CN115850474A

CN115850474A - IL-8 monoclonal antibody and application thereof

Info

Publication number: CN115850474A
Application number: CN202211499703.4A
Authority: CN
Inventors: 赖晨; 王然; 徐义; 陈志康; 袁伟杰; 龙凤英; 蔡开妹
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2022-11-28
Filing date: 2022-11-28
Publication date: 2023-03-28

Abstract

The invention belongs to the technical field of IL-8 monoclonal antibody preparation, and particularly relates to an anti-IL-8 monoclonal antibody and application thereof. The invention screens the monoclonal antibody which is combined with IL-8 through a phage antibody library. The anti-IL-8 monoclonal antibody of the invention can be specifically combined with IL-8, and can be used for diagnosing or treating diseases related to abnormal expression of IL-8.

Description

IL-8 monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of IL-8 monoclonal antibody preparation, and particularly relates to an anti-IL-8 monoclonal antibody and application thereof.

Background

Interleukin-8 (IL-8), also known as CXCL8, belongs to the CXC subtype and is the first chemotactic cytokine to be found. IL-8 is divided into two sub-groups of alpha and beta, the initial translation product of IL-8 is 99 amino acids, which are cleaved into 72 amino acids in monocytes and macrophages, with a molecular weight of 8KDa active substance. IL-8 is secreted primarily by monocytes and macrophages and functions to recruit and activate neutrophils, driving an inflammatory response and cell killing. In addition, tumor cells, fibroblasts, endothelial cells and the like influence the proliferation, invasion, metastasis and formation of tumor blood vessels through autocrine or paracrine under the action of proinflammatory factors such as IL-1 beta, tumor necrosis factor alpha, lipopolysaccharide and the like.

IL-8 acts by binding to the IL-8 receptor, IL-8 has 2 receptors, CXCR1 (IL-8 RA) and CXCR2 (IL-8 RB), these 2 receptors share 77% homology and are G protein coupled receptors. Because of the structural difference of the N end, the affinity of 2 receptors to IL-8 is different, and the affinity of CXCR1 to IL-8 is higher. In normal tissue cells, CXCR1 first binds to granulocyte chemotactic protein 2 (GCP-2) to form a complex, and then binds to IL-8 to play a role; CXCR2 has a low affinity for IL-8, and CXCR2 can bind to various ligands such as CXCL1, 2, 3, 5, and 7 in addition to IL-8. IL-8 has obvious chemotaxis and function activation effects on neutrophils, can also induce the chemotaxis of CD4+ and CD8+ T cells, and is involved in the development and tissue injury of various diseases, such as rheumatoid arthritis, psoriasis, ischemia-reperfusion syndrome (including myocardial infarction and multi-organ failure), glomerulonephritis and other various infectious diseases; IL-8 binding to the receptor can promote tumor development by activating multiple downstream signaling pathways such as PI3K/Akt, PLC/PKC and MAPK. Therefore, the screening of IL-8 antagonistic monoclonal antibodies can be used for effectively treating IL-8 mediated diseases.

IL-8 is used as immunogen to immunize rabbits, an anti-IL-8 phage antibody library is constructed, an antibody for antagonizing IL-8 can be effectively screened, and the antagonizing IL-8 antibody can be produced in a large scale through gene engineering and protein expression technology and is used for diagnosis and treatment of diseases related to IL-8 abnormality.

The invention content is as follows:

the invention mainly aims to provide an anti-IL-8 monoclonal antibody and application thereof; can be used for diagnosing and treating diseases caused by abnormal expression of IL-8.

In order to achieve the purpose, the invention is realized by the following technical scheme:

an anti-IL-8 monoclonal antibody, wherein the anti-IL-8 monoclonal antibody is named YX40; YX40 heavy chain CDR sequence:

CDRH1 amino acid sequence GFSLNNYA; see SEQ NO.1;

CDRH2 amino acid sequence IGSDGIP; see SEQ NO.2;

the CDRH3 amino acid sequence ASGYVGDDRYNI; see SEQ NO.3;

YX40 light chain CDR sequence:

CDRL1 amino acid sequence PSVYNNY; see SEQ NO.4;

CDRL2 amino acid sequence AAS; see SEQ NO.5;

CDRL3 amino acid sequence AGAYSSNDSDDG, see SEQ NO.6.

Further, the anti-IL-8 monoclonal antibody,

YX40 heavy chain variable region amino acid sequence:

<xnotran> GSTGDQTVKESGGRLVTPGTPLTLTCTVSGFSLNNYAMSWVRQAPGKGLEYIGVIGSDGIPYYATWAKGRFAISKTSTTVDLKITSPTTEDTATYFCASGYVGDDRYNIWGPGTLVTVSS; </xnotran> See SEQ NO.7;

YX40 light chain variable region amino acid sequence:

<xnotran> GSTGDADGVMTQTPSPVSAAVGGTVSISCQSSPSVYNNNYLSWYQQKPGQPPKLLIYAASTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCAGAYSNDSDDGFGGGTKVEIK, SEQ NO.8; </xnotran> .

Further, in the present invention,

YX40 heavy chain variable region nucleic acid sequence:

GGTTCCACTGGTGACCAGACAGTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACA

CTCACCTGCACCGTCTCTGGATTCTCCCTCAATAACTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGG

AAGGGGCTGGAATACATCGGAGTCATCGGCTCTGATGGTATTCCATACTACGCGACTTGGGCGAAAGGC

CGATTCGCCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACG

<xnotran> GCCACCTATTTCTGTGCCAGCGGATATGTTGGTGATGATAGATATAACATCTGGGGCCCAGGCACCCTCGTCACCGTCTCTTCA; </xnotran> See SEQ NO.9;

YX40 light chain variable region nucleic acid sequence:

GGTTCCACTGGTGACGCCGATGGCGTGATGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGC

ACAGTCAGCATCAGTTGCCAGTCCAGTCCGAGTGTTTATAATAACAACTACTTATCCTGGTATCAGCAG

AAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGG

TTCAAGGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCC

<xnotran> ACTTACTACTGTGCAGGCGCTTATAGTAATGATAGTGATGATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAA, SEQ NO.10. </xnotran>

The anti-IL-8 monoclonal antibody of the invention, the antibody molecules include but are not limited to: immunoglobulins, fabs, (Fab') 2 or single chain antibodies.

The invention also provides the application of the anti-IL-8 monoclonal antibody in preparing a preparation for diagnosing the human interleukin-8 abnormal expression disease.

The invention also provides the application of the anti-IL-8 monoclonal antibody in preparing medicines for treating diseases related to the abnormal expression of human interleukin-8.

Further, diseases associated with abnormal expression of human interleukin-8 include tumors.

The invention expresses IL-8 by pronucleus, the IL-8 is obtained according to 1 mg/rabbit dose hypodermic rabbit for immunization, 4 rounds of immunization are carried out, each round is separated for 2 weeks, after the immunization is finished, the rabbit is killed, the spleen is picked up, the spleen is ground into single cell suspension, the single cell is obtained by separation, the single cell is obtained and total RNA is extracted, cDNA is synthesized, the heavy chain variable region and the light chain variable region are merged by using a primer, the heavy chain variable region and the light chain variable region are amplified, the obtained heavy chain variable region and the light chain variable region are randomly combined and recombined with a phage vector, the recombined product is converted into XL1-Blue, an auxiliary phage VCSM13 is added to obtain the titer which is about 10 ¹³ Large and small phage antibody libraries. Using antigen IL-8, screening antibody of anti IL-8, obtaining heavy chain variable region and light chain variable region sequence capable of combining with IL-8 by sequencing, assembling Fc fragment of human IgG1 on heavy chain variable region, making heavy chain and light chain of antibody be simultaneously in lactationExpressing in the cell to express humanized and modified anti-IL-8 antibody. Through in vitro experiments, the anti-IL-8 monoclonal antibody screened by the invention can be specifically combined with IL-8, and can be used for detecting or treating diseases caused by abnormal expression of IL-8.

Description of the drawings:

FIG. 1IL-8 gene cloning;

FIG. 2 detection of IL-8SDS-PAGE after purification;

FIG. 3 cloning of anti-IL 8 antibody heavy chain variable region gene;

FIG. 4 cloning of anti-IL 8 antibody light chain variable region gene;

FIG. 5 anti-IL 8 antibody heavy and light chain variable region gene splicing;

FIG. 6 anti-IL 8 phage positive clone identification;

FIG. 7ELISA identification of 5 th round screening phage library;

FIG. 8 PCR identification of antibody heavy and light chains linked to pcDNA3.4;

FIG. 9 SDS-PAGE detection of purified product of antibody YX40;

FIG. 10 inhibition of neutrophil chemotaxis by anti-IL 8 antibodies;

FIG. 11 inhibition of HCT116 growth in vivo by anti-IL 8 antibodies.

The specific implementation mode is as follows:

the invention is further described with reference to specific examples, which are not intended to be limiting.

The IL-8 protein used in the examples of the present invention was prepared on its own in the laboratory.

The construction method of the phage antibody library of the invention is referred to as follows: dimitrov (ed.), generation and Selection of Rabbit Antibody Libraries by phase Display,2009, therapeutic μ tic Antibodies: methods and Protocols, vol.525, 101-128.

Example 1

1. IL-8 protein preparation takes human monocyte cDNA as a template, IL-8 fragments are cloned by using primers IL-8F and IL-8R, the fragments are introduced into pGEX-4T-1 vectors (28156ling, P0001), pGEX-4T-1-IL-8 which is successfully constructed is obtained by screening, expressed under IPTG induction conditions and purified to obtain IL-8 protein.

The method specifically comprises the following steps:

1. cloning of IL-8 fragments

Monocyte cDNA was diluted 10-fold

(1) The system was formulated as in table 1 below:

TABLE 1

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Note: cloning of IL8 gene primer

IL8-F: GTTCCGCGTGGATCCCCGGAAAGAGGTGCAGTTTTGCCAAG, see SEQ NO.11;

IL8-R: TCGAGTCGACCCGGGAATTTCATGAATTCTCAGCCCTCTTCA, see SEQ NO.12.

(2) The reaction conditions are shown in the following table

TABLE 2

Step1	98℃	15s
			Step2	98℃	10s
Step3	55℃	5s
			Step4	72℃	5s
		Go to Step 2x34
			Step5	72℃	1m 30s
Step6	4℃	Forever

(3) Electrophoresis

25 μ l of the DNA was subjected to 1% agarose gel electrophoresis to determine whether the desired band was amplified and had a size of about 240bp (see FIG. 1).

(4) The 240bp band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.

2. IL-8 fragment and vector pGEX-4T-1 are connected and transformed

The Uniclone step gelation kit, cat # SC612, from Kyoto Jinsha Biotechnology Limited is used

(1) Connection of

Architecture configuration

TABLE 3

Reaction conditions

50℃，15min

(2) Transformation of

Adding 2.5ul of the product obtained in the step (1) into 50ul DH5 alpha, standing on ice for 30min, performing heat shock at 42 ℃ for 90s, standing on ice for 2min, adding 1ml LB culture medium, culturing at 37 ℃ and 220rpm for 1h, uniformly smearing 100ul of the product on a plate containing ampicillin resistance, and culturing at 37 ℃ in a constant temperature box overnight.

(3) Positive clone identification

And (3) selecting the single clone in the step (2), carrying out colony PCR identification by using a primer IL-8F/IL-8R, identifying the positive clone, sending the positive clone to a sequencing company for further sequencing identification, and reserving the clone with the correct sequence.

3. IL-8 expression and purification

(1) Selecting a single clone, adding the single clone into 10ml of LB liquid culture medium containing ampicillin resistance, culturing the single clone at 37 ℃ and 220rpm overnight,

(2) Inoculating 1% of the bacterial liquid of step (1) into 100ml blank LB liquid culture medium containing benzyl amine resistance, culturing overnight at 37 ℃ and 220rpm, and adding 0.2mmol/L IPTG to induce for 4h when OD600 reaches 0.6.

(3) After induction, the bacterial liquid was poured into a 50ml centrifuge tube, centrifuged at 4000rpm for 15min, the supernatant was discarded, and 10ml of an equilibration solution (140mM NaCl,2.7mM KCl,10mM Na) was added ₂ HPO ₄ ，1.8mM KH ₂ PO ₄ pH 7.4) resuspend the cells. And (5) carrying out ultrasonic crushing until bacterial liquid is clear.

(4) Transferring the crushed bacteria liquid to a centrifuge tube, centrifuging for 20min at 15000g and 4 ℃, taking supernatant, filtering through a 0.45um filter, and placing filtrate on ice.

(5) Glutathlone Beads 4FF gravity column (Changzhou Tiandi and Biotech Co., ltd., product No. SA 010005) was equilibrated with 10-fold equilibration solution, the supernatant from step (4) was added, and the sample was allowed to pass through the column and repeated 4 times.

(6) The column was washed with 15 times of the equilibration solution.

(7) The protein eluted with 5 volumes of eluent (140mM NaCl, 2.7MKCl, 10mM Na) was added ₂ HPO ₄ ，1.8mM KH ₂ PO ₄ 10mM reduced glutathione, pH 7.4).

(8) The obtained target protein was concentrated with a 10Kd ultrafiltration tube, and the buffer was replaced with 10mM PBS.

(9) Determination of protein concentration by IL-8 after concentration and SDS-PAGE detection (see FIG. 2)

2. IL-8 immunized rabbit

(1) 500ul IL-8 (1 mg) was immunized for the first time and mixed with 500ul Freund's complete adjuvant, and 2 months old New Zealand white rabbits were injected subcutaneously at multiple sites on the back.

(2) Every 2 weeks, 500ul IL-8 (1 mg) was mixed with 500ul Freund's incomplete adjuvant

The back of the New Zealand white rabbit is injected subcutaneously at multiple points, and the immunization is carried out 3 times at intervals.

3. Construction of phage antibody libraries

After the immunization of the New Zealand white rabbits is finished, the rabbits are killed, spleens are picked, mononuclear cells are separated, total RNA is extracted and is reversely transcribed into cDNA, a heavy chain variable region and a light chain variable region fragment are obtained through amplification by using a heavy chain variable region and light chain variable region merger primer, the heavy chain variable region and the light chain variable region of an antibody are randomly spliced and connected with a phage vector, XL1-Blue bacteria (purchased from Toshiba Biotech Co., ltd.) are transformed, and the phage antibody display library is assembled under the condition that auxiliary phage VSCM13 (purchased from NTCC national type culture Collection) exists.

The method specifically comprises the following steps:

1. monocyte isolation

Mononuclear cells were isolated using human peripheral blood lymph isolate (LTS 1077-1, a tertiary ocean biologicals science and technology Limited company).

(1) Preparing a cell suspension: place spleen in cell sieve, place in culture dish with medium, use 2.5mL syringe needle push rod, squeeze gently until spleen is completely crushed, transfer cell suspension into 15mL centrifuge tube.

(2) A15 mL centrifuge tube containing the cell suspension was filled at 100g for 10min.

(3) Transferring the upper layer cell suspension into a 15ml centrifuge tube containing an equal volume of lymph separation liquid, carefully sucking the cell suspension with a pipette and adding the cell suspension on the liquid surface of the separation liquid, 500-1100g (1800 rpm), centrifuging for 10min, accelerating 5, and decelerating 4.

(4) After centrifugation, the centrifuge tube is divided into four layers from top to bottom; the second layer is a layer of annular milky white lymphocytes.

(5) Carefully pipette the second layer of circular opalescent lymphocytes with a pipette and transfer to a new 15mL centrifuge tube, add 10mL of pbs to the resulting centrifuge tube and mix the cells well.

(6) 1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.

(7) The supernatant was discarded, 5ml of erythrocyte lysate was added to each 15ml centrifuge tube, slightly mixed and allowed to stand at room temperature for 5min.

(8) 1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.

(9) The supernatant was discarded and 10mL of PBS was aspirated to resuspend the resulting cells.

(10) 1800rpm, 10min centrifugation, 5 acceleration, 4 deceleration.

(11) The supernatant was discarded and the mononuclear cells were precipitated.

2. Total RNA extraction

Subjecting the obtained mononuclear cells to a reaction at 5X10 ⁶ Total RNA was extracted by adding 0.75ml of Trizol (Thermo fisher) to each cell, and 18 portions of RNA were mixed in equal amounts each taking 1. Mu.g of RNA.

(1)5X10 ⁶ 0.75ml Trizol was added to each cell.

(2) Mix up and down several times, incubate for 5min on ice to ensure the cells are fully lysed.

(3) Centrifuge at 12000rpm for 10 minutes at 4 ℃ and transfer the supernatant to a clean centrifuge tube.

(4) 0.2ml of chloroform was added to 1ml of Trizol, and the mixture was mixed well.

(5) Incubate on ice for 2-3min.

(6) Centrifuge at 12000rpm for 15 minutes at 4 ℃.

(7) Transfer the supernatant to a clean centrifuge tube.

(8) Adding isopropanol with the same volume into the supernatant and mixing uniformly.

(9) Incubate at room temperature for 10min, centrifuge at 12000rpm at 4 ℃ for 10-15 min.

(10) The supernatant was discarded and the precipitate was retained.

(11) The precipitate was washed by adding 1ml of 75% ethanol.

(12) Centrifuge at 12000rpm for 5 minutes at 4 ℃.

(13) The supernatant was discarded as much as possible.

(15) Drying at room temperature for 5-10min, and no liquid can be seen on the tube wall.

(16) Add 20-50 μ l of RNAse free water to re-dissolve RNA.

(17) Taking 1. Mu.l of RNA for electrophoresis detection and determining the concentration of the RNA.

(18) RNA is subpackaged and stored in a refrigerator at the temperature of 80 ℃ below zero to avoid repeated freeze thawing.

3. Synthesis of cDNA

Mu.g of RNA was collected and used in a cDNA synthesis kit (K1612, thermo fisher).

(1) cDNA was synthesized in an amount of 5. Mu.g of RNA.

(2) The 10. Mu.l RNA/primer mix. Mu.re system is shown in Table 4 below, and includes:

TABLE 4

System of ingredients	Volume of
		RNA	5μg
50μM Oligo(dT) ₂₀	1μl
		Hex Random Primer	1μl
10mM dNTP	1μl
		DEPC-treated water	Total volume 10. Mu.l

(3) Incubate at 65 ℃ for 5min, place on ice and stand for 1min.

(4) The cDNA Synthesis Mix was prepared for the next reaction by adding additional reagents as in Table 5 below.

TABLE 5

Components of the System	1 reaction
		10xRT buffer	2μl
25mM MgCl ₂	4μl
		0.1M DTT	2μl
RNaseOΜT(40Μ/μl)	1μl
		Superscript III RT(200Μ/μl)	1μl

(5) Add 10. Mu.l of cDNA Synthesis Mix to the RNA/primer Mix gently mixed, centrifuge, and react according to the following conditions in Table 6:

TABLE 6

Step1	50℃	50min
			Step2	85℃	5min
		Cooling on ice, and centrifuging briefly
			Step3	RNaseH	1μl
Step4	37℃	20min

(6) The cDNA products were stored in aliquots at-20 ℃.

4. Amplification of antibody heavy chain variable region (VH) and light chain variable region (VL)

The synthesized cDNA was diluted 10-fold and used to amplify the templates for heavy and light chain variable regions.

(1) The system was formulated as in table 7 below, with VH as an example:

TABLE 7

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(2) The reaction conditions are shown in the following table

TABLE 8

(3) Electrophoresis

Mu.l of the DNA was subjected to 1% agarose gel electrophoresis to determine whether the desired band was amplified and the size was about 400bp (see FIG. 3).

(4) All amplified VH's were mixed in equal amounts, added with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol and mixed well, left overnight at-20 ℃.

(5) Centrifugation was carried out at 16000g for 15min at 4 ℃ and the supernatant removed, rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved in 200. Mu.l of water and gel-electrophoresed with 1% gelatin.

(6) The 400bp band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.

(7) Amplification and recovery of VL were performed according to steps (1) to (6), and the results are shown in FIG. 4.

(8) The nucleic acid concentration after purification of VH and VL was adjusted to a final concentration of 100 ng/. Mu.l and stored at-20 ℃.

5. Amplification of C kappa-pelB

(1) Amplification of C.kappa. -pelB and CL-pelB with the vectors pC. Kappa.and pCL (vectors from add gene) (100 ng/. Mu.l) as templates

For an example of amplification of C.kappa. -pelB, see Table 9.

TABLE 9

pCκ	10μl
		HCK (sequence see the aforementioned references)	60μl
Pelb (sequence see the aforementioned references)	60μl
		ddH ₂ O	370μl
PrimerSTAR	500μl
		Total	1000μl

(2) The reaction conditions are shown in Table 10

Watch 10

(3) 10 μ l of the product was electrophoretically detected to obtain a band of about 400bp in size.

(4) All amplified C.kappa. -pelB were mixed, added with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol, and mixed well, left overnight at-20 ℃.

(5) Centrifugation was carried out at 16000g for 15min at 4 ℃ and the supernatant removed, rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved in 200. Mu.l of water and subjected to gel electrophoresis using 1% gel.

(7) The purified C.kappa. -pelB was diluted to a final concentration of 100 ng/. Mu.l and stored at-20 ℃.

(8) CL-pelB was amplified and the template pC κ was changed to pCL by the procedure of (1) to (7).

6. Rab V kappa/Rab C kappa/CL/Rab VH fusions

(1) Mixing different amounts of VL, VH, ck-pelB according to Table 11 below

VL concentration 100 ng/. Mu.l, VH concentration 100 ng/. Mu.l, ckappa-pelB concentration 100 ng/. Mu.l

TABLE 11

VL	10μl
		VH	10μl
Ck-pelB	10μl
		Primerstar Mix	500μl
H ₂ O	470μl
		Total	1000μl

(2) The reaction conditions are shown in Table 12

TABLE 12

Step1	98℃	15s
			Step2	98℃	10s
Step3
		50℃	5s
Step4				72℃	5s
	Step5		Go to Step 2x10
Step6				72℃	1m 30s
	Step7	4℃	Forever

(3) Amplification of Rab VL/Rab Ck/Rab VH, see Table 13

Watch 13

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(4) The amplification conditions are shown in Table 14

TABLE 14

Step1	98℃	15s
			Step2	98℃	10s
Step3	55℃	5s
			Step4	72℃	5s(Go to Step2 x 39)
Step5	72℃	1m 30s
			Step6	4℃	Forever

(5) Electrophoretic detection

Taking 10. Mu.l of PCR product, and detecting by electrophoresis, wherein the size of the target gene band after fusion is about 1.2Kb. (see FIG. 5)

(6) All the amplified Rab VL/Rab C κ/Rab VH were mixed with 0.1 volume of 3M sodium acetate and 2.2 volumes of ethanol and mixed well and left overnight at-20 ℃.

(7) Centrifugation was carried out at 16000g for 15min at 4 ℃ and the supernatant removed, rinsed with 1ml of 70% ethanol (room temperature), dried at room temperature, redissolved in 200. Mu.l of water and subjected to gel electrophoresis using 1% gel.

(8) The 1.2Kb sized band was excised, recovered using QIAquick Gel Extraction Kit (QIAGEN, 28706), and the concentration was measured.

(9) Rab VL/Rab C.kappa.kappa.VH purified nucleic acid was diluted to a final concentration of 150 ng/. Mu.l and stored at-20 ℃.

(10) The Rab V lambda/Rab CL/Rab VH fusion was performed according to the steps (1) - (9).

7. SfiI enzyme digestion of Rab V kappa/Rab C kappa/CL/Rab VH and Rab V lambda/Rab CL/Rab VH

The Rab V kappa/Rab C kappa/CL/Rab VH enzyme digestion is taken as an example

(1) The enzyme digestion system is shown in Table 15

Watch 15

(2) Water bath at 50 ℃ and enzyme digestion reaction for 3h.

(3) The Gel was electrophoresed on a 1% Gel, and a 1.2kb band was excised and collected using QIAquick Gel Extraction Kit (QIAGEN, 28706).

(4) Detecting SfiI enzyme digestion, recovering Rab VL/Rab Ck/Rab VH segment, adjusting the concentration to 50 ng/mul, and storing at-20 ℃.

(5) The Rab V lambda/Rab CL/Rab VH is cleaved by enzyme, and the operation is carried out according to the steps (1) - (4).

8. SfiI restriction enzyme pC3C (vector from add gene)

(1) The enzyme digestion system is shown in Table 16

TABLE 16

pC3C 1μg/μl	50μl
		10Xbuffer	30μl
H ₂ O	208μl
		SfiI 40u/μl	12μl

(2) Water bath at 50 ℃ and enzyme digestion reaction for 3h.

(3) Electrophoresis using a 1% gel will cut two bands, a 3.5Kb and a 1.2Kb sized fragment.

(4) A3.5 Kb vector backbone was recovered using the QIAquick Gel Extraction Kit (QIAGEN, 28706).

(5) After Sfi enzyme digestion, the concentration of the recovered vector skeleton is adjusted to 100 ng/mul.

9. Ligation of pC3C (SfiI) and Rab VL/human Ck/Rab VH (SfiI)

(1) The connection system is shown in Table 17

TABLE 17

100 ng/. Mu.l SfiI digested pC3C	1.5μl
		50 ng/. Mu.l SfiI digested human VL/human Ck/human VH	2μl
10XT4 DNA ligase buffer	2μl
		H ₂ O	13.5μl
T4 DNA ligase 2000u/μl	1μl

The control group is shown in Table 18

Watch 18

100 ng/. Mu.l SfiI digested pC3C	1.5μl
		10XT4 DNA ligase buffer	2μl
H ₂ O	15.5μl
		T4 DNA ligase 2000u/μl	1μl

(2) The reaction was carried out at 16 ℃ overnight.

(3) All ligation reactions were combined and 1/10 volume of 3M sodium acetate solution was added.

(4) 2.2 times volume of precooled absolute ethyl alcohol is added, mixed evenly up and down, and precipitated at-20 ℃ overnight.

(5) 16000g, centrifuge at 4 deg.C for 30min, carefully discard the supernatant.

(6) Adding 70% ethanol, gently cleaning the precipitate, centrifuging at 16000g and 4 deg.C for 5min, and removing the supernatant.

(7) Drying at room temperature.

(8) Dissolution with appropriate amount of water, typically 1 ligation reaction with 1. Mu.l volume of ddH ₂ Dissolving O completely, and storing at-20 deg.C.

(9) The ligation of pC3C (SfiI) and Rab V.lamda/Rab CL/Rab VH was performed according to the procedures (1) to (8).

10. Conversion of ligation product to XL1-Blue

(1) XL1-Blue shock-receptive ice is placed for 10min for dissolution.

(2) And adding 19 mu l of the precipitated and concentrated ligation product into a 1.5ml centrifuge tube, carrying out ice bath, adding 300 mu l of XL1-Blue electric shock competence, mixing uniformly, quickly transferring into a 2mm electric shock cup, and standing on ice for 1min.

(3) Electric shock condition: 2.5KV,4ms

(4) After the electric conversion is finished, 5ml (1ml + 2ml) SOC culture medium is rapidly added, and the mixture is transferred into a 50ml conical centrifuge tube and cultured for 1h at 37 ℃ and 250 rpm. Mu.l of the bacterial liquid is added into 198 mu.l of LB, and meanwhile, the bacterial liquid is evenly coated on an LB plate containing 100 mu g/mu.l of carbenicillin, and the bacterial liquid is placed at 37 ℃ for overnight culture for calculating the transformation efficiency and the colony PCR identification positive rate. The PCR results are shown in FIG. 6.

(5) 10ml of SB medium, 3. Mu.l of 100. Mu.g/. Mu.l carbenicillin, and 30. Mu.l of 5. Mu.g/. Mu.l tetracycline were added. Incubated at 37 ℃ and 250rpm for 1h.

(6) Add 4.5. Mu.l of 100. Mu.g/. Mu.l carbenicillin, continue at 37 ℃,250rpm, incubate for 1-4h.

(7) The cultured bacteria were transferred to a 500ml culture flask, and 84ml of SB medium, 42.5. Mu.l of 100. Mu.g/. Mu.l carbenicillin, 170. Mu.l of 5. Mu.g/. Mu.l tetracycline, and 1ml of VCMS13 helper phage (10 ml) were added ¹¹ -10 ¹² pfu/ml)，37℃，275rpm，90min。

(8) Mu.l of 50. Mu.g/. Mu.l kanamycin was added thereto, and cultured at 37 ℃ and 275rpm overnight.

(9) 3000g, and centrifugation at 4 ℃ for 15min.

(10) Precipitated phages

The supernatant (200 ml) was transferred to a 500ml clean centrifuge tube, and 8g of PEG-8000 and 6g of NaCl were added, and the mixture was placed at 37 ℃ and 300rpm for 5min to promote dissolution. Standing on ice for 30min-1h.15000g, centrifuge at 4 ℃ for 15min. The supernatant was discarded, the flask was placed upside down on filter paper and allowed to dry for 10min, and excess liquid was carefully removed. The phage were resuspended in 2ml TBS containing 1% BSA (pipetted mix up and down), 16000g, centrifuged at 4 ℃ for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. Can be directly stored on ice in a short time, or added with 0.01 time of 2 percent sodium azide for 4 ℃ and added with 1 time of glycerol for long-time storage for-20 ℃.

2. Phage antibody library screening the example of IL-8 phage antibody screening

Using IL-8 protein coated 96-well plates, 5 rounds of screening were performed, and then single clones were selected to identify IL-8 specific antibodies.

1. Antigen coating

(1) IL-8 was added to a carbonate buffer at a concentration of 2. Mu.g/ml,

(2) Adding 50 μ l of IL-8 into each well of a corning high protein adsorption enzyme-linked immunosorbent assay 96 pore plate, and standing overnight at 4 ℃;

(3) Discarding supernatant, adding 5% skimmed milk powder 200 μ l/well, standing at 37 deg.C for 1 hr;

(4) The supernatant was discarded and washed 5 times with 0.05% TBST.

2. IL-8 antibody screening

(1) Diluting the phage antibody library with 2% skimmed milk powder by 10 times, taking 2 IL-8 antigen coated 96-well micropores, adding 100 μ l diluted phage, and standing at 37 deg.C for 1h.

(2) Discard the supernatant, wash 5 times with 0.05% TBST, add 100. Mu.l 100mM glycine, stand at 37 ℃ for 15min, blow-beat continuously, add 9. Mu.l 1M Tris.

(3) And (3) adding the phage eluted in the step (2) into 2ml of XL1-Blue bacteria, and standing for 15min at room temperature.

(4) 6ml of SB medium was added, 1.6. Mu.l of 100. Mu.g/. Mu.l carbenicillin and 12. Mu.g of 5. Mu.g/. Mu.l tetracycline were added, and the mixture was incubated at 37 ℃ and 250rpm for 1 hour.

(5) Adding 2.4 μ l of carbenicillin 100 μ g/μ l, and culturing for 1 hr;

(6) Add 1ml of helper phage VCSM13 (10) ¹¹ -10 ¹² pfu/ml), transferred to a 500ml flask, added with 91ml SB medium, added with 46. Mu.l of 100. Mu.g/. Mu.l carbenicillin, 184. Mu.l of 5. Mu.g/. Mu.l tetracycline, incubated at 37 ℃ for 1.5h at 250rpm, added with 140. Mu.l of 50. Mu.g/. Mu.l kanamycin, incubated at 37 ℃ overnight at 250 rpm.

(7) And (3) concentrating the phage antibody: centrifuging the bacterial liquid of the previous step to remove bacteria, and collecting supernatant (100 ml); 4g of PEG-8000 and 3g of NaCl were added thereto, and the mixture was heated at 37 ℃ and 300rpm for 5min to promote dissolution. Standing on ice for 30min-1h.15000g, centrifuge at 4 ℃ for 15min. The supernatant was discarded, the flask was placed upside down on filter paper and allowed to dry for 10min, and excess liquid was carefully removed. 2ml of TBS containing 1% BSA was used to resuspend the phage (pipetted up and down), 16000g, centrifuged at 4 ℃ for 5min, and the supernatant was passed through a 0.22 μm filter (Millipore) and transferred to a 2ml centrifuge tube. Can be directly stored on ice in a short time, or added with 0.01 time of 2 percent sodium azide for 4 ℃ and added with 1 time of glycerol for long-time storage for-20 ℃.

(8) And (5) taking the phage in the step (7), and performing 5 rounds of screening according to the steps (1) to (7).

(9) ELISA identification of five rounds of screened antibody libraries:

1) ELISA 96-well plates, each well coated with 100ng of IL-8;

2) Adding 100 μ l phage (diluted 10 times with 2% skimmed milk powder) and incubating at 37 deg.C for 1h;

3) Washing 5 times with 0.05% TBST;

4) Adding 100 μ l of anti-M13-HRP antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., ltd.) diluted 2000 times with 5% skimmed milk powder, and incubating at 37 deg.C for 1h;

5) Washing 5 times with 0.05% TBST;

6) Adding 100 μ l TMB, and developing in dark for 5min;

7) Add 50. Mu.l of 1M H ₂ SO ₄ The reaction was terminated.

8) Absorbance was measured at 450nm using a microplate reader.

The results are shown in FIG. 7 and Table 19.

TABLE 19ELISA identification of phage libraries from round 5 screening

3. IL-8 monoclonal antibody identification

(1) Taking the IL-8 phage selected in the 5 th round, diluting 10 ^-7 Adding 1 μ l into 200 μ l XL1-Blue bacteria, standing at room temperature for 15min;

(2) Completely coating the bacteria in the step (1) on an LB individual culture medium containing 100 mu g/ml carbenicillin, and culturing at 37 ℃ overnight;

(3) 200 monoclonals are selected and added into a liquid LB culture medium containing 10 mu g/ml tetracycline and 100 mu g/ml carbenicillin, the mixture is cultured for 4 hours at 37 ℃ and 250rpm, a small amount of bacterial liquid is taken for PCR identification of the bacterial liquid, and the clone with the band size of about 1200bp which can be amplified is selected.

The colony PCR amplification system is shown in table 20:

watch 20

Bacterial liquid	0.5μl
		VHSEQ (sequences see the aforementioned references)	0.2μl
vlseq (sequences see the aforementioned references)	0.2μl
		ddH ₂ O	4.1μl
PrimerSTAR	5μl
		Total	10μl

Colony PCR reaction conditions are shown in table 21:

TABLE 21

Step1	98℃	3min
			Step2	98℃	10s
Step3
		50℃	5s
Step4				72℃	15s
	Step5		Go to Step 2x34
Step6				72℃	1m 30s
	Step7	4℃	Forever

(4) Uniformly dividing the positive clone bacterial liquid obtained in the step (3) into 2 parts, and adding 3 mu l of helper phage VCSM13 (10) ¹¹ -10 ¹² pfu/ml), left at room temperature for 20min, 2.1. Mu.l of 50. Mu.g/. Mu.l kanamycin was added, and the other aliquot was incubated overnight at 37 ℃ and 250rpm without any substance;

(5) Centrifuging the bacteria added with the helper phage at 4000rpm for 10min, transferring the supernatant into a clean centrifuge tube, diluting the supernatant by 10 times, adding 100 μ l of the diluted supernatant into an enzyme-linked immunosorbent assay plate coated with IL-8, incubating at 37 ℃ for 1h, washing with 0.05% TBST for 5 times, and adding 2000 times of diluted supernatantThe anti-phage HRP-labeled antibody (purchased from Beijing Okawa Biotechnology Ltd.) was incubated at 37 ℃ for 1H, washed 5 times with 0.05% TBST, added with 100. Mu.l TMB, developed for 2min, and added with 50. Mu.l of 1M H ₂ SO ₄ The reaction is stopped, and the absorbance is measured at 450nm by using a microplate reader, and positive clones with the absorbance 2.1 times higher than that of the control are obtained.

(6) And (5) selecting the positive clones in the step (5), extracting plasmids of the bacteria without adding the helper phage, and sequencing to obtain sequences of the heavy chain variable region and the light chain variable region.

3. Expression and activity identification of fully humanized antibody

1. Construction of fully humanized vector

(1) Designing corresponding primers according to the obtained sequences of the heavy chain variable region or the light chain variable region of the antibody, respectively amplifying, adding secretion signal peptides at the N ends of the heavy chain and the light chain, and adding an Fc fragment of IgG1 at the C end of the heavy chain;

(2) The heavy chain variable region and the light chain variable region were homologously recombined into pcDNA3.4 vector, respectively. (see FIG. 8) (pcDNA3.4 vector purchased from Wuhan \28156lingBiotech, inc.

2. Expression of fully humanized antibodies

(1) Extracting corresponding heavy chain expression vector and light chain expression vector respectively by using endotoxin removing middle extraction kit (purchased from OMEGA);

(2) When the fusion level of 293T cells (purchased from Wuhan Punuoist Life technologies, inc.) reached 80%, transfection was performed using PEI;

(3) 12h after transfection, the medium was replaced with serum-free protein expression medium at 37 ℃ with 5% CO ₂ Culturing for 7 days;

(4) The antibody was purified using protein A/G packing. The results are shown in FIG. 9.

3. Identification of fully humanized antibody Activity

Exemplified by the identification of IL-8 antibody Activity

(1) ELISA identification

The obtained antibody was adjusted to a concentration of 1mg/ml, diluted 2000-fold with 5% skim milk powder, added to IL-8 antigen-coated ELISA wells, incubated at 37 ℃ for 1h, washed with 0.05% TBST5 times, diluting 2000 times of anti-human Fab-HRP secondary antibody (purchased from Beijing Solebao Biotech Co., ltd.) with 5% skimmed milk powder, incubating at 37 deg.C for 1h, washing with 0.05% TBST for 5 times, adding 100 μ l TMB, developing for 2min, adding 50 μ l1M H ₂ SO ₄ The reaction was terminated and absorbance was measured at 450nm using a microplate reader. The results are shown in Table 22.

TABLE 22 identification of YX40 binding to IL-8 after purification by ELISA

(2) The IL-8 protein is detected by using YX40 as an immobilized antibody and YX5, YX20 and YX40 as a detection antibody combination as follows:

the detection is carried out by adopting an ELISA sandwich method, a monoclonal fully-humanized antibody YX40 coats an ELISA plate, an antigen IL-8 is added, screened YX5, YX20 and YX40 biotin-labeled antibodies (biotin brand: thermo, cat # VF 300852) are added, different combinations are carried out, then streptavidin (brand: thermo Fisher, cat # VE 302151) with HRP labels is added, the detection color development is carried out, the deeper the color is, the stronger the IL-8 antigen capture capability is, and meanwhile, the pair of antibodies is suitable for the detection of the IL-8 antigen. Blank control means that no antibody was added, only HRP-labeled streptavidin was added.

The specific operation is as follows:

1) ELISA 96-well plates coated with 100ng of YX40 antibody per well;

2) Adding 100. Mu.l IL-8 (250 ng/ml), and incubating at 37 ℃ for 1h;

3) Washing 5 times with 0.05% TBST;

4) Adding 2000 times of YX5, YX20 and YX40 biotin labeled antibodies diluted by 5 percent of skimmed milk powder, and incubating for 1h at 37 ℃;

5) Washing 5 times with 0.05% TBST;

6) Adding 5% skimmed milk powder to dilute 5000 times streptavidin labeled with HRP, and incubating at room temperature for 10min;

7) Washing 5 times with 0.05% TBST;

8) Adding 100 μ l TMB, and developing in dark for 5min;

9) Add 50. Mu.l of 1M H ₂ SO ₄ The reaction was terminated.

10 Absorbance at 450nm using a microplate reader.

The results are shown in Table 23.

TABLE 23 Sandwich method for identifying antibody pairs for detecting IL8

Note: column coating phase, row detection phase, OD450 absorbance.

(3) Neutrophil chemotaxis inhibition

1) Neutrophils (tertiary tianjin, TBD LZS 11131) were isolated from human peripheral blood, and the neutrophil concentration was adjusted to 5X10 using 1640 ⁶ Per ml;

2) Using a 24-well Trans-well plate (Corning Costar, 3415), pore size 3um, experiments were divided into 3 groups: blank group, adding 400ul blank 1640 in lower chamber; for the positive control, 400ul of 1640 containing IL8 at a concentration of 10nM was added to the lower chamber; for the experimental group, 400ul 1640 containing IL8 antibody at a concentration of 10nM and 5ug/ml was added to the lower chamber; all 3 groups, 200ul (concentration 5X 10) were added to the upper chamber ⁶ Counts/ml) of neutrophils;

3) 5% of CO2, left at 37 ℃ for 2 hours, the upper chamber was discarded, and the cell count instrument calculated the cell amount in the lower chamber.

The results are shown in tables 24 and 25, FIG. 10.

TABLE 24 anti-IL 8 antibodies inhibit chemotaxis of neutrophils for IL8

TABLE 25 inhibition of neutrophil chemotaxis by anti-IL 8 antibody (unit:%)

	Multiple holes 1	Multiple holes 2	Multiple holes 3
				YX40	94.40298299	100	105.597017

(4) anti-IL 8 antibodies for tumor inhibition

1) Nude mice were divided into 2 groups, experimental group and control group, each group containing 3 mice, and each group was injected with 100ul 1X10 subcutaneous injection on the back before the start of the experiment ⁶ HCT116 tumor cells (colon tumor cells);

2) After 3 days, 200ul PBS is injected into the abdominal cavity of each nude mouse in the control group, and 200ul PBS containing 500ug of anti-IL 8 antibody is injected into the abdominal cavity of each nude mouse in the experimental group, and the injections are performed once every 3 days;

3) After 4 injections, the last 3 days of injection, nude mice were sacrificed and tumors were harvested and weighed. The results are shown in Table 26 and FIG. 11.

TABLE 26 tumor inhibition by anti-IL 8 antibodies (unit: g)

Numbering	1	2	3
				control	0.27	0.26	0.3
YX40	0.09	0.11	0.19

Claims

1. An anti-IL-8 monoclonal antibody, wherein said anti-IL-8 monoclonal antibody is designated YX40;

YX40 heavy chain CDR sequence:

CDRH1 amino acid sequence GFSLNNYA;

CDRH2 amino acid sequence IGSDGIP;

CDRH3 amino acid sequence ASGYVGDDRYNI;

YX40 light chain CDR sequence:

CDRL1 amino acid sequence PSVYNNY;

CDRL2 amino acid sequence AAS;

CDRL3 amino acid sequence AGAYSSNDSDDG.

2. The anti-IL-8 monoclonal antibody of claim 1, characterized by the YX40 heavy chain variable region amino acid sequence:

GSTGDQTVKESGGRLVTPGTPLTLTCTVSGFSLNNYAMSWVRQAPGKGLEYIGVIGSDGIPYYATWAKG RFAISKTSTTVDLKITSPTTEDTATYFCASGYVGDDRYNIWGPGTLVTVSS；

YX40 light chain variable region amino acid sequence:

GSTGDADGVMTQTPSPVSAAVGGTVSISCQSSPSVYNNNYLSWYQQKPGQPPKLLIYAASTLASGVPSR FKGSGSGTQFTLTISDVQCDDAATYYCAGAYSNDSDDGFGGGTKVEIK。

3. the anti-IL-8 monoclonal antibody of claim 1, characterized by the YX40 heavy chain variable region nucleic acid sequence:

GGTTCCACTGGTGACCAGACAGTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACCGTCTCTGGATTCTCCCTCAATAACTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAGTCATCGGCTCTGATGGTATTCCATACTACGCGACTTGGGCGAAAGGCCGATTCGCCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGCGGATATGTTGGTGATGATAGATATAACATCTGGGGCCCAGGCACCCTCGTCACCGTCTCTTCA；

YX40 light chain variable region nucleic acid sequence:

GGTTCCACTGGTGACGCCGATGGCGTGATGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTCCGAGTGTTTATAATAACAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGCTGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAGGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGCTTATAGTAATGATAGTGATGATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAA。

4. the anti-IL-8 monoclonal antibody of any of claims 1-3, characterized in that the antibody molecules include, but are not limited to: immunoglobulin, fab, (Fab') 2 or single chain antibody.

5. Use of an anti-IL-8 monoclonal antibody according to any one of claims 1 to 3 for the preparation of a preparation for the diagnosis of a disease in which the expression of human interleukin-8 is abnormal.

6. Use of an anti-IL-8 monoclonal antibody according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of a disease associated with an abnormal expression of human interleukin-8.

7. The use of an anti-IL-8 monoclonal antibody according to claim 6, characterized in that the diseases associated with an abnormal expression of human interleukin-8 comprise tumors.