CN116375865A

CN116375865A - anti-CD 70 antibodies with enhanced ADCP effect and uses thereof

Info

Publication number: CN116375865A
Application number: CN202210929990.1A
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: 2023-07-04
Also published as: CN113292653A; CN113292653B; WO2022262100A1

Abstract

The invention discloses an anti-CD 70 antibody with enhanced ADCP effect and application thereof. The application successfully prepares the antibody (fusion protein) capable of combining the human CD70 molecules, and the antibody has good specificity and higher affinity and combines the human CD70 expressed on the cell surface. Successful activation of signal transduction required for ADCP effects in reporter cells following CD70 binding. The antibodies may also bind efficiently to cynomolgus monkey CD70. The anti-human CD70 monoclonal antibody is a potential drug for tumor immunotherapy.

Description

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

Technical Field

The application belongs to the biomedical technology, and particularly relates to an anti-CD 70 antibody with enhanced ADCP effect and application thereof. The present invention is application number 202110673784.4, a divisional application entitled anti-CD 70 antibodies with enhanced ADCP effect and uses thereof.

Background

Antibody-dependent cell-mediated phagocytosis (ADCP) is an important property of antibodies, and macrophages (macrobay) and neutrophils (neutrophil) have this function, wherein macrophages have strong phagocytic activity and are the main effector cells of ADCP. Meanwhile, the macrophage has natural immune activity, and can play a role in regulating immunity after being activated, thereby playing a role in enhancing the whole immune function. In particular, the solid tumor has the characteristics of cell and tissue differentiation and compact structure, so that CART cells are difficult to infiltrate, and the phagocytic function of macrophages can play a role in counteracting the structure, so that the ADCP has more special practical significance for treating the solid tumor. Indeed, targeted therapies utilizing this feature of macrophages have attracted attention by researchers due to the bottleneck encountered in the application of CART therapy in solid tumors.

The antigen binds to the antibody, and the crystallizable fragment (fragment crystallizable, fc) of the antibody binds mainly to fcγiia (Fc gamma receptor iia) on the surface of the macrophage, and the antigen (particle) binds to the surface of the macrophage, causing actin at the uptake site to polymerize and pseudopodium to coat the particle, and actin depolymerizes after phagocytosis is completed. During this process, NFAT signaling pathways in macrophages are activated. With this early occurrence of signal transduction events, ADCP effects can be detected.

While ADCP has attracted great interest in tumor therapy, the ADCP effect is generally weaker in existing antibody drugs. In view of the important immunoregulatory function of CD70/CD27 molecules, the regulation and control of CD70 inevitably causes a series of T cells, even B cells, NK cells and other lymphocytes to signal transmission and physiological responses, is an ideal immunotherapeutic target, and can possibly mobilize various cellular immunity in reasonable application. The application uses human CD70 as a target point to develop an antibody with strong ADCP effect.

Current therapies targeting CD70 have CART and total antibodies with limited clinical efficacy, especially for solid tumors, both of which are almost ineffective. Starting from the ADCP effect, increasing the type of antibody mechanism of action is one way to increase the therapeutic effect of antibodies. Currently, antibodies in clinical trials still have weak ADCP effects and are still lacking in enhancing therapeutic efficacy.

Disclosure of Invention

The invention aims to: in view of the above-mentioned technical problems in the prior art, the present application provides a set of anti-CD 70 nanobodies with enhanced ADCP effect and uses thereof.

The technical scheme is as follows: a set of anti-CD 70 nanobodies with enhanced ADCP effect described herein, the amino acid sequence of the heavy chain variable region CDR1 is selected from the group consisting of SEQ ID NO:1-24, the amino acid sequence of cdr2 is selected from SEQ ID NO:25-48, the amino acid sequence of cdrd 3 is selected from SEQ ID NO:49-72.

Preferably, the anti-CD 70 nanobody with enhanced ADCP effect comprises a heavy chain variable region as shown in any one of the following:

the amino acid sequence of CDR1 is selected from SEQ ID NO:1, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 25, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 49;

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:26, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 50;

the amino acid sequence of CDR1 is selected from SEQ ID NO:3, cdr2 is selected from the amino acid sequence of SEQ ID NO:27, the amino acid sequence of cdr3 is selected from SEQ ID NO:51;

the amino acid sequence of CDR1 is selected from SEQ ID NO:4, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 28, the amino acid sequence of cdr3 is selected from SEQ ID NO:52;

the amino acid sequence of CDR1 is selected from SEQ ID NO:5, cdr2 is selected from the amino acid sequence of SEQ ID NO:29, the amino acid sequence of cdr3 is selected from SEQ ID NO:53;

the amino acid sequence of CDR1 is selected from SEQ ID NO:6, the amino acid sequence of cdr2 is selected from SEQ ID NO:30, the amino acid sequence of cdr3 is selected from SEQ ID NO:54;

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:31, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 55;

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:32, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 56;

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:33, cdr3 is selected from the amino acid sequence of SEQ ID NO:57;

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:34, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 58;

the amino acid sequence of CDR1 is selected from SEQ ID NO:11, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 35, the amino acid sequence of cdr3 is selected from SEQ ID NO:59;

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:36, the amino acid sequence of cdr3 is selected from SEQ ID NO:60;

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:37, cdr3 is selected from the amino acid sequence of SEQ ID NO:61;

the amino acid sequence of CDR1 is selected from SEQ ID NO:14, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 38, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 62;

the amino acid sequence of CDR1 is selected from SEQ ID NO:15, the amino acid sequence of cdr2 is selected from SEQ ID NO:39, cdr3 is selected from the amino acid sequence of SEQ ID NO:63;

the amino acid sequence of CDR1 is selected from SEQ ID NO:16, the amino acid sequence of cdr2 is selected from SEQ ID NO:40, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 64;

the amino acid sequence of CDR1 is selected from SEQ ID NO:17, the amino acid sequence of cdr2 is selected from SEQ ID NO:41, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 65.

The amino acid sequence of CDR1 is selected from SEQ ID NO:18, the amino acid sequence of cdr2 is selected from SEQ ID NO:42, cdr3 is selected from the amino acid sequence of SEQ ID NO:66;

the amino acid sequence of CDR1 is selected from SEQ ID NO:19, the amino acid sequence of cdr2 is selected from SEQ ID NO:43, the amino acid sequence of cdrd 3 is selected from SEQ ID NO:67;

the amino acid sequence of CDR1 is selected from SEQ ID NO:20, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 44, the amino acid sequence of cdr3 is selected from SEQ ID NO:68;

the amino acid sequence of CDR1 is selected from SEQ ID NO:21, the amino acid sequence of cdr2 is selected from SEQ ID NO:45, the amino acid sequence of cdr3 is selected from SEQ ID NO:69;

the amino acid sequence of CDR1 is selected from SEQ ID NO:22, the amino acid sequence of cdr2 is selected from SEQ ID NO:46, the amino acid sequence of cdrd 3 is selected from SEQ ID NO:70;

the amino acid sequence of CDR1 is selected from SEQ ID NO:23, the amino acid sequence of cdr2 is selected from SEQ ID NO:47, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 71;

the amino acid sequence of CDR1 is selected from SEQ ID NO:24, the amino acid sequence of cdr2 is selected from SEQ ID NO:48, the amino acid sequence of cdrd 3 is selected from SEQ ID NO:72.

SEQ ID NO:1-72 are shown in the following table:

the present application also discloses DNA molecules encoding the above anti-CD 70 nanobodies with enhanced ADCP effect, the heavy chain variable region SEQ ID NO:1-72, and the coding nucleotide sequences corresponding to the amino acids are shown in SEQ ID NO: 73-144.

SEQ ID NO:73-144 are shown in the following table:

the antibodies described herein target human or cynomolgus monkey CD70 molecules.

The application also discloses application of the anti-CD 70 nano-antibody with the enhanced ADCP effect to detection of CD70 molecules.

Further, the application of the anti-CD 70 nanobody with enhanced ADCP effect in preparing the medicine for treating tumor is also within the protection scope of the application. The present application uses commercial recombinant human CD70 biotin tag protein and phage display antibody library as starting materials. The CD70 biotin tag protein is incubated with streptavidin-coated magnetic beads, CD70 is fixed on the magnetic beads through the interaction between the streptavidin and the biotin, an antibody library is used for incubation, unbound/weakly bound phage are washed away, and phage bound with the CD70 protein are eluted. This was repeated 3 times, each time changing the amount of CD70 biotin tag protein used and the washing conditions, eliminating the weakly binding phage and retaining as much more strongly binding phage as possible. The host bacteria are infected with strongly bound phage, plated and cultured overnight to obtain monoclonal colonies, i.e., monoclonal antibodies to phage that bind CD70. ELISA screening was performed using the monoclonal culture supernatant, 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 were linked and read in frame to construct a mammalian expression vector. 293F cells are transfected, the supernatant is harvested after shaking culture for 5 days, and the fusion protein is purified from the supernatant by using protein A magnetic bead affinity chromatography.

Different concentrations of purified antibodies were incubated with cancer cell lines expressing human CD70 or cynomolgus monkey CD70 overexpressing cell lines, and antibodies bound to the cell surface were detected using fluorescently labeled secondary antibodies, and their binding ability to human or cynomolgus monkey CD70 on the cell surface was determined.

Target cells are cultured, cells displaying the luciferase reporter gene of ADCP signal transduction are added according to a certain proportion, the cells are incubated with a series of antibodies with gradient concentration, and the reporter gene is activated to different degrees. The final ADCP intensity is shown by the reporter activity.

The beneficial effects are that: the application successfully prepares the antibody (fusion protein) capable of combining the human CD70 molecules, and the antibody has good specificity and higher affinity and combines the human CD70 expressed on the cell surface. Successful activation of signal transduction required for ADCP effects in reporter cells following CD70 binding. The antibodies may also bind efficiently to cynomolgus monkey CD70. 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 phage crude culture with human CD 70;

FIG. 2 shows the results of FACS detection of the binding of the phage crude culture with human CD 70;

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

FIG. 4 is SDS-PAGE of purified antibodies;

FIG. 5 is a graph showing the detection of the binding properties of purified antibodies to cell surface CD 70;

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

FIG. 7 is a purified antibody binding assay to cynomolgus monkey CD70.

Detailed Description

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

(1) Phage display antibody library panning

1) Activation of host strain TG 1: preparation of mini agar Medium plates [1 XM 9 salts, 2% glucose, 2mM MgSO ] ₄ ，0.1mM CaCl ₂ 1mM vitamin B1]The streaking method was used to culture TG1 overnight in an incubator at 37 ℃.

2) Washing and sealing magnetic beads: 50ul of magnetic beads (available from Invitrogen) were pipetted on a magnetic rack, the liquid was aspirated after adsorption, resuspended in 1ml PBS, washed twice, blocked with 1ml 1.5% nonfat milk powder+1.5% BSA blocking agent (increasing concentration of blocking agent during the second and third rounds of panning) for 1 hour, and the liquid was removed.

3) Antigen binding: human CD70 protein (purchased from Acro Biosystem, gradually decreasing antigen concentration at each subsequent round of panning) was diluted to 1ml in PBS (pH 7.2-7.4) at a concentration of 16ug/ml, and the beads were resuspended and incubated for 1 hour with spin.

4) Library sealing: with antigensBinding to magnetic bead synchronization, take 10 ¹¹ pfu phage virus particles (from original antibody pool or panning amplification products) were spin incubated with 1ml of 1.0% nonfat milk powder+1.0% BSA blocking agent for 1 hour.

5) Phage binding: and placing the magnetic beads on a magnetic frame to remove the liquid. The blocked pool was added to the beads, resuspended and incubated for 1 hour with spin to remove the liquid.

6) Washing: washed with 1ml PBST[0.01M PBS (pH 7.4), 0.1% Tween-20 (second, third, tween-20 concentrations 0.2%, 0.3%, respectively) and then 0.01M PBS (pH 7.4).

7) Eluting: the liquid was sucked off, eluted with 300ul of 0.2M glycine-HCl (pH 2.2) for 10 minutes, and 20ul of neutralization solution [1M Tris-Cl (pH 9.0) ] was added and mixed well, and temporarily stored at 4 ℃.

8) Titer was measured: 2ul, 0.2ul (stock solution was diluted 10 times with 2 XYT medium and 2ul was obtained), 0.02ul (stock solution was diluted 100 times with 2 XYT medium and 2ul was obtained) of the eluate was mixed with 0.2ml of TG1 in mid-log phase (OD 600 = 0.5), incubated at room temperature for 30 minutes, spread uniformly on 2 XYT-GA 100[ 2% glucose, 100ug/ml ampicillin ] plates, cultured overnight at 37℃and the number of clones on plates of about 50 clones was counted, and titer was calculated from the dilution.

9) Phage amplification: while panning was in progress, TG1 monoclonal on mini agar plates was picked and inoculated into 10ml of 2 xyt medium and shake-cultured at 37 ℃ at 250rpm to mid-log phase (od600=0.5). 200ul of the eluates obtained by panning were added and incubated at 37℃for 30 minutes. Helper phage M13KO7 was added and incubated at 37℃for an additional 30 minutes, and shaking culture was performed at 250rpm at 37℃for 1 hour. The supernatant was removed by centrifugation, resuspended in 20ml of 2 XYT containing ampicillin at a working concentration of 100ug/ml and kanamycin at a working concentration of 50ug/ml, and incubated overnight at 30℃with shaking at 220 rpm.

10 Phage precipitation: the cells were removed by centrifugation at 10000rpm for 15 minutes, and 1/5 volume of 2.5M NaCl/20% PEG8000 was added to the supernatant, followed by ice-bath for 2 hours. Centrifugation at 10000rpm for 10min gave phage pellet, the residue was removed cleanly and the pellet was resuspended in 0.2ml of 0.01M PBS (pH 7.4) as measured for titer above.

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

(2) Monoclonal ELISA

1) 0.3ug/ml streptavidin 4 ℃ overnight coated ELISA plate, 2%BSA/PBS blocking solution treatment 2 hours, and PBS washing 3 times.

2) The monoclonal colonies picked from the 2 XYT-GA 100 plates were shake-cultured to mid-log phase, added with helper phage M13KO7 and incubated at 37℃for 30 minutes. The culture was incubated at 37℃with 220rpm for 1 hour and centrifuged at 4000rpm for 15 minutes. The culture was resuspended in 400ul of 2 XYT containing ampicillin at a working concentration of 100ug/ml and kanamycin at a working concentration of 50ug/ml, and incubated overnight at 30℃with shaking at 220 rpm. Centrifugation at 4000rpm for 15 minutes precipitated the cells.

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

4) The liquid was removed, washed 5 times with 0.1% pbst, 3 times with PBS, and the liquid was removed.

5) HRP-labeled anti-M13 phage antibody (purchased from beijing, san) was diluted 3000-fold with 2% bsa, 100ul was added to the elisa plate, incubated for 1 hour, liquid removed, 0.1% pbst washed 3 times, and the residue was patted dry.

6) 100ul of TMB chromogenic solution was added and incubated at 37℃for 10min or until the blue color developed sufficiently, the reaction was stopped by adding 100ul of 1M sulfuric acid and the OD450 was read on a microplate reader. The results are shown in FIG. 1, and 50ul of 2 XYT medium containing phage virus particles was used for detection during phage panning. The raw data are shown in Table 1, wherein the values are the absorbance at 450nm and the ratio thereof.

Table 1: ELISA detection result of phage crude culture solution combined with human CD70

No.ID	Blank group	Experimental group	Experiment/blank	No.ID	Blank group	Experimental group	Experiment/blank
								1	0.3393	1.5412	4.5423	13	0.7909	2.7965	3.5358
2	0.3801	0.6504	1.7111	14	0.5959	2.3806	3.9949
								3	0.9456	1.8750	1.9829	15	0.5943	2.4666	4.1504
4	1.2482	2.9280	2.3458	16	0.8776	2.5020	2.8510
								5	1.1651	2.1283	1.8267	17	0.8653	2.2645	2.6170
6	1.1849	2.2273	1.8797	18	0.6055	1.5305	2.5277
								7	0.7467	1.5409	2.0636	19	0.9782	2.6730	2.7326
8	1.1179	2.0062	1.7946	20	1.0186	2.0023	1.9657
								9	0.9789	1.9353	1.9770	21	0.5214	2.2278	4.2727
10	1.1326	2.8815	2.5441	22	0.5376	2.1932	4.0796
								11	0.8119	1.6906	2.0823	23	1.0740	2.2402	2.0858
12	0.5712	2.3354	4.0886	24	0.5522	1.7882	3.2383

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

1) Monoclonal colonies picked from 2 XYT-GA 100 plates were liquid shake cultured overnight and phagemid (phagemid) was extracted by plasmid extraction.

2) Primers were synthesized and the coding region of the antibody gene displayed by phage was PCR amplified.

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

4) Sterile endotoxin-free plasmids were prepared. 23ug was diluted with 0.75mL of diluent (e.g., OPM-293CD05 medium) and 70 ul of transfection reagent (e.g., PEI solution) was added to 0.75mL of diluent (e.g., OPM-293CD05 medium) and gently mixed. Adding PEI diluent into plasmid diluent, immediately and gently mixing with a gun, standing at room temperature for 15min, and avoiding disturbance.

5) Adding into 25ml of 293F cells and culture solution thereof, 80rpm,37 ℃ and 5% CO ₂ Culturing under the condition for 24 hours, adding 25mL fresh growth medium (such as OPM-293CD 05), 80rpm,37 ℃ and 5% CO ₂ Culturing was continued for 72 hours.

6) Centrifugation was carried out at 10000rpm for 10 minutes, and the supernatant was taken. And (3) incubating the mixture with the balanced proteonA affinity magnetic beads for 1 hour in a rotating way, placing the mixture on a magnetic rack, and removing the supernatant.

7) Washing the mixture with 30ml PBS 3 times, adding 5ml0.1M glycine (pH 3.0) for eluting for 10 minutes, placing the mixture on a magnetic rack, and immediately absorbing the supernatant with 1M Tris-HCl buffer solution (pH 8.5) to be neutral to obtain the purified antibody.

8) SDS-PAGE detects purified antibodies, while concentration measurements are performed. By reducing electrophoresis, disulfide bonds in the antibody molecules are opened, and the molecules undergo electrophoretic migration in the state of extended single peptide chains, and the results are shown in FIG. 4.

The purified antibodies obtained from the screening were sequenced to give antibodies numbered 1-24 as shown in the following table:

wherein the full-length amino acid sequence of the variable region of the antibody with the number 1 is shown in SEQ ID NO:145, the nucleic acid sequence is shown in SEQ ID NO: shown at 146; the full-length amino acid sequence of the variable region of the antibody with the number 2 is shown in SEQ ID NO:147, the nucleic acid sequence is shown in SEQ ID NO: indicated at 148; the full-length amino acid sequence of the variable region of the antibody with the number 3 is shown in SEQ ID NO:149, the nucleic acid sequence is shown in SEQ ID NO: 150.

SEQ ID NO：145

QLQLVESGGGLVQPGGSLRLSCAASVSIFTFYFIGWYRQVPGKQRELVALITSNGRTNYADSVEGRFTISTDTARNTVFLEMKSLKPEDTAVYYCNADPSTDWGQGAQVTVSS

SEQ ID NO：146

CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGTTAGCATCTTCACTTTCTATTTCATTGGCTGGTACCGCCAGGTTCCAGGGAAGCAGCGCGAGTTGGTCGCGCTTATTACTAGTAATGGTCGCACAAACTATGCAGATTCTGTGGAGGGCCGATTCACCATCTCCACAGACACAGCCAGGAACACGGTGTTTCTGGAAATGAAGAGCCTGAAACCGGAGGACACGGCCGTCTATTACTGTAATGCAGACCCTTCGACGGACTGGGGCCAGGGGGCCCAGGTCACCGTCTCCTCA

SEQ ID NO：147

QLQLVESGGGLVQPGGSLRLSCAASENFSIYAMGWYRQAPGKQRELVAAVTSGGMTNYADSVEGRFTISRDNTKNTVYLQMNSLKPEDTAVYYCNVVPSLNYGLGYEYTTYWGPGTQVTVSS

SEQ ID NO：148

CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGAAAACTTCAGTATTTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAGCCGTTACTAGTGGTGGTATGACAAACTATGCAGACTCCGTGGAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACACGGTGTATCTCCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGTAGTCCCTAGTTTAAACTACGGATTGGGGTACGAGTACACCACCTACTGGGGCCCGGGGACCCAGGTCACCGTCTCCTCC

SEQ ID NO：149

QLQLVESGGGLVQPGGSLRLSCAASGNIFSIYAMAWYQQAPGKQRELVAQITNAGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVLHESTEVAGPVGEKRYTYQEIWGQGTLVTVSS

SEQ ID NO：150

CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAACATCTTCAGTATCTATGCCATGGCCTGGTACCAGCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCACAAATTACTAACGCTGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGTCCTCCATGAGAGTACGGAGGTAGCTGGTCCCGTGGGCGAGAAGCGCTACACGTATCAAGAAATTTGGGGCCAGGGCACCCTGGTCACTGTCTCCTCA

9) Flow cytometry to detect the cell binding capacity of purified antibodies

(1) Cell lines expressing CD70 (e.g., 786-0) were thoroughly digested with 0.25% pancreatin, the digestion was terminated with serum, the cells were collected by centrifugation, and PBS was gently blown to prepare a single cell suspension.

(2) Cells were washed 1 time with 10ml PBS, centrifuged at 1000rpm for 5min, and then suspended with 1ml PBS and counted.

(3) Taking 2.5X10 ⁵ Cells were collected by centrifugation in 96-well cell culture plates.

(4) Add 100. Mu.l of the phase supernatant from step (2) 2) or the purified antibody from step (3) 8) to a concentration gradient of 100ul (see FIG. 5 or Table 3), mix well and incubate at room temperature for 30 minutes to 1 hour.

(5) Cells were collected by centrifugation and washed 1 time with 300ul of PBS.

(6) If the detection is phage detection, 100ul of anti-M13 phage antibody (purchased from Beijing Yiqiao Shenzhou) diluted by 1000 times with PBS is added, incubation is carried out for 30min, PBS is washed once, 100ul of fluorescent marker (such as FITC and APC) antibody diluted by 100 times with PBS is added, and the reaction is carried out for 20min at room temperature in a dark place; for detection of purified antibodies, 100. Mu.l of fluorescence-labeled (e.g.FITC, APC) antibodies diluted 200-fold with PBS were added and reacted at room temperature in the absence of light for 20min.

(7) The cells were washed 1 time with 1ml PBS and centrifuged at 1000rpm for 8min, and the supernatant was removed.

(8) 100 μl PBS was added to resuspend the suspension as a single cell suspension, which was detected on-machine by flow cytometry. The phasage flow is shown in FIG. 2, wherein the left and right bar graphs represent binding signals to wild CHO-K1 and CD70 overexpressing CHO-K1 cell lines, respectively. The raw data are shown in Table 2, wherein the values are the Median Fluorescence Intensity (MFI) and the ratio thereof.

Table 2: detection result of binding of phage crude culture solution and human CD70 by FACS

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

Table 3: purified antibodies and cell surface CD70 binding assay

(4) Purified antibody ADCP reporter activation assay

1) Target cells raji and effector cells (ADCP reporter cells) ADCP fcγ RIIa Jurkat Effector Cell Line (purchased from gemini organisms) were cultured.

2) Target cells were suspended in 1% FBS+RPMI-1640 medium, 20 ul/well was taken and 20000 were ensured to be taken and added to 384 well plates.

3) Effector cells were suspended in 1% FBS+RPMI-1640 medium, 20 ul/well was taken, and 50000 were ensured to be taken and added to 384 well plates.

3) The antibodies were diluted to a series of stock solutions at 5-fold working concentration in 1% FBS+RPMI-1640 medium, and 10 ul/well was added to the cells. ARGX-110, negative control IgG1 and detection antibody working concentrations were reduced by 8 gradients 4-fold starting at 10 ug/ml.

4) Placed in 5% CO ₂ Incubate in incubator at 37℃for 6 hours.

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

6) The reaction was carried out for 3 minutes and read with a microplate reader (completed within 15 minutes). As a result, as shown in FIG. 6, when ADCP signal transduction was activated by the antibody using the luciferase reporter gene system, the reporter gene was transcriptionally activated to express luciferase, wherein ARGX-110 was used as a positive control and hIgG1 was used as a negative control. The raw data are shown in Table 4, where the values are MFI.

Table 4: purified antibody ADCP reporter activation assay

(5) Purified antibodies and cynomolgus monkey CD70 binding assays

1) CHO-K1 cells overexpressing cynomolgus CD70 were cultured, fully digested with 0.25% pancreatin, serum stopped from digestion, cells were collected by centrifugation, and single cell suspensions were prepared by gently beating with PBS.

2) 200000 cells were added to a 96-well plate per well and 50ul per well was ensured.

3) Negative, positive and control antibodies were prepared as mother liquors (e.g., 10, 1, 0.1 ug/ml) at 2-fold working concentrations, 50ul was added to the cells, mixed well and incubated for 30min at 4 ℃.

4) 100ul PBS was added, gently shaken, 500g centrifuged for 5 minutes, the supernatant removed, 200ul PBS was added, gently shaken, 500g centrifuged for 5 minutes, and the supernatant removed.

5) 100ul of diluted fluorescent secondary antibody was added, the 96-well plate was shaken to suspend the cells, and incubated at 4℃for 30 minutes.

6) Step 4) is repeated to wash the cells.

7) Cells were resuspended with 80ul PBS and fluorescence was detected by flow cytometry. The results are shown in FIG. 7, where the antibody was incubated with cells that overexpressed cynomolgus monkey CD70 on the cell surface, and the fluorescence-labeled secondary antibody detected cell-bound antibody. Wherein hIgG1 is a negative control and ARGX-110 is a positive control. The raw data are shown in Table 5, where the values are MFI.

Table 5: purified antibodies and cynomolgus monkey CD70 binding assays

Concentration ug- ml	hIgG1	ARGX- 110	1	2	3	4	5	6	7	8	9	10	11
														5	24.0	4206.2	8432.1	10224. 9	8617.7	9493.3	11788. 0	10944. 4	11575. 3	10282. 5	10006. 7	10468. 3	8251.8
0.5	19.3	5598.7	6492.6	6535.4	8171.1	10537. 0	8351.3	9531.3	10230. 6	9909.7	11604. 4	10292. 2	10978. 5
														0.05	21.2	1290.5	1149.2	953.2	1385.4	2178.1	1336.2	1870.6	1668.9	1726.1	2376.3	1927.1	2318.0
Concentration ug- ml	12	13	14	15	16	17	18	19	20	21	22	23	24
														5	9559. 4	9494.5	10531. 9	10524. 0	9478.1	11981. 5	11160. 2	11793. 0	11737. 5	10785. 2	11248. 8	11363. 8	9229.7
0.5	8446. 9	10682.1	13776. 9	12954. 9	10935. 0	11218. 7	11713. 6	12145. 2	9547.0	11574. 3	5144.4	9611.6	10997. 8
														0.05	1395. 8	2768.0	5826.8	3783.9	3277.0	1978.4	1815.8	2589.1	1717.6	3379.3	785.7	1696.4	2247.5

Claims

1. A set of anti-CD 70 antibodies having an enhanced ADCP effect, characterized in that the amino acid sequence of CDR1 of the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 2, the amino acid sequence of cdr2 is selected from SEQ ID NO:26, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 50.

2. a DNA molecule encoding an anti-CD 70 antibody with enhanced ADCP effect according to claim 1, characterized in that its heavy chain variable region SEQ ID NO: 2. 26, 50 are respectively shown in SEQ ID NO: 74. 98, 122.

3. The anti-CD 70 antibody with enhanced ADCP effect according to claim 1, characterized in that the antibody targets human or cynomolgus monkey CD70 molecules.

4. Use of an anti-CD 70 antibody with enhanced ADCP effect according to claim 1 for the preparation of a diagnostic reagent for the detection of CD70 molecules.

5. Use of an anti-CD 70 antibody with enhanced ADCP effect according to claim 1 for the manufacture of a medicament for the treatment of tumors.