CN108752473B

CN108752473B - anti-C5 fully human single-chain antibody C5B3 and application thereof

Info

Publication number: CN108752473B
Application number: CN201810414275.8A
Authority: CN
Inventors: 闫亚平; 朱文莉
Original assignee: Shaanxi Normal University
Current assignee: Xi'an Tianhong Biotechnology Co.,Ltd.
Priority date: 2018-05-03
Filing date: 2018-05-03
Publication date: 2021-08-31
Anticipated expiration: 2038-05-03
Also published as: CN108752473A

Abstract

The invention discloses an anti-C5 fully human single-chain antibody C5B3 and application thereof, wherein the DNA sequence of the anti-C5 fully human single-chain antibody C5B3 is shown in SEQ ID NO 1. Can obviously inhibit the formation of membrane attack complex in the inflammatory reaction process, thereby reducing the damage in the inflammatory reaction process. The C5 fully human single-chain antibody C5B3 is applied to the preparation of medicines for treating autoimmune diseases such as neuromyelitis optica, multiple sclerosis, systemic lupus erythematosus and myasthenia gravis and/or Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical hemolytic toxemia syndrome (AHU), inflammation caused by trauma and related characteristics of the inflammation.

Description

anti-C5 fully human single-chain antibody C5B3 and application thereof

Technical Field

The invention belongs to the field of bioengineering, and relates to an anti-C5 fully human single-chain antibody C5B3 and application thereof.

Background

C5 is an important component of complement, the complement system comprises more than 30 components, and is widely present on the surface of serum, tissue fluid and cell membrane, complement is not only an important component of the body's inherent immune defense system, but also one of the important mechanisms of antibody to exert immune effect, and participates in adaptive immune response and its regulation in different links. During the inflammatory reaction, C5 convertase breaks down C5 into C5a and C5b, C5a is free in the liquid phase and is an important inflammatory mediator, and C5b forms a Membrane Attack Complex (MAC) through a series of complement reactions, leading to cell lysis death.

Complement activation is a highly ordered cascade of reactions that exerts a wide range of biological effects. Normally, complement activation and its end effects are under tight regulation, thus effectively maintaining the body's homeostasis, but can cause damage to its own tissues when immune dysfunction or uncontrolled complement activation occurs. This is closely related to the occurrence and development of many diseases, such as systemic lupus erythematosus, rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria, neuromyelitis optica, and the like. Therefore, the treatment methods related to C5 have a great relationship to the treatment and prognosis of diseases.

The hybridoma technology was created in 1975 by Kohler and Milstein, and monoclonal antibodies specifically recognizing and specifically binding to a single epitope of an antigen were obtained for the first time, opening a new era in which antibodies are widely used as therapeutic drugs. Compared with polyclonal antibodies, the monoclonal antibodies have the advantages of high specificity, high affinity, small side effect and the like, and a large amount of mouse monoclonal antibodies are researched, developed and applied and are quickly used in clinical experiments. However, in many clinical trials, it has been found that the mouse monoclonal antibody is heterologous to human body, and causes strong human anti-mouse antibody reaction (HAMA) of patients, and cannot be used for many times; and intact antibodies are of large molecular weight and rarely reach the target organ or tissue. Therefore, they are molecularly engineered from the origin of the amino acid residue sequence. The antibody humanization technology brings about a great reduction in the immunogenicity of the therapeutic genetically engineered antibody, but even the humanized antibody of the murine mab with a very high degree of humanization, which still contains at least 1% to 5% of heterologous components, may still cause an anti-antibody response in the patient. Although humanized antibodies currently dominate the clinical applications of antibodies, with the progress of human antibody technology, fully human antibodies are gradually coming and coming, and occupy the leading position for the development of new antibody drugs.

At present, monoclonal antibodies for blocking the effect of C5 complement are mainly obtained by performing humanized transformation on a murine source, the humanized process is complicated, and HAMA reaction still exists after humanized, the reaction affects the therapeutic effect of the antibodies, secondary harm can be caused to patients, such as anaphylactic reaction, kidney damage and the like, and the life can be threatened in serious cases. Therefore, human antibodies are becoming the mainstream trend of antibody development with their unique advantages. Based on the research background, the present phage display technology is widely applied to monoclonal antibody screening, but is different for different target targets and different phage antibody libraries.

Disclosure of Invention

Aiming at the defects and shortcomings in the prior art, the invention aims to provide an anti-C5 fully human single-chain antibody C5B3 which can obviously inhibit the formation of a membrane attack complex in the inflammatory reaction process, thereby reducing the damage in the inflammatory reaction process and possibly providing antibody-based therapy for autoimmune diseases and diseases characterized by complement correlation.

The second purpose of the invention is to provide the application of the C5 fully human single-chain antibody C5B3 in preparing the medicines for treating autoimmune diseases and/or diseases with complement as relevant characteristics.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

an anti-C5 fully human single-chain antibody C5B3, wherein the DNA sequence of the anti-C5 fully human single-chain antibody C5B3 is selected from the following sequences: 1 is shown in SEQ ID NO; a sequence having more than 90% homology with SEQ ID NO. 1.

Optionally, the amino acid sequence of the anti-C5 fully human single-chain antibody C5B3 is selected from the coding sequence of one of the following proteins: the sequence shown as SEQ ID NO. 2; a protein sequence with more than 90 percent of homology with SEQ ID NO. 2.

Optionally, the amino acid sequence of the heavy chain variable region VHCDR1 of the anti-C5 fully human single-chain antibody C5B3 is shown in SEQ ID NO. 3; the amino acid sequence of VH CDR2 in the heavy chain variable region is shown in SEQ ID NO. 4; the amino acid sequence of the heavy chain variable region VHCDR3 is shown in SEQ ID NO. 5;

the amino acid sequence of the light chain variable region V LCDR1 of the anti-C5 fully human single-chain antibody C5B3 is shown in SEQ ID NO. 6, the amino acid sequence of the light chain variable region VLCDR2 is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region VLCDR3 is shown in SEQ ID NO. 8.

A carrier containing anti-C5 fully human single-chain antibody C5B3 is pET30a, and the anti-C5 fully human single-chain antibody C5B3 is inserted into the carrier.

A recombinant host cell BL21, said recombinant host cell BL21 contains said vector.

The anti-C5 fully human single-chain antibody C5B3 is applied to the preparation of medicines for treating autoimmune diseases and/or diseases with C5 complement as related characteristics.

The anti-C5 fully human single-chain antibody C5B3 is applied to preparation of medicines for treating autoimmune diseases caused by formation of membrane attack complexes in inflammatory reaction processes and/or diseases with C5 complement as relevant characteristics.

Optionally, the autoimmune disease includes neuromyelitis optica, multiple sclerosis, systemic lupus erythematosus, and myasthenia gravis.

Optionally, the diseases characterized by C5 complement include paroxysmal nocturnal hemoglobinuria, atypical hemolytic syndrome, and inflammation caused by trauma.

The invention has the beneficial effects that:

the single-chain antibody of the invention can obviously inhibit the formation of a membrane attack complex in the inflammatory reaction process aiming at complement C5, thereby reducing the damage in the inflammatory reaction process and possibly providing an antibody-based therapy for autoimmune diseases and complement-related diseases.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 shows the specificity of the elisa detection phage C5B3 antibody;

FIG. 2 is a schematic structural diagram of a recombinant expression vector pET30a-C5B 3;

FIG. 3 is a purified C5B3 single chain antibody, approximately 30Kd in size;

FIG. 4 is a (left) C5B3 graph showing a reduced complement deposition and (right) a reduced complement deposition statistic graph;

FIG. 5 shows the functional verification of the single-chain antibody of the present invention, and the left panel shows a negative control; the right panel shows that dead cells (red) are significantly reduced when the single-chain antibody is added;

FIG. 6 shows the dose-effect relationship of the anti-C5 scFv function verification, wherein the number of dead cells decreased with the increase of the amount of the scFv added;

FIG. 7 shows the results of in vivo experiments on the antibodies of the present invention, in which the C5B3mAb group is added to provide significant protection, and the lesions are significantly smaller;

FIG. 8 is a graph of statistical analysis of in vivo experimental data;

detailed description of the invention

The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The invention is based on the fully human scFv single-chain antibody library, and the fully human anti-C5 single-chain antibody is obtained by screening through a phage display method, so that HAMA reaction is avoided, and functional verification is performed; compared with the CDR region of the prior anti-C5 single-chain antibody, the difference is large, and the monoclonal antibody is a brand-new monoclonal antibody drug. The invention is a single-chain antibody, has the size of about 30KD and small protein molecular weight, is easier to enter a focus part to take effect, can pass through a blood brain barrier particularly for brain diseases, shortens the time of reaching the focus, and can play a better treatment effect.

The DNA sequence of the fully human anti-complement C5 single-chain antibody C5B3 and the fully human anti-complement C5 single-chain antibody C5B3 is shown as follows:

ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACTTTCTCTATGTATGATATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTTCTTCTTCTGGTGGCATTACTTCGTACGCAGA CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATG AACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACGACGCGGTTTTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAG CGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA GGAGACAGAGTCACCATCACTTGCCGGGCAAGTGGCTCGAACATCGGAAGTAATATTTTAAATT GGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAATGACCATCGTTTGCAAAG TGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT CTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCTCGGACTAGTCCTTCTACGTTCG GCCAAGGGACCAAGGTGGAAATCAAACGG。

the amino acid sequence of the fully human anti-complement C5 single-chain antibody C5B3 is shown as follows: MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSMYDMSWVRQAPGKGLEWVSSSSSGGITSYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTTRFFDYWGQGTLVTVSSGGGGSGGGGSG GGGSTDIQMTQSPSSLSASVGDRVTITCRASGSNIGSNILNWYQQKPGKAPKLLIYNDHRLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQARTSPSTFGQGTKVEIKR are provided.

The fully human anti-complement C5 single-chain antibody C5B3 contains a complete antibody heavy chain variable region VH and a light chain variable region VL, and the amino acid sequence of the heavy chain variable region VHCDR1 is as follows: GFTFSMYD, the amino acid sequence of VH CDR2 of the heavy chain variable region is SSSGIT, and the amino acid sequence of VHCDR3 of the heavy chain variable region is: AKTTRFFDY, respectively; the amino acid sequence of the light chain variable region VLCDR1 is as follows: GSNIGSNI, amino acid sequence of light chain variable region VLCDR 2: NDH, amino acid sequence of the light chain variable region VLCDR 3: QQARTSPST are provided.

The invention recombines an expression vector pET30a-C5B3 based on pET30a, contains the complete nucleic acid sequence in the step (3), and can express C5B3 in a large amount by being transformed into BL 21.

Autoimmune diseases and/or complement-associated diseases, wherein the autoimmune diseases include optic neuromyelitis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis and other autoimmune diseases, and the C5 complement-associated diseases include Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical hemolytic toxemia syndrome (AHU), and trauma-induced inflammation.

1. Interpretation of terms:

antibodies (antibodies), also called immunoglobulins (igs), are globulins with immune function synthesized and secreted by B cells after immune cells of the body are activated by antigens and the B cells differentiate into plasma cells, which are specifically bound with corresponding antigens. The basic structure of an Ig molecule is composed of four polypeptide chains. Namely, the polypeptide consists of two identical peptide chains with relatively large molecular mass (called heavy chains, H chains) and two identical peptide chains with relatively small molecular mass (called light chains, L chains), and V regions of the L chains and the H chains are respectively called VL and VH. There is a higher degree of variation in the amino acid composition and sequence of certain local regions in VL and VH, and where antibodies bind antigen, and are therefore referred to as complementarity-determining regions (CDRs), including three distinct sites CDR1, CDR2 and CDR3, and generally CDR3 is more variable. Hypervariable regions are also the sites where the unique determinants (idiotopic determinants) of the Ig molecule are predominantly present. In most cases the H chain plays a more important role in binding to the antigen. The amino acid composition and arrangement of the non-CDR regions in the V region is relatively conserved, called the Framework Regions (FRs). The hypervariable regions in VL are three FR1, FR2 and FR 3.

Murine monoclonal antibodies: early therapeutic antibodies were mostly murine monoclonal antibodies directed against only one epitope prepared by B-lymphocyte hybridoma technology. Due to the difference of genetic backgrounds between human and mice, when a mouse antibody is used in a human body, the mouse antibody can cause human anti-mouse antibody reaction because of exogenous protein antigens, and human effect functions are fully activated, so that the application and development of the monoclonal antibody in disease treatment are greatly limited. In contrast, murine monoclonal antibody therapeutics are generally difficult to achieve successfully. After the murine antibody is used for a human body, the half-life period of the antibody molecule is short, the antibody molecule needs to be repeatedly applied, and HAMA response can be generated after repeated large-scale application, so that the murine antibody is eliminated by the human body, and the drug effect is reduced.

Humanized monoclonal antibodies: humanized antibodies include reshaped antibodies and veneered antibodies, and are characterized by reduced HAMA response and relatively long half-life in blood. The reshaped antibody is also called CDR grafting antibody, which is to graft the C DR of the mouse monoclonal antibody to the frame area of the human monoclonal antibody, so that the human monoclonal antibody has the same antigen specificity as the mouse monoclonal antibody and can reduce the heterogeneity of the mouse monoclonal antibody to the maximum. The reshaped antibody further reduces the proportion of murine portions in the antibody, reducing the HAMA response, as compared to the chimeric antibody.

Fully human antibodies: although the content of mouse components of the humanized antibody is obviously reduced, the immunogenicity of the humanized antibody cannot achieve a satisfactory result, and the immunogenicity cannot be ignored or even cannot be tolerated. Therefore, the research of fully human antibodies is naturally becoming a current trend, and fully human antibodies can be realized by using human hybridoma cells, transgenic animals, phage antibody libraries, and the like. The utilization of phage display technology, transgenic mice and other molecular biology techniques allows the rapid development of the research of fully human monoclonal antibodies.

2. Phage display

(1) The antibody phage library is subjected to antigen-specific panning by using a solid phase panning method, which comprises the following specific operations:

the resuscitated phage library (from CreactiveBiolabs) was grown in 2ml of 2 × TY medium at 37 ℃ in a shaker until OD was 0.4, and inoculated into 50ml of medium for expansion culture for 2 h. Adding M13KO7 helper phage (Invitrogen); standing at 37 deg.C for 30-60min, incubating in shaker for 30-60min, centrifuging at 4500r/min for 5-10min, discarding supernatant, resuspending cells in culture medium containing 100ug/ml ampicillin and 50ug/ml kanamycin, and culturing in shaker at 30 deg.C overnight; the phage library was precipitated by centrifugation at 8000r/min for 3-5min and the supernatant was transferred to a clean 50ml centrifuge tube, 25 ml/tube. Adding 1/5 volume of PEG/NaCl (20% PEG-8000, 2.5mol/L NaCl, the same below) into each centrifugal tube, mixing, placing on ice for at least 2h, centrifuging at 12000r/min for 20min, and discarding supernatant; the phage were resuspended in 2ml sterile PBS, centrifuged, 5000r/min, 10min, and the remaining cell debris was removed.

The specific screening process is as follows:

(1) diluting complement C5 with 0.1mol/L sodium bicarbonate solution with pH9.6 sodium carbonate, coating 96-well plate with antigen amount of 300-800ng per round, coating system with 50-100 μ L per well, and standing overnight at 4 deg.C;

(2) washing off unadsorbed antigen in the plate the next day, blocking with 2% BSA (PBS) or 5% skimmed milk Powder (PBS) at room temperature for 1-2 h;

(3) and (4) removing the confining liquid, adding 50-70 mu l of phage antibody library into each hole, and incubating for 2h at normal temperature in the first round and the second round. Incubating for 1 hour in the third round, incubating for 40min in the fourth round and the fifth round, reducing incubation time, reducing non-specific adsorption and phage antibodies with weak affinity, discarding unbound phage in the wells, washing each well with 0.1% PBST buffer (0.1% Tween-20 is added into 1. multidot. PBS) and PBS washing solution for 6-8 times, increasing times in sequence for several rounds to improve screening stringency, and fully washing off non-specific adsorption phage;

(4) adding 50 μ l glycine hydrochloric acid elution buffer solution with pH of 2.2 into each well, incubating at room temperature for 10min for amplification in the first, second and third rounds, pre-eluting with glycine for 5min in the fourth and fifth rounds, discarding the eluate, and eluting with 50ul glycine for 15 min. During this period, the pipette was used to blow several times, but care was taken not to blow too many bubbles. Adding 2mol/L Tris-HCl PH8.8 to lead the pH value of the solution to be about 7.0 so as to neutralize the eluted phage solution;

(5) the eluate was immediately added to 3ml of freshly prepared OD₆₀₀About 0.6 E.coli TG1 strain, incubated at 37 ℃ for 1-2 h. Adding 100ug/ml ampicillin and 2% glucose at 37 deg.C and 220r/min overnight;

(6) adding 10¹²The helper phage M13KO7 of pfu, 1-2h at 37 ℃. 4500r 10min, discarding supernatant, adding 100ug/ml ampicillin and 50ug/ml kanamycin, and shake culturing at 37 deg.C overnight;

(7) collecting supernatant at 8000r/min for 5min, adding PEG/NaCl, ice-drying for 2-4h, collecting phage at 12000r20-30min, discarding supernatant, adding 1ml sterile PBS to dissolve phage for next round of screening, adding 20% glycerol, and preserving at-80 deg.C for a long period. The invention carries out 5 rounds of screening;

(8) after 5 rounds of enrichment screening, the eluted phage was infected with freshly prepared TG1 bacteria, cultured overnight at 37 ℃, and 265 clones were randomly selected and shaken, and helper phage were added to obtain phage antibody, which was used for Elisa detection.

Elisa analysis and sequencing analysis

Diluting complement C5 with carbonate coating solution (pH9.6), adding 50ul into each reaction well, and coating overnight at 4 deg.C; discarding the liquid in the reaction wells, washing with 1 × PBS buffer solution for 3 times, adding 200ul of 2% BSA blocking solution to each well, and blocking at room temperature for 1-2 h; washing 3 times with 1 × PBS buffer; adding phage supernatant containing scFv antibody and negative control 50 ul/well, incubating at room temperature for 1-2h, and washing with 1 × PBS buffer solution for 5 times; adding 50 ul/well of Anti-M13(HRP) monoclonal antibody (Creative Diagnostics) diluted with blocking buffer (1:1000), incubating at room temperature for 1h, and washing 5 times with 1 × PBS buffer; adding TMB substrate solution (50 ul/hole) into each reaction hole for reaction for 5-10 min; stop solution (2M sulfuric acid, 50 ul/well) was added to stop the reaction; reading the light absorption value of 450nm by using an enzyme-labeling instrument; elisa was repeated, 50 clones with higher affinity were selected for sequencing according to the results of Elisa, homology analysis was performed on the sequencing results, and the sequences included 9 sequences, among which C5B3, C5A6, C5B35, C5A102 appeared at a higher frequency, C5B3 appeared at the highest frequency, and 9 clones were C5B3, and Elisa was strongly positive and had good specificity (FIG. 1).

Construction and transfection of the C5B3 plasmid

The single-chain antibody C5B3, C5A6 and C5B35 genes of the anti-complement C5 are amplified in the form of phage vectors, C5B3 is taken as an example for illustration, BamHI and NotI are selected as enzyme cutting sites, upstream BamHI-R: AGGATCCGCCGAGGTGCAGCTGTTG downstream primer NotI-F: TGCGGCCGCCCGTTTGATTTCCACCTT, PCR is designed to be amplified to obtain the C5B3 single-chain antibody gene. The target fragment, pET28a and PGEX-4T vector were digested with BamHI and NotI (Takara), the target fragment and vector were ligated with T4DNA ligase, and a ligation reaction system was prepared in a molecular molar ratio of foreign gene to vector DNA of 3: 1:10 XT 4DNA Ligase Buffer (Takara) 1uL DNA fragment about 5ul vector DNA about 2ul T4DNA Ligase 1uL 4 ℃ for overnight connection, adding the connection product into E.coli DH5 alpha competence (Thermo), incubating overnight at 37 ℃ for colony appearance, carrying out colony PCR identification, sequencing the positive result of PCR identification, transferring the correct sequencing result into expression bacteria E.coli BL21(DE3), pET28a-C5B3 and PGEX-4T-C5B3 can not express. The single chain antibody C5B3 gene was attempted to be ligated to pET-30a vector to form pET30a-C5B3 (FIG. 2), transformed into E.coli BL21(DE 3). The transformants were plated and positive clones were picked on LB plates containing 70. mu.g/ml kanamycin and sequenced. And comparing and sequencing results by using DNAMAN, and transferring the successfully constructed clone into E.coli BL21(DE3) strain.

Randomly selecting a recombinant host bacterium BL21(DE3) which is transferred into pET30a-C5B3 for cloning and expanding culture. The correctly sequenced high expression monoclonal was inoculated into 10mL LB medium containing 70. mu.g/mL kanamycin and cultured at 37 ℃ at 220rpm overnight. The next day, the bacterial suspension was added to 500mL of LB medium containing 70ug/mL kanamycin in two flasks on average, and shaken under the same conditions until the OD600 reached about 0.6. 1M IPTG was added to a final concentration of 0.2mM, and the mixture was induced at 16 ℃ and 180rpm for 20 h. After the induction expression is finished, the bacterial liquid is transferred to a 50ml centrifuge tube. Freeze-centrifuging at 8000rpm and 4 deg.C for 5min, and collecting for several times. The pellet was resuspended in 10mL of precooled PBS. The bacteria are crushed by ultrasonic waves, and the parameters are as follows: the power is 400W, the working time is 5s, the intermission is 6s, each circulation is 99 times, and the circulation is repeated for 4 times. Centrifuging at 4 deg.C at 10000rpm for 30min, filtering the supernatant with 0.22um filter membrane for sterilization, and obtaining single chain antibody protein crude solution. The nickel column was washed with double distilled water for 10min and then equilibrated with PBS for 10min at a flow rate of 2 mL/min. The crude single-chain antibody protein solution was applied to the column with a flow rate of 2 mL/min. The effluent was collected, re-loaded and after completion the nickel column was equilibrated with 20ml PBS. Next, the proteins on the nickel column were eluted with elution buffers containing 25mM, 100mM, 300mM, 600mM imidazole, respectively, filling 6ml tubes per concentration gradient. 50ul of each tube was subjected to denaturing SDS-PAGE, the gel concentration was 12% and the protein was selected for the highest purity for subsequent experiments (FIG. 3). And finally washing the nickel column with double distilled water for 10min, and storing in 20% ethanol at 4 ℃.

5. Functional verification

In vitro experiment verification: CHO cells (expressing M23-AQP4) were seeded at a density of 20,000 cells on 96-well microplates (Corning, USA) and at 37 ℃ in 5% CO₂In a humid atmosphere. Cells were washed with PBS and incubated with 20% heat inactivated NMO serum for 30min at room temperature. A solution of 2% pooled normal human complement serum (Innovative Research, USA) and C5mAb was incubated for 30min and then added to CHO cells. After addition of human complement, heat-inactivated NMO serum and C5B3, CHO cells were then incubated at 27 ℃ for 60 min. The amount of membrane attack complex formed is shown by the cellular fluorescence of C5B-9, the experimental group with the addition of C5B3mAbCompound was significantly reduced (figure 4); dead and live cells were labeled with a live/dead cell staining kit (Invitrogen, USA), and dead cells (red) and live cells (green) were counted. Compared with the control group, the C5A6 and the C5B35 have no protective effect, and the C5B3 experimental group has obvious protective effect on the neuromyelitis optica model (figure 5 and figure 6). Can be used for preventing autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, etc., Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical hemolytic toxemia syndrome (AHU), and inflammation caused by trauma.

In vivo experiment verification: induction in animal models: female C57BL/6(8-12 weeks old) mice matched in weight were anesthetized with 10% chloral hydrate (3ml/kg, i.p.) and placed in a stereotactic frame (RWD Life Science, Shenzhen, China). A midline scalp incision was made exposing bregma. For intraparenchymal injection (intracerebral injection of neuromyelitis optica immunoglobulin G and human complement produced neuromuscular injury in mouse brain parenchyma), a burr hole high speed drill (RWD) with a diameter of 0.7mm was used at the cranial position 2mm to the right of bregma. A33-g needle connected to a 25-ll airtight glass syringe (Hamilton, Reno, NV) was inserted 3mm deep to inject 6ul NM0-IgG, 4ul human complement (with or without) 100ugC5B3mAb in a total volume of 15ul (1 ul/min). Rectal temperature of the mice was maintained at 37-38 ℃ during anaesthesia. After disinfecting the surface, the scalp is sutured. After 7 days, mice were anesthetized (10% chloral hydrate) and heart perfused with Phosphate Buffered Saline (PBS) followed by left ventricular perfusion with 4% Paraformaldehyde (PFA). The brain was post-fixed in 4% PFA overnight, dehydrated with 30% sucrose solution, and then embedded into frozen sections with embedding medium.

Frozen sections (6mm) were selected through the needle track. Frozen sections were infiltrated with cold acetone and blocked (5% BSA), then immunostained with the next primary antibody overnight at 4 ℃: rabbit polyclonal anti-aquaporin AQP4 (1:100, Santa Cruz Biotechnology), goat polyclonal anti-astrocyte GFAP (1:1000, Abcam), rat anti-Myelin Basic Protein (MBP) (1: 200, aa 82-87, Millipore), PBS wash primary antibody 5 times followed by a suitable Alexa Fluor secondary antibody (1:1000, Invitrogen). The tissue sections were examined by Leica DM 6000B microscopy. After 7 days, intracerebral injection of IgG-NMO and human complement produced significant loss of GFAP, AQP4 and MBP. In the experimental group added with the C5B3mAb, GFAP, AQP4 and MBP around the injection part of the mouse are obviously reduced (figure 7 and figure 8), the focus caused by autoantibodies and complement of neuromyelitis optica is obviously reduced, and the protective effect on autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus, myasthenia gravis and the like and/or Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical hemolytic toxemia syndrome (AHU) and inflammation caused by trauma can be achieved.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. In order to avoid unnecessary repetition, various possible combinations will not be separately described in this disclosure.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Nucleotide sequence list electronic file

<110> university of Shanxi university

<120> anti-C5 fully human single-chain antibody C5B3 and application thereof

<160>10

<210>1

<211>729

<212>DNA

<213> Whole human resources

<220> fully human anti-complement C5 Single chain antibody C5B3

<400>1

ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCT

CTGGATTCACTTTCTCTATGTATGATATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTTC

TTCTTCTGGTGGCATTACTTCGTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACG

CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACGACGCGGTTTTTTGACT

ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTC

GACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA

AGTGGCTCGAACATCGGAAGTAATATTTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATA

ATGACCATCGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAG

CAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCTCGGACTAGTCCTTCTACGTTCGGCCAAGGGACC

AAGGTGGAAATCAAACGG

<210>2

<211>243

<212>PRT

<213> Whole human resources

<220> amino acid sequence of fully human anti-complement C5 single-chain antibody C5B3

<400>2

MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSMYDMSWVRQAPGKGLEWVSSSSSGGITSYADSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAKTTRFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRA

SGSNIGSNILNWYQQKPGKAPKLLIYNDHRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQARTSPSTFGQGT

KVEIKR

<210>3

<211>8

<212>PRT

<213> Whole human resources

<220> amino acid sequence of VHCDR1 in heavy chain variable region of fully human anti-complement C5 single-chain antibody C5B3

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GFTFSMYD

<210>4

<211>7

<212>PRT

<213> Whole human resources

<220> amino acid sequence of VHCDR2 in heavy chain variable region of fully human anti-complement C5 single-chain antibody C5B3

<400>4

SSSGGIT

<210>5

<211>9

<212>PRT

<213> Whole human resources

<220> amino acid sequence of VHCDR3 in heavy chain variable region of fully human anti-complement C5 single-chain antibody C5B3

<400>5

AKTTRFFDY

<210>6

<211>8

<212>PRT

<213> Whole human resources

<220> amino acid sequence of VLCDR1 in light chain variable region of fully human anti-complement C5 single-chain antibody C5B3

<400>6

GSNIGSNI

<210>7

<211>3

<212>PRT

<213> Whole human resources

<220> amino acid sequence of VLCDR2 in light chain variable region of fully human anti-complement C5 single-chain antibody C5B3

<400>7

NDH

<210>8

<211>9

<212>PRT

<213> Whole human resources

<220> amino acid sequence of VLCDR3 in light chain variable region of fully human anti-complement C5 single-chain antibody C5B3

<400>8

QQARTSPST

<210>9

<211>25

<212>DNA

<213> Whole human resources

<220> upstream BamHI-R

<400>9

AGGATCCGCCGAGGTGCAGCTGTTG

<210>10

<211>27

<212>DNA

<213> Whole human resources

<220> downstream primer NotI-F

<400>10

TGCGGCCGCCCGTTTGATTTCCACCTT

Claims

1. The anti-C5 fully human single-chain antibody C5B3 is characterized in that the DNA sequence of the anti-C5 fully human single-chain antibody C5B3 is as follows: SEQ ID NO. 1.

2. An anti-C5 fully human single-chain antibody C5B3, wherein the amino acid sequence of the anti-C5 fully human single-chain antibody C5B3 is as follows: SEQ ID NO. 2.

3. An anti-C5 fully human single-chain antibody C5B3, characterized in that the amino acid sequence of the heavy chain variable region VHCDR1 of the anti-C5 fully human single-chain antibody C5B3 is shown as SEQ ID NO. 3; the amino acid sequence of the heavy chain variable region VHCDR2 is shown in SEQ ID NO. 4; the amino acid sequence of the heavy chain variable region VHCDR3 is shown in SEQ ID NO. 5;

the amino acid sequence of the light chain variable region VLCDR1 of the anti-C5 fully humanized single chain antibody C5B3 is shown in SEQ ID NO. 6, the amino acid sequence of the light chain variable region VLCDR2 is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region VLCDR3 is shown in SEQ ID NO. 8.

4. A vector containing an anti-C5 fully human single-chain antibody C5B3 is characterized in that the vector is pET30a, and a DNA sequence shown in SEQ ID NO 1 is inserted into the vector.

5. A recombinant host E.coli BL21, wherein said recombinant host E.coli BL21 comprises the vector of claim 4.

6. Use of the anti-C5 fully human single chain antibody C5B3 of any one of claims 1 to 3 in the preparation of a medicament for the treatment of autoimmune diseases caused by the formation of membrane attack complexes during inflammatory response and/or diseases characterized by the association with C5 complement;

the autoimmune diseases comprise neuromyelitis optica, multiple sclerosis, systemic lupus erythematosus and myasthenia gravis;

the diseases characterized by C5 complement include paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome and inflammation caused by trauma.