WO2022122020A1 - Anti-c5 antibody and application thereof - Google Patents

Abstract

An anti-C5 antibody and an application thereof. A variable region of the anti-C5 antibody comprises amino acid sequences shown in SEQ ID NOs: 19-20, SEQ ID NOs: 21-22 or SEQ ID NOs: 23-24. Anti-C5 antibodies CR3#24, CR3#30 and CR3#75, especially CR3#75, have a significant binding capacity to a recombinant human C5 protein, show an obvious CDC inhibitory effect, have better binding activity to C5 than eculizumab, are long in half-life period and good in solubility and thermostability, and have a potential application value in the development of druggability.

Description

Anti-C5 antibody and its application technical field

The present application belongs to the technical field of biomedicine, and relates to anti-C5 antibodies and applications thereof.

Background technique

Complement system is composed of more than 30 kinds of plasma proteins and membrane proteins, which widely exist in blood, tissue fluid and cell surface. It promotes the ability of phagocytes to phagocytose and dissolve target cells. Among them, C5 is an important complement. In the process of inflammatory reaction, C5 convertase decomposes C5 into C5a and C5b. C5a is free in the liquid phase and is an important inflammatory mediator. C5b undergoes a series of complement reactions to form membrane attack complex ( MAC), resulting in cell lytic death.

Under normal circumstances, the activation and inhibition mechanisms of complement work together to maintain a healthy body state, but when C5 is overactivated due to certain conditions, abnormal autoimmunity occurs. For example, paroxysmal nocturnal hemoglobinuria (PNH) results from impaired C5 inhibition due to CD59 deficiency in PNH erythrocytes, and atypical hemolytic uremic syndrome (aHUS) results from overactivation of the complement pathway leading to membrane It is caused by the formation of attack complexes and damage to microvascular endothelial cells, and their common mechanism of action is the over-activation of C5.

SUMMARY OF THE INVENTION

The present application provides an anti-C5 antibody and an application thereof. The anti-C5 antibody can bind to C5 with high affinity, prevent it from being cleaved by C5 convertase, and is beneficial to block the formation of membrane attack complexes, thereby achieving remission and improving clinical outcomes. Purpose of symptoms.

In a first aspect, the application provides an anti-C5 antibody comprising a heavy chain variable region and a light chain variable region;

The heavy chain variable region includes the CDR3 shown in SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:15;

The light chain variable region includes the CDR3 shown in SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:18.

Preferably, the heavy chain variable region further comprises the CDR1 shown in SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:13.

Preferably, the heavy chain variable region further comprises the CDR2 shown in SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:14.

Preferably, the light chain variable region further comprises the CDR1 shown in SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:16.

Preferably, the light chain variable region further comprises the CDR2 shown in SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:17.

In the present application, CDR1-3 of the heavy chain variable region of the antibody and CDR1-3 of the light chain variable region jointly determine the specific recognition and binding ability of the antibody to the antigen, including SEQ ID NO: 1-6, SEQ ID NO: The antibodies against the CDRs of 7-12 and SEQ ID NOs: 13-18 have significant binding ability to C5 protein, showing obvious CDC inhibitory 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, and CDR2 shown in SEQ ID NO:3 CDR3;

The light chain variable region of the anti-C5 antibody CR3#24 includes CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and 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: QVWDNSDHVI.

In the present application, the anti-C5 antibody CR3#24 comprising the heavy chain variable region CDRs of SEQ ID NOs: 1 to 3 and the light chain variable region CDRs of SEQ ID NOs: 4 to 6 has C5 protein binding activity, showing significant CDC inhibition.

Preferably, the heavy chain variable region of the anti-C5 antibody CR#24 includes the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 20;

SEQ ID NO: 19:

SEQ ID NO: 20:

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, and CDR2 shown in SEQ ID NO:9 CDR3;

The light chain variable region of the anti-C5 antibody CR3#30 includes CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and 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 present application, the anti-C5 antibody CR3#30 comprising the heavy chain variable region CDRs of SEQ ID NOs: 7 to 9 and the light chain variable region CDRs of SEQ ID NOs: 10 to 12 has C5 protein binding activity, showing significant CDC inhibition.

Preferably, the heavy chain variable region of the anti-C5 antibody CR3#30 includes the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 22;

SEQ ID NO: 21:

SEQ ID NO: 22:

In a specific embodiment, the heavy chain variable region of the anti-C5 antibody CR3#75 comprises CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR2 shown in SEQ ID NO:15 CDR3;

The light chain variable region of the anti-C5 antibody CR3#75 includes CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and 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 present application, the anti-C5 antibody CR3#75 comprising the heavy chain variable region CDRs of SEQ ID NOs: 13 to 15 and the light chain variable region CDRs of SEQ ID NOs: 16 to 18 was compared to iculizumab (Eculizumab, Soliris/Alexion) has better binding activity to C5, showing very strong CDC inhibitory effect, long half-life, long-term retention, and reducing the frequency and dosage of administration can also achieve therapeutic purposes.

Preferably, the heavy chain variable region of the anti-C5 antibody CR3#75 includes the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 24;

SEQ ID NO: 23:

SEQ ID NO: 24:

Preferably, the anti-C5 antibody further comprises a constant region.

Preferably, the constant region is derived from any one of IgG4, IgG1 or IgG2, preferably IgG4.

Preferably, the IgG4 has the S228P mutation, which helps to improve the stability of the IgG4.

Preferably, the IgG1 has the N297A and/or K322A mutation.

Preferably, the IgG2 has the S257A mutation.

In this application, the mutation sites N297A and K322A on IgG1 were used to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), respectively.

Preferably, the IgG2 has lower antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and may have the S257A mutation.

Preferably, the anti-C5 antibody molecule can be modified at the N-terminus, internally or at the C-terminus by oligomerization, glycosylation or conjugation with a label, thereby modulating the function of the antibody.

In a second aspect, the present application provides a nucleic acid molecule comprising a DNA fragment encoding the anti-C5 antibody of the first aspect.

In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.

In a fourth aspect, the present application provides a recombinant cell expressing the anti-C5 antibody of the first aspect.

Preferably, the nucleic acid molecule of the second aspect is integrated into the genome of the recombinant cell.

Preferably, the recombinant cell comprises the expression vector of the third aspect.

In a fifth aspect, the application provides a method for preparing the anti-C5 antibody described in the first aspect, the preparation method comprising the following steps:

(1) connecting the nucleic acid encoding the anti-C5 antibody into a plasmid, transferring it into competent cells, and picking monoclonal cells for screening after culturing;

(2) Extracting the expression vector of the screened positive clone, transferring it into a host cell, culturing and collecting the supernatant, and separating and purifying to obtain the antibody.

In a sixth aspect, the present application provides a pharmaceutical composition comprising the anti-C5 antibody of the first aspect.

Preferably, the pharmaceutical composition further includes an antitumor drug.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two pharmaceutically acceptable carriers, diluents or excipients.

In the seventh aspect, the present application provides the anti-C5 antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect, or the sixth aspect. Application of the pharmaceutical composition in the preparation of disease detection reagents and/or disease treatment drugs.

Preferably, the disease comprises an autoimmune disease caused by excessive activation of C5.

Compared with the prior art, the present application has the following beneficial effects:

(1) The anti-C5 antibodies CR3#24, CR3#30 and CR3#75 of the present application, especially CR3#75, have significant binding ability to human recombinant C5 protein, and show obvious CDC inhibitory effect, CR3#75 The dissociation equilibrium constant KD is 0.366-0.722nM, which has better binding activity to C5 than the control antibody Eculizumab;

(2) The anti-C5 antibody CR3#75 of the present application exhibits excellent pharmacokinetics in a mouse model, with a half-life of up to 15 days;

(3) The CR3#75/IgG4 monoclonal antibody of this application is structurally stable, with an isoelectric point of 6.0 to 6.9, good solubility at low concentrations (>1 mg/mL), good thermal stability, and a Tm value of 69.83°C ; CR3#75/IgG1 (N297A/K322A) has superior thermal stability compared to CR3#75/IgG4; and

(4) The anti-C5 antibody of the present application has potential application value in preventing and/or treating autoimmune diseases caused by excessive activation of C5.

Description of drawings

Figure 1 shows the ELISA affinity detection results of 128 C5-specific binding peptide phages obtained after 3 rounds of screening.

Fig. 2 is the SDS-PAGE electrophoresis image of C5-specific antibody containing IgG constant region, wherein, M-protein molecular weight 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 is the detection result of the activity of the screened C5-specific antibodies in binding to the C5 protein.

FIG. 4A shows the results of the CDC inhibition effect of the screened C5-specific antibodies, and FIG. 4B shows the results of the CDC inhibition effects of different concentrations of the C5-specific antibodies.

Figure 5A is the dissociation curve of Eculizumab, and Figure 5B is the dissociation curve of CR3#75.

Figure 6 is the metabolic curve of CR3#75/IgG4 in mice.

Figure 7 shows the results of the integrity analysis of CR3#75/IgG4, in the figure, Figure I - under non-reducing conditions, and Figure II - under reducing conditions.

Figure 8 is the isoelectric point detection result of CR3#75/IgG4, wherein, M-protein molecular weight Marker, 1-CR3#75/IgG4 monoclonal antibody.

Figure 9 shows the results of SEC-HPLC analysis of low concentrations of CR3#75/IgG4 (>1 mg/mL).

Figure 10 shows the results of thermal stability analysis of CR3#75/IgG4.

Figure 11 shows the results of SEC-HPLC analysis of high concentrations of CR3#75/IgG4 (>10 mg/mL).

Fig. 12A is a statistical picture of the deletion area of AQP4 lesions in mouse brain tissue, and Fig. 12B is a statistical picture of the deletion area of GFAP lesions in mouse brain tissue.

Detailed ways

In order to further illustrate the technical means adopted in the present application and its effects, the present application will be further described below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through regular channels.

Example 1 Screening of C5 Antibody Active Peptides

In order to obtain the C5 antibody with therapeutic effect, this example firstly screened the peptide fragment with specific binding activity to C5 from the phage antibody library OmniMab. Hundreds of B cells from healthy donors were collected in the phage antibody library OmniMab (AP Biosciences Inc.), and antibody display was performed based on Hyperphage (M13K07ΔρΙΙΙ, Progen, Heidelberg, Germany). The full-length C5 referenced NCBI Reference Sequence: NP_001726.2 antigen, and was recombinantly expressed in HEK293 cells (Sino Biologica, Cat#13416-H18H) for enrichment and isolation of specific binding peptides from the OmniMab library, and with different HEK293 cells expressing C5 served as controls.

After three rounds of screening (CR3) and enrichment, C5-specific binding peptides were identified and isolated by ELISA. As shown in Figure 1, based on the clone-specific recognition of C5 activity, sequencing analysis was performed on CR3#1 to CR3#128 to confirm the sequence information and diversity of the heavy and light chains, and 15 candidate samples were selected for subsequent screening. . Wherein, the heavy chain variable region and light chain variable region of CR3#24 are shown in SEQ ID NO:19～20, and the heavy chain variable region and light chain variable region of CR3#30 are shown in SEQ ID NO:21～ 22, the heavy chain variable region and light chain variable region of CR3#75 are shown in SEQ ID NOs: 23-24.

Example 2 Expression and purification of C5-specific antibodies containing IgG constant regions

In order to further evaluate the physicochemical properties of C5-specific binding peptides after forming intact antibodies, the heavy and light chains of the 15 samples obtained in Example 1 were subcloned into an IgG expression vector (AP) carrying an IgG4 (S228P) constant region. Biosciences Inc.), wherein the mutation site on IgG4 is used to improve the stability of IgG4.

After the recombinant plasmid was identified by sequencing as a positive clone with the target fragment inserted correctly, a plasmid extraction kit was used to prepare a sufficient amount of sterile recombinant plasmid, which was transfected into 6×10 ⁶ ExpiCHO cells by transfection reagent (Invitrogen) for antibody expression. Recombinant ExpiCHO cells were cultured at 37°C in 5% _CO for 6 days, the antibody was secreted into the serum-free cell culture supernatant, and the antibody was affinity purified from the cell culture supernatant by Protein A chromatography. After concentration, it was placed in DPBS buffer for lysis, and the protein concentration was detected by NanoDrop2000. The purity and integrity were detected by SDS-PAGE under non-reducing and reducing conditions. The amount of protein loaded 5 μg/lane. After the gel was stained with Coomassie brilliant blue, it was washed with ddH ₂ O. A control group (C5 Ref) of Eculizumab (Soliris/Alexion) was also set up.

As shown in Figure 2, the integrity and purity of the C5 antibody secreted by ExpiCHO cells reached more than 95%, and there were almost no visible fragments in the SDS-PAGE gel, indicating that the screening, subcloning, expression, and purification steps of the antibody will not affect the antibody protein. structural stability.

Example 3 Detection of binding activity of C5-specific antibodies based on direct ELISA

In this example, the direct ELISA method was used to detect the binding activity of C5-specific antibodies to human recombinant C5 protein, and an eculizumab (Eculizumab, Soliris/Alexion) control group (C5 Ref) was set. Proceed as follows:

Human C5 protein (Sino Biologica, Cat#13416-H18H) was diluted to 1 μg/mL with 1×DPBS, added to the ELISA plate at 100 μL/well, incubated at 4°C overnight, and then coated; the coating solution was discarded Then, add 350 μL of 1% BSA blocking solution to each well, and block at room temperature for 1 h;

Prepare a gradient dilution of the antibody solution to be detected (3-fold dilution starting from 30 nM, 10 dilutions, and adding a solution without antibody, a total of 12 concentrations for detection), add 100 μL of different concentrations to each well of the prepared ELISA plate The concentration of the antibody solution to be detected was incubated at room temperature for 1 h under shaking conditions; the supernatant was discarded, 350 μL/well washing solution (0.05% Tween-20+1×DPBS) was added, and washed 3 times;

Add 100 μL/well of HRP-labeled antibody anti-Fc-Ab-HRP (0.4 μg/mL) (Jackson Immuno Research, cat#109-006-098), incubate at room temperature for 30 min under shaking conditions; discard the supernatant and add 350 μL/well washing solution, washed 3 times;

After adding TMB to develop color for 1 to 5 minutes, add 50 μL of stop solution (1N HCl) to stop the reaction, and place it on a microplate reader to read the absorbance value at 450 nm.

The results are shown in Figure 3. The screened CR3#75 has a C5 protein binding activity very close to C5 Ref, and CR3#24, CR3#30, and CR3#53 also have a significant C5 protein binding activity.

Example 4 Functional verification of C5-specific antibodies based on CDC inhibition experiments

C5-specific antibodies can block CDC activation, and this example detects the function of C5-specific antibodies based on CDC experiments. Proceed as follows:

10 μg/mL Rituximab was added to 2×10 ⁴ Raji cells, incubated at room temperature for 20 min, followed by 20 μg/mL anti-C5 antibody and complement-activated human serum (12.5%) to a final volume of 200 μL ;

After incubation at 37°C for 2 h, dead cells were stained with propidium iodide, and flow cytometry was performed (Attune NxT Flow Cytometer system, ThermoFisher SCIENTIFIC).

The results are shown in Fig. 4A and Fig. 4B, CR3#24, CR3#30, CR3#75 showed obvious CDC inhibitory effect, among them, CR3#75 had the strongest neutralizing activity, and the control antibody (C5 Ref, Soliris) ) were comparable in neutralizing activity.

Example 5 Affinity detection of C5-specific antibody and human recombinant C5 protein

This embodiment adopts

(Menlo Park, Calif.) Biosensors analyzed the affinity of CR3#75 and C5 and set up an Eculizumab control group (C5 Ref). Proceed as follows:

Briefly, the antibody was 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, and monitored on-line until the signal reached approx. 0.5nm;

Antibody-loaded sensors were exposed to different concentrations (0.4688nM, 0.9375nM, 1.88nM, 3.75nM, 7.5nM, 15nM and 30nM) of C5 (Sino Biologica, cat#13416-H18H) followed by 1 min incubation in PBST to determine baseline , incubate in different concentrations of C5 solution for 5 min to detect the binding rate of antibody and C5, and incubate in PBST for 5 min to detect the dissociation rate of antibody and C5. Kinetic parameters were analyzed using Octet Data software.

The results are shown in Table 1, Table 2, Figure 5A and Figure 5B. Compared with the control antibody Eculizumab (KD=0.371-0.553nM), CR3#75 has better binding activity to C5, and the dissociation equilibrium constant KD is 0.366 ~0.722nM.

Table 1 Binding ability of Eculizumab to C5

Numbering	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 capacity of CR3#75 and C5

Numbering	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 detection

This example evaluates the half-life, AUC (area under the curve), Cmax, Cl (clearance) and Vd (volume of distribution) of C5-specific antibody CR3#75/IgG4 in mice.

SCID-Bg mice (CB.17/.Cg-PrkdcscidLystbg-J/CrlBltw) were injected intravenously with 5 mg/kg 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 detected by direct ELISA, and the pharmacokinetic parameters were calculated by PKSolver (ver.2.0).

The results are shown in Table 3, Table 4 and Figure 6. CR3#75 showed excellent pharmacokinetics in the mouse model, with a half-life of up to 361.5h (equivalent to 15 days), indicating that the antibody is metabolized slowly in mice It can be retained for a long time, the frequency of administration can be reduced, and low-dose antibodies can be administered in animal experiments to achieve the purpose of treatment.

table 3

time (h)	Concentration (μg/mL)
0.5	141.692
twenty four	59.527
72	53.084
168	38.222
240	42.075

336	28.307
504	12.811
672	23.378

Table 4

parameter	unit	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 Integrity analysis of CR3#75/IgG4 monoclonal antibody

In this example, LabChip GXII was used to detect the integrity of the purified CR3#75/IgG4 monoclonal antibody. Proceed as follows:

The purified CR3#75/IgG4 monoclonal antibody was diluted to 1 mg/mL with DPBS, and 2.5 μL of protein sample dilution was mixed with 18 μL of sample buffer (containing 1 M DTT or 250 mM NEM), incubated at 70 °C for 10 min, and then cooled to room temperature. 35 μL of deionized water was added to the system and then loaded into LabChip GXII (Perkin Elmer, Inc., Cat. CLS138160) for integrity analysis.

The results are shown in Table 5 and Figure 7. The complete CR3#75/IgG4 monoclonal antibody (M.W.172.75kDa) can be detected under non-reducing conditions. The purity of the CR3#75/IgG4 monoclonal antibody was 100%, indicating that the heavy chain and light chain of the antibody were correctly paired, and the antibody could maintain structural stability during the preparation and purification process. Under reducing conditions, the sum of heavy and light chains (HC+LC) also reached 96.67%, indicating that most impurities can be removed by a single purification step.

table 5

Example 8 Isoelectric point detection

In this example, IEF gel electrophoresis was used to detect the charge of CR3#75/IgG4 monoclonal antibody. Proceed as follows:

Mix 5 μL of protein sample and 5 μL of 2× sample buffer, and the upper and lower buffer chambers of the electrophoresis device XCell SureLock ^™ Mini-Cell (Invitrogen) were filled with 200 mL of 1× IEF cathode buffer or 600 mL of 1× IEF anode buffer, respectively. The electrophoresis conditions were 100V electrophoresis for 1h, 200V electrophoresis for 1h, and 500V electrophoresis for 30min. After electrophoresis, the gel was fixed with 12% TCA for 30 min, and the gel was imaged after Coomassie brilliant blue staining.

The results are shown in FIG. 8 , the charge distribution of CR3#75/IgG4 is not uniform, the isoelectric point detected by IEF gel electrophoresis is 6.0-6.9, and the predicted isoelectric point is 5.92.

Example 9 Aggregation analysis

After purification and concentration of the CR3#75/IgG4 antibody, SEC-HPLC analysis was performed. Proceed as follows:

The concentrated protein sample (>1 mg/mL) was mixed with an equal volume of mobile phase buffer (25 mM phosphate, 200 mM NaCl, pH 6.8), filtered through a 0.22 μm filter (Millipore, Cat#SLGP003RB), and loaded onto XBridge Protein BEH SEC Column (Waters, Cat#186007640), SEC separation was performed at 25 °C, the flow rate was set to 0.4 mL/min, the sample added volume was 10 μL, and the absorbance was measured at 280 nm. Finally, Empower 2 was used for data analysis.

The results are shown in Figure 9. The main peak at 17.839 min shows that the purity of the protein sample is 99.9% and the concentration is 1.014 mg/mL, indicating that CR3#75/IgG4 maintains good solubility in DPBS and basically does not aggregate and precipitate.

Example 10 Thermal stability analysis

In this example, differential scanning calorimetry (differential scanning calorimetry, DSC) was used to detect the thermal stability of the antibody. Proceed as follows:

Prepare a protein sample with a concentration of 0.2 to 1.0 mg/mL, use MicroCal PEAQ-DSC system (Northampton, Malvern) to perform DSC detection at a rate of 200 °C/h from 10 to 100 °C, and draw a thermogram. Analytical Figures Perform a thermal stability analysis of the antibody.

Figure 10 is the melting curve of CR3#75/IgG4, the Tm value is 69.83°C, indicating that the thermal stability of this antibody is normal.

Example 11 Solubility Analysis

After purification and concentration of the CR3#75/IgG4 antibody, SEC-HPLC analysis was performed. Proceed as follows:

Concentrated protein samples (>10 mg/mL) were mixed with an equal volume of mobile phase buffer (25 mM phosphate, 200 mM NaCl, pH 6.8), filtered through a 0.22 μm filter (Millipore, Cat#SLGP003RB), and loaded onto XBridge Protein BEH SEC Column (Waters, Cat#186007640), SEC separation was performed at 25°C, the flow rate was set to 0.4 mL/min, the sample addition volume was 2 μL, and the absorbance was measured at 280 nm. Finally, Empower 2 was used for data analysis.

The results are shown in Figure 11, the CR3#75/IgG4 antibody can be concentrated to 10mg/mL without producing visible precipitation, 97.15% of the protein produced a single peak at 18.005min, and only <4% of the protein formed a precipitate, indicating this The solubility of the antibody is normal, and drug development can continue.

Example 12 In vivo efficacy

This example verifies the in vivo efficacy of CR3#75/IgG4 antibody in the NMO mouse model. The steps are as follows:

C57 female mice aged 6 to 8 weeks were anesthetized with chloral hydrate as the spare skin, and the head of the mouse was fixed on the brain stereotaxic instrument. After adjusting the positioning, the hole was drilled, and the micro-injection needle was vertically inserted into the mouse brain tissue. The syringe pump was turned on, and the mixed solution of CR3#75/IgG4 antibody, complement and pathogenic antibody was injected. Seven days after modeling, the brain tissue was taken out to make frozen tissue sections, and the expression of AQP4 and GFAP in astrocytes was stained by immunofluorescence technique.

Results As shown in Figures 12A and 12B, CR3#75/IgG4 antibody inhibited the formation of typical NMO lesions at concentrations of 0.04 mg/mL, 0.156 mg/mL, 0.625 mg/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, forming 6 new Antibody, the amino acid sequence is as follows:

CR3#30/IgG1 (N297A/K322A) heavy chain (SEQ ID NO: 25):

CR3#30/IgG1 (N297A) heavy chain (SEQ ID NO: 26):

CR3#30/IgG2 heavy chain (SEQ ID NO:27):

Above-mentioned CR3#30/IgG1 (N297A/K322A), CR3#30/IgG1 (N297A) and CR3#30/IgG2 all select CR3#30/lambda light chain, the sequence is as shown in SEQ ID NO:28:

SEQ ID NO: 28:

CR3#75/IgG1 (N297A/K322A) heavy chain (SEQ ID NO: 29):

CR3#75/IgG1 (N297A) heavy chain (SEQ ID NO:30):

CR3#75/IgG2 heavy chain (SEQ ID NO:31):

Above-mentioned CR3#75/IgG1 (N297A/K322A), CR3#75/IgG1 (N297A) and CR3#75/IgG2 all select CR3#75/kappa light chain, the sequence is as shown in SEQ ID NO:32.

SEQ ID NO: 32:

Gene synthesis of the above antibody was carried out to construct a recombinant plasmid. After the recombinant plasmid was identified by sequencing as a positive clone with the correct insertion of the target fragment, a plasmid extraction kit was used to prepare a sufficient amount of sterile recombinant plasmid for transient transfection to produce protein samples.

Example 14 Different constant region antibody production

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 total 9 kinds of sterile recombinant plasmids were transfected with transfection reagent (Invitrogen) into 6×10 ⁶ ExpiCHO for antibody expression.

After culturing the recombinant ExpiCHO cells at 37°C in 5% CO ₂ for 9-11 days, the antibody was secreted into the serum-free cell culture supernatant. Protein A-HPLC was performed on the fermentation broth to detect protein expression. Proceed as follows:

The fermentation broth sample was filtered with a 0.22 μm membrane filter (Sartorius, Cat#17C07FT), and then loaded onto a Poros-20A Column (Thermo Scientific, Cat#1-5024-12), and then detected by ProteinA-HPLC at 25°C. Chromatographic parameters See Table 6. Finally, Empower 3 was used for data acquisition and analysis, and the results are shown in Table 7.

Table 6 ProteinA-HPLC chromatographic parameters

Table 7 Detection of protein expression in fermentation broth by ProteinA-HPLC

After 9 batches of CHO cells were cultured for 9 to 11 days, all fermentation broths were tested for protein purity by SEC-HPLC. The steps were as follows:

After filtering the protein sample with a 0.22 μm filter (Sartorius, Cat#17C07FT), the sample was loaded on a TSKgel G3000 SWXL SEC Column (Tosoh, Cat#008541), and the SEC was separated at 25°C. The equipment was Waters e2695 and the detector was Waters 2998PDA detector, the mobile phase is 50mM phosphate + 400mM sodium chloride (pH 7.0) buffer, the flow rate is set to 0.5mL/min, the injection volume is 10μL, and the absorbance of each separated component is measured at 280nm. Finally, Empower 3 was used for data acquisition and analysis, and the results are shown in Table 8.

Table 8 SEC-HPLC detects the purity of fermentation broth

Sample serial number	sample name	High molecular weight (%)	Main peak (%)	Low molecular weight (%)
S125(38#)-1	CR3#24/IgG4	30.2	19.1	50.7
S125(38#)-2	CR3#30/IgG4	31.6	19.7	48.6
S125(38#)-3	CR3#75/IgG4	28.4	24.8	46.8
S125(38#)-4	CR3#30/IgG1(N297A/K322A)	29.7	22.7	47.6
S125(38#)-5	CR3#30/IgG1(N297A)	29.3	25.5	45.2
S125(38#)-6	CR3#30/IgG2	34.6	10.8	54.7
S125(38#)-7	CR3#75/IgG1(N297A/K322A)	30.0	19.0	51.0
S125(38#)-8	CR3#75/IgG1(N297A)	29.6	19.8	50.5
S125(38#)-9	CR3#75/IgG2	27.1	13.5	59.5

After the fermentation broth of 9 candidate molecules was captured by PrisMA, the protein concentration was determined by UV spectrophotometer. The steps were as follows:

Take the test solution and take the same batch of solvent used to prepare the test solution as the blank control. Using a 1 cm quartz absorption cell, the sample was diluted with a blank solvent, and the quartz absorption cell containing the sample was placed in the detection cell for detection. The UV spectrophotometer was IMPLEN, model NP80.

The formula for calculating protein concentration is as follows:

protein concentration

In the formula, X: dilution ratio;

The absorption coefficient of the protein to be tested .

The results are shown in Table 9.

Table 9 Purified sample concentration

Sample serial number	sample name	Protein concentration (mg/mL)
S125(38#)-1	CR3#24/IgG4	2.5
S125(38#)-2	CR3#30/IgG4	2.6
S125(38#)-3	CR3#75/IgG4	2.5
S125(38#)-4	CR3#30/IgG1(N297A/K322A)	4.4
S125(38#)-5	CR3#30/IgG1(N297A)	2.4
S125(38#)-6	CR3#30/IgG2	1.4
S125(38#)-7	CR3#75/IgG1(N297A/K322A)	3.0
S125(38#)-8	CR3#75/IgG1(N297A)	2.0
S125(38#)-9	CR3#75/IgG2	1.4

The fermentation broth of the 9 candidate molecules was captured by PrisMA and then analyzed by SEC-HPLC. The steps are as follows:

After filtering the protein sample with a 0.22 μm filter (Sartorius, Cat#17C07FT), the sample was loaded on a TSKgel G3000 SWXL SEC Column (Tosoh, Cat#008541), and the SEC was separated at 25°C. The equipment was Waters e2695 and the detector was Waters 2998 PDA detector, the mobile phase is 50mM phosphate + 400mM sodium chloride (pH 7.0) buffer, the flow rate is set to 0.5mL/min, the injection volume is 10μL, and the absorbance value is measured at 280nm. Finally, Empower 3 was used for data collection and analysis, and the results are shown in Table 10.

Table 10 Purity of purified samples detected by SEC-HPLC

Based on the above results, the sample production of 9 kinds of antibodies was successfully completed, the concentrations were all greater than 1 mg/mL, and the purity was higher than 90%, and subsequent experiments could be carried out.

Example 15 Aggregation Analysis

The present embodiment utilizes iCIEF to measure the isoelectric point of each candidate molecule, and the steps are as follows:

After the sample was desalted, it was diluted with distilled water to a protein concentration of about 2 mg/mL, and prepared according to the following system (200 μL system): 8 μL 2% pharmalyte (pH 3～10) (GE, Cat#17045601), 70 μL 1% MC (ProteinSimple, Cat#1018761), 1 μL Low pI (ProteinSimple, Cat#102222), 1 μL High pI (ProteinSimple, Cat#101996), 100 μL 8M Urea (Sigma, Cat#U0631-500G1), 20 μL sample.

The system was placed in a centrifuge tube, followed by centrifugation at 10,000g for 5min, and the supernatant (60-120μL) was added to the inner tube. The inner tube was placed in the centrifuge tube and centrifuged at 10,000g for 5min. Machine detection, the device is ProteinSimple iCE3. The results are shown in Table 11, the actual isoelectric point is slightly higher than the predicted isoelectric point.

Table 11 Isoelectric points of candidate molecules

Sample serial number	sample name	predicted isoelectric point	iCIEF - isoelectric point
S125(38#)-1	CR3#24/IgG4	5.97	6.13
S125(38#)-2	CR3#30/IgG4	6.18	6.30
S125(38#)-3	CR3#75/IgG4	5.92	5.89
S125(38#)-4	CR3#30/IgG1(N297A/K322A)	7.24	7.33
S125(38#)-5	CR3#30/IgG1(N297A)	7.56	7.57

S125(38#)-6	CR3#30/IgG2	6.50	6.97
S125(38#)-7	CR3#75/IgG1(N297A/K322A)	6.36	6.58
S125(38#)-8	CR3#75/IgG1(N297A)	6.51	6.76
S125(38#)-9	CR3#75/IgG2	5.97	6.18

Example 16 Capillary electrophoresis analysis

After the fermentation broth of 9 candidate molecules was captured by PrisMA, CE-SDS analysis was performed. The steps were as follows:

The protein sample was ultrafiltered with a 30μm ultrafiltration centrifuge tube (Sartorius, Cat#VS0122), then the sample was prepared, heated at 70°C, loaded, and separated by CE-SDS at 25°C. The equipment was SCIEX PA800 Plus, and the detector was PDA detection. The absorbance was measured at 220 nm. Finally, 32karat was used for data collection and analysis, and the results are shown in Table 12, in which the purity of CR3#75/IgG2 was low.

Table 12 Purity of purified samples detected by capillary electrophoresis

Example 17 Detection of protein stability of C5-specific antibodies

This example adopts a protein stability analysis system

(Unchained Labs, Calif.) Tm&Tagg with optional DLS program to measure protein size and stability. By analyzing the result parameters Tm (protein melting temperature), Tagg266 (protein aggregation temperature), Tagg473 (protein aggregation temperature), Z-Ave.Dia (protein average particle size), PDI (polydispersity coefficient), Pk1 Mode Dia, Pk1 Mass (%), Pk2 Mode Dia and Pk2 Mass (%) 9 parameters, multi-angle analysis of protein particle size and particle size distribution and thermal stability. Proceed as follows:

Take 9 μL of the antibody and add it to a uni tube (Unchained Labs, Calif.), repeat 2 replicate wells for each antibody, and select Tm&Tagg with optional DLS program for analysis.

The particle size results at 25°C and 95°C are shown in Table 13 and Table 14, respectively, and the Tm & Tagg results are shown in Table 15. Among them, Z-Ave.Dia - the average hydration kinetic diameter of the sample, reflecting the overall particle size of the sample; PDI - polydispersity coefficient, PDI<0.1 indicates a narrow particle size distribution, and the larger the value, the more inhomogeneous and discrete the particle size distribution. The higher the degree; the Fit Var- fitting deviation, less than 0.01 indicates high reliability of Z-Ave.Dia and PDI, the larger the value, the lower the reliability; Pk1 Mode Dia- When the light intensity distribution shows multiple peaks, the first The mode particle size of the main peak; Pk 1 Mass (%) - the mass ratio of the first peak.

It can be seen from Table 13 that the average hydration kinetic diameter and Pk1 Mode Dia. of S125(38#)-3/6/7/9 are both around 11nm, without large particle size components, and PDI<0.1, indicating that the particle size is uniform. It is presumed to be a protein monomer; the remaining samples in the group all have larger particle size components, which are presumed to be aggregates. As can be seen from Table 14, it can be seen from the average hydration kinetic diameter and PDI that the protein molecules aggregated significantly after heating. It can be seen from Table 15 that S125(38#)-4 and S125(38#)-7Tm are higher, and it is speculated that the conformational thermal stability is higher. S125(38#)-2/4/5/6/7Tagg266 are Above 60°C, it is presumed that the thermal stability of the colloid is high.

Table 13 Particle size results at 25°C

Table 14 95℃ particle size results

Table 15 Tm&Tagg results

Based on the above results, it can be seen that the comprehensive thermal stability of the S125(38#)-7 group is higher, that is, the thermal stability of CR3#75/IgG1 (N297A/K322A) is better than that of CR3#75/IgG4.

In summary, the anti-C5 antibody of the present application has significant binding ability to human recombinant C5 protein, long half-life, good solubility, and good thermal stability, and is expected to be developed as a protein drug for the prevention and/or treatment of C5 excess. activation in autoimmune diseases.

The applicant declares that the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation. Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

Claims (13)

1. an anti-C5 antibody comprising a heavy chain variable region and a light chain variable region;

   The heavy chain variable region includes the CDR3 shown in SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:15;

   The light chain variable region includes the CDR3 shown in SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:18.
2. The anti-C5 antibody of claim 1, wherein the heavy chain variable region further comprises the CDR1 shown in SEQ ID NO: 1, SEQ ID NO: 7 or SEQ ID NO: 13.
3. The anti-C5 antibody of claim 1, wherein the heavy chain variable region further comprises the CDR2 shown in SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 14.
4. The anti-C5 antibody of claim 1, wherein the light chain variable region further comprises the CDR1 shown in SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:16.
5. The anti-C5 antibody of claim 1, wherein the light chain variable region further comprises the CDR2 shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 17.
6. The anti-C5 antibody according to any one of claims 1-5, wherein the heavy chain variable region of the anti-C5 antibody 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 includes CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

   Optionally, the heavy chain variable region of the anti-C5 antibody comprises CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9;

   The light chain variable region of the anti-C5 antibody includes CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

   Optionally, the heavy chain variable region of the anti-C5 antibody comprises CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 15;

   The light chain variable region of the anti-C5 antibody includes CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 18.
7. The anti-C5 antibody according to any one of claims 1-6, wherein the heavy chain variable region of the anti-C5 antibody comprises the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region comprises SEQ ID NO: : the amino acid sequence shown in 20;

   Optionally, the heavy chain variable region of the anti-C5 antibody includes the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 22;

   Optionally, the heavy chain variable region of the anti-C5 antibody includes the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:24;

   Optionally, the anti-C5 antibody further comprises a constant region;

   Optionally, the constant region is derived from any one of IgG4, IgG1 or IgG2;

   Optionally, the IgG4 has the S228P mutation;

   Optionally, the IgG1 has N297A and/or K322A mutations;

   Optionally, the IgG2 has the S257A mutation.
8. A nucleic acid molecule comprising a DNA fragment encoding the anti-C5 antibody of any one of claims 1-7.
9. An expression vector comprising the nucleic acid molecule of claim 8.
10. A recombinant cell expressing the anti-C5 antibody of any one of claims 1-7;

    Optionally, the nucleic acid molecule of claim 8 is integrated into the genome of the recombinant cell;

    Optionally, the recombinant cell comprises the expression vector of claim 9.
11. A preparation method of the anti-C5 antibody of any one of claims 1-7, comprising the following steps:

    (1) connecting the nucleic acid encoding the anti-C5 antibody into a plasmid, transferring it into competent cells, and picking monoclonal cells for screening after culturing;

    (2) Extracting the expression vector of the screened positive clone, transferring it into a host cell, culturing and collecting the supernatant, and separating and purifying to obtain the antibody.
12. A pharmaceutical composition comprising the anti-C5 antibody of any one of claims 1-7;

    Optionally, the pharmaceutical composition further includes an antitumor drug;

    Optionally, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.
13. The anti-C5 antibody of any one of claims 1-7, the nucleic acid molecule of claim 8, the expression vector of claim 9, the recombinant cell of claim 10, or the drug combination of claim 12 The application of the substance in the preparation of disease detection reagents and/or disease treatment drugs;

    Optionally, the disease comprises an autoimmune disease caused by overactivation of C5.

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