variable region and the CDRs 1-3 of the light chain variable region of the antibody jointly determine the specific recognition binding capacity of the antibody to antigen, and the antibody containing the CDRs of SEQ ID NO. 1-6, SEQ ID NO. 7-12 and SEQ ID NO. 13-18 has remarkable binding capacity to C5 protein and shows remarkable CDC inhibition effect.
In a specific embodiment, the heavy chain variable region of the anti-C5 antibody CR3# 24 comprises CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
the light chain variable region of the anti-C5 antibody CR3# 24 comprises a CDR1 shown in SEQ ID NO. 4, a CDR2 shown in SEQ ID NO. 5 and a CDR3 shown in SEQ ID NO. 6;
SEQ ID NO:1：DYYMH；
SEQ ID NO:2：VVDPEDGEIIYAEKFQG；
SEQ ID NO:3：SDYGAGSYMVDP；
SEQ ID NO:4：GGNNLESKIVS；
SEQ ID NO:5：YDTDRPS；
SEQ ID NO:6：QVWDSNSDHVI。
in the invention, the anti-C5 antibody CR3# 24 comprising the heavy chain variable region CDR of SEQ ID NO 1-3 and the light chain variable region CDR of SEQ ID NO 4-6 has the C5 protein binding activity and shows obvious CDC inhibition effect.
Preferably, the heavy chain variable region of the anti-C5 antibody CR# 24 comprises the amino acid sequence shown in SEQ ID NO. 19, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 20;
SEQ ID NO:19：
EVQLVQSGAEVKKPGAAVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGVVDPEDGEIIYAEKFQGRITITADTSTDTVYMELSSLRSEDTAVYYCARSDYGAGSYMVDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；
SEQ ID NO:20：
SYVLTQPPSVSVTPGKTARIACGGNNLESKIVSWYQQKPGQAPVLVIFYDTDRPSGISERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSNSDHVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVATAECS。
in a specific embodiment, the heavy chain variable region of the anti-C5 antibody CR3# 30 comprises CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8, CDR3 shown in SEQ ID NO. 9;
the light chain variable region of the anti-C5 antibody CR3# 30 comprises a CDR1 shown in SEQ ID NO. 10, a CDR2 shown in SEQ ID NO. 11 and a CDR3 shown in SEQ ID NO. 12;
SEQ ID NO:7：SYYMH；
SEQ ID NO:8：IINPSGGSTSYAQKFQG；
SEQ ID NO:9：GTVDDAFDI；
SEQ ID NO:10：TGTSSDVGGYDLVS；
SEQ ID NO:11：DVSKRPS；
SEQ ID NO:12：SSFADSNNWEVV。
in the invention, the anti-C5 antibody CR3# 30 comprising the heavy chain variable region CDR of SEQ ID NO 7-9 and the light chain variable region CDR of SEQ ID NO 10-12 has the C5 protein binding activity and shows obvious CDC inhibition effect.
Preferably, the heavy chain variable region of the anti-C5 antibody CR3# 30 comprises the amino acid sequence shown in SEQ ID NO. 21, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 22;
SEQ ID NO:21：
QVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGTVDDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；
SEQ ID NO:22：
QSALTQPPSASGSPGQSVTISCTGTSSDVGGYDLVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTVSGLQADDEADYYCSSFADSNNWEVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWESHRSYSCQVTHEGSTVEKTVAPTECS。
in a specific embodiment, the heavy chain variable region of anti-C5 antibody CR3# 75 comprises CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 15;
the light chain variable region of the anti-C5 antibody CR3# 75 comprises a CDR1 shown in SEQ ID NO. 16, a CDR2 shown in SEQ ID NO. 17 and a CDR3 shown in SEQ ID NO. 18.
SEQ ID NO:13：DYYTH；
SEQ ID NO:14：LVDPEGGETIYAEKFQG；
SEQ ID NO:15：GSFLAAPDY；
SEQ ID NO:16：KSSQSLLYNSNNKNYLA；
SEQ ID NO:17：WASTRES；
SEQ ID NO:18：QQYYSTPLT。
In the invention, the anti-C5 antibody CR3# 75 comprising the heavy chain variable region CDR of SEQ ID NO 13-15 and the light chain variable region CDR of SEQ ID NO 16-18 has better binding activity with C5 than that of Izorimab (Soliris/Alexion), shows very strong CDC inhibition effect, has long half-life, can stay for a long time, and can reduce the administration frequency and the administration dosage and achieve the treatment purpose.
Preferably, the heavy chain variable region of the anti-C5 antibody CR3# 75 comprises the amino acid sequence shown in SEQ ID NO. 23, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 24;
SEQ ID NO:23：
EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYTHWVQQAPGKGLEWMGLVDPEGGETIYAEKFQGRVTITADASTDTAYMELSSLRSEDTAVYYCATGSFLAAPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；
SEQ ID NO:24：
DIVMTQAPDSLAVSLGERATVNCKSSQSLLYNSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYSTPLTLGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
preferably, the anti-C5 antibody further comprises a constant region.
Preferably, the constant region is derived from any of IgG4, igG1 or IgG2, preferably IgG4.
Preferably, the IgG4 has an S228P mutation, which helps to improve the stability of IgG4.
Preferably, the IgG1 has the N297A and/or K322A mutation.
Preferably, the IgG2 has an S257A mutation.
In the present invention, mutation sites N297A and K322A on IgG1 are used to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), respectively.
Preferably, the IgG2 has low antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and may have the S257A mutation.
Preferably, the anti-C5 antibody molecule may be oligomerized, glycosylated or modified conjugated to a label at the N-terminus, the internal or the C-terminus, thereby modulating the function of the antibody.
In a second aspect, the invention provides a nucleic acid molecule comprising a DNA fragment encoding the anti-C5 antibody of the first aspect.
In a third aspect, the invention provides an expression vector comprising a nucleic acid molecule according to the second aspect.
In a fourth aspect, the invention provides a recombinant cell expressing an anti-C5 antibody according to the first aspect.
Preferably, the recombinant cell has integrated into its genome the nucleic acid molecule of the second aspect.
Preferably, the recombinant cell comprises the expression vector of the third aspect.
In a fifth aspect, the present invention provides a method for preparing the anti-C5 antibody according to the first aspect, the method comprising the steps of:
(1) Connecting the encoding nucleic acid of the anti-C5 antibody into a plasmid, transferring into competent cells, culturing, and selecting monoclonal cells for screening;
(2) Extracting the expression vector of the screened positive clone, transferring the expression vector into host cells, culturing, collecting supernatant, and separating and purifying to obtain the antibody.
In a sixth aspect, the invention provides a pharmaceutical composition comprising an anti-C5 antibody according to the first aspect.
Preferably, the pharmaceutical composition further comprises an anti-tumor drug.
Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.
In a seventh aspect, the invention provides the use of an anti-C5 antibody according to the first aspect, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fourth aspect or a pharmaceutical composition according to the sixth aspect for the preparation of a disease detection agent and/or a disease treatment medicament.
Preferably, the disease comprises an autoimmune disease caused by excessive activation of C5.
Compared with the prior art, the invention has the following beneficial effects:
(1) The anti-C5 antibodies CR3# 24, CR3# 30 and CR3# 75, especially CR3# 75, have remarkable binding capacity with human recombinant C5 protein, show remarkable CDC inhibition effect, and have better binding activity compared with the control antibody Eculizumab and C5, wherein the dissociation equilibrium constant KD of the CR3# 75 is 0.366-0.722 nM;
(2) The anti-C5 antibody cr3# 75 of the present invention shows excellent pharmacokinetics in a mouse model with a half-life of up to 15 days;
(3) The CR3# 75/IgG4 monoclonal antibody has stable structure, isoelectric point of 6.0-6.9, good solubility (1 mg/mL) at low concentration level, good thermal stability and Tm value of 69.83 ℃; the CR3# 75/IgG1 (N297A/K322A) has better thermal stability than the CR3# 75/IgG 4;
(4) The anti-C5 antibody has potential application value in preventing and/or treating autoimmune diseases caused by excessive activation of C5.
Drawings
FIG. 1 shows ELISA affinity detection results of 128C 5 specific binding peptide fragments obtained by 3 rounds of screening;
FIG. 2 is a SDS-PAGE electrophoresis of C5-specific antibodies containing IgG constant regions, wherein M-protein molecular weights Marker,1-CR3# 24,2-CR3# 30,3-C5 Ref,4-CR3# 25,5-CR3#29,6-CR3#37,7-CR3#47,8-CR3# 53,9-CR3# 56, 10-CR3# 74, 11-CR3# 75, 12-CR3#79, 13-CR3# 110, 14-CR3# 112, 15-CR3# 115;
FIG. 3 shows the results of the detection of the activity of the screened C5-specific antibodies in binding to the C5 protein;
FIG. 4A shows the result of the CDC inhibitory effect of the selected C5-specific antibodies, and FIG. 4B shows the result of the CDC inhibitory effect of different concentrations of the C5-specific antibodies;
FIG. 5A is a dissociation curve of Eculizumab, and FIG. 5B is a dissociation curve of CR3# 75;
FIG. 6 is a metabolic profile of CR3# 75/IgG4 in mice;
FIG. 7 shows the results of an integrity analysis of CR3# 75/IgG4, in which FIG. I is a graph of non-reducing conditions and FIG. II is a graph of reducing conditions;
FIG. 8 shows the isoelectric point of CR3# 75/IgG4, wherein the M-protein has a molecular weight Marker,1-CR3# 75/IgG4 monoclonal antibody;
FIG. 9 is a SEC-HPLC analysis result of low concentration CR3# 75/IgG4 (> 1 mg/mL);
FIG. 10 shows the results of a thermal stability analysis of CR3# 75/IgG 4;
FIG. 11 is a SEC-HPLC analysis result of high concentration CR3# 75/IgG4 (> 10 mg/mL);
fig. 12A is a statistical picture of the area of missing AQP4 lesions in the brain tissue of mice, and fig. 12B is a statistical picture of the area of missing GFAP lesions in the brain tissue of mice.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
EXAMPLE 1 screening of active peptide fragments of C5 antibodies
In order to obtain a C5 antibody having a therapeutic effect, the present example first screens peptide fragments having an activity of specifically binding to C5 from phage antibody library OmniMab. B cells from hundreds of healthy donors were collected in phage antibody library Omnimab (AP Biosciences Inc.), and antibody display was performed based on Hyperphage (M13K07.DELTA.ρI I, progen, heidelberg, germany). Full length C5 reference NCBI Reference Sequence NP-001726.2 antigen and recombinant expression using HEK293 cells (Sino Biologica, cat # 13416-H18H) was used to enrich and isolate specific binding peptides from the Omnimab pool and HEK293 cells not expressing C5 were used as controls.
Through 3 rounds of screening (CR 3) and enrichment, C5-specific binding peptides were identified and isolated by ELISA. As shown in fig. 1, from clone-specific recognition of C5 activity, sequence analysis was performed on cr3# 1 to cr3#128, confirming sequence information and diversity of heavy and light chains, and 15 candidate samples were selected therefrom for subsequent screening. Wherein the heavy chain variable region and the light chain variable region of the CR3# 24 are shown in SEQ ID NOS.19-20, the heavy chain variable region and the light chain variable region of the CR3# 30 are shown in SEQ ID NOS.21-22, and the heavy chain variable region and the light chain variable region of the CR3# 75 are shown in SEQ ID NOS.23-24.
EXAMPLE 2 expression and purification of C5-specific antibodies containing IgG constant regions
To further evaluate physicochemical properties of the C5-specific binding peptide after formation of the intact antibody, the heavy and light chains of the 15 samples obtained in example 1 were subcloned onto an IgG expression vector (AP Biosciences inc.) carrying an IgG4 (S228P) constant region, wherein the mutation sites on IgG4 were used to increase the stability of IgG4.
After sequencing the recombinant plasmid to identify positive clones with the correct insertion of the fragment of interest, a sufficient amount of sterile recombinant plasmid was prepared using a plasmid extraction kit and transfected to 6X 10 with a transfection reagent (Invitrogen) 6 Antibody expression was performed in individual expcho. Recombinant ExpiCHO cells were cultured at 37℃with 5% CO 2 The antibody was secreted into serum-free cell culture supernatant for 6 days, the antibody was affinity purified from the cell culture supernatant by protein A chromatography (Protein A chromatography), concentrated and then placed in DPBS buffer for lysis, protein concentration was detected by Nanodrop2000, purity and integrity were detected by SDS-PAGE under non-reducing and reducing conditions, and protein loading was 5. Mu.g/lane. After coomassie brilliant blue staining of the gel, ddH was used 2 And (3) cleaning. A control group (C5 Ref) of Icalicheamicin (Eculizumab, soliris/Alexion) was also set.
As shown in FIG. 2, the integrity and purity of the C5 antibody secreted by the ExpiCHO cells reached 95% or more, and there were few visible fragments in SDS-PAGE gel, indicating that the screening, subcloning, expression, and purification steps of the antibody did not affect the structural stability of the antibody protein.
Example 3 detection of binding Activity of C5-specific antibodies based on direct ELISA
In this example, the binding activity of the C5-specific antibody to the human recombinant C5 protein was detected by direct ELISA, and a control group (C5 Ref) of Icalicheazumab (Eculizumab, soliris/Alexion) was set. The method comprises the following steps:
human C5 protein (Sino biological, cat # 13416-H18H) was diluted to 1. Mu.g/mL with 1 XPBS, added to ELISA plates at 100. Mu.L/well, incubated overnight at 4℃for coating treatment; after the coating solution was removed, 350. Mu.L of 1% BSA blocking solution was added to each well, and the wells were blocked at room temperature for 1 hour;
preparing gradient diluted antibody solution to be detected (3 times of dilution is carried out from 30nM, 10 times of dilution is carried out, and solution without antibody is added for detection, and total concentration is 12), adding 100 mu L of antibody solution to be detected with different concentrations into each hole of the prepared ELISA plate, and incubating for 1h under the condition of room temperature oscillation; the supernatant was discarded, 350. Mu.L/well of wash solution (0.05% Tween-20+1 XDPBS) was added and washed 3 times;
100. Mu.L/well of HRP-labeled antibody anti-Fc-Ab-HRP (0.4. Mu.g/mL) (Jackson Immuno Research, cat# 109-006-098) was added and incubated for 30min at room temperature with shaking; discarding the supernatant, adding 350 mu L/hole of washing liquid, and washing for 3 times;
after TMB is added for color development for 1-5 min, 50 mu L of stop solution (1N HCl) is added for stopping the reaction, and the reaction is placed on an enzyme-labeled instrument for reading the light absorption value at 450 nm.
As a result, as shown in fig. 3, the screened cr3# 75 had activity to bind to C5 protein very close to C5 Ref, and cr3# 24, cr3# 30, cr3# 53 also had significant C5 protein binding activity.
Example 4 functional validation of C5-specific antibodies based on CDC inhibition experiments
The C5-specific antibodies are capable of blocking CDC activation, and this example detects the function of the C5-specific antibodies based on CDC experiments.
The method comprises the following steps:
to 2X 10 4 mu.g/mL Rituximab (Rituximab) was added to each Raji cell, incubated at room temperature for 20min, followed by 20. Mu.g/mL of anti-C5 antibody and complement activation(12.5%) to a final volume of 200 μl;
incubation was carried out at 37℃for 2h, staining of dead cells with propidium iodide (propidium iodide) and flow detection (Attune NxT Flow Cytometer system, thermoFisher SCIENTIFIC).
As shown in fig. 4A and 4B, cr3# 24, cr3# 30, cr3# 75 exhibited significant CDC inhibition, with cr3# 75 having the strongest neutralizing activity, comparable to that of the control antibody (C5 Ref, soliris).
Example 5 affinity detection of C5-specific antibodies with human recombinant C5 protein
The embodiment adopts
(Menlo Park, calif.) biosensors were analyzed for affinity for CR3# 75 and C5, and an Icalicheamicin (Eculizumab) control (C5 Ref) was set. The method comprises the following steps:
briefly, antibodies were loaded onto an AHC (Anti-Human IgG Fc Capture) biosensor (Cat # 18-5060) at a concentration of 5 μg/mL, the sensor was equilibrated off-line in assay buffer, monitored on-line until the signal reached about 0.5nm;
the antibody-loaded sensors were exposed to different concentrations (0.4688 nM, 0.9375nM, 1.88nM, 3.75nM, 7.5nM, 15nM and 30 nM) of C5 (Sino Biologica, cat# 13416-H18H), followed by incubation in PBST for 1min to determine the baseline, incubation in different concentrations of C5 solution for 5min to detect the binding rate of the antibody to C5, and incubation in PBST for 5min to detect the dissociation rate of the antibody from C5. Kinetic parameters were analyzed using the Octet Data software.
The results are shown in tables 1, 2, 5A and 5B, and compared with the control antibody ecllizumab (kd=0.371 to 0.553 nM), cr3# 75 has better binding activity to C5, and dissociation equilibrium constant KD is 0.366 to 0.722nM.
TABLE 1 binding force of Eculizumab to C5
| Numbering device | KD(M) | kon(1/Ms) | koff(1/s) |
| 1 | 5.53E-10 | 5.44E+05 | 3.00E-04 |
| 2 | 3.77E-10 | 3.94E+05 | 1.49E-04 |
| 3 | 3.99E-10 | 3.81E+05 | 1.52E-04 |
| 4 | 4.35E-10 | 3.77E+05 | 1.64E-04 |
| 5 | 3.71E-10 | 3.51E+05 | 1.30E-04 |
TABLE 2 binding force of CR3# 75 to C5
| Numbering device | KD(M) | kon(1/Ms) | koff(1/s) |
| 1 | 3.66E-10 | 4.13E+05 | 1.51E-04 |
| 2 | 6.14E-10 | 4.88E+05 | 3.00E-04 |
| 3 | 6.16E-10 | 4.52E+05 | 2.79E-04 |
| 4 | 7.22E-10 | 4.25E+05 | 3.07E-04 |
| 5 | 7.20E-10 | 4.29E+05 | 3.09E-04 |
EXAMPLE 6 pharmacokinetic testing
This example evaluates the half-life, AUC (area under the curve), cmax, cl (clearence) and Vd (volume of distribution) of the C5-specific antibody CR3# 75/IgG4 in mice.
SCID-Bg mice (CB.17/. Cg-PrkdcsccidLystbg-J/CrlBltw) were intravenously injected with 5mg/kg of CR3# 75/IgG4 monoclonal antibody, and blood samples were collected from the tail vein at various time points. The concentration of cr3# 75 in serum was measured by direct ELISA and pharmacokinetic parameters were calculated by PKSolver (ver.2.0).
The results are shown in tables 3, 4 and 6, and the cr3# 75 shows excellent pharmacokinetics in a mouse model, and has a half-life as long as 361.5h (15 days in a fold), which indicates that the antibody can be metabolized slowly in the mouse body for a long time, the administration frequency can be reduced, and the therapeutic purpose can be achieved by administering a low dose of the antibody in animal experiments.
TABLE 3 Table 3
| Time (h) | Concentration (μg/mL) |
| 0.5 | 141.692 |
| 24 | 59.527 |
| 72 | 53.084 |
| 168 | 38.222 |
| 240 | 42.075 |
| 336 | 28.307 |
| 504 | 12.811 |
| 672 | 23.378 |
TABLE 4 Table 4
| Parameters (parameters) | Unit (B) | Numerical value |
| t1/2 | h | 361.451 |
| Cmax | μg/mL | 141.69 |
| AUC 0-t | μg/mL×h | 22283.99 |
| Vz_obs | (mg/kg)/(μg/mL) | 0.0756 |
| Cl_obs | (mg/kg)/(μg/mL)/h | 0.000145 |
| Vss_obs | (mg/kg)/(μg/mL) | 0.0838 |
EXAMPLE 7 analysis of the integrity of the CR3# 75/IgG4 monoclonal antibody
This example uses LabChip GXII to detect the integrity of purified CR3# 75/IgG4 monoclonal antibody. The method comprises the following steps:
As a result, as shown in Table 5 and FIG. 7, the complete CR3# 75/IgG4 monoclonal antibody (M.W.172.75 kDa) was detected under non-reducing conditions, and the purity of the complete CR3# 75/IgG4 monoclonal antibody was 100% as determined by protein A chromatography (Protein A chromatography), indicating that the heavy and light chains of the antibody were properly paired and that the antibody maintained structural stability during preparation and purification. Under reducing conditions, the sum of heavy and light chains (hc+lc) also reached 96.67%, indicating that most of the impurities could be removed by a single purification step.
TABLE 5
Example 8 isoelectric point detection
This example uses IEF gel electrophoresis to detect the charge properties of the CR3# 75/IgG4 monoclonal antibody. The method comprises the following steps:
mu.L of protein sample and 5 mu.L of 2 Xsample buffer are mixed, 200mL of 1 XIEF cathode buffer or 600mL of 1 XIEF anode buffer are respectively filled in the upper layer buffer chamber and the lower layer buffer chamber of the electrophoresis device XCell SureLockTMMini-Cell (Invitrogen), and electrophoresis conditions are 100V electrophoresis for 1h,200V electrophoresis for 1h and 500V electrophoresis for 30min. After electrophoresis, the gel was fixed with 12% TCA for 30min, and after staining with Coomassie brilliant blue, gel imaging was performed.
As a result, as shown in FIG. 8, the charge distribution of CR3# 75/IgG4 was not uniform, and the isoelectric point detected by IEF gel electrophoresis was 6.0 to 6.9, and the predicted isoelectric point was 5.92.
EXAMPLE 9 aggregation analysis
After purification and concentration of the CR3# 75/IgG4 antibody, SEC-HPLC analysis was performed. The method comprises the following steps:
concentrated protein samples (> 1 mg/mL) were mixed with an equal volume of mobile phase buffer (25 mM phosphate, 200mM NaCl,pH 6.8), filtered through a 0.22 μm filter (Millipore, cat#SLGP003 RB), loaded to XBridge Protein BEH SEC Column (Waters, cat# 186007640), SEC separated at 25℃with a flow rate of 0.4mL/min, sample addition volume of 10. Mu.L, and absorbance measured at 280 nm. And finally, adopting Empower 2 to analyze data.
As shown in FIG. 9, the main peak at 17.839min showed 99.9% purity of the protein sample at a concentration of 1.014mg/mL, indicating that CR3# 75/IgG4 remained well soluble in DPBS with substantially no aggregation precipitation.
Example 10 thermal stability analysis
This example uses differential scanning calorimetry (differential scanning calorimetry, DSC) to detect the thermal stability of antibodies. The method comprises the following steps:
protein samples at a concentration of 0.2 to 1.0mg/mL were prepared, DSC detection was performed at a rate of 200 ℃/h from 10 to 100℃using a MicroCal PEAQ-DSC system (Northampton, malvern), a thermogram was drawn, and the thermal stability analysis of the antibody was performed based on the drawn thermogram.
FIG. 10 shows the melting curve of CR3# 75/IgG4 with a Tm value of 69.83℃indicating that the antibody has normal thermal stability.
EXAMPLE 11 solubility analysis
After purification and concentration of the CR3# 75/IgG4 antibody, SEC-HPLC analysis was performed. The method comprises the following steps:
concentrated protein samples (> 10 mg/mL) were mixed with an equal volume of mobile phase buffer (25 mM phosphate, 200mM NaCl,pH 6.8), filtered through a 0.22 μm filter (Millipore, cat#SLGP003 RB), loaded to XBridge Protein BEH SEC Column (Waters, cat# 186007640), SEC separated at 25℃with a flow rate of 0.4mL/min, sample addition volume of 2. Mu.L, and absorbance measured at 280 nm. And finally, adopting Empower 2 to analyze data.
As shown in FIG. 11, the CR3# 75/IgG4 antibody could be concentrated to 10mg/mL without macroscopic precipitation, 97.15% protein produced a single peak at 18.005min, with only < 4% protein forming a precipitate, indicating that the antibody was normally soluble and continued to be developed as a pharmaceutical.
EXAMPLE 12 in vivo efficacy
This example demonstrates the in vivo efficacy of a cr3# 75/IgG4 antibody in a NMO mouse model, as follows:
the 6-8 week old C57 female mice are anesthetized with chloral, the heads of the mice are fixed on a brain stereotactic instrument, holes are drilled after alignment and positioning, a microscale sample injection needle is vertically inserted into brain tissues of the mice, an injection pump is started, a CR3# 75/IgG4 antibody, complement and pathogenic antibody mixed solution is injected, the needle is pulled out after injection is completed, the skin is sutured, and the mice are placed in a cage for feeding. And taking out brain tissues after 7 days of molding to prepare frozen tissue slices, staining the AQP4 and GFAP expressed by astrocytes by adopting an immunofluorescence technology, and counting the missing areas of focuses of the AQP4 and GFAP in each group.
As shown in FIGS. 12A and 12B, the CR3# 75/IgG4 antibody inhibited NMO-typical lesion formation at concentrations of 0.04mg/mL, 0.156mg/mL, 0.625mg/mL, and 2.5 mg/mL.
EXAMPLE 13 antibody constant region replacement
In this example, while maintaining the variable regions of CR3# 30/IgG4 and CR3# 75/IgG4 unchanged, the heavy chain constant regions were replaced with IgG1 (N297A/K322A), igG1 (N297A) and IgG2, resulting in 6 completely novel antibodies with the following amino acid sequences:
QVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGTVDDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
QVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGTVDDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
QVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGTVDDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
the CR3# 30/IgG1 (N297A/K322A), the CR3# 30/IgG1 (N297A) and the CR3# 30/IgG2 all adopt CR3# 30/lambda light chains, and the sequences are shown as SEQ ID NO: 28:
SEQ ID NO:28：
QSALTQPPSASGSPGQSVTISCTGTSSDVGGYDLVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTVSGLQADDEADYYCSSFADSNNWEVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS。
EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYTHWVQQAPGKGLEWMGLVDPEGGETIYAEKFQGRVTITADASTDTAYMELSSLRSEDTAVYYCATGSFLAAPDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYTHWVQQAPGKGLEWMGLVDPEGGETIYAEKFQGRVTITADASTDTAYMELSSLRSEDTAVYYCATGSFLAAPDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；
EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYTHWVQQAPGKGLEWMGLVDPEGGETIYAEKFQGRVTITADASTDTAYMELSSLRSEDTAVYYCATGSFLAAPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
the CR3# 75/IgG1 (N297A/K322A), the CR3# 75/IgG1 (N297A) and the CR3# 75/IgG2 all adopt CR3# 75/kappa light chains, and the sequences are shown as SEQ ID NO. 32.
SEQ ID NO:32：
DIVMTQAPDSLAVSLGERATVNCKSSQSLLYNSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYYSTPLTLGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
And (3) carrying out gene synthesis on the antibody to construct a recombinant plasmid, and preparing a sufficient amount of sterile recombinant plasmid by using a plasmid extraction kit after the recombinant plasmid is sequenced and identified to be a positive clone correctly inserted with a target fragment for producing a protein sample by transient transfection.
EXAMPLE 14 different constant region antibody production
A sterile recombinant plasmid of 9 antibodies in total of CR3# 24/IgG4, CR3# 30/IgG4, CR3# 75/IgG4, CR3# 30/IgG1 (N297A/K322A), CR3# 30/IgG1 (N297A), CR3# 30/IgG2, CR3# 75/IgG1 (N297A/K322A), CR3# 75/IgG1 (N297A) and CR3# 75/IgG2 was transfected to 6X 10 by a transfection reagent (Invitrogen) 6 Antibody expression was performed in individual expcho.
Recombinant ExpiCHO cells were cultured at 37℃with 5% CO 2 After 9-11 days of culture, the antibodies were secreted into serum-free cell culture supernatants. Protein A-HPLC was performed on the fermentation broth to detect the protein expression level. The method comprises the following steps:
after filtration of the broth samples through a 0.22 μm filter (Sartorius, cat#17C07 FT), they were loaded onto a Poros-20A Column (Thermo Scientific, cat#1-5024-12) and subjected to ProteinA-HPLC at 25℃with chromatographic parameters as shown in Table 6. Finally, data acquisition and analysis were performed using Empower 3, with the results shown in table 7.
TABLE 6 ProteinA-HPLC chromatography parameters
TABLE 7 ProteinA-HPLC detection of protein expression level of fermentation broth
After 9 batches of CHO cells were cultured for 9-11 days, the protein purity was detected by SEC-HPLC on the whole broth, as follows:
protein samples were filtered through a 0.22 μm filter (Sartorius, cat#17c07 FT), loaded onto a TSKgel G3000 SWXL SEC Column (Tosoh, cat#008541), SEC separated at 25 ℃ with a Waters e2695 detector, a Waters 2998PDA detector, a mobile phase of 50mM phosphate+400 mM sodium chloride (pH 7.0) buffer, a flow rate of 0.5mL/min, a sample volume of 10 μl, and the absorbance of each component separated was measured at 280 nm. Finally, data acquisition and analysis were performed using Empower 3, and the results are shown in table 8.
TABLE 8 SEC-HPLC detection of fermentation broth purity
| Sample numbering | Sample name | High molecular weight (%) | Main peak (%) | Low molecular weight (%) |
| S125(38#)-1 | |
30.2 | 19.1 | 50.7 |
| S125(38#)-2 | |
31.6 | 19.7 | 48.6 |
| S125(38#)-3 | |
28.4 | 24.8 | 46.8 |
| S125(38#)-4 | |
29.7 | 22.7 | 47.6 |
| S125(38#)-5 | |
29.3 | 25.5 | 45.2 |
| S125(38#)-6 | |
34.6 | 10.8 | 54.7 |
| S125(38#)-7 | |
30.0 | 19.0 | 51.0 |
| S125(38#)-8 | |
29.6 | 19.8 | 50.5 |
| S125(38#)-9 | |
27.1 | 13.5 | 59.5 |
After prism A capture was performed on the fermentation broth of 9 candidate molecules, the protein concentration was determined using an ultraviolet spectrophotometer, as follows:
taking the sample solution, and taking the same batch of solvent for preparing the sample solution as a blank control. And diluting the sample by using a 1cm quartz absorption cell, and placing the quartz absorption cell filled with the sample into a detection cell for detection. The ultraviolet spectrophotometer is IMPLEN, model NP80.
The protein concentration calculation formula is as follows:
wherein X: dilution factor;
test proteinIs a coefficient of absorption of (a).
The results are shown in Table 9.
TABLE 9 purification sample concentration
| Sample numbering | Sample name | Protein concentration (mg/mL) |
| S125(38#)-1 | |
2.5 |
| S125(38#)-2 | |
2.6 |
| S125(38#)-3 | |
2.5 |
| S125(38#)-4 | |
4.4 |
| S125(38#)-5 | |
2.4 |
| S125(38#)-6 | |
1.4 |
| S125(38#)-7 | |
3.0 |
| S125(38#)-8 | |
2.0 |
| S125(38#)-9 | |
1.4 |
After prism a capture of the 9 candidate molecule fermentation broth, SEC-HPLC analysis was performed as follows:
protein samples were filtered through a 0.22 μm filter (Sartorius, cat#17c07 FT), loaded onto a TSKgel G3000 SWXL SEC Column (Tosoh, cat#008541), SEC separated at 25 ℃, with a Waters 2695 detector, waters 2998PDA detector, 50mM phosphate+400 mM sodium chloride (pH 7.0) buffer in mobile phase, set flow rate of 0.5mL/min, sample volume of 10 μl, and absorbance at 280 nm. Finally, data acquisition and analysis were performed using Empower 3, and the results are shown in table 10.
TABLE 10 SEC-HPLC detection of purity of purified samples
In view of the above results, 9 antibodies successfully completed sample production, the concentrations were all greater than 1mg/mL, the purities were all greater than 90%, and subsequent experiments were possible.
EXAMPLE 15 aggregation analysis
In this example, the isoelectric point of each candidate molecule was determined using iCIEF, as follows:
after desalting the sample, it was diluted with distilled water to a protein concentration of about 2mg/mL and was prepared as follows (200. Mu.L system): 8 μL 2% pharmamalyte (pH 3-10) (GE, cat# 17045601), 70 μL1% MC (ProteinSimple, cat# 1018761), 1 μL Low pI (ProteinSimple, cat# 102222), 1 μL High pI (ProteinSimple, cat#101996), 100 μL 8M urea (Sigma, cat#U0631-500G 1), 20 μL sample.
The system is arranged in a centrifuge tube, 10000g of the centrifuge tube is centrifuged for 5min, the supernatant (60-120 mu L) is added into the inner cannula, the inner cannula is placed in the centrifuge tube, 10000g of the centrifuge tube is centrifuged for 5min, the inner cannula is taken out, the inner cannula is placed in a sample injection bottle, and the sample injection bottle is checked by a machine, wherein the equipment is ProteinSimple iCE. The results are shown in Table 11, where the actual isoelectric point is slightly higher than the predicted isoelectric point.
TABLE 11 isoelectric points of candidate molecules
| Sample numbering | Sample name | Prediction of isoelectric point | iCIEF-isoelectric point |
| S125(38#)-1 | |
5.97 | 6.13 |
| S125(38#)-2 | |
6.18 | 6.30 |
| S125(38#)-3 | |
5.92 | 5.89 |
| S125(38#)-4 | |
7.24 | 7.33 |
| S125(38#)-5 | |
7.56 | 7.57 |
| S125(38#)-6 | |
6.50 | 6.97 |
| S125(38#)-7 | |
6.36 | 6.58 |
| S125(38#)-8 | |
6.51 | 6.76 |
| S125(38#)-9 | |
5.97 | 6.18 |
Example 16 capillary electrophoresis analysis
After prism A capture of the fermentation broth of 9 candidate molecules, CE-SDS analysis was performed, as follows:
the protein samples were ultrafiltered with a 30 μm ultrafiltration centrifuge tube (Sartorius, cat#VS0122), then the samples were prepared, heated at 70℃and loaded for CE-SDS separation at 25℃with the apparatus SCIEX PA800 Plus, detector PDA detector, absorbance at 220 nm. Data were collected and analyzed using a 32karat and the results are shown in Table 12, where CR3# 75/IgG2 was of lower purity.
TABLE 12 capillary electrophoresis detection of purified sample purity
Example 17 protein stability detection of C5-specific antibodies
This example employs a protein stability analysis system
Tm of (Unchained Labs, calif.)&Tagg with optional DLS procedure, protein particle size and stability were tested. Protein particle size and particle size distribution and thermostability were analyzed at multiple angles by analyzing the resulting parameters Tm (protein melting temperature), tagg266 (protein aggregation temperature), tagg473 (protein aggregation temperature), Z-ave. Dia (protein average particle size), PDI (polydispersity index), pk1 Mode Dia, pk 1Mass (%), pk2 Mode Dia and Pk2 Mass (%). The method comprises the following steps:
mu.L of antibody was added to a uni tube (Unchained Labs, calif.), 2 duplicate wells were repeated for each antibody, and the Tm & Tagg with optional DLS program was selected for analysis.
Particle size results at 25℃and 95℃are shown in Table 13 and Table 14, respectively, and Tm & Tagg results are shown in Table 15. Wherein, the average hydration kinetic diameter of the Z-Ave. Dia-sample reflects the overall particle size of the sample; PDI-polydispersity index, PDI <0.1 representing a narrow particle size distribution, a larger value representing a more non-uniform particle size distribution, a higher degree of dispersion; fit Var-fitting deviation less than 0.01 indicates high confidence in Z-Ave. Dia and PDI, with larger values indicating lower confidence; when the Pk1 Mode Dia-light intensity distribution shows multiple peaks, the Mode particle size of the first main peak; pk 1Mass (%) -Mass ratio of the first peak.
As can be seen from Table 13, the average hydration kinetic diameter of S125 (38#) -3/6/7/9 and Pk1 Mode Dia are both about 11nm, no large particle size component exists, PDI is less than 0.1, the particle size is uniform, and the protein monomer is presumed; the remaining samples in the group all had larger particle size components, presumably aggregates. From Table 14, it can be seen that the protein molecules significantly aggregate upon heating, as seen from the average hydration kinetic diameter and PDI. As can be seen from Table 15, S125 (38#) -4 and S125 (38#) -7Tm are higher, the thermal stability of the presumed conformation is higher, S125 (38#) -2/4/5/6/7Tagg266 is above 60 ℃, and the thermal stability of the presumed gel is higher.
Table 13 particle size results at 25℃
Table 14 particle size results at 95℃
TABLE 15 Tm & Tagg results
From the above results, it can be seen that the S125 (38#) -7 group has higher overall thermal stability, i.e., CR3# 75/IgG1 (N297A/K322A) has thermal stability superior to CR3# 75/IgG 4.
In conclusion, the anti-C5 antibody has remarkable binding capacity with human recombinant C5 protein, long half-life, good solubility and good thermal stability, and is expected to be developed into a protein drug to be applied to preventing and/or treating autoimmune diseases caused by excessive activation of C5.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Sequence listing
<110> Tianshili biological medicine Co., ltd
<120> anti-C5 antibody and use thereof
<130> 2021
<160> 32
<170> PatentIn version 3.3
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130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 20
<211> 214
<212> PRT
<213> artificial sequence
<400> 20
Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Lys
1 5 10 15
Thr Ala Arg Ile Ala Cys Gly Gly Asn Asn Leu Glu Ser Lys Ile Val
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Phe
35 40 45
Tyr Asp Thr Asp Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp His
85 90 95
Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
100 105 110
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
115 120 125
Ala Asn Lys Ala Thr Leu Val Cys Leu Val Ser Asp Phe Tyr Pro Gly
130 135 140
Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Val Gly
145 150 155 160
Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
165 170 175
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser
180 185 190
Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
195 200 205
Ala Thr Ala Glu Cys Ser
210
<210> 21
<211> 445
<212> PRT
<213> artificial sequence
<400> 21
Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Val Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 22
<211> 218
<212> PRT
<213> artificial sequence
<400> 22
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asp Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
65 70 75 80
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Phe Ala Asp Ser
85 90 95
Asn Asn Trp Glu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
130 135 140
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
145 150 155 160
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Glu
180 185 190
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
195 200 205
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 23
<211> 445
<212> PRT
<213> artificial sequence
<400> 23
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Thr His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Leu Val Asp Pro Glu Gly Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Ala Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Phe Leu Ala Ala Pro Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 24
<211> 220
<212> PRT
<213> artificial sequence
<400> 24
Asp Ile Val Met Thr Gln Ala Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Val Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Asn
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Leu Thr Leu Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
<210> 25
<211> 448
<212> PRT
<213> artificial sequence
<400> 25
Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Val Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 26
<211> 448
<212> PRT
<213> artificial sequence
<400> 26
Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Val Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 27
<211> 443
<212> PRT
<213> artificial sequence
<400> 27
Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Thr Val Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
210 215 220
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
290 295 300
Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 28
<211> 218
<212> PRT
<213> artificial sequence
<400> 28
Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asp Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu
65 70 75 80
Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Phe Ala Asp Ser
85 90 95
Asn Asn Trp Glu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
130 135 140
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
145 150 155 160
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
180 185 190
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
195 200 205
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 29
<211> 448
<212> PRT
<213> artificial sequence
<400> 29
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Thr His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Leu Val Asp Pro Glu Gly Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Ala Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Phe Leu Ala Ala Pro Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 30
<211> 448
<212> PRT
<213> artificial sequence
<400> 30
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Thr His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Leu Val Asp Pro Glu Gly Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Ala Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Phe Leu Ala Ala Pro Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 31
<211> 443
<212> PRT
<213> artificial sequence
<400> 31
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Thr His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Leu Val Asp Pro Glu Gly Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Ala Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Phe Leu Ala Ala Pro Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
210 215 220
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe
260 265 270
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr
290 295 300
Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320
Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
340 345 350
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser
385 390 395 400
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440
<210> 32
<211> 220
<212> PRT
<213> artificial sequence
<400> 32
Asp Ile Val Met Thr Gln Ala Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Val Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Asn
20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln
85 90 95
Tyr Tyr Ser Thr Pro Leu Thr Leu Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
Claims (17)
1. An anti-C5 antibody, wherein the anti-C5 antibody comprises a heavy chain variable region and a light chain variable region;
the heavy chain variable region of the anti-C5 antibody comprises a CDR1 shown as SEQ ID NO. 1, a CDR2 shown as SEQ ID NO. 2 and a CDR3 shown as SEQ ID NO. 3; the light chain variable region of the anti-C5 antibody comprises a CDR1 shown as SEQ ID NO. 4, a CDR2 shown as SEQ ID NO. 5 and a CDR3 shown as SEQ ID NO. 6;
or, the heavy chain variable region of the anti-C5 antibody comprises a CDR1 shown as SEQ ID NO. 7, a CDR2 shown as SEQ ID NO. 8 and a CDR3 shown as SEQ ID NO. 9; the light chain variable region of the anti-C5 antibody comprises a CDR1 shown as SEQ ID NO. 10, a CDR2 shown as SEQ ID NO. 11 and a CDR3 shown as SEQ ID NO. 12;
or, the heavy chain variable region of the anti-C5 antibody comprises a CDR1 shown as SEQ ID NO. 13, a CDR2 shown as SEQ ID NO. 14, and a CDR3 shown as SEQ ID NO. 15; the light chain variable region of the anti-C5 antibody comprises a CDR1 shown as SEQ ID NO. 16, a CDR2 shown as SEQ ID NO. 17 and a CDR3 shown as SEQ ID NO. 18.
2. The anti-C5 antibody of claim 1, wherein the heavy chain variable region of the anti-C5 antibody comprises the amino acid sequence set forth in SEQ ID No. 19 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID No. 20.
3. The anti-C5 antibody of claim 1, wherein the heavy chain variable region of the anti-C5 antibody comprises the amino acid sequence set forth in SEQ ID No. 21 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID No. 22.
4. The anti-C5 antibody of claim 1, wherein the heavy chain variable region of the anti-C5 antibody comprises the amino acid sequence set forth in SEQ ID No. 23 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID No. 24.
5. The anti-C5 antibody of claim 1, wherein the anti-C5 antibody further comprises a constant region.
6. The anti-C5 antibody of claim 5, wherein the constant region is derived from any one of IgG4, igG1 or IgG 2.
7. A nucleic acid molecule comprising a DNA fragment encoding the anti-C5 antibody of any one of claims 1-6.
8. An expression vector comprising the nucleic acid molecule of claim 7.
9. A recombinant cell expressing the anti-C5 antibody of any one of claims 1-6.
10. The recombinant cell of claim 9, wherein the recombinant cell has integrated into its genome the nucleic acid molecule of claim 7.
11. The recombinant cell of claim 9, wherein the recombinant cell comprises the expression vector of claim 8.
12. A method of preparing an anti-C5 antibody according to any one of claims 1 to 6, comprising the steps of:
(1) Connecting the encoding nucleic acid of the anti-C5 antibody into a plasmid, transferring into competent cells, culturing, and selecting monoclonal cells for screening;
(2) Extracting the expression vector of the screened positive clone, transferring the expression vector into host cells, culturing, collecting supernatant, and separating and purifying to obtain the antibody.
13. A pharmaceutical composition comprising an anti-C5 antibody according to any one of claims 1-6.
14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition further comprises an anti-tumor drug.
15. The pharmaceutical composition of claim 14, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent, or excipient.
16. Use of an anti-C5 antibody according to any one of claims 1-6, a nucleic acid molecule according to claim 7, an expression vector according to claim 8, a recombinant cell according to any one of claims 9-11 or a pharmaceutical composition according to any one of claims 13-15 for the preparation of a disease detection reagent, said disease being an autoimmune disease resulting from overactivation of C5.
17. Use of an anti-C5 antibody according to any one of claims 1-6, a nucleic acid molecule according to claim 7, an expression vector according to claim 8, a recombinant cell according to any one of claims 9-11 or a pharmaceutical composition according to any one of claims 13-15 for the preparation of a medicament for the treatment of a disease, which is an autoimmune disease resulting from overactivation of C5.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2021/137116 WO2022122020A1 (en) | 2020-12-11 | 2021-12-10 | Anti-c5 antibody and application thereof |
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| CN2020114596190 | 2020-12-11 | ||
| CN202011459619 | 2020-12-11 |
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| WO (1) | WO2022122020A1 (en) |
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| WO2010015608A1 (en) * | 2008-08-05 | 2010-02-11 | Novartis Ag | Compositions and methods for antibodies targeting complement protein c5 |
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| US9079949B1 (en) * | 2014-03-07 | 2015-07-14 | Alexion Pharmaceuticals, Inc. | Anti-C5 antibodies having improved pharmacokinetics |
| CN110603054A (en) * | 2017-03-06 | 2019-12-20 | 宾夕法尼亚大学理事会 | anti-C5 antibodies and uses thereof |
| WO2020051418A1 (en) * | 2018-09-06 | 2020-03-12 | The Trustees Of The University Of Pennsylvania | Humanized anti-c5 antibodies and uses thereof |
| CN111234016A (en) * | 2020-02-23 | 2020-06-05 | 北京康普美特创新医药科技有限责任公司 | Fully human monoclonal antibody of anti-complement C5 molecule and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2113516B1 (en) * | 2000-10-10 | 2014-05-21 | Genentech, Inc. | Antibodies against C5 inhibiting type II endothelial cell activation |
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2021
- 2021-11-30 CN CN202111444150.8A patent/CN114149501B/en active Active
- 2021-12-10 WO PCT/CN2021/137116 patent/WO2022122020A1/en active Application Filing
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| WO2010015608A1 (en) * | 2008-08-05 | 2010-02-11 | Novartis Ag | Compositions and methods for antibodies targeting complement protein c5 |
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| WO2020051418A1 (en) * | 2018-09-06 | 2020-03-12 | The Trustees Of The University Of Pennsylvania | Humanized anti-c5 antibodies and uses thereof |
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| CN114149501A (en) | 2022-03-08 |
| WO2022122020A1 (en) | 2022-06-16 |
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