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

Anti-c5 antibody and application thereof Download PDF

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WO2022122020A1
WO2022122020A1 PCT/CN2021/137116 CN2021137116W WO2022122020A1 WO 2022122020 A1 WO2022122020 A1 WO 2022122020A1 CN 2021137116 W CN2021137116 W CN 2021137116W WO 2022122020 A1 WO2022122020 A1 WO 2022122020A1
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seq
antibody
variable region
chain variable
optionally
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PCT/CN2021/137116
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French (fr)
Chinese (zh)
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吕明启
李剑
黄瑞晶
于永生
王根辈
王轶博
李文蕾
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天士力生物医药股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present application belongs to the technical field of biomedicine, and relates to anti-C5 antibodies and applications thereof.
  • 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.
  • C5 is an important complement.
  • 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.
  • MAC membrane attack complex
  • PNH paroxysmal nocturnal hemoglobinuria
  • aHUS atypical hemolytic uremic syndrome
  • 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.
  • 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.
  • the heavy chain variable region further comprises the CDR1 shown in SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:13.
  • the heavy chain variable region further comprises the CDR2 shown in SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:14.
  • the light chain variable region further comprises the CDR1 shown in SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:16.
  • the light chain variable region further comprises the CDR2 shown in SEQ ID NO:5, SEQ ID NO:11 or SEQ ID NO:17.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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;
  • the anti-C5 antibody further comprises a constant region.
  • the constant region is derived from any one of IgG4, IgG1 or IgG2, preferably IgG4.
  • the IgG4 has the S228P mutation, which helps to improve the stability of the IgG4.
  • the IgG1 has the N297A and/or K322A mutation.
  • the IgG2 has the S257A mutation.
  • the mutation sites N297A and K322A on IgG1 were used to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), respectively.
  • the IgG2 has lower antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and may have the S257A mutation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • 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.
  • the present application provides a nucleic acid molecule comprising a DNA fragment encoding the anti-C5 antibody of the first aspect.
  • the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.
  • the present application provides a recombinant cell expressing the anti-C5 antibody of the first aspect.
  • the nucleic acid molecule of the second aspect is integrated into the genome of the recombinant cell.
  • the recombinant cell comprises the expression vector of the third aspect.
  • the application provides a method for preparing the anti-C5 antibody described in the first aspect, the preparation method comprising the following steps:
  • the present application provides a pharmaceutical composition comprising the anti-C5 antibody of the first aspect.
  • the pharmaceutical composition further includes an antitumor drug.
  • the pharmaceutical composition further comprises any one or a combination of at least two pharmaceutically acceptable carriers, diluents or excipients.
  • 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.
  • the disease comprises an autoimmune disease caused by excessive activation of C5.
  • the dissociation equilibrium constant KD is 0.366-0.722nM, which has better binding activity to C5 than the control antibody Eculizumab;
  • 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;
  • 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;
  • 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.
  • 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.
  • M-protein molecular weight Marker 1-CR3#24, 2-CR3#30, 3-C5 Ref
  • 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
  • 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
  • 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
  • Fig. 12B is a statistical picture of the deletion area of GFAP lesions in mouse brain tissue.
  • 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 (M13K07 ⁇ , Progen, Heidelberg, Germany).
  • C5-specific binding peptides were identified and isolated by ELISA.
  • 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.
  • the heavy chain variable region and light chain variable region of CR3#24 are shown in SEQ ID NO:19 ⁇ 20
  • 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.
  • 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.
  • IgG expression vector AP
  • IgG4 S228P
  • 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 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 ⁇ g/mL with 1 ⁇ DPBS, added to the ELISA plate at 100 ⁇ L/well, incubated at 4°C overnight, and then coated; the coating solution was discarded Then, add 350 ⁇ L of 1% BSA blocking solution to each well, and block at room temperature for 1 h;
  • 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.
  • C5-specific antibodies can block CDC activation, and this example detects the function of C5-specific antibodies based on CDC experiments. Proceed as follows:
  • the antibody was loaded onto an AHC (Anti-Human IgG Fc Capture) biosensor (Cat# 18-5060) at a concentration of 5 ⁇ g/mL, the sensor was equilibrated off-line in assay buffer, and monitored on-line until the signal reached approx. 0.5nm;
  • AHC Anti-Human IgG Fc Capture
  • 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.
  • 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).
  • 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.
  • 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 ⁇ L of protein sample dilution was mixed with 18 ⁇ L 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 ⁇ L of deionized water was added to the system and then loaded into LabChip GXII (Perkin Elmer, Inc., Cat. CLS138160) for integrity analysis.
  • sample buffer containing 1 M DTT or 250 mM NEM
  • 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.
  • 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.
  • IEF gel electrophoresis was used to detect the charge of CR3#75/IgG4 monoclonal antibody. 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 ⁇ m 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 ⁇ L, and the absorbance was measured at 280 nm. Finally, Empower 2 was used for data analysis.
  • mobile phase buffer 25 mM phosphate, 200 mM NaCl, pH 6.8
  • filtered through a 0.22 ⁇ m filter Millipore, Cat#SLGP003RB
  • XBridge Protein BEH SEC Column Waters, Cat#186007640
  • differential scanning calorimetry differential scanning calorimetry, DSC was used to detect the thermal stability of the antibody. Proceed as follows:
  • 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.
  • 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 ⁇ m 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 ⁇ L, and the absorbance was measured at 280 nm. Finally, Empower 2 was used for data analysis.
  • mobile phase buffer 25 mM phosphate, 200 mM NaCl, pH 6.8
  • filtered through a 0.22 ⁇ m filter Millipore, Cat#SLGP003RB
  • XBridge Protein BEH SEC Column Waters, Cat#186007640
  • 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.
  • mice 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.
  • 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.
  • 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):
  • CR3#30/IgG1 N297A/K322A
  • CR3#30/IgG1 N297A
  • CR3#30/IgG2 all select CR3#30/lambda light chain, the sequence is as shown in 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/IgG1 N297A/K322A
  • CR3#75/IgG1 N297A
  • CR3#75/IgG2 all select CR3#75/kappa light chain, the sequence is as shown in SEQ ID NO:32.
  • transfection reagent Invitrogen
  • the fermentation broth sample was filtered with a 0.22 ⁇ m 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.
  • the UV spectrophotometer was IMPLEN, model NP80.
  • X dilution ratio
  • the fermentation broth of the 9 candidate molecules was captured by PrisMA and then analyzed by SEC-HPLC.
  • the steps are as follows:
  • the present embodiment utilizes iCIEF to measure the isoelectric point of each candidate molecule, and the steps are as follows:
  • the system was placed in a centrifuge tube, followed by centrifugation at 10,000g for 5min, and the supernatant (60-120 ⁇ L) 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.
  • Table 11 the actual isoelectric point is slightly higher than the predicted isoelectric point.
  • CE-SDS analysis was performed. The steps were as follows:
  • the protein sample was ultrafiltered with a 30 ⁇ m 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.
  • 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.
  • This example adopts a protein stability analysis system (Unchained Labs, Calif.) Tm&Tagg with optional DLS program to measure protein size and stability.
  • 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.
  • 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.
  • 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 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.

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

抗C5抗体及其应用Anti-C5 antibody and its application 技术领域technical field
本申请属于生物医药技术领域，涉及抗C5抗体及其应用。The present application belongs to the technical field of biomedicine, and relates to anti-C5 antibodies and applications thereof.
背景技术Background technique
补体***由30多种血浆蛋白和膜蛋白组成，广泛存在于血液、组织液和细胞表面，促进吞噬细胞的吞噬和溶解靶细胞能力，是机体免疫防御机制的重要组成部分。其中，C5是一种重要的补体，在炎症反应过程中C5转化酶将C5分解成C5a和C5b，C5a游离于液相，是重要的炎症介质，C5b经过一系列补体反应形成膜攻击复合物(MAC)，导致细胞裂解死亡。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.
正常情况下补体的激活和抑制机制共同作用维持健康的机体状态，而当由于某些情况导致C5过度激活时，则产生异常的自身免疫。例如，阵发性夜间血红蛋白尿(PNH)是由于PNH红细胞的CD59缺陷导致C5抑制作用受损引起的，非典型性溶血性***综合征(aHUS)则是由于补体旁路的过度激活导致膜攻击复合物形成并损伤微血管内皮细胞引起的，其共同的作用机理均为C5的过度激活。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.
发明内容SUMMARY OF THE INVENTION
本申请提供了抗C5抗体及其应用，所述抗C5抗体能够高亲和性地结合C5，阻止其被C5转化酶裂解，有利于阻断膜攻击复合物的形成，最终达到缓解并改善临床症状的目的。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.
第一方面，本申请提供了抗C5抗体，所述抗C5抗体包括重链可变区和轻链可变区；In a first aspect, the application provides an anti-C5 antibody comprising a heavy chain variable region and a light chain variable region;
所述重链可变区包括SEQ ID NO:3、SEQ ID NO:9或SEQ ID NO:15所示的CDR3；The heavy chain variable region includes the CDR3 shown in SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:15;
所述轻链可变区包括SEQ ID NO:6、SEQ ID NO:12或SEQ ID NO:18所示的CDR3。The light chain variable region includes the CDR3 shown in SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:18.
优选地，所述重链可变区还包括SEQ ID NO:1、SEQ ID NO:7或SEQ ID NO:13所示的CDR1。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.
优选地，所述重链可变区还包括SEQ ID NO:2、SEQ ID NO:8或SEQ ID NO:14所示的CDR2。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.
优选地，所述轻链可变区还包括SEQ ID NO:4、SEQ ID NO:10或SEQ ID NO:16所示的CDR1。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.
优选地，所述轻链可变区还包括SEQ ID NO:5、SEQ ID NO:11或SEQ ID NO:17所示的CDR2。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.
本申请中，抗体的重链可变区的CDR1～3和轻链可变区的CDR1～3共同决定抗体对抗原的特异性识别结合能力，含有SEQ ID NO:1～6、SEQ ID NO:7～12以及SEQ ID NO:13～18的CDR的抗体对C5蛋白具有显著的结合能力，表现出明显的CDC抑制作用。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.
在一个具体实施例中，所述抗C5抗体CR3#24的重链可变区包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、SEQ ID NO:3所示的CDR3；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;
所述抗C5抗体CR3#24的轻链可变区包括SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2、SEQ ID NO:6所示的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: 1: DYYMH;
SEQ ID NO:2：VVDPEDGEIIYAEKFQG；SEQ ID NO: 2: VVDPEDGEIIYAEKFQG;
SEQ ID NO:3：SDYGAGSYMVDP；SEQ ID NO: 3: SDYGAGSYMVDP;
SEQ ID NO:4：GGNNLESKIVS；SEQ ID NO: 4: GGNNLESKIVS;
SEQ ID NO:5：YDTDRPS；SEQ ID NO: 5: YDTDRPS;
SEQ ID NO:6：QVWDSNSDHVI。SEQ ID NO: 6: QVWDNSDHVI.
本申请中，包含SEQ ID NO:1～3的重链可变区CDR和SEQ ID NO:4～6的轻链可变区CDR的抗C5抗体CR3#24具有C5蛋白结合活性，表现出明显的CDC抑制作用。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.
优选地，所述抗C5抗体CR#24的重链可变区包括SEQ ID NO:19所示的氨基酸序列，轻链可变区包括SEQ ID NO:20所示的氨基酸序列；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: 19:
Figure PCTCN2021137116-appb-000001
Figure PCTCN2021137116-appb-000001
SEQ ID NO:20：SEQ ID NO: 20:
Figure PCTCN2021137116-appb-000002
Figure PCTCN2021137116-appb-000002
在一个具体实施例中，所述抗C5抗体CR3#30的重链可变区包括SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2、SEQ ID NO:9所示的CDR3；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;
所述抗C5抗体CR3#30的轻链可变区包括SEQ ID NO:10所示的CDR1、SEQ ID NO:11所示的CDR2、SEQ ID NO:12所示的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: 7: SYYMH;
SEQ ID NO:8：IINPSGGSTSYAQKFQG；SEQ ID NO: 8: IINPSGGSTSYAQKFQG;
SEQ ID NO:9：GTVDDAFDI；SEQ ID NO: 9: GTVDDAFDI;
SEQ ID NO:10：TGTSSDVGGYDLVS；SEQ ID NO: 10: TGTSSDVGGYDLVS;
SEQ ID NO:11：DVSKRPS；SEQ ID NO: 11: DVSKRPS;
SEQ ID NO:12：SSFADSNNWEVV。SEQ ID NO: 12: SSFADSNNWEVV.
本申请中，包含SEQ ID NO:7～9的重链可变区CDR和SEQ ID NO:10～12的轻链可变区CDR的抗C5抗体CR3#30具有C5蛋白结合活性，表现出明显的CDC抑制作用。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.
优选地，所述抗C5抗体CR3#30的重链可变区包括SEQ ID NO:21所示的氨基酸序列，轻链可变区包括SEQ ID NO:22所示的氨基酸序列；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: 21:
Figure PCTCN2021137116-appb-000003
Figure PCTCN2021137116-appb-000003
SEQ ID NO:22：SEQ ID NO: 22:
Figure PCTCN2021137116-appb-000004
Figure PCTCN2021137116-appb-000004
在一个具体实施例中，所述抗C5抗体CR3#75的重链可变区包括SEQ ID NO:13所示的CDR1、SEQ ID NO:14所示的CDR2、SEQ ID NO:15所示的CDR3；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;
所述抗C5抗体CR3#75的轻链可变区包括SEQ ID NO:16所示的CDR1、SEQ ID NO:17所示的CDR2、SEQ ID NO:18所示的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: 13: DYYTH;
SEQ ID NO:14：LVDPEGGETIYAEKFQG；SEQ ID NO: 14: LVDPEGGETIYAEKFQG;
SEQ ID NO:15：GSFLAAPDY；SEQ ID NO: 15: GSFLAAPDY;
SEQ ID NO:16：KSSQSLLYNSNNKNYLA；SEQ ID NO: 16: KSSQSLLYNSNNKNYLA;
SEQ ID NO:17：WASTRES；SEQ ID NO: 17: WASTRES;
SEQ ID NO:18：QQYYSTPLT。SEQ ID NO: 18: QQYYSTPLT.
本申请中，包含SEQ ID NO:13～15的重链可变区CDR和SEQ ID NO:16～18的轻链可变区CDR的抗C5抗体CR3#75相比于伊库利珠单抗(Eculizumab，Soliris/Alexion)与C5具有更优的结合活性，表现出非常强的CDC抑制作用，半衰期长，可以长时间滞留，降低给药频次和施用计量也可达到治疗目的。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.
优选地，所述抗C5抗体CR3#75的重链可变区包括SEQ ID NO:23所示的氨基酸序列，轻链可变区包括SEQ ID NO:24所示的氨基酸序列；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: 23:
Figure PCTCN2021137116-appb-000005
Figure PCTCN2021137116-appb-000005
Figure PCTCN2021137116-appb-000006
Figure PCTCN2021137116-appb-000006
SEQ ID NO:24：SEQ ID NO: 24:
Figure PCTCN2021137116-appb-000007
Figure PCTCN2021137116-appb-000007
优选地，所述抗C5抗体还包括恒定区。Preferably, the anti-C5 antibody further comprises a constant region.
优选地，所述恒定区来源于IgG4、IgG1或IgG2中的任意一种，优选为IgG4。Preferably, the constant region is derived from any one of IgG4, IgG1 or IgG2, preferably IgG4.
优选地，所述IgG4具有S228P突变，有助于提高IgG4的稳定性。Preferably, the IgG4 has the S228P mutation, which helps to improve the stability of the IgG4.
优选地，所述IgG1具有N297A和/或K322A突变。Preferably, the IgG1 has the N297A and/or K322A mutation.
优选地，所述IgG2具有S257A突变。Preferably, the IgG2 has the S257A mutation.
本申请中，IgG1上的突变位点N297A和K322A分别用于消除抗体依赖的细胞介导的细胞毒性作用(ADCC)和补体依赖的细胞毒性(CDC)。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.
优选地，所述IgG2具有较低的抗体依赖的细胞介导的细胞毒性作用(ADCC)和补体依赖的细胞毒性(CDC)，可具有S257A突变。Preferably, the IgG2 has lower antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and may have the S257A mutation.
优选地，所述抗C5抗体分子可以在N端、内部或C端进行寡聚化、糖基化或与标记物缀合的修饰，从而调节抗体的功能。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.
第二方面，本申请提供了核酸分子，所述核酸分子包括编码第一方面所述的抗C5抗体的DNA片段。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.
第三方面，本申请提供了表达载体，所述表达载体包括第二方面所述的核酸分子。In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.
第四方面，本申请提供了重组细胞，所述重组细胞表达第一方面所述的抗C5抗体。In a fourth aspect, the present application provides a recombinant cell expressing the anti-C5 antibody of the first aspect.
优选地，所述重组细胞的基因组中整合有第二方面所述的核酸分子。Preferably, the nucleic acid molecule of the second aspect is integrated into the genome of the recombinant cell.
优选地，所述重组细胞包括第三方面所述的表达载体。Preferably, the recombinant cell comprises the expression vector of the third aspect.
第五方面，本申请提供了一种第一方面所述的抗C5抗体的制备方法，所述制备方法包括以下步骤：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)将抗C5抗体的编码核酸连接入质粒，转入感受态细胞，培养后挑取单克隆细胞进行筛选；(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)提取筛选的阳性克隆的表达载体，转入宿主细胞，培养并收集上清液，分离纯化得到所述抗体。(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.
第六方面，本申请提供了药物组合物，所述药物组合物包括第一方面所述的抗C5抗体。In a sixth aspect, the present application provides a pharmaceutical composition comprising the anti-C5 antibody of the first aspect.
优选地，所述药物组合物还包括抗肿瘤药物。Preferably, the pharmaceutical composition further includes an antitumor drug.
优选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。Preferably, the pharmaceutical composition further comprises any one or a combination of at least two pharmaceutically acceptable carriers, diluents or excipients.
第七方面，本申请提供了第一方面所述的抗C5抗体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组细胞或第六方面所述的药物组合物在制备疾病检测试剂和/或疾病治疗药物中的应用。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.
优选地，所述疾病包括C5过度激活导致的自身免疫疾病。Preferably, the disease comprises an autoimmune disease caused by excessive activation of C5.
与现有技术相比，本申请具有如下有益效果：Compared with the prior art, the present application has the following beneficial effects:
(1)本申请的抗C5抗体CR3#24、CR3#30和CR3#75、尤其是CR3#75，与人源重组C5蛋白具有显著的结合能力，表现出明显的CDC抑制作用，CR3#75的解离平衡常数KD为0.366～0.722nM，相比于对照抗体伊库利珠单抗(Eculizumab)与C5具有更优的结合活性；(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)本申请的抗C5抗体CR3#75在小鼠模型中表现出优异的药代动力学，半衰期长达15天；(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)本申请的CR3#75/IgG4单克隆抗体结构稳定，等电点为6.0～6.9，在低浓度水平下(>1mg/mL)溶解性良好，热稳定性良好，Tm值达69.83℃；与CR3#75/IgG4相比，CR3#75/IgG1(N297A/K322A)具有更优的热稳定性；以及(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)本申请的抗C5抗体在预防和/或治疗C5过度激活导致的自身免疫疾病方面具有潜在的应用价值。(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.
附图说明Description of drawings
图1为经过3轮筛选获得的128条C5特异性结合肽段噬菌体ELISA亲和力检测结果。Figure 1 shows the ELISA affinity detection results of 128 C5-specific binding peptide phages obtained after 3 rounds of screening.
图2为含有IgG恒定区的C5特异性抗体的SDS-PAGE电泳图，其中，M-蛋白分子量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. 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.
图3为筛选的C5特异性抗体结合C5蛋白的活性的检测结果。FIG. 3 is the detection result of the activity of the screened C5-specific antibodies in binding to the C5 protein.
图4A为筛选的C5特异性抗体的CDC抑制效果结果，图4B为不同浓度的C5特异性抗体的CDC抑制效果结果。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.
图5A为伊库利珠单抗(Eculizumab)的解离曲线，图5B为CR3#75的解离曲线。Figure 5A is the dissociation curve of Eculizumab, and Figure 5B is the dissociation curve of CR3#75.
图6为CR3#75/IgG4在小鼠体内的代谢曲线。Figure 6 is the metabolic curve of CR3#75/IgG4 in mice.
图7为CR3#75/IgG4的完整性分析结果，图中，I图-非还原性条件下，II图-还原性条件下。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.
图8为CR3#75/IgG4的等电点检测结果，其中，M-蛋白分子量Marker，1-CR3#75/IgG4单克隆抗体。Figure 8 is the isoelectric point detection result of CR3#75/IgG4, wherein, M-protein molecular weight Marker, 1-CR3#75/IgG4 monoclonal antibody.
图9为低浓度CR3#75/IgG4(>1mg/mL)的SEC-HPLC分析结果。Figure 9 shows the results of SEC-HPLC analysis of low concentrations of CR3#75/IgG4 (>1 mg/mL).
图10为CR3#75/IgG4的热稳定性分析结果。Figure 10 shows the results of thermal stability analysis of CR3#75/IgG4.
图11为高浓度CR3#75/IgG4(>10mg/mL)的SEC-HPLC分析结果。Figure 11 shows the results of SEC-HPLC analysis of high concentrations of CR3#75/IgG4 (>10 mg/mL).
图12A为小鼠脑组织AQP4病灶缺失面积统计图片，图12B为小鼠脑组织GFAP病灶缺失面积统计图片。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.
具体实施方式Detailed ways
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。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.
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。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.
实施例1 C5抗体活性肽段的筛选Example 1 Screening of C5 Antibody Active Peptides
为了获得具有治疗效果的C5抗体，本实施例首先从噬菌体抗体库OmniMab中筛选具有特异性结合C5活性的肽段。噬菌体抗体库OmniMab(AP Biosciences Inc.)中收集有上百个健康供者的B细胞，并基于Hyperphage(Μ13K07ΔρΙΙΙ，Progen，Heidelberg，Germany)进行抗体展示。全长C5参考NCBI Reference Sequence:NP_001726.2抗原，并采用HEK293细胞(Sino Biologica，Cat#13416-H18H)进行重组表达，用于从OmniMab库中富集和分离特异性结合肽段，并以不表达C5的HEK293细胞作为对照。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.
经过3轮筛选(CR3)和富集，C5特异性结合肽段采用ELISA进行鉴定和分离。如图1所示，根据克隆特异性识别C5活性，对CR3#1至CR3#128进行测序分析，确认重链和轻链的序列信息和多样性，并从中选出15个候选样品进行后续筛选。其中，CR3#24的重链可变区和轻链可变区如SEQ ID NO:19～20所示，CR3#30的重链可变区和轻链可变区如SEQ ID NO:21～22所示，CR3#75的重链可变区和轻链可变区如SEQ ID NO:23～24所示。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.
实施例2 表达和纯化含有IgG恒定区的C5特异性抗体Example 2 Expression and purification of C5-specific antibodies containing IgG constant regions
为了进一步评估C5特异性结合肽段形成完整抗体后的理化性质，将实施例1中获得的15个样品的重链和轻链亚克隆至携带有IgG4(S228P)恒定区的IgG表达载体(AP Biosciences Inc.)上，其中，IgG4上的突变位点用于提高IgG4的稳定性。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.
重组质粒经测序鉴定为正确***有目的片段的阳性克隆后，使用质粒提取试剂盒制备足量无菌重组质粒，经转染试剂(Invitrogen)转染至6×10 6个ExpiCHO中进行抗体表达。将重组ExpiCHO细胞在37℃、5％CO 2中培养6天，抗体分泌至无血清细胞培养上清中，采用蛋白质A色谱法(Protein A chromatography)从细胞培养上清中亲和纯化抗体，经浓缩后置于DPBS缓冲液中进行裂解，采用NanoDrop2000检测蛋白浓度，采用SDS-PAGE、在非还原性(non-reducing)和还原性(reducing)条件下进行纯度和完整性检测，蛋白上样量为5μg/泳道。凝胶进行考马斯亮蓝染色后，采用ddH 2O清洗。同时设置伊库利珠单抗(Eculizumab， Soliris/Alexion)对照组(C5 Ref)。 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.
如图2所示，ExpiCHO细胞分泌的C5抗体的完整性和纯度达到95％以上，SDS-PAGE胶中几乎没有可见的片段，说明抗体的筛选、亚克隆、表达、纯化步骤不会影响抗体蛋白的结构稳定性。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.
实施例3 基于直接ELISA的C5特异性抗体的结合活性检测Example 3 Detection of binding activity of C5-specific antibodies based on direct ELISA
本实施例利用直接ELISA法，检测C5特异性抗体与人源重组C5蛋白的结合活性，并设置伊库利珠单抗(Eculizumab，Soliris/Alexion)对照组(C5 Ref)。步骤如下：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:
将人源C5蛋白(Sino Biologica，Cat#13416-H18H)用1×DPBS稀释至1μg/mL，以100μL/孔加入到ELISA板中，4℃孵育过夜，进行包被处理；弃去包被液后，向每孔中加入350μL1％BSA封闭液，室温封闭1h；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;
配制梯度稀释的待检测抗体溶液(从30nM开始进行3倍稀释，稀释10次，并添加不含抗体的溶液，共12个浓度进行检测)，向制备好的ELISA板的每孔中加入100μL不同浓度的待检测抗体溶液，室温振荡条件下孵育1h；弃上清，加入350μL/孔洗涤液(0.05％Tween-20+1×DPBS)，洗涤3次；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;
加入100μL/孔的HRP标记抗体anti-Fc-Ab-HRP(0.4μg/mL)(Jackson Immuno Research，cat#109-006-098)，室温振荡条件下孵育30min；弃上清，加入350μL/孔的洗涤液，洗涤3次；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;
加入TMB显色1～5min后，加入50μL终止液(1N HCl)终止反应，置于酶标仪上读取450nm处的吸光值。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.
结果如图3所示，筛选的CR3#75具有与C5 Ref非常接近的结合C5蛋白的活性，CR3#24、CR3#30、CR3#53也具有显著的C5蛋白结合活性。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.
实施例4 基于CDC抑制实验的C5特异性抗体的功能验证Example 4 Functional verification of C5-specific antibodies based on CDC inhibition experiments
C5特异性抗体能够阻断CDC激活，本实施例基于CDC实验检测C5特异性抗体的功能。步骤如下：C5-specific antibodies can block CDC activation, and this example detects the function of C5-specific antibodies based on CDC experiments. Proceed as follows:
向2×10 4个Raji细胞中加入10μg/mL利妥昔单抗(Rituximab)，室温孵育20min，随后加入20μg/mL抗C5抗体和补体激活的人血清(12.5％)，至终体积为200μL； 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 ;
37℃孵育2h，采用碘化丙锭(propidium iodide)进行死细胞染色，进行流式检测(Attune NxT Flow Cytometer system，ThermoFisher SCIENTIFIC)。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).
结果如图4A和图4B所示，CR3#24、CR3#30、CR3#75表现出明显的CDC抑制作用，其中，CR3#75具有最强的中和活性，与对照抗体(C5 Ref，Soliris)的中和活性相当。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.
实施例5 C5特异性抗体与人源重组C5蛋白的亲和力检测Example 5 Affinity detection of C5-specific antibody and human recombinant C5 protein
本实施例采用
Figure PCTCN2021137116-appb-000008
(Menlo Park，Calif.)生物传感器分析CR3#75和C5的亲和力，并设置伊库利珠单抗(Eculizumab)对照组(C5 Ref)。步骤如下： This embodiment adopts
Figure PCTCN2021137116-appb-000008
(Menlo Park, Calif.) Biosensors analyzed the affinity of CR3#75 and C5 and set up an Eculizumab control group (C5 Ref). Proceed as follows:
简言之，将抗体以5μg/mL的浓度装载到AHC(Anti-Human IgG Fc Capture)生物传感器(Cat#18-5060)上，将传感器在测定缓冲液中离线平衡，在线监测直至信号达到约0.5nm；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;
将装载抗体的传感器暴露于不同浓度(0.4688nM、0.9375nM、1.88nM、3.75nM、7.5nM、15nM和30nM)C5(Sino Biologica，cat#13416-H18H)中，随后在PBST中孵育1min确定基线，在不同浓度的C5溶液中孵育5min检测抗体与C5的结合速率，在PBST中孵育5min检测抗体与C5的解离速率。动力学参数采用Octet Data软件进行分析。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.
结果如表1、表2、图5A和图5B所示，相比于对照抗体Eculizumab(KD＝0.371～0.553nM)，CR3#75与C5具有更优的结合活性，解离平衡常数KD为0.366～0.722nM。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.
表1 Eculizumab与C5的结合力Table 1 Binding ability of Eculizumab to C5
编号Numbering KD(M)KD(M) kon(1/Ms)kon(1/Ms) koff(1/s)koff(1/s)
11 5.53E-105.53E-10 5.44E+055.44E+05 3.00E-043.00E-04
22 3.77E-103.77E-10 3.94E+053.94E+05 1.49E-041.49E-04
33 3.99E-103.99E-10 3.81E+053.81E+05 1.52E-041.52E-04
44 4.35E-104.35E-10 3.77E+053.77E+05 1.64E-041.64E-04
55 3.71E-103.71E-10 3.51E+053.51E+05 1.30E-041.30E-04
表2 CR3#75与C5的结合力Table 2 Binding capacity of CR3#75 and C5
编号Numbering KD(M)KD(M) kon(1/Ms)kon(1/Ms) koff(1/s)koff(1/s)
11 3.66E-103.66E-10 4.13E+054.13E+05 1.51E-041.51E-04
22 6.14E-106.14E-10 4.88E+054.88E+05 3.00E-043.00E-04
33 6.16E-106.16E-10 4.52E+054.52E+05 2.79E-042.79E-04
44 7.22E-107.22E-10 4.25E+054.25E+05 3.07E-043.07E-04
55 7.20E-107.20E-10 4.29E+054.29E+05 3.09E-043.09E-04
实施例6 药代动力学检测Example 6 Pharmacokinetic detection
本实施例评价C5特异性抗体CR3#75/IgG4在小鼠体内的半衰期、AUC(area under the curve)、Cmax、Cl(clearance)和Vd(volume of distribution)。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.
向SCID-Bg鼠(CB.17/.Cg-PrkdcscidLystbg-J/CrlBltw)静脉注射5mg/kg CR3#75/IgG4单克隆抗体，在不同时间点从尾静脉收集血液样本。采用直接ELISA法检测血清中的CR3#75浓度，并采用PKSolver(ver.2.0)计算药代动力学参数。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).
结果如表3、表4和图6所示，CR3#75在小鼠模型中表现出优异的药代动力学，半衰期长达361.5h(折合15天)，表明抗体在小鼠体内代谢较慢可以长时间滞留，可以降低给药频次并在动物实验中施用低剂量的抗体即可达到治疗的目的。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.
表3table 3
时间(h)time (h) 浓度(μg/mL)Concentration (μg/mL)
0.50.5 141.692141.692
24twenty four 59.52759.527
7272 53.08453.084
168168 38.22238.222
240240 42.07542.075
336336 28.30728.307
504504 12.81112.811
672672 23.37823.378
表4Table 4
参数parameter 单位unit 数值Numerical value
t1/2t1/2 hh 361.451361.451
CmaxCmax μg/mLμg/mL 141.69141.69
AUC 0-tAUC 0-t μg/mL×hμg/mL×h 22283.9922283.99
Vz_obsVz_obs (mg/kg)/(μg/mL)(mg/kg)/(μg/mL) 0.07560.0756
Cl_obsCl_obs (mg/kg)/(μg/mL)/h(mg/kg)/(μg/mL)/h 0.0001450.000145
Vss_obsVss_obs (mg/kg)/(μg/mL)(mg/kg)/(μg/mL) 0.08380.0838
实施例7 CR3#75/IgG4单克隆抗体的完整性分析Example 7 Integrity analysis of CR3#75/IgG4 monoclonal antibody
本实施例利用LabChip GXII检测纯化的CR3#75/IgG4单克隆抗体的完整性。步骤如下：In this example, LabChip GXII was used to detect the integrity of the purified CR3#75/IgG4 monoclonal antibody. Proceed as follows:
将纯化的CR3#75/IgG4单克隆抗体用DPBS稀释至1mg/mL，吸取2.5μL蛋白样品稀释液与18μL样品缓冲液(含1M DTT或250mM NEM)混合，70℃下孵育10min，随后冷却至室温。向体系中加入35μL去离子水后上样至LabChip GXII(Perkin Elmer,Inc.，Cat.CLS138160)，进行完整性分析。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.
结果如表5和图7所示，在非还原性条件下可以检测到完整的CR3#75/IgG4单克隆抗体(M.W.172.75kDa)，经蛋白质A色谱法(Protein A chromatography)分析发现，完整的CR3#75/IgG4单克隆抗体的纯度达100％，说明抗体的重链和轻链正确配对，抗体在制备和纯化过程中可以保持结构的稳定性。在还原性条件下，重链和轻链之和(HC+LC)也达到96.67％，说明采用一步纯化步骤即可去除大部分杂质。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.
表5table 5
Figure PCTCN2021137116-appb-000009
Figure PCTCN2021137116-appb-000009
实施例8 等电点检测Example 8 Isoelectric point detection
本实施例利用IEF凝胶电泳检测CR3#75/IgG4单克隆抗体的电荷性。步骤如下：In this example, IEF gel electrophoresis was used to detect the charge of CR3#75/IgG4 monoclonal antibody. Proceed as follows:
将5μL蛋白样品和5μL 2×样品缓冲液混合，电泳装置XCell SureLock TM Mini-Cell(Invitrogen)的上层和下层缓冲室中分别充有200mL 1×IEF阴极缓冲液或600mL 1×IEF阳极缓冲液，电泳条件为100V电泳1h，200V电泳1h，500V电泳30min。电泳结束后采用12％TCA固定凝胶30min，考马斯亮蓝染色后进行凝胶成像。 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.
结果如图8所示，CR3#75/IgG4的电荷分布不均一，IEF凝胶电泳检测的等电点为6.0～6.9， 预测的等电点为5.92。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.
实施例9 聚集性分析Example 9 Aggregation analysis
将CR3#75/IgG4抗体进行纯化和浓缩后，进行SEC-HPLC分析。步骤如下：After purification and concentration of the CR3#75/IgG4 antibody, SEC-HPLC analysis was performed. Proceed as follows:
将浓缩蛋白样品(>1mg/mL)与等体积流动相缓冲液(25mM磷酸盐、200mM NaCl，pH 6.8)混合，经0.22μm滤膜(Millipore，Cat#SLGP003RB)过滤后，上样至XBridge Protein BEH SEC Column(Waters，Cat#186007640)，25℃下进行SEC分离，设置流速为0.4mL/min，样品加入体积为10μL，在280nm处测定吸光值。最后采用Empower 2进行数据分析。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.
结果如图9所示，17.839min处的主峰显示蛋白样品的纯度为99.9％，浓度为1.014mg/mL，说明CR3#75/IgG4在DPBS中保持良好的溶解性，基本未发生聚集沉淀。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.
实施例10 热稳定性分析Example 10 Thermal stability analysis
本实施例采用差示扫描量热法(differential scanning calorimetry，DSC)检测抗体的热稳定性。步骤如下：In this example, differential scanning calorimetry (differential scanning calorimetry, DSC) was used to detect the thermal stability of the antibody. Proceed as follows:
配制浓度为0.2～1.0mg/mL的蛋白样品，采用MicroCal PEAQ-DSC system(Northampton，Malvern)从10～100℃、以200℃/h的速率进行DSC检测，绘制热分析图，根据绘制的热分析图进行抗体的热稳定性分析。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.
图10为CR3#75/IgG4的熔解曲线，Tm值为69.83℃，说明此抗体的热稳定性正常。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.
实施例11 溶解性分析Example 11 Solubility Analysis
将CR3#75/IgG4抗体进行纯化和浓缩后，进行SEC-HPLC分析。步骤如下：After purification and concentration of the CR3#75/IgG4 antibody, SEC-HPLC analysis was performed. Proceed as follows:
将浓缩蛋白样品(>10mg/mL)与等体积流动相缓冲液(25mM磷酸盐、200mM NaCl，pH 6.8)混合，经0.22μm滤膜(Millipore，Cat#SLGP003RB)过滤后，上样至XBridge Protein BEH SEC Column(Waters，Cat#186007640)，25℃下进行SEC分离，设置流速为0.4mL/min，样品加入体积为2μL，在280nm处测定吸光值。最后采用Empower 2进行数据分析。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.
结果如图11所示，CR3#75/IgG4抗体可以浓缩至10mg/mL而不产生肉眼可见的沉淀，97.15％蛋白在18.005min处产生单一峰，仅有＜4％的蛋白形成沉淀，说明此抗体可溶性正常，可以继续成药性开发。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.
实施例12 体内药效Example 12 In vivo efficacy
本实施例验证CR3#75/IgG4抗体在NMO小鼠模型的体内药效，步骤如下：This example verifies the in vivo efficacy of CR3#75/IgG4 antibody in the NMO mouse model. The steps are as follows:
6～8周龄的C57雌性小鼠用水合氯醛麻醉后备皮，将小鼠头部固定于脑立体定位仪，调正定位后钻孔，垂直将微量进样针***小鼠脑组织中，开启注射泵，注入CR3#75/IgG4抗体、补体、致病性抗体混合溶液，注射完成后拔针，缝合皮肤，放入笼内饲养。造模7天后取出脑组织制作冰冻组织切片，采用免疫荧光技术对星形胶质细胞表达的AQP4和GFAP进行染色，对各组AQP4和GFAP病灶缺失面积进行统计。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.
结果如图12A和图12B所示，CR3#75/IgG4抗体在0.04mg/mL、0.156mg/mL、0.625mg/mL和2.5mg/mL的浓度下，可抑制NMO典型病灶的形成。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.
实施例13 抗体恒定区替换Example 13 Antibody constant region replacement
本实施例在维持CR3#30/IgG4和CR3#75/IgG4可变区不变的情况下，将重链恒定区替换为IgG1(N297A/K322A)、IgG1(N297A)和IgG2，形成6个全新抗体，氨基酸序列如下：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)重链(SEQ ID NO:25)：CR3#30/IgG1 (N297A/K322A) heavy chain (SEQ ID NO: 25):
Figure PCTCN2021137116-appb-000010
Figure PCTCN2021137116-appb-000010
CR3#30/IgG1(N297A)重链(SEQ ID NO:26)：CR3#30/IgG1 (N297A) heavy chain (SEQ ID NO: 26):
Figure PCTCN2021137116-appb-000011
Figure PCTCN2021137116-appb-000011
CR3#30/IgG2重链(SEQ ID NO:27)：CR3#30/IgG2 heavy chain (SEQ ID NO:27):
Figure PCTCN2021137116-appb-000012
Figure PCTCN2021137116-appb-000012
上述CR3#30/IgG1(N297A/K322A)、CR3#30/IgG1(N297A)和CR3#30/IgG2均选用CR3#30/lambda轻链，序列如SEQ ID NO:28所示：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：SEQ ID NO: 28:
Figure PCTCN2021137116-appb-000013
Figure PCTCN2021137116-appb-000013
CR3#75/IgG1(N297A/K322A)重链(SEQ ID NO:29)：CR3#75/IgG1 (N297A/K322A) heavy chain (SEQ ID NO: 29):
Figure PCTCN2021137116-appb-000014
Figure PCTCN2021137116-appb-000014
Figure PCTCN2021137116-appb-000015
Figure PCTCN2021137116-appb-000015
CR3#75/IgG1(N297A)重链(SEQ ID NO:30)：CR3#75/IgG1 (N297A) heavy chain (SEQ ID NO:30):
Figure PCTCN2021137116-appb-000016
Figure PCTCN2021137116-appb-000016
CR3#75/IgG2重链(SEQ ID NO:31)：CR3#75/IgG2 heavy chain (SEQ ID NO:31):
Figure PCTCN2021137116-appb-000017
Figure PCTCN2021137116-appb-000017
上述CR3#75/IgG1(N297A/K322A)、CR3#75/IgG1(N297A)和CR3#75/IgG2均选用CR3#75/kappa轻链，序列如SEQ ID NO:32所示。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：SEQ ID NO: 32:
Figure PCTCN2021137116-appb-000018
Figure PCTCN2021137116-appb-000018
将上述抗体进行基因合成，构建重组质粒，重组质粒经测序鉴定为正确***有目的片段的阳性克隆后，使用质粒提取试剂盒制备足量无菌重组质粒，用于瞬时转染生产蛋白样品。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.
实施例14 不同恒定区抗体生产Example 14 Different constant region antibody production
将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)和CR3#75/IgG2共计9种抗体的无菌重组质粒经转染试剂(Invitrogen)转染至6×10 6个 ExpiCHO中进行抗体表达。 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.
将重组ExpiCHO细胞在37℃、5％CO 2中培养9～11天后，抗体分泌至无血清细胞培养上清中。对发酵液进行ProteinA-HPLC检测蛋白表达量。步骤如下： 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:
将发酵液样品用0.22μm滤膜(Sartorius，Cat#17C07FT)过滤后，上样至Poros-20A Column(Thermo Scientific，Cat#1-5024-12)，25℃下进行ProteinA-HPLC检测，色谱参数见表6。最后用Empower 3进行数据采集和分析，结果如表7所示。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.
表6 ProteinA-HPLC色谱参数Table 6 ProteinA-HPLC chromatographic parameters
Figure PCTCN2021137116-appb-000019
Figure PCTCN2021137116-appb-000019
表7 ProteinA-HPLC检测发酵液蛋白表达量Table 7 Detection of protein expression in fermentation broth by ProteinA-HPLC
Figure PCTCN2021137116-appb-000020
Figure PCTCN2021137116-appb-000020
9批CHO细胞培养9～11天后，对全部发酵液进行SEC-HPLC检测蛋白纯度，步骤如下：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:
将蛋白样品用0.22μm滤膜(Sartorius，Cat#17C07FT)过滤后，上样至TSKgel G3000 SWXL SEC Column(Tosoh，Cat#008541)，25℃下进行SEC分离，设备为Waters e2695，检测器为 Waters 2998PDA检测器，流动相为50mM磷酸盐+400mM氯化钠(pH 7.0)缓冲液，设置流速为0.5mL/min，进样体积为10μL，在280nm处测定分离的各组分吸光值。最后采用Empower 3进行数据采集和分析，结果如表8所示。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.
表8 SEC-HPLC检测发酵液纯度Table 8 SEC-HPLC detects the purity of fermentation broth
样品编号Sample serial number 样品名称sample name 高分子量(％)High molecular weight (%) 主峰(％)Main peak (%) 低分子量(％)Low molecular weight (%)
S125(38#)-1S125(38#)-1 CR3#24/IgG4 CR3#24/IgG4 30.230.2 19.119.1 50.750.7
S125(38#)-2S125(38#)-2 CR3#30/IgG4 CR3#30/IgG4 31.631.6 19.719.7 48.648.6
S125(38#)-3S125(38#)-3 CR3#75/IgG4 CR3#75/IgG4 28.428.4 24.824.8 46.846.8
S125(38#)-4S125(38#)-4 CR3#30/IgG1(N297A/K322A) CR3#30/IgG1(N297A/K322A) 29.729.7 22.722.7 47.647.6
S125(38#)-5S125(38#)-5 CR3#30/IgG1(N297A) CR3#30/IgG1(N297A) 29.329.3 25.525.5 45.245.2
S125(38#)-6S125(38#)-6 CR3#30/IgG2 CR3#30/IgG2 34.634.6 10.810.8 54.754.7
S125(38#)-7S125(38#)-7 CR3#75/IgG1(N297A/K322A) CR3#75/IgG1(N297A/K322A) 30.030.0 19.019.0 51.051.0
S125(38#)-8S125(38#)-8 CR3#75/IgG1(N297A) CR3#75/IgG1(N297A) 29.629.6 19.819.8 50.550.5
S125(38#)-9S125(38#)-9 CR3#75/IgG2 CR3#75/IgG2 27.127.1 13.513.5 59.559.5
9个候选分子的发酵液进行PrismA捕获后，利用紫外分光光度计测定蛋白浓度，步骤如下：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:
取供试品溶液，以配制供试品溶液的同批溶剂为空白对照。采用1cm的石英吸收池，将样品用空白溶剂进行稀释，将装有样品的石英吸收池放入检测池检测。紫外分光光度计为IMPLEN，型号NP80。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.
蛋白浓度计算公式如下：The formula for calculating protein concentration is as follows:
蛋白质浓度
Figure PCTCN2021137116-appb-000021
protein concentration
Figure PCTCN2021137116-appb-000021
式中，X：稀释倍数；
Figure PCTCN2021137116-appb-000022
供试品蛋白的吸收系数。 In the formula, X: dilution ratio;
Figure PCTCN2021137116-appb-000022
The absorption coefficient of the protein to be tested .
结果如表9所示。The results are shown in Table 9.
表9 纯化样品浓度Table 9 Purified sample concentration
样品编号Sample serial number 样品名称sample name 蛋白浓度(mg/mL)Protein concentration (mg/mL)
S125(38#)-1S125(38#)-1 CR3#24/IgG4 CR3#24/IgG4 2.52.5
S125(38#)-2S125(38#)-2 CR3#30/IgG4 CR3#30/IgG4 2.62.6
S125(38#)-3S125(38#)-3 CR3#75/IgG4 CR3#75/IgG4 2.52.5
S125(38#)-4S125(38#)-4 CR3#30/IgG1(N297A/K322A) CR3#30/IgG1(N297A/K322A) 4.44.4
S125(38#)-5S125(38#)-5 CR3#30/IgG1(N297A) CR3#30/IgG1(N297A) 2.42.4
S125(38#)-6S125(38#)-6 CR3#30/IgG2 CR3#30/IgG2 1.41.4
S125(38#)-7S125(38#)-7 CR3#75/IgG1(N297A/K322A) CR3#75/IgG1(N297A/K322A) 3.03.0
S125(38#)-8S125(38#)-8 CR3#75/IgG1(N297A) CR3#75/IgG1(N297A) 2.02.0
S125(38#)-9S125(38#)-9 CR3#75/IgG2 CR3#75/IgG2 1.41.4
将9个候选分子的发酵液进行PrismA捕获后，进行SEC-HPLC分析，步骤如下：The fermentation broth of the 9 candidate molecules was captured by PrisMA and then analyzed by SEC-HPLC. The steps are as follows:
将蛋白样品用0.22μm滤膜(Sartorius，Cat#17C07FT)过滤后，上样至TSKgel G3000 SWXL SEC Column(Tosoh，Cat#008541)，25℃下进行SEC分离，设备为Waters e2695，检测器为Waters 2998 PDA检测器，流动相为50mM磷酸盐+400mM氯化钠(pH 7.0)缓冲液，设置流速为0.5mL/min，进样体积为10μL，在280nm处测定吸光值。最后采用Empower 3进行数据采集和分析，结果如表10所示。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.
表10 SEC-HPLC检测纯化样品纯度Table 10 Purity of purified samples detected by SEC-HPLC
Figure PCTCN2021137116-appb-000023
Figure PCTCN2021137116-appb-000023
综合上述结果，9种抗体顺利完成样品生产，浓度均大于1mg/mL，纯度均高于90％，可以进行后续实验。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.
实施例15聚集性分析Example 15 Aggregation Analysis
本实施例利用iCIEF测定各候选分子等电点，步骤如下：The present embodiment utilizes iCIEF to measure the isoelectric point of each candidate molecule, and the steps are as follows:
样品脱盐后，用蒸馏水稀释至蛋白浓度约为2mg/mL，按以下体系配置(200μL体系)：8μL 2％pharmalyte(pH 3～10)(GE，Cat#17045601)，70μL 1％MC(ProteinSimple，Cat#1018761)，1μL Low pI(ProteinSimple，Cat#102222)，1μL High pI(ProteinSimple，Cat#101996)，100μL 8M尿素(Sigma，Cat#U0631-500G1)，20μL样品。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.
体系配置于离心管中，随后10000g离心5min，取上清(60～120μL)加入内插管，内插管置于离心管中10000g离心5min，取出内插管，放于进样瓶中，上机检测，设备为ProteinSimple iCE3。结果如表11所示，实际等电点略高于预测的等电点。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.
表11 候选分子等电点Table 11 Isoelectric points of candidate molecules
样品编号Sample serial number 样品名称sample name 预测等电点predicted isoelectric point iCIEF-等电点iCIEF - isoelectric point
S125(38#)-1S125(38#)-1 CR3#24/IgG4 CR3#24/IgG4 5.975.97 6.136.13
S125(38#)-2S125(38#)-2 CR3#30/IgG4 CR3#30/IgG4 6.186.18 6.306.30
S125(38#)-3S125(38#)-3 CR3#75/IgG4 CR3#75/IgG4 5.925.92 5.895.89
S125(38#)-4S125(38#)-4 CR3#30/IgG1(N297A/K322A) CR3#30/IgG1(N297A/K322A) 7.247.24 7.337.33
S125(38#)-5S125(38#)-5 CR3#30/IgG1(N297A) CR3#30/IgG1(N297A) 7.567.56 7.577.57
S125(38#)-6S125(38#)-6 CR3#30/IgG2 CR3#30/IgG2 6.506.50 6.976.97
S125(38#)-7S125(38#)-7 CR3#75/IgG1(N297A/K322A) CR3#75/IgG1(N297A/K322A) 6.366.36 6.586.58
S125(38#)-8S125(38#)-8 CR3#75/IgG1(N297A) CR3#75/IgG1(N297A) 6.516.51 6.766.76
S125(38#)-9S125(38#)-9 CR3#75/IgG2 CR3#75/IgG2 5.975.97 6.186.18
实施例16 毛细管电泳分析Example 16 Capillary electrophoresis analysis
将9个候选分子的发酵液进行PrismA捕获后，进行CE-SDS分析，步骤如下：After the fermentation broth of 9 candidate molecules was captured by PrisMA, CE-SDS analysis was performed. The steps were as follows:
将蛋白样品用30μm超滤离心管(Sartorius，Cat#VS0122)超滤，然后制备样品，70℃加热，上样，25℃下进行CE-SDS分离，设备为SCIEX PA800 Plus，检测器为PDA检测器，在220nm处测定吸光值。最后采用32karat进行数据采集和分析，结果如表12所示，其中CR3#75/IgG2纯度较低。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.
表12 毛细管电泳检测纯化样品纯度Table 12 Purity of purified samples detected by capillary electrophoresis
Figure PCTCN2021137116-appb-000024
Figure PCTCN2021137116-appb-000024
实施例17 C5特异性抗体的蛋白稳定性检测Example 17 Detection of protein stability of C5-specific antibodies
本实施例采用蛋白稳定性分析***
Figure PCTCN2021137116-appb-000025
(Unchained Labs，Calif.)的Tm&Tagg with optional DLS程序，检测蛋白粒径和稳定性。通过分析结果参数Tm(蛋白熔解温度)、Tagg266(蛋白聚集温度)、Tagg473(蛋白聚集温度)、Z-Ave.Dia(蛋白平均粒径)、PDI(多分散性系数)、Pk1 Mode Dia、Pk1 Mass(％)、Pk2 Mode Dia和Pk2 Mass(％)9个参数，多角度分析蛋白粒度及粒度分布和热稳定性。步骤如下： This example adopts a protein stability analysis system
Figure PCTCN2021137116-appb-000025
(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:
取抗体9μL加入uni管(Unchained Labs，Calif.)中，每种抗体重复2个复孔，选定Tm&Tagg with optional DLS程序进行分析。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.
25℃和95℃粒度结果分别如表13和表14所示，Tm&Tagg结果如表15所示。其中，Z-Ave.Dia-样品平均水化动力学直径，反映样品整体粒径情况；PDI-多分散性系数，PDI<0.1表示粒度窄分布，值越大表示粒径分布越不均一，离散程度越高；Fit Var-拟合偏差，小于0.01表示Z-Ave.Dia和PDI高可信度，值越大表示可信度越低；Pk1 Mode Dia-光强分布显示多峰时，第一个主峰的众数粒径；Pk 1 Mass(％)-第一个峰的质量占比。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.
从表13可以看出，S125(38#)-3/6/7/9平均水化动力学直径与Pk1 Mode Dia.均在11nm左右，无大粒径成分，PDI<0.1，表明粒度均一，推测为蛋白单体；组中其余样品均存在较大粒径成分，推测为聚集体。从表14可以看出，由平均水化动力学直径和PDI可知，经过加热后蛋白分子产生明显聚集。从表15可以看出，S125(38#)-4和S125(38#)-7Tm较高，推测构象热稳定性较高，S125(38#)-2/4/5/6/7Tagg266均在60℃以上，推测胶体热稳定性较高。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.
表13 25℃粒度结果Table 13 Particle size results at 25°C
Figure PCTCN2021137116-appb-000026
Figure PCTCN2021137116-appb-000026
Figure PCTCN2021137116-appb-000027
Figure PCTCN2021137116-appb-000027
表14 95℃粒度结果Table 14 95℃ particle size results
Figure PCTCN2021137116-appb-000028
Figure PCTCN2021137116-appb-000028
Figure PCTCN2021137116-appb-000029
Figure PCTCN2021137116-appb-000029
表15 Tm&Tagg结果Table 15 Tm&Tagg results
Figure PCTCN2021137116-appb-000030
Figure PCTCN2021137116-appb-000030
综合上述结果，可以看出S125(38#)-7组的综合热稳定性较高，即CR3#75/IgG1(N297A/K322A)具有优于CR3#75/IgG4的热稳定性。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.
综上所述，本申请的抗C5抗体与人源重组C5蛋白具有显著的结合能力，半衰期长，溶解性良好，热稳定性良好，有望开发为蛋白药物，应用于预防和/或治疗C5过度激活导致的自身免疫疾病中。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.
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。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. 抗C5抗体，其包括重链可变区和轻链可变区；an anti-C5 antibody comprising a heavy chain variable region and a light chain variable region;
    所述重链可变区包括SEQ ID NO:3、SEQ ID NO:9或SEQ ID NO:15所示的CDR3；The heavy chain variable region includes the CDR3 shown in SEQ ID NO:3, SEQ ID NO:9 or SEQ ID NO:15;
    所述轻链可变区包括SEQ ID NO:6、SEQ ID NO:12或SEQ ID NO:18所示的CDR3。The light chain variable region includes the CDR3 shown in SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:18.
  2. 根据权利要求1所述的抗C5抗体，其中，所述重链可变区还包括SEQ ID NO:1、SEQ ID NO:7或SEQ ID NO:13所示的CDR1。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. 根据权利要求1所述的抗C5抗体，其中，所述重链可变区还包括SEQ ID NO:2、SEQ ID NO:8或SEQ ID NO:14所示的CDR2。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. 根据权利要求1所述的抗C5抗体，其中，所述轻链可变区还包括SEQ ID NO:4、SEQ ID NO:10或SEQ ID NO:16所示的CDR1。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. 根据权利要求1所述的抗C5抗体，其中，所述轻链可变区还包括SEQ ID NO:5、SEQ ID NO:11或SEQ ID NO:17所示的CDR2。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. 根据权利要求1-5任一项所述的抗C5抗体，其中，所述抗C5抗体的重链可变区包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、SEQ ID NO:3所示的CDR3；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;
    所述抗C5抗体的轻链可变区包括SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2、SEQ ID NO:6所示的CDR3；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;
    任选地，所述抗C5抗体的重链可变区包括SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2、SEQ ID NO:9所示的CDR3；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;
    所述抗C5抗体的轻链可变区包括SEQ ID NO:10所示的CDR1、SEQ ID NO:11所示的CDR2、SEQ ID NO:12所示的CDR3；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;
    任选地，所述抗C5抗体的重链可变区包括SEQ ID NO:13所示的CDR1、SEQ ID NO:14所示的CDR2、SEQ ID NO:15所示的CDR3；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;
    所述抗C5抗体的轻链可变区包括SEQ ID NO:16所示的CDR1、SEQ ID NO:17所示的CDR2、SEQ ID NO:18所示的CDR3。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. 根据权利要求1-6任一项所述的抗C5抗体，其中，所述抗C5抗体的重链可变区包括SEQ ID NO:19所示的氨基酸序列，轻链可变区包括SEQ ID NO:20所示的氨基酸序列；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;
    任选地，所述抗C5抗体的重链可变区包括SEQ ID NO:21所示的氨基酸序列，轻链可变区包括SEQ ID NO:22所示的氨基酸序列；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;
    任选地，所述抗C5抗体的重链可变区包括SEQ ID NO:23所示的氨基酸序列，轻链可变区包括SEQ ID NO:24所示的氨基酸序列；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;
    任选地，所述抗C5抗体还包括恒定区；Optionally, the anti-C5 antibody further comprises a constant region;
    任选地，所述恒定区来源于IgG4、IgG1或IgG2中的任意一种；Optionally, the constant region is derived from any one of IgG4, IgG1 or IgG2;
    任选地，所述IgG4具有S228P突变；Optionally, the IgG4 has the S228P mutation;
    任选地，所述IgG1具有N297A和/或K322A突变；Optionally, the IgG1 has N297A and/or K322A mutations;
    任选地，所述IgG2具有S257A突变。Optionally, the IgG2 has the S257A mutation.
  8. 核酸分子，其包括编码权利要求1-7任一项所述的抗C5抗体的DNA片段。A nucleic acid molecule comprising a DNA fragment encoding the anti-C5 antibody of any one of claims 1-7.
  9. 表达载体，其包括权利要求8所述的核酸分子。An expression vector comprising the nucleic acid molecule of claim 8.
  10. 重组细胞，其表达权利要求1-7任一项所述的抗C5抗体；A recombinant cell expressing the anti-C5 antibody of any one of claims 1-7;
    任选地，所述重组细胞的基因组中整合有权利要求8所述的核酸分子；Optionally, the nucleic acid molecule of claim 8 is integrated into the genome of the recombinant cell;
    任选地，所述重组细胞包括权利要求9所述的表达载体。Optionally, the recombinant cell comprises the expression vector of claim 9.
  11. 一种权利要求1-7任一项所述的抗C5抗体的制备方法，其包括以下步骤：A preparation method of the anti-C5 antibody of any one of claims 1-7, comprising the following steps:
    (1)将抗C5抗体的编码核酸连接入质粒，转入感受态细胞，培养后挑取单克隆细胞进行筛选；(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)提取筛选的阳性克隆的表达载体，转入宿主细胞，培养并收集上清液，分离纯化得到所述抗体。(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. 药物组合物，其包括权利要求1-7任一项所述的抗C5抗体；A pharmaceutical composition comprising the anti-C5 antibody of any one of claims 1-7;
    任选地，所述药物组合物还包括抗肿瘤药物；Optionally, the pharmaceutical composition further includes an antitumor drug;
    任选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂中的任意一种或至少两种的组合。Optionally, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.
  13. 权利要求1-7任一项所述的抗C5抗体、权利要求8所述的核酸分子、权利要求9所述的表达载体、权利要求10所述的重组细胞或权利要求12所述的药物组合物在制备疾病检测试剂和/或疾病治疗药物中的应用；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;
    任选地，所述疾病包括C5过度激活导致的自身免疫疾病。Optionally, the disease comprises an autoimmune disease caused by overactivation of C5.
PCT/CN2021/137116 2020-12-11 2021-12-10 Anti-c5 antibody and application thereof WO2022122020A1 (en)

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