CN113292652A

CN113292652A - anti-CD 70 antibodies with enhanced ADCC effect and uses thereof

Info

Publication number: CN113292652A
Application number: CN202110673783.XA
Authority: CN
Inventors: 张军锋; 郭志刚
Original assignee: Nanjing Landun Biotechnology Co ltd
Current assignee: Nanjing Landun Biotechnology Co ltd
Priority date: 2021-06-17
Filing date: 2021-06-17
Publication date: 2021-08-24
Also published as: WO2022262101A1

Abstract

The invention discloses an anti-CD 70 antibody with an enhanced ADCC effect and application thereof. The application successfully prepares an antibody (fusion protein) capable of binding to the human CD70 molecule, wherein the antibody has good specificity and high affinity and can bind to the human CD70 expressed on the surface of cells. Binding to CD70 not only activates the signaling required for ADCC effects, but ultimately kills the target cells. The anti-human CD70 monoclonal antibody is a potential drug for tumor immunotherapy.

Description

anti-CD 70 antibodies with enhanced ADCC effect and uses thereof

Technical Field

The application belongs to the biotechnology, and particularly relates to an anti-CD 70 antibody with an enhanced ADCC effect and application thereof.

Background

Because CD70/CD27 molecules have important immunoregulation functions, regulation and control of CD70 inevitably causes a series of signal transmission and physiological reactions of T cells, even B cells, NK cells and other lymphocytes, so that the CD70/CD27 molecule is an ideal immunotherapy target, and reasonable application of the CD70 molecule can possibly mobilize multiple cellular immunity. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important property of antibodies, and is generally killed by natural killer cells (NK) or by macrophages. Through the ADCC effect, not only can direct killing be carried out, but also the killed cells release antigens to activate acquired immunity. Therefore, ADCC not only can directly kill cells, but also mobilizes the opsonization of natural immunity, has more diverse action mechanisms, and has more important practical significance for the treatment of solid tumors. Therefore, antibody molecules having an ADCC effect are used in most antibody drugs.

The application takes human CD70 as a target point to develop an antibody with strong ADCC effect. The expression level of CD70 in cancer cells is much higher than that in normal cells, and ADCC effect mainly acts on cancer cells, thereby playing a role in treating cancer. However, not all antibodies have ADCC, even antibodies with similar structures do not necessarily have ADCC effect, and the specific spatial structure of the antibody variable region has an important influence on the ADCC effect. Starting from the ADCC effect, increasing the type of antibody mechanism of action is one way to improve the therapeutic effect of antibodies. At present, the ADCC effect of antibodies in clinical experiments is still weak, and the enhancement of therapeutic effects is still insufficient.

Disclosure of Invention

The purpose of the invention is as follows: in view of the above technical problems in the prior art, the present application provides a group of anti-CD 70 antibodies with enhanced ADCC effect and uses thereof.

The technical scheme is as follows: the group of anti-CD 70 antibodies with enhanced ADCC effect described herein have the amino acid sequence of CDR1 of the heavy chain variable region selected from the group consisting of SEQ ID NO: 1-14, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 15-28, the amino acid sequence of CDR3 is selected from SEQ ID NOs: 29-42.

Preferably, the anti-CD 70 antibody with enhanced ADCC effect described herein comprises the heavy chain variable region of any one of the following:

the amino acid sequence of CDR1 is selected from SEQ ID NO: 1, the amino acid sequence of CDR2 is selected from SEQ ID NO: 15, the amino acid sequence of CDR3 is selected from SEQ ID NO: 29;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 2, the amino acid sequence of CDR2 is selected from SEQ ID NO: 16, the amino acid sequence of CDR3 is selected from SEQ ID NO: 30, of a nitrogen-containing gas;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 3, the amino acid sequence of CDR2 is selected from SEQ ID NO: 17, the amino acid sequence of CDR3 is selected from SEQ ID NO: 31;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 4, the amino acid sequence of CDR2 is selected from SEQ ID NO: 18, the amino acid sequence of CDR3 is selected from SEQ ID NO: 32, a first step of removing the first layer;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 5, the amino acid sequence of CDR2 is selected from the group consisting of SEQ ID NO: 19, the amino acid sequence of CDR3 is selected from SEQ ID NO: 33;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 6, the amino acid sequence of CDR2 is selected from the group consisting of SEQ ID NO: 20, the amino acid sequence of CDR3 is selected from SEQ ID NO: 34;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 7, the amino acid sequence of CDR2 is selected from SEQ ID NO: 21, the amino acid sequence of CDR3 is selected from SEQ ID NO: 35;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 8, the amino acid sequence of CDR2 is selected from SEQ ID NO: 22, the amino acid sequence of CDR3 is selected from SEQ ID NO: 36;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 9, the amino acid sequence of CDR2 is selected from SEQ ID NO: 23, the amino acid sequence of CDR3 is selected from SEQ ID NO: 37;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 10, the amino acid sequence of CDR2 is selected from SEQ ID NO: 24, the amino acid sequence of CDR3 is selected from SEQ ID NO: 38;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 11, the amino acid sequence of CDR2 is selected from SEQ ID NO: 25, the amino acid sequence of CDR3 is selected from SEQ ID NO: 39;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 12, the amino acid sequence of CDR2 is selected from SEQ ID NO: 26, the amino acid sequence of CDR3 is selected from SEQ ID NO: 40;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 13, the amino acid sequence of CDR2 is selected from SEQ ID NO: 27, the amino acid sequence of CDR3 is selected from SEQ ID NO: 41;

the amino acid sequence of CDR1 is selected from SEQ ID NO: 14, the amino acid sequence of CDR2 is selected from SEQ ID NO: 28, the amino acid sequence of CDR3 is selected from SEQ ID NO: 42.

SEQ ID NO: 1-42 are shown in the following table:

the application also discloses a DNA molecule encoding the above VHH antibody against CD70 with enhanced ADCC effect, the heavy chain variable region of which SEQ ID NO: 1-42 are shown as SEQ ID NO: 43-84.

SEQ ID NO: 43-84 are shown in the following table:

the application also discloses application of the anti-CD 70 with the enhanced ADCC effect to detection of CD70 molecules.

Further, the use of the anti-CD 70 antibody with enhanced ADCC effect for the preparation of a medicament for the treatment of tumors is also within the scope of the present application.

The anti-CD 70 antibody with enhanced ADCC effect has variable region capable of targeting to specific antigen and Fc region capable of binding to Fc receptor (FcR) on the surface of immune cell to activate the killing function of immune cell and kill cell expressing antigen recognized by the variable region.

The present application starts from a commercial recombinant human CD70 biotin-tagged protein and a phage display antibody library. The CD70 biotin-tagged protein was co-incubated with streptavidin-coated magnetic beads, CD70 was immobilized on the magnetic beads by interaction between streptavidin and biotin, incubated with an antibody library, unbound/weakly bound phage were washed away, and phage bound to the CD70 protein were eluted. Repeating the steps for 3 times, changing the use amount and washing conditions of the CD70 biotin-labeled protein each time, and gradually eliminating the phage with weak binding and keeping more phage with strong binding as much as possible. The host bacteria are infected with strongly bound phage, plated and cultured overnight to obtain a monoclonal colony, i.e., phage antibodies that bind CD70 are monoclonal. ELISA screening was performed using monoclonal culture supernatants, and nucleic acid sequencing was performed on the monoclonal.

The coding sequences of the signal peptide, the anti-CD 70 antibody and the human IgG1 Fc are connected and read in frame to construct a mammalian expression vector. 293F cells are transfected, supernatant is harvested after shaking culture for 5 days, fusion protein is obtained by purifying the supernatant through protein A magnetic bead affinity chromatography, and the binding property of the fusion protein and CD70 is identified.

Culturing target cells, adding cells capable of displaying an ADCC signal transduction luciferase reporter gene according to a certain proportion, and incubating with a series of antibodies with gradient concentrations, wherein the reporter gene is activated to different degrees. And finally expressed by reporter gene activity.

Culturing target cells over-expressing firefly luciferase, adding Peripheral Blood Mononuclear Cells (PBMC) according to a certain proportion, co-incubating with a series of antibodies with gradient concentrations, activating ADCC function of effector cells in the PBMC, and killing the target cells. The stronger the ADCC effect of the antibody, the fewer the target cells that survive, and the lower the luciferase activity. The ADCC effect of the antibody is finally known by detecting the total luciferase activity of the target cells.

The technical effects are as follows: the application successfully prepares an antibody (fusion protein) capable of binding to the human CD70 molecule, wherein the antibody has good specificity and high affinity and can bind to the human CD70 expressed on the surface of cells. Binding to CD70 not only activates the signaling required for ADCC effects, but ultimately kills the target cells. The anti-human CD70 monoclonal antibody is a potential drug for tumor immunotherapy.

Drawings

FIG. 1 shows the results of ELISA detection of the combination of the coarse culture solution of phase and human CD 70;

FIG. 2 shows the results of FACS detection of the combination of the coarse culture solution of phase and human-derived CD 70;

FIG. 3 is a schematic diagram showing the structure of a CD70 antibody expression vector, MCS being a multiple cloning site into which a target gene is inserted;

FIG. 4 is a SDS-PAGE electrophoresis of purified antibodies;

FIG. 5 is a graph showing the performance of purified antibody binding to cell surface CD 70;

FIG. 6 is a purified antibody ADCC reporter gene activation assay;

FIG. 7 is a purified antibody ADCC killing effect assay.

Detailed Description

The present application will be described in detail with reference to examples.

(1) Phage display antibody library panning

1) Activation of host bacterium TG 1: preparation of mini agar Medium plate [1 XM 9 salt, 2% glucose, 2mM MgSO₄，0.1mM CaCl₂1mM vitamin B1]The TG1 was streaked overnight in a 37 ℃ incubator.

2) Magnetic bead washing and sealing: 50ul of magnetic beads (purchased from Invitrogen) were pipetted and placed on a magnetic rack, the liquid was pipetted after adsorption, 1ml of PBS was resuspended and washed twice, and 1ml of a blocking agent consisting of 1.5% nonfat dry milk and 1.5% BSA (the concentration of blocking agent gradually increased during the second and third rounds of panning) was used for 1 hour to remove the liquid.

3) Antigen binding: human CD70 protein (purchased from Acro biosystems, and the antigen concentration gradually decreased in each subsequent round of panning) was diluted to 1ml in PBS (pH7.2-7.4) at a concentration of 16ug/ml, and the beads were resuspended and incubated for 1 hour with rotation.

4) And (4) closing the library: taking 10 simultaneous with antigen binding to magnetic beads¹¹pfu phage virus particles (from the original antibody library or panning amplification products), with 1ml of 1.0% skim milk powder + 1.0% BSA blocking agent spin incubation for 1 hours.

5) Phage binding: and (5) placing the magnetic beads on a magnetic frame, and removing the liquid. The sealed pool was added with magnetic beads, resuspended and incubated for 1 hour with rotation, and the liquid removed.

6) Washing: washed with 1ml of PBST [0.01M PBS (pH7.4), 0.1% Tween-20 (second and third rounds of Tween-20 concentrations 0.2%, 0.3%, respectively) ], and then washed with 0.01M PBS (pH7.4).

7) And (3) elution: the liquid was aspirated, eluted with 300ul of 0.2M glycine-hydrochloric acid (pH2.2) for 10 minutes, mixed with 20ul of a neutralizing solution [1M Tris-Cl (pH9.0) ] and stored temporarily at 4 ℃.

8) And (3) measuring the titer: 2ul and 0.2ul (2 ul after the stock solution was diluted 10 times with 2 XYT medium) and 0.02ul (2 ul after the stock solution was diluted 100 times with 2 XYT medium) were mixed with 0.2ml of TG1 in the middle logarithmic phase (OD 600. about.0.5), incubated at room temperature for 30 minutes, spread on 2 XYT-GA 100 (containing 2% glucose and 100ug/ml ampicillin) plates, cultured overnight at 37 ℃ and the number of clones on about 50 plates was counted, and the titer was calculated from the dilution factor.

9) Phage amplification: while panning was performed, TG1 single clones on mini agar plates were picked and inoculated into 10ml of 2 XYT medium, and cultured at 37 ℃ with shaking at 250rpm until mid-log phase (OD 600. RTM. 0.5). 200ul of eluted product from panning was added and incubated at 37 ℃ for 30 minutes. The helper phage M13KO7 was added and incubated at 37 ℃ for an additional 30 minutes and incubated at 37 ℃ for 1 hour with shaking at 250 rpm. The supernatant was centrifuged off and resuspended in 20ml of 2 XYT containing 100ug/ml ampicillin and 50ug/ml kanamycin at 30 ℃ overnight with shaking at 220 rpm.

10) Phage precipitation: the cells were removed by centrifugation at 10000rpm for 15 minutes, and 1/5 volumes of 2.5M NaCl/20% PEG8000 were added to the supernatant, which was then cooled in ice for 2 hours. The phage pellet was centrifuged at 10000rpm for 10 minutes, the residue was removed and 0.2ml of 0.01M PBS (pH7.4) was added to resuspend the pellet, and the titer was determined as above.

11) Repeating the steps 2) -10) for two or three times to obtain the phage display antibody with strong binding force.

(2) Monoclonal ELISA

1)0.3ug/ml streptavidin was coated onto the elisa plate overnight at 4 ℃, treated with 2% BSA/PBS blocking solution for 2h, and washed 3 times with PBS.

2) Single colonies picked from 2 XYT-GA 100 plates were cultured with shaking to the middle logarithmic phase, and the helper phage M13KO7 was added and incubated at 37 ℃ for 30 minutes. The cells were cultured at 37 ℃ for 1 hour with shaking at 220rpm, and centrifuged at 4000rpm for 15 minutes. 400ul containing working concentration 100ug/ml ampicillin and working concentration 50ug/ml kanamycin 2 XYT heavy suspension, 30 degrees C, 220rpm shaking culture overnight. The cells were centrifuged at 4000rpm for 15 minutes to precipitate the cells.

3) 50ul of 4% BSA/PBS was added to the ELISA plate, and 50ul of phage supernatant was added, mixed well and incubated for 1 hour.

4) Remove liquid, wash 5 times with 0.1% PBST, wash 3 times with PBS, remove liquid.

5) An HRP-labeled anti-M13 phage antibody (purchased from Beijing Yiqiao Shenzhou) is diluted 3000 times with 2% BSA, 100ul of the diluted antibody is added into an enzyme label plate, the plate is incubated for 1 hour, the liquid is removed, the plate is washed 3 times with 0.1% PBST, and the residual liquid is drained.

6)100 ul of TMB developing solution is added, the mixture is incubated at 37 ℃ for 10min or until blue color is fully developed, 100ul of 1M sulfuric acid is added to stop the reaction, and OD450 is read on a microplate reader. As shown in FIG. 1, in the phage panning, 50ul of 2-virion medium containing phage virions was used for detection. The raw data are shown in Table 1, where the values are the absorbance at 450nm and its ratio.

Table 1: detection result of combination of phase crude culture solution and human-derived CD70 by ELISA

(3) Preparation and flow detection of humanized CD70 monoclonal antibody

1) Single colonies picked from 2 XYT-GA 100 plates were cultured overnight with shaking in liquid, and phagemids (phagemid) were extracted by the plasmid extraction method.

2) Synthesizing primer, PCR amplifying the antibody gene coding region displayed by phage.

3) The above nucleic acid fragments were inserted into the MCS region of the eukaryotic expression vector Abexp-uIgG1 in the order (FIG. 3) to encode a fusion protein having a signal peptide at the N-terminus, an antibody at the middle, and an Fc tag at the C-terminus.

4) Sterile endotoxin-free plasmids were prepared. 23ug was diluted with 0.75mL of a diluent (e.g., OPM-293CD05 medium) and 70 medium was added to the 0.75mL of the diluent (e.g., OPM-293CD05 medium) and gently mixed. The PEI diluent was added to the plasmid diluent, immediately mixed gently with a gun, and allowed to stand at room temperature for 15min to avoid disturbance.

5) Adding into 25ml 293F cell and its culture solution at 80rpm, 37 deg.C, and 5% CO₂Culturing under the conditions of 24 hr, adding 25mL of fresh growth medium (such as OPM-293CD05), 80rpm, 37 deg.C, and 5% CO₂The culture was continued for 72 hours.

6) Centrifugation was carried out at 10000rpm for 10 minutes, and the supernatant was collected. And rotationally incubating the mixture with equilibrated proteinA affinity magnetic beads for 1 hour, placing the mixture on a magnetic frame, and removing supernatant.

7) The mixture was washed 3 times with 30ml of PBS, eluted with 5ml of 0.1M glycine (pH 3.0) for 10 minutes, placed on a magnetic frame, and the supernatant was immediately aspirated to neutrality with 1M Tris-HCl buffer (pH 8.5) to obtain a purified antibody.

8) The purified antibody was examined by SDS-PAGE and the concentration was determined. By using reduction electrophoresis, disulfide bonds in antibody molecules are opened, and the molecules are electrophoretically migrated in an extended single-peptide chain state, as shown in FIG. 4.

Sequencing the purified antibodies obtained by screening to obtain antibodies with numbers 1-14 shown in the following table:

wherein, the whole-length amino acid sequence of the variable region of the antibody with the number 1 is shown as SEQ ID NO: 85, and the nucleic acid sequence is shown as SEQ ID NO: 86, respectively; the full-length amino acid sequence of the variable region of the antibody with the number 2 is shown as SEQ ID NO: 87, and the nucleic acid sequence is shown as SEQ ID NO: 88; the full-length amino acid sequence of the variable region of the numbered 3 antibody is shown as SEQ ID NO: 89, and the nucleic acid sequence is shown as SEQ ID NO: shown at 90.

SEQ ID NO：85：

QLQLVESGGGLVQPGGSLRLSCAASGSIFSIYLMAWYRQAPGKERELVAVITSDGIANYADSVKGRFTVSRDDAKRTVYLQMNSLKPEDTADYYCNAQSRATREQVGIKGWGQGTQVTVSS

SEQ ID NO：86：

CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTATCTATCTCATGGCCTGGTACCGCCAGGCTCCAGGGAAGGAGCGCGAGCTGGTCGCAGTTATTACTAGTGATGGTATTGCAAACTATGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACGACGCCAAGAGAACCGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGACTATTACTGTAATGCTCAGTCTAGAGCGACTCGCGAACAAGTCGGTATTAAAGGCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

SEQ ID NO：87：

QLQLVESGGGLVQPGGSLRLSCAASAGTFRFKTMGWHRQVQGKQRELLALITSGGSTNYADFVEGRFTISRDNAKNLVYLQMNSLKPEDTAVYYCNAYYDDIWGQGTQVTVSS

SEQ ID NO：88：

CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCCGCAGGAACCTTCAGATTCAAAACTATGGGCTGGCACCGCCAGGTTCAAGGGAAACAGCGCGAATTGCTCGCATTAATTACCAGTGGTGGTAGTACAAATTATGCAGACTTCGTCGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACCTGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTATTGTAATGCTTATTACGACGATATTTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

SEQ ID NO：89：

QLQLVESGGGLVQPGGSLRLSCAASGFTTSTYDMRWYRQAPGKERELVAIIDSVHGITNYADSVKGRFTISRDNARNTVYLQMNNLKPEDTAVYYCNTSPRVPRLRWGQGTQVTVSS

SEQ ID NO：90

CAGTTGCAGCTCGTCGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCACCAGTACCTATGACATGAGGTGGTACCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCAATTATCGATAGTGTTCATGGTATCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAGGAACACGGTGTATCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTGTATTACTGTAACACAAGTCCTAGGGTGCCCCGTCTACGCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

9) Flow cytometry for detecting cell binding capacity of purified antibody

Cell lines (such as 786-0) expressing CD70 are fully digested by 0.25% pancreatin, the digestion is stopped by serum, cells are collected by centrifugation, and a single cell suspension is prepared by gently blowing PBS.

② 10ml PBS washing cells 1 times, 1000rpm centrifugation for 5min, then 1ml PBS suspension cells, cell count.

③ take 2.5X 10⁵The cells were collected by centrifugation in 96-well cell culture plates.

And fourthly, adding 100 mu l of the phage supernatant obtained in the step (2)2) or 100ul of the purified antibody obtained in the step (3)9) into the mixture, uniformly mixing the mixture, and incubating the mixture for 30 minutes to 1 hour at room temperature.

Fifthly, centrifugally collecting the cells, and washing the cells 1 time by 300ul PBS.

Sixthly, if the detection is for the phase, 100ul of an anti-M13 phage antibody (purchased from Beijing Yiqianzhou) diluted by 1000 times by PBS is added, the incubation is carried out for 30min, the PBS is washed once, 100ul of a fluorescence labeling (such as FITC and APC) antibody diluted by 100 times by PBS is added, and the reaction is carried out for 20min at room temperature in a dark place; for antibody purification detection, 100. mu.l of fluorescence-labeled (e.g., FITC, APC) antibody diluted 200-fold with PBS was added and the reaction was carried out at room temperature for 20min in the absence of light.

Seventhly, washing the cells with 1ml of PBS for 1 time, centrifuging at 1000rpm for 8min, and removing the supernatant.

Adding 100 mul PBS to resuspend into single cell suspension, and detecting on a flow cytometer. The phase flow is shown in FIG. 2, and the left and right column graphs in the figure represent the binding signals of wild-type CHO-K1 and CD70 overexpression CHO-K1 cell lines, respectively. The raw data are shown in Table 2, where the values are the Median Fluorescence Intensity (MFI) and its ratio.

Table 2: FACS detection result combining phage rough culture solution and human-derived CD70

No.ID	Control group	Experimental group	Experiment/control	No.ID	Control group	Experimental group	Experiment/control
								1	978.80	19548.80	19.97	8	543.20	7898.20	14.54
2	522.60	5463.60	10.45	9	538.80	7974.30	14.80
								3	820.50	6673.90	8.13	10	544.50	9048.30	16.62
4	545.10	8022.20	14.72	11	572.70	3829.30	6.69
								5	551.10	7572.80	13.74	12	614.50	4377.20	7.12
6	527.80	7748.60	14.68	13	551.80	5303.90	9.61
								7	539.90	7151.70	13.25	14	547.80	7865.30	14.36

The purified antibody flow was as shown in FIG. 5, and flow cytometry was used for this assay, with ARGX-110 as a positive control and hIgG1 as a negative control. The raw data are shown in Table 3, where the values are MFI.

Table 3: detection of binding Performance of purified antibody to cell surface CD70

(4) Purified antibody ADCC reporter gene activation detection

1) Culturing target cells raji and effector cells (ADCC reporter gene cells) ADCC Fc, and detecting on a flow cytometer. The phagemids were extracted by warm-standing extraction (resuspending Cell Line (purchased from Ginkly organisms).

2) The target cells were suspended in RPMI-1640 medium at 20 ul/well and 20000/well and added to 384 well plates.

3) Effector cells were suspended in RPMI-1640 medium at 20 ul/well, and 50000 cells/well were added to 384 well plates.

3) The antibody was diluted in RPMI-1640 medium to a series of 5-fold working concentration mother liquors, taken 10 ul/well and added to the cells. Wherein the ARGX-110 working concentration starts from 0.2ug/ml, and 2 times reduces 8 gradients; the working concentration of the detection antibody starts from 0.2ug/ml, and is reduced by 8 gradients by 4 times; the working concentration of the negative control IgG1 was started at 0.2ug/ml, with a 4-fold reduction of 7 gradients.

4) Placing in 5% CO₂Incubate at 37 ℃ for 6 hours in an incubator.

5) The One-Lite Luciferase Assay System (from Novozam) was returned to room temperature in advance and added at 30 ul/well.

6) The reaction was carried out for 3 minutes and read by a microplate reader (completed in 15 minutes). The results are shown in FIG. 6, and when the antibody activates ADCC signal transduction using the luciferase reporter gene system, the reporter gene is transcriptionally activated, and luciferase is expressed, wherein ARGX-110 is a positive control and hIgG1 is a negative control. The raw data are shown in Table 4, where the values are MFI.

Table 4: purified antibody ADCC reporter gene activation detection

(5) Detection of ADCC killing effect of purified antibody

1) Culturing target cells-raji cells over expressing firefly luciferase. The suspension was suspended in RPMI-1640 medium containing 10% bovine serum, 20 ul/well and 20000/well were secured and added to 384 well plates.

2) Effector PBMC (purchased from Confucian herboria) were washed with PBS, collected by centrifugation at 1000g for 10 minutes, suspended in RPMI-1640 medium containing 10% bovine serum, and stained with Trypol blue for viability (above 90%). 20 ul/well was taken and 50000/well was ensured and added to 384 well plates.

3) The antibody was diluted with 10% bovine serum in RPMI-1640 medium to a series of 5-fold working concentration mother liquors, and 10 ul/well was added to the cells. Wherein the ARGX-110 working concentration starts from 2ug/ml, and is reduced by 10 gradients by 3 times; the working concentration of the detection antibody starts from 2ug/ml, and is reduced by 10 gradients by 4 times; the working concentration of the negative control IgG1 was reduced by 10 gradients starting at 20ug/ml, 4-fold.

4) Placing in 5% CO₂Incubate at 37 ℃ for 48 hoursThe 384 well plate was removed and allowed to cool to room temperature.

6) The reaction was carried out for 3 minutes and read by a microplate reader (completed in 15 minutes). As a result, as shown in FIG. 7, PBMC were used as effector cells, and raji cells expressing a luciferase reporter gene were used as target cells. The effector cells and target cells treated with the antibody are combined to form a detection group, and the effector cells and target cells without the antibody are combined to form a reference group. And (3) measuring the fluorescence value of the living cells, and subtracting the fluorescence value of the detection group from the fluorescence value of the reference group to obtain the decrease of the fluorescence value caused by the addition of the antibody with the corresponding concentration, so as to map the logarithm of the concentration of the antibody. The raw data are shown in Table 5, where the values are MFI.

Table 5: detection of ADCC killing effect of purified antibody

Sequence listing

<110> Nanjing Landun Biotech Co., Ltd

<120> anti-CD 70 antibody having enhanced ADCC effect and use thereof

<160> 90

<170> SIPOSequenceListing 1.0

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Gly Phe Thr Thr Ser Thr Tyr Asp

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Gly Phe Thr Phe Arg Ser Ala Val

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Gly Ser Ile Phe Ser Ile Tyr Ala

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Gly Ser Ile Phe Ser Ile Tyr Ala

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Gly Phe Thr Leu Asp Tyr Tyr Ala

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Gly Phe Thr Leu Ser Ser Tyr Ala

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Ile Asp Ser Val His Gly Ile Thr

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Ile Thr Ser Gly Gly Asp Val

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<400> 19

Ile Thr Asn Ala Gly Gly Thr

1 5

<210> 20

<211> 7

<212> PRT

<213> Homo sapiens

<400> 20

Ile Thr Asp Gly Gly Val Thr

1 5

<210> 21

<211> 7

<212> PRT

<213> Homo sapiens

<400> 21

Ile Thr Asn Ala Gly Gly Thr

1 5

<210> 22

<211> 7

<212> PRT

<213> Homo sapiens

<400> 22

Val Thr Asn Ala Gly Ser Thr

1 5

<210> 23

<211> 7

<212> PRT

<213> Homo sapiens

<400> 23

Ile Thr Asn Val Gly Ser Thr

1 5

<210> 24

<211> 7

<212> PRT

<213> Homo sapiens

<400> 24

Ile Val Tyr Gly Gly Arg Ile

1 5

<210> 25

<211> 8

<212> PRT

<213> Homo sapiens

<400> 25

Ile Ser Ser Ser Asp Gly Ser Thr

1 5

<210> 26

<211> 8

<212> PRT

<213> Homo sapiens

<400> 26

Ile Ser Ser Ser Asp Val Ser Thr

1 5

<210> 27

<211> 7

<212> PRT

<213> Homo sapiens

<400> 27

Ile Thr Asn Ser Gly Ser Thr

1 5

<210> 28

<211> 7

<212> PRT

<213> Homo sapiens

<400> 28

Val Thr Asn Val Gly Ser Thr

1 5

<210> 29

<211> 15

<212> PRT

<213> Homo sapiens

<400> 29

Asn Ala Gln Ser Arg Ala Thr Arg Glu Gln Val Gly Ile Lys Gly

1 5 10 15

<210> 30

<211> 7

<212> PRT

<213> Homo sapiens

<400> 30

Asn Ala Tyr Tyr Asp Asp Ile

1 5

<210> 31

<211> 10

<212> PRT

<213> Homo sapiens

<400> 31

Asn Thr Ser Pro Arg Val Pro Arg Leu Arg

1 5 10

<210> 32

<211> 7

<212> PRT

<213> Homo sapiens

<400> 32

Asn Ala Tyr Tyr Leu Asp Tyr

1 5

<210> 33

<211> 19

<212> PRT

<213> Homo sapiens

<400> 33

Asn Ala Ile Glu Glu Glu Val Glu Pro Gly Thr Asp Arg Tyr Arg Tyr

1 5 10 15

Leu Glu Val

<210> 34

<211> 7

<212> PRT

<213> Homo sapiens

<400> 34

Asn Ala Tyr Tyr Leu Asp Tyr

1 5

<210> 35

<211> 23

<212> PRT

<213> Homo sapiens

<400> 35

Asn Val Leu His Glu Ser Thr Val Val Ala Gly Pro Val Gly Glu Asn

1 5 10 15

Arg Tyr Glu Tyr Gln Glu Ile

20

<210> 36

<211> 23

<212> PRT

<213> Homo sapiens

<400> 36

Asn Val Ile Glu Glu Arg Thr Val Asp Ser Gly Asp Thr Gln Arg Thr

1 5 10 15

Arg Tyr Lys Tyr Phe Glu Val

20

<210> 37

<211> 23

<212> PRT

<213> Homo sapiens

<400> 37

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

1 5 10 15

Arg Tyr Asn Tyr Glu Glu Ile

20

<210> 38

<211> 18

<212> PRT

<213> Homo sapiens

<400> 38

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

1 5 10 15

Glu Val

<210> 39

<211> 18

<212> PRT

<213> Homo sapiens

<400> 39

Ala Ala Arg Phe Arg Ser Asp Tyr Val Cys Tyr Pro Glu Gly Ser Tyr

1 5 10 15

Asp Tyr

<210> 40

<211> 18

<212> PRT

<213> Homo sapiens

<400> 40

Ala Ala Arg Tyr Ile Thr Asp Tyr Met Cys Tyr Pro Glu Gly Gly Tyr

1 5 10 15

Asp Tyr

<210> 41

<211> 23

<212> PRT

<213> Homo sapiens

<400> 41

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

1 5 10 15

Arg Tyr Arg Tyr Asp Glu Val

20

<210> 42

<211> 23

<212> PRT

<213> Homo sapiens

<400> 42

Tyr Val Leu His Glu Ser Thr Val Val Asp Asn Pro Val Gly Glu Lys

1 5 10 15

Arg Tyr Lys Tyr Glu Glu Ile

20

<210> 43

<211> 24

<212> DNA

<213> Homo sapiens

<400> 43

ggaagcatct tcagtatcta tctc 24

<210> 44

<211> 24

<212> DNA

<213> Homo sapiens

<400> 44

gcaggaacct tcagattcaa aact 24

<210> 45

<211> 24

<212> DNA

<213> Homo sapiens

<400> 45

ggattcacca ccagtaccta tgac 24

<210> 46

<211> 24

<212> DNA

<213> Homo sapiens

<400> 46

ggattcacct tcagaagcgc ggtc 24

<210> 47

<211> 24

<212> DNA

<213> Homo sapiens

<400> 47

ggaagcatct tcagtatcta tgcc 24

<210> 48

<211> 24

<212> DNA

<213> Homo sapiens

<400> 48

ggattcacct tcagtagcgt agtc 24

<210> 49

<211> 24

<212> DNA

<213> Homo sapiens

<400> 49

ggaagcatct tcagtatcta tgcc 24

<210> 50

<211> 30

<212> DNA

<213> Homo sapiens

<400> 50

gcctctggaa gcacgagcag tatctatgcc 30

<210> 51

<211> 24

<212> DNA

<213> Homo sapiens

<400> 51

ggaagcagct tcagttccta tgcc 24

<210> 52

<211> 24

<212> DNA

<213> Homo sapiens

<400> 52

ggcagcatct tcaatatcta tgcc 24

<210> 53

<211> 24

<212> DNA

<213> Homo sapiens

<400> 53

ggattcagtt tggattatta tttc 24

<210> 54

<211> 24

<212> DNA

<213> Homo sapiens

<400> 54

ggattcactt tggattatta tgcc 24

<210> 55

<211> 24

<212> DNA

<213> Homo sapiens

<400> 55

ggaagcatct ccagtatcta tgcc 24

<210> 56

<211> 24

<212> DNA

<213> Homo sapiens

<400> 56

ggattcacct tgagtagcta tgcc 24

<210> 57

<211> 21

<212> DNA

<213> Homo sapiens

<400> 57

attactagtg atggtattgc a 21

<210> 58

<211> 21

<212> DNA

<213> Homo sapiens

<400> 58

attaccagtg gtggtagtac a 21

<210> 59

<211> 24

<212> DNA

<213> Homo sapiens

<400> 59

atcgatagtg ttcatggtat caca 24

<210> 60

<211> 21

<212> DNA

<213> Homo sapiens

<400> 60

attactagtg gtggcgacgt a 21

<210> 61

<211> 21

<212> DNA

<213> Homo sapiens

<400> 61

attactaatg ctggtggcac a 21

<210> 62

<211> 21

<212> DNA

<213> Homo sapiens

<400> 62

attactgatg gtggcgtcac a 21

<210> 63

<211> 21

<212> DNA

<213> Homo sapiens

<400> 63

attactaacg ctggtggcac a 21

<210> 64

<211> 21

<212> DNA

<213> Homo sapiens

<400> 64

gttactaatg ctggtagtac a 21

<210> 65

<211> 21

<212> DNA

<213> Homo sapiens

<400> 65

ataactaatg ttggtagtac a 21

<210> 66

<211> 21

<212> DNA

<213> Homo sapiens

<400> 66

atagtatatg gtggtaggat a 21

<210> 67

<211> 24

<212> DNA

<213> Homo sapiens

<400> 67

attagtagta gtgatggtag caca 24

<210> 68

<211> 24

<212> DNA

<213> Homo sapiens

<400> 68

attagtagta gtgatgtgag caca 24

<210> 69

<211> 21

<212> DNA

<213> Homo sapiens

<400> 69

attactaata gtggtagcac t 21

<210> 70

<211> 21

<212> DNA

<213> Homo sapiens

<400> 70

gttactaatg ttggtagcac a 21

<210> 71

<211> 45

<212> DNA

<213> Homo sapiens

<400> 71

aatgctcagt ctagagcgac tcgcgaacaa gtcggtatta aaggc 45

<210> 72

<211> 21

<212> DNA

<213> Homo sapiens

<400> 72

aatgcttatt acgacgatat t 21

<210> 73

<211> 30

<212> DNA

<213> Homo sapiens

<400> 73

aacacaagtc ctagggtgcc ccgtctacgc 30

<210> 74

<211> 21

<212> DNA

<213> Homo sapiens

<400> 74

aatgcttact atttggacta c 21

<210> 75

<211> 57

<212> DNA

<213> Homo sapiens

<400> 75

aatgcaattg aggaggaggt agaacctggt actgaccgct acaggtatct cgaagtt 57

<210> 76

<211> 21

<212> DNA

<213> Homo sapiens

<400> 76

aacgcatact atttggatta c 21

<210> 77

<211> 69

<212> DNA

<213> Homo sapiens

<400> 77

aatgtcctcc atgagagtac ggtagtagct ggtcccgtgg gcgagaatcg ctacgagtat 60

caagaaatt 69

<210> 78

<211> 69

<212> DNA

<213> Homo sapiens

<400> 78

aatgtcatag aggaacgtac ggtagatagt ggtgataccc aaagaactcg ctacaagtat 60

ttcgaagtt 69

<210> 79

<211> 69

<212> DNA

<213> Homo sapiens

<400> 79

aatgtcctcc atgagagtac ggtagctgct ggtcccgtgg gcgagaagcg ctacaattac 60

gaagaaatt 69

<210> 80

<211> 54

<212> DNA

<213> Homo sapiens

<400> 80

aatgcagttg acgaaatccc gtcggagggt gagcgctaca ggtatctcga agtt 54

<210> 81

<211> 54

<212> DNA

<213> Homo sapiens

<400> 81

gcagctcgat ttcgttcaga ctatgtgtgt tatcccgaag gatcgtatga ctac 54

<210> 82

<211> 54

<212> DNA

<213> Homo sapiens

<400> 82

gcagcacgct acataacaga ctatatgtgt tacccagagg gtgggtatga ctac 54

<210> 83

<211> 69

<212> DNA

<213> Homo sapiens

<400> 83

tatgtcctcc atgagagtac ggtaacagct ggtcccgtgg gcgagaagcg ctacaggtat 60

gatgaagtt 69

<210> 84

<211> 69

<212> DNA

<213> Homo sapiens

<400> 84

tatgtcctcc atgagagtac ggtagtagat aatcccgtgg gcgagaagcg ctacaagtat 60

gaagaaatt 69

<210> 85

<211> 121

<212> PRT

<213> Homo sapiens

<400> 85

Gln Leu Gln Leu Val 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 Ser Ile Phe Ser Ile Tyr

20 25 30

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

35 40 45

Ala Val Ile Thr Ser Asp Gly Ile Ala Asn Tyr Ala Asp Ser Val Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr Cys Asn

85 90 95

Ala Gln Ser Arg Ala Thr Arg Glu Gln Val Gly Ile Lys Gly Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 86

<211> 363

<212> DNA

<213> Homo sapiens

<400> 86

cagttgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catcttcagt atctatctca tggcctggta ccgccaggct 120

ccagggaagg agcgcgagct ggtcgcagtt attactagtg atggtattgc aaactatgca 180

gactccgtga agggccgatt caccgtctcc agagacgacg ccaagagaac cgtgtatctg 240

caaatgaaca gcctgaaacc tgaggacacg gccgactatt actgtaatgc tcagtctaga 300

gcgactcgcg aacaagtcgg tattaaaggc tggggccagg ggacccaggt caccgtctcc 360

tca 363

<210> 87

<211> 113

<212> PRT

<213> Homo sapiens

<400> 87

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Gly Thr Phe Arg Phe Lys

20 25 30

Thr Met Gly Trp His Arg Gln Val Gln Gly Lys Gln Arg Glu Leu Leu

35 40 45

Ala Leu Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Phe Val Glu

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Tyr Tyr Asp Asp Ile Trp Gly Gln Gly Thr Gln Val Thr Val Ser

100 105 110

Ser

<210> 88

<211> 339

<212> DNA

<213> Homo sapiens

<400> 88

cagttgcagc tcgtggagtc tgggggaggc ttggtgcagc cgggggggtc tctgagactc 60

tcctgtgcag cctccgcagg aaccttcaga ttcaaaacta tgggctggca ccgccaggtt 120

caagggaaac agcgcgaatt gctcgcatta attaccagtg gtggtagtac aaattatgca 180

gacttcgtcg agggccgatt caccatctcc agagacaacg ccaagaacct ggtgtatttg 240

caaatgaaca gcctgaaacc tgaggacacg gccgtctatt attgtaatgc ttattacgac 300

gatatttggg gccaggggac ccaggtcacc gtctcctca 339

<210> 89

<211> 117

<212> PRT

<213> Homo sapiens

<400> 89

Gln Leu Gln Leu Val 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 Thr Ser Thr Tyr

20 25 30

Asp Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Ile Ile Asp Ser Val His Gly Ile Thr Asn Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Thr Ser Pro Arg Val Pro Arg Leu Arg Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 90

<211> 351

<212> DNA

<213> Homo sapiens

<400> 90

cagttgcagc tcgtcgagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt caccaccagt acctatgaca tgaggtggta ccgccaggct 120

ccagggaagg agcgcgagtt ggtcgcaatt atcgatagtg ttcatggtat cacaaactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca atgccaggaa cacggtgtat 240

ctacaaatga acaacctgaa acctgaggac acggccgtgt attactgtaa cacaagtcct 300

agggtgcccc gtctacgctg gggccagggg acccaggtca ccgtctcctc a 351

Claims

1. A group of anti-CD 70 antibodies with enhanced ADCC effect, characterized in that the amino acid sequence of CDR1 in the heavy chain variable region is selected from the group consisting of SEQ ID NO: 1-14, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 15-28, the amino acid sequence of CDR3 is selected from SEQ ID NOs: 29-42.

2. The anti-CD 70 antibody having an enhanced ADCC effect according to claim 1, comprising a heavy chain variable region selected from any one of the following:

3. a DNA molecule encoding the anti-CD 70 antibody having enhanced ADCC effect of claim 1, wherein the heavy chain variable region thereof is SEQ ID NO: 1-42 are shown as SEQ ID NO: 43-84.

4. Use of the anti-CD 70 antibody with enhanced ADCC effect according to claim 1 or 2 for the detection of a CD70 molecule.

5. Use of an anti-CD 70 antibody having an enhanced ADCC effect according to claim 1 or 2 for the preparation of a medicament for the treatment of a tumor.