CN115286713B

CN115286713B - Fully human antagonistic antibody taking connective tissue growth factor as target spot and application thereof

Info

Publication number: CN115286713B
Application number: CN202111581655.9A
Authority: CN
Inventors: 王建光; 吴淦; 金胜威; 杨新宇
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2024-07-23
Anticipated expiration: 2040-10-27
Also published as: CN115304675A; CN115304675B; CN112266417A; CN115286713A; CN115286714B; CN112266417B; CN115286714A

Abstract

The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a fully human antagonistic antibody taking connective tissue growth factor as a target spot and application thereof. The fully human antagonistic antibody taking connective tissue growth factor as a target point is a single-chain antibody, and the amino acid sequence of the fully human antagonistic antibody is shown as SEQ ID NO. 6. The invention screens out the fully human anti-CTGF monoclonal antibody through the ScFv phage antibody library of the rheumatoid arthritis patient, and respectively determines the nucleotide sequence and the amino acid sequence of the fully human anti-CTGF monoclonal antibody, and the fully human antagonistic antibody obtained through the method has the specificity of targeting human connective tissue growth factor.

Description

Fully human antagonistic antibody taking connective tissue growth factor as target spot and application thereof

The application relates to a full human antagonistic antibody taking connective tissue growth factor as a target spot and application thereof, which are divisional applications of application number 202011159590.4 and application date 2020, 10 and 27.

Technical Field

The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a fully human antagonistic antibody taking connective tissue growth factor as a target spot and application thereof.

Background

Rheumatoid arthritis is an autoimmune disease with complex etiology and recurrent attacks, and its pathological features are chronic inflammation of joint synovium, cartilage absorption, bone destruction and fibrosis, which cause great inconvenience and pain for patients. Because the etiology is not completely clear, the pathogenesis involves a plurality of aspects such as heredity, environment, immunity and the like, the traditional medicine is difficult to radically cure patients, and only symptomatic treatment can be realized, namely, the red swelling and pain of the joints of the patients caused by joint inflammation are reduced or eliminated, the development of the diseases is controlled, the joint damage is reduced, and the functions of the affected joints are ensured as much as possible.

The medicines for treating rheumatoid arthritis at home and abroad comprise nonsteroidal anti-inflammatory drugs (NSAIDs), disease-improving antirheumatic drugs (DMARDs), biological preparations, traditional Chinese medicines, traditional Chinese medicine preparations and the like. Wherein, the antibody biological agent usually takes TNF-alpha, IL-1, IL-6 and the like as targets. Because these proteins play an important role in maintaining normal immune function of the organism, adverse reactions of treatment by adopting inhibitors thereof as medicines are remarkable. The natural ligand of cell membrane receptor can be selected as target inhibitor or activator from human combined antibody library by phage display, etc. the chemical nature of said inhibitor or activator is antibody. Not only has stable structure (prevents the drug from being degraded in advance in the body before reaching the focus), but also has good targeting (can greatly reduce toxic and side effects), more importantly: the produced antibody can overcome the defects of easy rejection reaction, complex preparation process, low fusion efficiency, low antibody yield, unstable antibody and weak penetrability of antibody produced by hybridoma technology, can realize the aim of high-flux and rapid antibody screening, and has low price.

Human connective tissue growth factor is a protein which is closely related to the onset and progression of rheumatoid arthritis and is discovered at present, but no documents and products for screening inhibitor antibodies targeting human connective tissue growth factor from a human combined antibody library by using phage display and other technologies are known up to the present. Compared with the existing antibody medicine for treating rheumatoid arthritis, the antibody inhibitor medicine taking human connective tissue growth factor as a target point is still in a young ascending stage, and has a large development space.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art, and provides an anti-human connective tissue growth factor fully human monoclonal single-chain antibody ScFv, which is specifically combined with human connective tissue growth factor by panning and identifying the human connective tissue growth factor fully human single-chain antibody scFv in a human phage antibody library of a rheumatoid arthritis patient by using a genetic engineering method.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

The fully human antagonistic antibody taking connective tissue growth factor as a target point is a single-chain antibody, and the amino acid sequence of the fully human antagonistic antibody is shown as SEQ ID NO. 1, SEQ ID NO. 2, 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.

Genes encoding fully human antagonistic antibodies targeting connective tissue growth factor as described above.

The nucleotide sequences of the genes are SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19 or SEQ ID NO. 20, and the corresponding amino acid sequences are SEQ ID NO. 1, SEQ ID NO. 2, 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.

An expression vector comprising the gene as described above. These expression vectors may be selected from plasmids or viruses.

A recombinant cell comprising an expression vector as described above. These recombinant cells may be eukaryotic or prokaryotic cells.

An enzymatic fragment of a fully human antagonistic antibody targeting connective tissue growth factor as described above, said enzymatic fragment being a Fab, fab ', F (ab') 2 or Fv fragment.

A coding gene of the enzymatic hydrolysis fragment.

The use of a fully human antagonist antibody targeting connective tissue growth factor as described above for the preparation of an inhibitor targeting human connective tissue growth factor.

The application of the fully human antagonistic antibody taking connective tissue growth factor as a target spot in preparing the medicine for treating or relieving rheumatoid arthritis is provided.

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

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are required in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that it is within the scope of the invention to one skilled in the art to obtain other drawings from these drawings without inventive faculty.

FIG. 1 is a PCR amplified light chain variable region vκ gene;

FIG. 2 shows PCR amplification of heavy chain variable region VH genes;

FIG. 3 is a PCR amplified light chain variable region V.lambda.gene;

FIG. 4 shows SOE-PCR amplification of scFv genes;

FIG. 5 is a graph showing the results of a full human antibody panel in a monoclonal phage ELISA assay;

FIG. 6 is a heavy and light chain sequence diagram of a fully human antagonistic antibody with targeting specificity;

FIG. 7 is a comparison of joint injury and disease progression in CIA mice treated with CTGF antagonist antibodies.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.

The invention provides a fully human antagonistic antibody taking connective tissue growth factor as a target, which is a single-chain antibody, has the specificity of targeting human connective tissue growth factor, and has the amino acid sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, 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 taking connective tissue growth factor as a target point can be fused with other antibody medicines for treating rheumatoid arthritis, and the formed antibody can play a role of targeting human connective tissue growth factor on one hand and can play a role of treating rheumatoid arthritis on the other hand.

The DNA of the above single-chain antibody can be obtained by a conventional gene recombination technique. The DNA sequences encoding the single-chain antibodies are obtained by PCR and cloned into vectors, which may be plasmids, viruses or gene fragments commonly used in molecular biology. A protein secretion signal peptide sequence is added at the front end of the DNA sequence for encoding the antibody so as to ensure that the antibody can be secreted from cells. Included among the vector sequences are promoters for gene expression, protein translation initiation and termination signals, and polyadenylation (PolyA) sequences. The vectors contain antibiotic resistance genes to facilitate replication and expression of the vectors in host cells such as bacteria and eukaryotic cells. In addition, eukaryotic cell-selective genes are included in the vector for stable transfection of choice of host cell lines.

The single chain antibodies of the invention may be further fused to other effector molecules to achieve other additional effects without affecting their targeting properties, all of which are within the scope of the invention.

After construction of the plasmid containing the DNA sequence encoding the above antibody, the recombinant vector may be used to transfect or transform a 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, and the like. Since prokaryotic cells express antibodies to form inclusion bodies easily, mammalian cells are the preferred system for expressing the protein. There are various mammalian cells that can be used for large-scale expression of antibodies, such as CHO cells, 293 cells, NS0 cells, COS cells, etc., all of which are included among the cells that can be used in the present invention. The recombinant plasmid containing the gene encoding the antibody can be transfected into host cells, and the methods for transfecting cells are various, including electroporation, liposome transfection, calcium phosphate transfection, etc.

A preferred method of protein expression is by use of stably transfected host cells. For example, after stably transfecting a host cell without Neomycin resistance with a recombinant vector containing a Neomycin (Neomycin) resistance gene, the concentration of Neomycin may be increased in the cell culture broth to select a stable cell strain with high expression; for example, after stably transfecting a host cell lacking DHFR with a recombinant vector containing the dihydrofolate reductase (DHFR) gene, the concentration of Methotrexate (MTX) may be increased in the cell culture medium to select for stable cell lines with high expression.

Other expression systems besides mammalian cells, such as insect cells, yeast, bacteria, etc., may also be used to express the antibodies of the invention, and they are also encompassed by the host cells that can be used in accordance with the invention. The protein yield of these expression systems is higher than that of mammalian cells, but inclusion bodies are easily formed, and thus further protein renaturation is required.

Antibodies of the invention may also be carried and expressed using viral vectors including, but not limited to, adenovirus vector (adenoviralvectors), adeno-associated virus vector (adeno-associatedviralvectors), retrovirus vector (retroviralvectors), herpes simplex virus vector (herpessimplexvirus-basedvectors), lentivirus vector (lentiviralvectors).

The antibodies of the invention may be formulated in a variety of pharmaceutical formulations, more preferably as injections, most preferably as freeze-dried injections, according to conventional techniques of pharmacy.

The antibodies of the invention may be formed into pharmaceutical compositions with other drugs that may be used in the treatment of diseases with other therapeutic methods including chemotherapy, radiation therapy, biological therapy.

The fully human antagonistic antibody taking connective tissue growth factor as a target point is screened out by a ScFv phage antibody library of a rheumatoid arthritis patient. The specific process is as follows:

Description of Experimental demonstration information

Antigen CTF-H82E6 (Biotinylated Human CTGF/CCN2 Protein, available from Beijing Baiposi Biotech Co., ltd.);

phage library for screening, namely ScFv phage antibody library of rheumatoid arthritis patient;

Illustrative purposes antagonistic antibodies targeting human connective tissue growth factor were screened from the ScFv phage antibody library of rheumatoid arthritis patients.

2. Experimental details

2.1 Construction of ScFv phage antibody library for rheumatoid arthritis patients

(1) Preparation of cDNA: 5 ml of 45 human peripheral blood was collected from rheumatoid arthritis patients, EDTA-K ₂ was anticoagulated and mononuclear cells (PBMC) were isolated using lymphocyte separation fluid, and RNA was extracted from each of the 5 patients PBMC mixed together and then reversed to cDNA.

(2) PCR amplification and ligation of VH and VL genes: primers were designed to synthesize amplified human antibody genes. PCR amplification of the VH and VL genes was performed using the first strand of cDNA as template. And the VH and VL genes were linked to the ScFv gene by a linker peptide (Gly 4 Ser) 3. The agarose electrophoresis results are shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4.

(3) Construction of phage antibody library: mixing the amplified products in proper proportion by taking VH and VL gene fragments as templates, splicing by overlapping PCR, amplifying ScFv genes, cloning into a pCANTAB-5E vector, carrying out electroporation to transform TG1, counting and cloning to determine the stock capacity as 10 ⁷, and obtaining a phage antibody library by auxiliary phage super-infection after bacterial proliferation.

2.2 Liquid phase-affinity panning

2.2.1 Blocking phage libraries and magnetic beads

(1) Blocking the phage library. Adding the phage library of 1X 10 ¹¹ into 3%M-PBS blocking solution (namely PBS containing 3% skimmed milk powder), mixing uniformly by inversion, and standing at 37 ℃ for 1-2 h.

(2) Sealing magnetic beads, namely placing a magnetic bead bottle on a vortex oscillator, vibrating and re-suspending for 20s, preparing two new centrifuge tubes A and B, respectively taking 50ul magnetic beads into the new centrifuge tubes by using a pipettor, adding 1ml of 0.05% PBST, vibrating and re-suspending for 20s, magnetically separating for 2min, and removing supernatant. (washing 2 times in total)

(3) Adding 1ml of sealing liquid into the tube A-negative panning and the tube B-positive panning respectively, shaking and re-suspending for 20s, magnetically separating for 5min, removing supernatant, adding 1ml of sealing liquid again, mixing uniformly upside down, placing on a rotary mixer, and placing for 1h at room temperature of 20 rpm.

2.2.2 Binding of antigen proteins to magnetic beads, phage library negative panning

(1) Antigen-magnetic bead binding "B tube-positive panning", transient magnetic separation for 2min, removal of supernatant, washing 2 times with 0.05% PBST, adding 1ml biotinylated antigen Protein (CTF-H82E 6, biotinylated Human CTGF/CCN2 Protein) 0.05% PBST for dilution, making the magnetic bead concentration lmg/ml, mixing uniformly upside down, and placing on a rotary mixer at room temperature of 20rpm for 1H.

(2) Phage library negative panning, "tube A-negative panning," transient separation, magnetic separation for 2min, removal of supernatant, washing 3 times with 0.05% PBST, adding the blocked phage library into tube A, mixing well upside down, placing on a rotary mixer, and placing at room temperature at 20rpm for 1h. After 1 hour, the A-tube was magnetically separated for 2min and the transfer supernatant was added to the new EP-tube, the phage library after blocking and negative panning.

2.2.3 Panning

(1) Antigen-antibody combination is that B tube-positive panning, instantaneous separation, magnetic separation for 2min, removing supernatant, washing with 0.05% PBST for 3 times, adding phage library of negative panning, reversing and mixing uniformly, placing on a rotary mixer, and placing at room temperature of 20rpm for 0.5-1 h.

(2) Washing, wherein 'B tube-positive panning' is magnetically separated by 5min, the supernatant is sucked off, 1ml of 0.1% -0.5% PBST is added, shaking is carried out for 20s, the magnetic separation is carried out for 2min, and the supernatant is sucked off. This step is repeated 10 to 20 times.

(3) And (3) eluting and neutralizing, namely, a pipe B, magnetically separating for 2min, sucking the supernatant, washing with PBST for 2 times, adding 400ul of eluting buffer (glycine-hydrochloric acid pH2.2), reversely and uniformly mixing, placing on a rotary mixer, and placing at room temperature for 10min at 20 rpm. The supernatant was magnetically separated for 2min and the new 1.5ml EP tube (200 ul of neutralization buffer (1M Tris HCl pH 8.0) was added in advance) and kept at 4℃for further use, details are given in Table 1.

2.3 Amplification of eluted products

(1) TG1 monoclonal on the plate was picked, added to 20ml 2yt, and incubated at 37 ℃ at 220rpm until log phase od600=0.6-0.8.

(2) Adding the eluted antibody library, mixing, and standing at 37deg.C for 60min.

(3) 4Ul Amp was added and incubated at 37℃for 60min at 180 rpm.

(4) M13KO7 (helper phage: TG1> =10:1) was added to the culture and left at 37℃for 30min.

(5) 30Ml of 2YT and 6ul of Amp were added and incubated at 37℃for 60min at 180 rpm.

(6) Centrifugation at 5000rpm for 10min at room temperature, removal of supernatant, collection of bacterial pellet, addition of new 50ml 2YTAK resuspended pellet, and cultivation in 200ml Erlenmeyer flask at 30℃at 220rpm overnight. The following day was precipitated and recovered, and the amplification product titer was measured.

(7) The culture was transferred to a 100ml centrifuge tube, centrifuged at 12000rpm for 20min at 4℃and the supernatant transferred to a new centrifuge tube, centrifuged again at 12000rpm for 20min, and the supernatant transferred to a new centrifuge tube.

(8) 1/4 Volume of PEG/NaCl was added to the supernatant, and ice bath 4h was added.

(9) Centrifuging at 12000rpm at 4deg.C for 20min, discarding supernatant, and centrifuging again briefly to remove supernatant.

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

(11) Titer (pool of amplified antibodies, gradient diluted to 10E10-10E 11) was determined.

(12) To 200ul of log phase TG1 was added a 10-9/10-10 dilution of the antibody pool, which was placed at 37℃for 30min, spread on a 2YTAG plate, incubated overnight at 37℃and the colony count was calculated the next day.

2.4Phage ELISA

Preparation of monoclonal Phage supernatant

(1) Single colony is selected from the phage library plate eluted in the last round of screening and inoculated into a 96-well deep hole plate containing 300ul of 2YTAG, and cultured at 37C and 700rpm until the logarithmic phase;

(2) Sucking 100ul of bacterial liquid from each well into a sterilized 96-well plate, and preserving at 4 ℃;

(3) 100ul of M13KO7 (titer 2X10 ¹⁰, about 10 times bacterial concentration) diluted with 2YT was added, and incubated at 37℃for 30min, followed by incubation at 37℃for 60min at 600 rpm.

(4) Centrifuging 96-well deep-hole plate at 4000rpm for 10 min, discarding supernatant, adding 300ul 2TYAK resuspended cells per well, culturing at 700rpm at 30deg.C overnight

Monoclonal PHAGE ELISA

(5) Coating target protein: the biotinylated target molecules were diluted to lug/ml with 0.05% PBST solution, added to streptavidin plates, 100 ul/well, and mixed well for 1h at 37 ℃.

(6) Washing: removing the coating supernatant, beating to dry, and cleaning with 0.05% PBST for 3 times each for 1min;

(7) Closing: adding sealing solution 4% skimmed milk Powder (PBS) 300 ul/hole, standing at 37deg.C for 1 hr;

(8) Washing: discarding the blocking solution, beating to dry, and cleaning with 0.05% PBST for 3 times each for 1min;

(9) Mixing 2% skimmed milk Powder (PBS) and monoclonal supernatant 50ul each, adding into corresponding enzyme-labeled plate holes at 37deg.C, and shaking at 100rpm for 1 hr;

(10) Washing: the supernatant was discarded, patted dry, and washed 5 times with 0.1% pbst:

(11) Secondary antibody binding: anti-M13-HRP antibody was used at 1:5000 dilution in 1% skim milk Powder (PBS) as a blocking solution, 100 ul/well, addition to ELISA plate, shaking slowly at 37deg.C and 100rpm for 1h;

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

(13) 100ul of TMB is added, the reaction is carried out for 5 to 10 minutes in a dark place, 100ul of 2M hydrochloric acid is added for stopping the reaction, and the OD450 value is read in an enzyme labeling instrument.

As a result of ELISA detection of the monoclonal phage, see FIG. 5, 92 clones specifically binding to antigen protein (human connective tissue growth factor protein) were obtained. We selected 10 clones labeled with background color in fig. 5 (i.e., with different a 450. Ag values) for further sequencing.

Finally, 10 antigen binding protein sequences (respectively shown as SEQ ID NO:1, 2,3, 4, 5, 6, 7, 8, 9 or 10) and nucleotide sequences (respectively shown as SEQ ID NO:11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) with targeting specificity to human Connective Tissue Growth Factor (CTGF) are obtained, and the antigen binding protein is shown in the heavy and light chain sequences shown in the accompanying figure 6.

2.5 Treatment of CIA mouse arthritis with CTGF antagonist antibodies

(1) Expressing and purifying the single-chain antibody in escherichia coli, taking a No. 5 phage, amplifying a single-chain antibody DNA fragment by a PCR method, cloning into an expression vector pGEX-6P-1 after double digestion by EcoR I and Xho I, and converting escherichia coli BL 21; culturing recombinant strain, adding IPTG to a final concentration of 0.5mM, inducing expression at 30deg.C for 5h, ultrasonic crushing, centrifuging at 12000rpm/10min, collecting supernatant, purifying with GST column, eluting target protein with equilibration buffer, obtaining electrophoresis purified single chain antibody, and measuring protein concentration by BCA method.

(2) Constructing a CIA mouse model: a Complete Freund's Adjuvant (CFA) emulsion containing 4mg/ml of Mycobacterium tuberculosis with 6mg/ml of collagen was used for one immunization per DBA/1 mouse injection of 200 ul. After 21 days, each mice was injected with 100ul of collagen and Incomplete Freund's Adjuvant (IFA) for a second immunization. Joint scoring was performed every three days, and a CIA mouse model was selected for successful construction.

(3) Two groups of CIA mice (10 per group) constructed with DBA/1 were intraperitoneally injected weekly with 200 μg of CTGF antagonist antibody and purified mouse IgG (Sigma-Aldrich) 4 times from day 22 to day 43. Three groups of mice were then sacrificed compared to normal mice, the legs were removed, fixed, decalcified, and joint embedded sections were sectioned for HE staining. The morphology of the tissue was observed under a microscope and the results are shown in FIG. 7. The results show that CTGF antagonist antibodies do have therapeutic effect on CIA mouse arthritis.

The foregoing disclosure is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.

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

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Glu Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asp Pro Ser Thr Tyr Tyr Asp Ile Leu Thr Gly Ser Arg Leu

100 105 110

Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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 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 attactgtgc 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 attactgtgc 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 ggccagggga 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 gtggaggcac 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 attactgtgc gagagggggc 300

tacgtttggg gttactacta 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 attactgtgc 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 attactgtgc 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 gcctggagtg ggtctcaggt attagttgga atagtggtag cataggctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240

ctgcaaatga acagtctgag agctgaggac acggccttgt attactgtgc aaaagatata 300

gcgtattgta gtggtggtag ctgctactct gtctactact actacggtat ggacgtctgg 360

ggccaaggga ccacggtcac cgtctcctca ggtggtggcg gttcaggcgg aggtggctct 420

ggcggtggcg gatcgcagcc tgtgctgact cagcccccct cgatgtctgc agccccccgg 480

cagagggtcg 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 atcaacacca acactgggaa cccaacgtat 180

gcccagggct tcacaggacg gtttgtcttc tccttggaca cctctgtcag aacggcatat 240

ctgcagatca gcagcctaaa ggctgaggac actgccgtct attactgtgc 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 attactgtgc 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 attggggaat 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

1. A fully human antagonistic antibody targeting connective tissue growth factor, characterized in that: the fully human antagonistic antibody is a single-chain antibody, and the amino acid sequence of the fully human antagonistic antibody is shown as SEQ ID NO. 6.

2. A gene encoding the fully human antagonistic 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. 16.

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

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

6. The use of a 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 a disease of rheumatoid arthritis.