CN115286714A - Fully human antagonistic antibody with connective tissue growth factor as target and application thereof - Google Patents

Abstract

The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a fully human antagonistic antibody targeting connective tissue growth factor and its application. The fully human antagonistic antibody targeting connective tissue growth factor is a single-chain antibody whose amino acid sequence is shown in SEQ ID NO: 3. In the present invention, the fully human anti-CTGF monoclonal antibody is screened out through the ScFv phage antibody library of patients with rheumatoid arthritis, and its nucleotide sequence and amino acid sequence are determined respectively. The fully human antagonistic antibody obtained by this method has the ability to target human Specificity of connective tissue growth factor.

Description

A fully human antagonistic antibody targeting connective tissue growth factor and its application

This application is a divisional application with application number 202011159590.4, application date October 27, 2020, and the title of the invention "a fully human antagonistic antibody targeting connective tissue growth factor and its application".

technical field

The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a fully human antagonistic antibody targeting connective tissue growth factor and its application.

Background technique

Rheumatoid arthritis is an autoimmune disease with complex etiology and recurrent attacks. Its pathological features are chronic inflammation of joint synovium, cartilage absorption, bone destruction and fibrosis, which has caused great inconvenience and pain to the majority of patients. . Because its etiology is not yet fully clarified, and its pathogenesis involves many aspects such as genetics, environment, immunity, etc., it is difficult for current drugs to cure patients, and only symptomatic treatment can be achieved, that is, to reduce or eliminate joint redness, swelling and pain caused by joint inflammation. Control the development of the disease, reduce joint damage, and ensure the function of the affected joints as much as possible.

Drugs commonly used to treat rheumatoid arthritis at home and abroad include non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), biological agents, traditional Chinese medicines, and traditional Chinese medicine preparations. Antibody biological agents often target TNF-α, IL-1, IL-6, etc. Because these proteins play an important role in maintaining the normal immune function of the body, the adverse reactions of using their inhibitors as drugs for treatment are more significant. Inhibitors or activators targeting the natural ligands of cell membrane receptors can be screened from the human combined antibody library by using phage display and other technologies. The chemical nature of these inhibitors or activators is antibodies. Not only is the structure stable (preventing the drug from being degraded in vivo before it reaches the lesion), but it also has good targeting (can greatly reduce toxic and side effects), and more importantly, the antibody produced in this way can overcome the easy-to-cause of hybridoma technology to produce antibodies. The shortcomings of rejection, complex preparation process, low fusion efficiency, low antibody yield, antibody instability, and weak penetration can also achieve high-throughput and rapid antibody screening, and the price is low.

Human connective tissue growth factor is currently discovered as a protein closely related to the pathogenesis and progression of rheumatoid arthritis, but so far no inhibitors targeting human connective tissue growth factor have been screened from human combinatorial antibody libraries using phage display and other technologies. Literature and products of anti-antibodies. Compared with the existing antibody drugs for the treatment of rheumatoid arthritis, antibody inhibitor drugs targeting human connective tissue growth factor are still in the young rising stage, and there is a lot of room for development.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a fully human monoclonal single-chain antibody ScFv against human connective tissue growth factor, using genetic engineering methods, in rheumatoid joints Panning and identifying the human connective tissue growth factor fully human single-chain antibody scFv from the fully human phage antibody library of patients with inflammation, which specifically binds to human connective tissue growth factor.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A fully human antagonistic antibody targeting connective tissue growth factor, the fully human antagonistic antibody is a single-chain antibody, and its amino acid sequence is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5. Shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

A gene encoding a fully human antagonistic antibody targeting connective tissue growth factor as described above.

The nucleotide sequences of the genes are respectively SEQIDNO:11, SEQIDNO:12, SEQIDNO:13, SEQIDNO:14, SEQIDNO:15, SEQIDNO:16, SEQIDNO:17, SEQIDNO:18, SEQIDNO:19 or SEQIDNO:20, The corresponding amino acid sequences are SEQIDNO:1, SEQIDNO:2, SEQIDNO:3, SEQIDNO:4, SEQIDNO:5, SEQIDNO:6, SEQIDNO:7, SEQIDNO:8, SEQIDNO:9 or SEQIDNO:10 fully human antagonistic antibody .

An expression vector containing the above-mentioned gene. These expression vectors can be selected from plasmids or viruses.

A recombinant cell containing the above-mentioned expression vector. These recombinant cells can be eukaryotic or prokaryotic.

An enzymatic fragment of the above-mentioned fully human antagonistic antibody targeting connective tissue growth factor, the enzymatic fragment is a Fab, Fab', F(ab')2 or Fv fragment.

A gene encoding the above-mentioned enzymatic fragment.

Application of the above-mentioned fully human antagonistic antibody targeting connective tissue growth factor in the preparation of an inhibitor targeting human connective tissue growth factor.

Application of the above-mentioned fully human antagonistic antibody targeting connective tissue growth factor in the preparation of drugs for treating or alleviating rheumatoid arthritis.

The beneficial effects of the present invention are as follows: the present invention screens out the fully human anti-CTGF monoclonal antibody through the ScFv phage antibody library of patients with rheumatoid arthritis, and determines its nucleotide sequence and amino acid sequence respectively, and the fully human monoclonal antibody obtained by this method The source antagonistic antibody has specificity for targeting human connective tissue growth factor.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, obtaining other drawings based on these drawings still belongs to the scope of the present invention without any creative effort.

Figure 1 is the PCR amplification of the light chain variable region Vκ gene;

Figure 2 is the PCR amplification of the heavy chain variable region VH gene;

Fig. 3 is PCR amplification light chain variable region V lambda gene;

Fig. 4 is SOE-PCR amplified scFv gene;

Fig. 5 is the result figure of the fully human antibody experimental group in the monoclonal phage ELISA detection;

Figure 6 is a sequence diagram of the heavy and light chains of a fully human antagonistic antibody with target specificity;

Fig. 7 is a comparison of joint injury and disease progression in CIA mice treated with CTGF antagonistic antibody.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings.

The present invention provides a fully human antagonistic antibody targeting connective tissue growth factor, which is a single-chain antibody, which has the specificity of targeting human connective tissue growth factor, and its amino acid sequence is SEQ ID NO: 1, SEQ ID NO: 2, Shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

The fully human antagonistic antibody targeting connective tissue growth factor of the present invention can be fused with other antibody drugs for treating rheumatoid arthritis, and the formed antibody can play the role of targeting human connective tissue growth factor on the one hand, and on the other On the one hand, it can play a role in the treatment of rheumatoid arthritis.

The DNA of the above-mentioned single-chain antibody can be obtained by conventional gene recombination technology. The DNA sequences encoding the above-mentioned single-chain antibodies are obtained by PCR and cloned into vectors respectively, and the vectors used can be plasmids, viruses or gene fragments commonly used in molecular biology. A protein secretion signal peptide sequence is added to the front of the DNA sequence encoding the above antibody to ensure that the antibody can be secreted from the cells. The vector sequence includes a promoter for gene expression, protein translation initiation and termination signals, and polyadenylic acid (PolyA) sequence. The vector contains antibiotic resistance genes to facilitate the replication and expression of the vector in host cells such as bacteria and eukaryotic cells. In addition, the vector also includes a eukaryotic cell selection gene for selection of stable transfected host cell lines.

The single-chain antibody of the present invention can be further fused with other effector molecules to achieve other additional functions without affecting its targeting, and they all belong to the scope of the present invention.

After the construction of the plasmid containing the DNA sequence encoding the above antibody is completed, the recombinant vector can be used to transfect or transform the host cell to express the corresponding protein. There are various expression systems that can be used to express antibodies, which can be eukaryotic cells or prokaryotic cells, including mammalian cells, insect cells, yeast, bacteria, etc. Mammalian cells are the preferred system for expressing the protein due to the tendency of prokaryotic expression of antibodies to form inclusion bodies. There are many kinds of mammalian cells that can be used for large-scale expression of antibodies, such as CHO cells, 293 cells, NSO cells, COS cells, etc., all of which are included in the cells that can be used in the present invention. The recombinant plasmids containing the genes encoding the above antibodies can be transfected into host cells. There are many methods for transfecting cells, including electroporation, liposome transfection and calcium phosphate transfection.

A preferred method of protein expression is expression by stably transfected host cells. For example, after stably transfecting host cells without neomycin resistance with a recombinant vector containing a neomycin (Neomycin) resistance gene, the concentration of neomycin can be increased in the cell culture medium to select stable cells with high expression For another example, after stably transfecting a host cell lacking DHFR with a recombinant vector containing a dihydrofolate reductase (DHFR) gene, the concentration of methotrexate (MTX) can be increased in the cell culture medium to screen out a stable cell line with high expression .

Other expression systems other than mammalian cells, such as insect cells, yeast, bacteria, etc., can also be used to express the antibodies of the present invention, and they are also included in the list of host cells that can be used in the present invention. These expression systems have higher protein yields than mammalian cells, but are prone to formation of inclusion bodies, thus requiring further protein renaturation.

The antibodies of the present invention can also be carried and expressed by viral vectors, which include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, herpes simplex virus Vector (herpessimplexvirus-basedvectors), lentiviral vectors (lentiviralvectors).

The antibody of the present invention can be prepared into various forms of pharmaceutical preparations according to conventional techniques of pharmacy, more preferably injections, most preferably freeze-dried injections.

The antibody of the present invention can form a pharmaceutical composition with other drugs, and the composition can treat diseases together with other treatment methods, and the other treatment methods include chemotherapy, radiation therapy, and biological therapy.

The fully human antagonistic antibody targeting connective tissue growth factor of the present invention is screened from a ScFv phage antibody library of patients with rheumatoid arthritis. The specific process is as follows:

1. Explanation of experimental illustration information

Antigen: CTF-H82E6 (Biotinylated Human CTGF / CCN2 Protein, purchased from Beijing Baipu Saisi Biotechnology Co., Ltd.);

Phage library used for screening: ScFv phage antibody library of patients with rheumatoid arthritis;

Exemplary purpose: To screen antagonistic antibodies targeting human connective tissue growth factor from the ScFv phage antibody library of patients with rheumatoid arthritis.

2. Experimental content

2.1 Construction of ScFv phage antibody library in patients with rheumatoid arthritis

(1) Preparation of cDNA: Collect 5 ml of peripheral blood from 45 patients with rheumatoid arthritis, anticoagulate with EDTA-K ₂ , separate mononuclear cells (PBMC) with lymphocyte separation medium, and extract PBMC from every 5 patients RNA, then reversed to cDNA.

(2) PCR amplification and connection of VH and VL genes: design and synthesize primers for amplifying human antibody genes. Using the first strand of cDNA as a template, the VH and VL genes were amplified by PCR. And the VH and VL genes are connected into ScFv gene by encoding connecting peptide (Gly4Ser)3. The results of agarose electrophoresis are shown in Figure 1, Figure 2, Figure 3 and Figure 4.

(3) Construction of phage antibody library: using VH and VL gene fragments as templates, the amplified products were mixed in an appropriate ratio, spliced by overlapping PCR, amplified ScFv gene, cloned into pCANTAB-5E vector, electroporated to transform TG1, The library capacity was determined to be 10 ⁷ by counting clones, and the phage antibody library was obtained by superinfection with helper phage after bacterial proliferation.

2.2 Liquid phase-affinity panning

2.2.1 Blocking phage library and magnetic beads

(1) Blocking the phage library. Add 1×10 ¹¹ phage library into 3% M-PBS blocking solution (that is, PBS containing 3% skimmed milk powder), invert and mix evenly, and place at 37°C for 1~2h.

(2) Seal the magnetic beads: place the magnetic bead bottle on a vortex shaker, shake and resuspend for 20s, prepare two new centrifuge tubes A and B, and take 50ul magnetic beads into the new centrifuge tubes with a pipette, Add 1ml of 0.05% PBST, shake and resuspend for 20s, magnetically separate for 2min, and remove the supernatant. (total wash 2 times)

(3) Add 1ml of blocking solution to "Tube A-Negative Panning" and "Tube B-Positive Panning", shake and resuspend for 20s, magnetically separate for 5min, remove supernatant: add 1ml of blocking solution again, mix evenly by inversion, Place it on a rotary mixer and place it at room temperature at 20rpm for 1h.

2.2.2 Binding of antigenic protein to magnetic beads, negative panning of phage library

(1) Antigen-magnetic bead binding: "Tube B-positive panning", transient magnetic separation for 2 minutes, remove the supernatant, wash twice with 0.05% PBST, add 1ml of biotinylated antigen protein (CTF-H82E6, Biotinylated Human CTGF / CCN2Protein) was diluted with 0.05% PBST to make the concentration of magnetic beads 1mg/ml, mixed evenly by inversion, and placed on a rotary mixer at room temperature 20rpm for 1h.

(2) Negative panning of phage library: "Tube A-Negative panning", instant centrifugation, magnetic separation for 2 minutes, remove supernatant, wash 3 times with 0.05% PBST, add the blocked phage library to tube A, mix evenly by inversion, Place it on a rotary mixer and place it at room temperature at 20rpm for 1h. After 1 hour, tube A was magnetically separated for 2 min, and the supernatant was transferred to a new tube EP, which was the phage library that had undergone blocking and negative panning.

2.2.3 Panning

(1) Antigen-antibody binding: "Tube B-Positive Panning", spin off, magnetically separate for 2 minutes, remove the supernatant, wash 3 times with 0.05% PBST, add the phage library from the negative panning, mix evenly by inversion, place in a rotating mixer Place on a homogenizer at room temperature 20rpm for 0.5~1h.

(2) Washing: "Tube B-Positive panning" magnetic separation for 5 minutes, aspirate the supernatant, add 1ml of 0.1%~0.5% PBST, shake and resuspend for 20s, magnetically separate for 2 minutes, aspirate the supernatant. Repeat this step 10-20 times.

(3) Elution and neutralization: Tube B, magnetically separated for 2 minutes, sucked off the supernatant, washed twice with PBST, added 400ul elution buffer (glycine hydrochloride pH 2.2), mixed evenly by inversion, and placed on a rotary mixer , placed at room temperature 20rpm for 10min. After magnetic separation for 2 minutes, absorb the supernatant and add it to a new 1.5ml EP tube (add 200ul neutralization buffer (1M Tris hydrochloric acid pH8.0) in advance, set aside at 4°C. See Table 1 for details.

2.3 Amplification of eluted products

(1) Pick the TG1 monoclonal on the plate, add it to 20ml 2YT, and culture it at 37°C and 220rpm until the logarithmic phase OD600=0.6-0.8.

(2) Add the eluted antibody library, mix well, and place at 37°C for 60 minutes.

(3) Add 4ul Amp and incubate at 37°C 180rpm for 60min.

(4) Add M13KO7 (helper phage: TG1>=10: 1) to the culture, and place it at 37°C for 30 minutes.

(5) Add 30ml 2YT and 6ul Amp, and incubate at 37°C and 180rpm for 60min.

(6) Centrifuge at room temperature at 5000rpm for 10min, remove the supernatant, collect the bacterial pellet, add new 50ml 2YTAK to resuspend the pellet, and culture overnight at 30°C and 220rpm in a 200ml Erlenmeyer flask. Precipitate and recover the next day, and measure the amplification product titer.

(7) Transfer the culture into a 100ml centrifuge tube, centrifuge at 12000rpm at 4°C for 20min, transfer the supernatant to a new centrifuge tube, centrifuge again at 12000rpm for 20min, and transfer the supernatant to a new centrifuge tube.

(8) Add 1/4 volume of PEG/NaCl to the supernatant, and ice-bath for 4 h.

(9) Centrifuge at 12000 rpm at 4°C for 20 min, discard the supernatant, and centrifuge briefly again to remove the supernatant.

(10) The pellet was resuspended in 1ml PBS, centrifuged at 12000rpm at 4°C for 10min, and the supernatant was transferred to a new centrifuge tube.

(11) Determination of titer (amplified antibody library, serially diluted to 10E10-10E11).

(12) Add antibody library at 10-9/10-10 dilution to 200ul logarithmic phase TG1, place at 37°C for 30min, spread on 2YTAG plate, culture overnight at 37°C, count the number of colonies the next day.

2.4 Phage ELISA

Monoclonal Phage Supernatant Preparation

(1) Pick a single colony from the phage library plate eluted in the last round of screening and inoculate it in a 96-well deep-well plate containing 300ul 2YTAG, culture at 37C, 700rpm until the logarithmic growth phase;

(2) Pipette 100ul of bacterial solution from each well into a sterilized 96-well plate and store at 4°C;

(3) Add 100ul of 2YT diluted M13KO7 (titer 2x10 ¹⁰ , about 10 times the bacterial concentration), incubate at 37°C for 30min, then incubate at 37°C and 600rpm for 60min.

(4) Centrifuge the 96-well deep-well plate at 4000rpm for 10 minutes, discard the supernatant, add 300ul 2TYAK resuspended bacteria to each well, culture at 700rpm at 30°C overnight

Monoclonal Phage ELISA

(5) Coating target protein: dilute the biotinylated target molecule with 0.05% PBST solution to lug/ml, add to the streptavidin microtiter plate, 100ul/well, mix at 37°C for 1h.

(6) Washing: Discard the coating supernatant, pat dry, wash 3 times with 0.05% PBST, 1 min each time;

(7) Sealing: Add 300ul/well of blocking solution 4% skimmed milk powder (PBS), let stand at 37°C for 1h;

(8) Washing: Discard the blocking solution, pat dry, wash 3 times with 0.05% PBST, 1 min each time;

(9) Binding of phage supernatant: 50ul of 2% skimmed milk powder (PBS) and monoclonal supernatant were mixed at a ratio of 1:1, added to the corresponding microplate well, and shaken slowly at 100rpm at 37°C for 1h;

(10) Washing: Discard the supernatant, pat dry, and wash 5 times with 0.1% PBST:

(11) Secondary antibody binding: Dilute Anti-M13-HRP antibody 1:5000 in blocking solution 1% skimmed milk powder (PBS), add 100ul/well to the microtiter plate, shake slowly at 100rpm at 37°C for 1h;

(12) Washing: discard the liquid, pat dry, and wash 5 times with 0.1% PBST;

(13) Add 100ul TMB, react in the dark for 5-10 minutes, add 100ul 2M hydrochloric acid to terminate the reaction, and read the OD450 value in a microplate reader.

The results of the monoclonal phage ELISA are shown in Figure 5, from which we obtained 92 clones that specifically bind to the antigenic protein (human connective tissue growth factor protein). We selected 10 clones marked with background color (that is, with different A450.Ag values) in Fig. 5 for the next step of sequencing.

Finally, 10 antigen-binding protein sequences (denoted as SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) with targeting specificity to human connective tissue growth factor (CTGF) were obtained. ) and nucleotide sequence (respectively denoted as SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, the antigen-binding protein sees the heavy and light chain sequences shown in Figure 6.

2.5 CTGF antagonistic antibody treatment of arthritis in CIA mice

(1) Expression and purification of single-chain antibody in Escherichia coli Take No. 5 phage, amplify the DNA fragment of the single-chain antibody by PCR method, and clone it into the expression vector pGEX-6P-1 after double digestion with EcoR I and Xho I. Transform Escherichia coli BL21; culture the recombinant strain, add IPTG to a final concentration of 0.5mM, induce expression at 30°C for 5h, ultrasonically disrupt, centrifuge at 12,000rpm/10min, take the supernatant and pass it through a GST column for purification, and use equilibration buffer The target protein was eluted to obtain electrophoresis-purified single-chain antibody, and the protein concentration was determined by BCA method.

(2) Construction of CIA mouse model: each DBA/1 mouse was injected with 200ul of emulsion containing 4mg/ml Mycobacterium tuberculosis complete Freund's adjuvant (CFA) and 6mg/ml collagen for primary immunization. After 21 days, collagen and incomplete Freund's adjuvant (IFA) were used to inject 100ul of each mouse for the second immunization. Joint scoring was performed every three days, and a successful CIA mouse model was selected.

(3) Two groups of CIA mice (10 in each group) constructed by intraperitoneally injecting DBA/1 with CTGF antagonistic antibody and 200 μg of purified mouse IgG (Sigma-Aldrich) were co-injected from the 22nd day to the 43rd day 4 times. Compared with normal mice, the three groups of mice were killed, and the legs of the mice were taken, fixed, decalcified, embedded in the joints and sectioned for HE staining. The tissue morphology was observed under a microscope, and the results are shown in Figure 7. The results show that CTGF antagonistic antibody does have a therapeutic effect on arthritis in CIA mice.

The above disclosures are only preferred embodiments of the present invention, and certainly cannot limit the scope of rights of the present invention. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope of the present invention.

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Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg

145 150 155 160

Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr

165 170 175

Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile

180 185 190

Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser

210 215 220

Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn

225 230 235 240

Gly Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu

245 250

<210> 7

<211> 243

<212> PRT

<213> Human ScFv Phage Antibody Library

<400> 7

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Pro Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Pro Gly Tyr Tyr Tyr Ala Leu Tyr Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Pro

130 135 140

Leu Ser Val Ser Val Ala Leu Gly Gln Thr Ala Arg Ile Thr Cys Gly

145 150 155 160

Gly Asn Asn Ile Gly Ser Lys Asn Val His Trp Tyr Gln Gln Lys Pro

165 170 175

Gly Gln Ala Pro Val Leu Val Ile Tyr Arg Asp Ser Asn Arg Pro Ser

180 185 190

Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr

195 200 205

Leu Thr Ile Ser Arg Ala Gln Ala Gly Asp Glu Ala Asp Tyr Tyr Cys

210 215 220

Gln Val Trp Asp Ser Ser Thr Val Val Phe Gly Gly Gly Thr Lys Val

225 230 235 240

Thr Val Leu

<210> 8

<211> 255

<212> PRT

<213> Human ScFv Phage Antibody Library

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Ala Tyr Cys Ser Gly Gly Ser Cys Tyr Ser Val Tyr

100 105 110

Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

115 120 125

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gln Pro Val Leu Thr Gln Pro Pro Ser Met Ser Ala Ala Pro Arg

145 150 155 160

Gln Arg Val Ala Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Glu

165 170 175

Asn Thr Val Ser Trp Tyr Gln Gln Phe Pro Gly Lys Pro Pro Lys Leu

180 185 190

Leu Ile Leu Phe Asp Asp Val Leu Ser Ser Gly Val Ser Asp Arg Phe

195 200 205

Ser Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

210 215 220

Gln Ser Glu Asp Glu Ala Val Tyr Phe Cys Ala Thr Trp Asp Asp Ser

225 230 235 240

Leu Asn Gly Val Ile Phe Gly Gly Gly Thr Lys Val Thr Val Leu

245 250 255

<210> 9

<211> 237

<212> PRT

<213> Human ScFv Phage Antibody Library

<400> 9

Glu Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Ser Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asn Tyr

20 25 30

Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe

50 55 60

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Arg Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Arg Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser

130 135 140

Pro Gly Gln Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Leu Arg Ser

145 150 155 160

Lys Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu

165 170 175

Val Ile Tyr Gln Asp Thr Lys Arg Pro Ser Gly Val Pro Glu Arg Phe

180 185 190

Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr

195 200 205

Gln Ala Met Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Asn

210 215 220

Thr Val Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu

225 230 235

<210> 10

<211> 247

<212> PRT

<213> Human ScFv Phage Antibody Library

<400> 10

Glu Val His Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Arg Leu Tyr Cys Ser Gly Gly Ser Cys Leu Asn Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val

130 135 140

Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln Thr Val Ser

145 150 155 160

Ile Thr Cys Ser Gly Asp Lys Leu Gly Asn Lys Tyr Ala Ser Trp Tyr

165 170 175

Gln Gln Arg Pro Gly Gln Ser Pro Ile Leu Val Ile Tyr Gln Asp Thr

180 185 190

Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly

195 200 205

Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met Asp Glu Ala

210 215 220

Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Asn Thr Ala Phe Phe Gly Ala

225 230 235 240

Gly Thr Gln Leu Thr Val Leu

245

<210> 11

<211> 711

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 11

gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat 180

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240

atggagctga gcaggctgag atctgacgac acggccatat attackgtgc gagagtagga 300

gggtccgccc actactgggg ccagggaacc ctggtcaccg tctcctcagg tggtggcggt 360

tcaggcggag gtggctctgg cggtggcgga tcgcagcctg tgctgactca gcccccctca 420

gtgtccgtgt ccccagggca gacagccagc atcacctgct ctggagataa attgggacat 480

aagtatgctt cctggtatca acagaagcca ggccagtccc ctgtgctggt catctatcaa 540

gacaggaagc ggccctcagg gatccctgag cgattctctg gctccaactc tgggaacaca 600

gccactctga ccatcagcgg gacccaggct atggatgagg ctgactatta ctgtcaggcg 660

tgggacagca gccatgtggt attcggcgga ggcaccaagg tgaccgtcct c 711

<210> 12

<211> 735

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 12

gaggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc gactactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaacccta acagtggtgg cacaaactat 180

gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240

atggagctga gcaggctgag atctgacgac acggccgtgt attackgtgc gagagctagg 300

tatggttcgg ggagctccca gaactactgg ggccagggaa ccacggtcac cgtctcctca 360

ggtggtggcg gttcgggcgg aggtggctct ggcggtggcg gatcgcagtc tgtgctgact 420

cagccaccct cagcgtctgg gacccccggg cagaggatca ccatctcttg ttctggaagc 480

agatccaaca tcggaagtaa tggtgtttac tggtaccagc agctcccagg gacggccccc 540

aaactcctca tctctaggaa cgatcagcgg ccctcagggg tccctgagcg attctctggc 600

tccaagtctg gcacctcagc ctccctggcc atcagtgggc tccggtccga ggatgaggct 660

gattattatt gtgctgtatg ggatgacagc ctaaatggtt gggtgttcgg cggaggcacc 720

cagctgaccg ccctc 735

<210> 13

<211> 720

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 13

caaatgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg ggtgggaagg atcatcccta tccttggtat agcaaactac 180

gcacaggagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attattgtgc gagagacgat 300

atagcagtgg ctggcaactt tgactactgg ggccaggggga ccctggtcac cgtctcctca 360

ggtggtggcg gttcaggcgg aggtggctct ggcggtggcg gatcgtccta tgtgctgact 420

cagccatcct cagtgacagt gtctccggga cagacagcca ggatcacctg ctcaggagat 480

ctactggcaa aaaaatatgt tcggtggctc cagcagaagc cgggccaggc ccctgtattg 540

ttgatttata aagacagtga gcgcctgtca gggatccctg agcgattctc tggctccaac 600

tcggggaaca tggccaccct gaccatcagc agagcccaag ccggagatga ggctgactat 660

tactgtcagg tgtgggacag cagttgggtg ttcggcggag ggacggagct gaccgtccta 720

<210> 14

<211> 708

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 14

caggtgcagc tacaacagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctc tctatgacgg gtccttcagt gattactatt ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa gtcaatcgta gtgggaggcac caactacaac 180

ccgtccctca agagtcgagt caccgtatca gtagacacgt ccaagaacca gttctccctg 240

aagttgaggt ctatgaccgc cgcggacacg gctgtgtatt attgtgcgag acgctacggt 300

tcatttgagt attggggcca ggggaccacg gtcaccgtct cctcaggtgg tggcggttca 360

ggcggaggtg gctctggcgg tggcggatcg tcctatgtgc tgactcagcc accctcagtg 420

tccgtgtccc caggacagac agtcagcata acctgctctg gagataaatt ggggaataaa 480

tacgcttcct ggtaccagca gaggccaggc cagtccccta tactggtcat ctatcaagat 540

accaagcggc cctcagggat ccctgagcga ttctctggct ccaactctgg gaacacagcc 600

actctgacca tcagcgggac ccaggctatg gatgaggctg actattactg tcaggcgtgg 660

gacagcaaca ctgccttctt cggagctggg accaaggtca ccgtccta 708

<210> 15

<211> 729

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 15

gaggtgcaac tggtggagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180

gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attackgtgc gagaggggggc 300

tacgtttggg gttactactacta ctacggtatg gacgtctggg gccaaggaac cctggtcacc 360

gtctcctcag gtggtggcgg ttcaggcgga ggtggctctg gcggtggcgg atcgcagcct 420

gtgctgactc agccactctc agtgtcagtg gccctgggac agacggccag gcttacctgt 480

gggggaaaca acattggaag taaaaatgtt cactggtacc agcagaagcc aggccaggcc 540

cctgtgctgg tcatctatag ggattccatc cggccctctg ggatccctga gcgattctct 600

ggctccaact cggggaacac ggccaccctg accatcagca gagcccaagt cggggatgag 660

gctgactatt actgtcaggt gtgggacagc ggcactgtct tcggaactgg caccaaggtg 720

accgtcctc 729

<210> 16

<211> 759

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 16

caggtgcagc tggtgcaatc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg ggtgggaagg atcatcccta tccttggtat agcaaactac 180

gcacaggagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attackgtgc gagagatccc 300

tcaacgtatt acgatatttt gactggttca cgactccgtg cttttgatat ctggggccaa 360

gggaccacgg tcaccgtctc ttcaggtggt ggcggttcag gcggaggtgg ctctggcggt 420

ggcggatcgc agtctgtgct gacgcagccg ccctcagcgt ctgggacccc cgggcagagg 480

gtcaccatct cttgttctgg aagcagctcc aacatcggaa gtaatactgt aaactggtac 540

cagcagctcc caggaacggc ccccaaactc ctcatctata gtaataatca gcggccctca 600

ggggtccctg accgattctc tggctccaag tctggcacct cagcctccct ggccatcagt 660

gggctccagt ctgaggatga ggctgattat tactgtgcag catgggatga cagcctgaat 720

ggttgggtgt tcggcggagg gaccaaggtc accgtccta 759

<210> 17

<211> 729

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 17

gaggtccagc tggtacagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg ctcctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat 180

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240

atggagctga ggagcctgag atctgacgac acggccgtgt attackgtgc gagagtgccc 300

gggtattact atgctctcta tgcttttgat atctggggcc aagggaccac ggtcaccgtc 360

tcctcaggtg gtggcggttc aggcggaggt ggctctggcg gtggcggatc gcagcctgtg 420

ctgactcagc cgctctcagt gtcagtggcc ctgggacaga cggccaggat tacctgtggg 480

ggaaacaaca ttggaagtaa aaatgtgcac tggtaccagc agaagccagg ccaggcccct 540

gtgctggtca tctataggga tagcaaccgg ccctctggga tccctgagcg attctctggc 600

tccaactcgg ggaacacggc caccctgacc atcagcagag cccaagccgg ggatgaggct 660

gactattact gtcaggtgtg ggacagcagc actgtggtat tcggcggagg caccaaggtg 720

accgtcctc 729

<210> 18

<211> 765

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 18

caggtgcagc tgcaggagtc ggggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct 120

ccagggaagg gcctggagtgggtctcaggt attagttgga atagtggtag cataggctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240

ctgcaaatga acagtctgag agctgaggac acggccttgt attackgtgc aaaagatata 300

gcgtattgta gtggtggtag ctgctactct gtctactact actacggtat ggacgtctgg 360

ggccaaggga ccacggtcac cgtctcctca ggtggtggcg gttcaggcgg aggtggctct 420

ggcggtggcg gatcgcagcc tgtgctgact cagcccccct cgatgtctgc agccccccgg 480

cagaggtcg ccatctcctg ttctggaggc agctccaaca tcggagagaa cactgtgagc 540

tggtatcagc agttcccagg aaagcctccc aaactcctca tcctttttga tgatgtcttg 600

tcctcagggg tctctgaccg cttctctgcc tccaagtcag gcacctcagc ctccctggcc 660

atcagtggcc tccaatctga ggatgaggct gtttatttct gtgctacatg ggacgacagc 720

ctgaatggtg tgattttcgg cggagggacc aaggtcaccg tccta 765

<210> 19

<211> 711

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 19

gaagtgcagc tggtgcagtc tgggtctgaa ttgaagaagt ctggggcctc agtgaaagtt 60

tcctgcaagg cttctggata caccttcaat aactatgcta tccattgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaaccacca acactgggaa cccaacgtat 180

gcccagggct tcacaggacg gtttgtcttc tccttggaca cctctgtcag aacggcatat 240

ctgcagatca gcagcctaaa ggctgaggac actgccgtct attackgtgc gagagtgagg 300

tacggtatgg acgtctgggg ccaagggaca atggtcaccg tctcttcagg tggtggcggt 360

tcaggcggag gtggctctgg cggtggcgga tcgtcctatg tgctgactca gccaccctca 420

gtgtccgtgt ccccaggaca gacagccagc atcacctgct ctggagataa cttgaggagt 480

aaatatgctt cctggtatca gcagaagcca ggccagtcgc ctgtgctggt catctatcaa 540

gataccaagc ggccctcagg ggtccctgag cgattctctg gctccaactc tgggaacaca 600

gccactctga ccatcagcgg gacccaggct atggatgagg cagactatta ctgtcaggcg 660

tgggacagta acactgtggt attcggcgga gggaccgagc tgaccgtcct c 711

<210> 20

<211> 741

<212>DNA

<213> Human ScFv Phage Antibody Library

<400> 20

gaggtgcatc tggtggagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180

gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attackgtgc gagagggacg 300

cgattatatt gtagtggtgg tagctgcttg aacggtatgg acgtctgggg ccaagggacc 360

acggtcaccg tctcctcagg tggtggcggt tcaggcggag gtggctctgg cggtggcgga 420

tcgtcctatg tgctgactca gccaccctca gtgtccgtgt ccccaggaca gacagtcagc 480

ataacctgct ctggagataa attgggaat aaatacgctt cctggtatca gcagaggcca 540

ggccagtccc ctatactggt catctatcaa gataccaagc ggccctcagg gatccctgag 600

cgattctctg gctccaactc tgggaacaca gccactctga ccatcagcgg gacccaggct 660

atggatgagg ctgactatta ctgtcaggcg tgggacagca acactgcctt cttcggagct 720

ggcacccagc tgaccgtcct c 741

Claims (7)

1. A fully human antagonist antibody targeting connective tissue growth factor, comprising: the fully human antagonistic antibody is a single-chain antibody, and the amino acid sequence of the fully human antagonistic antibody is shown in SEQ ID NO. 3.

2. A gene encoding the fully human antagonist antibody targeting connective tissue growth factor according to claim 1.

3. The gene according to claim 2, characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 13.

4. An expression vector comprising the gene of claim 2.

5. A recombinant cell comprising the expression vector of claim 4.

6. Use of a fully human antagonist antibody targeting connective tissue growth factor according to claim 1 for the preparation of an inhibitor targeting human connective tissue growth factor.

7. Use of the fully human antagonist antibody targeting connective tissue growth factor according to claim 1 for the preparation of a medicament for the treatment or alleviation of rheumatoid arthritis.

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