CN116120451A

CN116120451A - anti-CD 70 internalizing antibodies, antibody conjugates and uses thereof

Info

Publication number: CN116120451A
Application number: CN202210942655.5A
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: 2023-05-16
Also published as: CN113444178B; WO2022262099A1; CN113444178A

Abstract

The invention discloses an anti-CD 70 internalizing antibody, an antibody conjugate and application thereof. The application takes the human CD70 as a target point, successfully develops and obtains the antibody which can be combined with the human CD70 molecule, has good specificity and higher affinity, combines the human CD70 expressed on the cell surface, and can be efficiently transported into the cell by the cell; because the antibody has an internalization function, toxin molecules can be carried into cells to kill the cells, and the effect of treating cancer is achieved. Therefore, the antibody has novel characteristics and a drug development direction, and is a potential drug for tumor immunotherapy.

Description

anti-CD 70 internalizing antibodies, antibody conjugates and uses thereof

Technical Field

The invention relates to the field of biotechnology, in particular to an anti-CD 70 internalizing antibody, an antibody conjugate and application thereof. The invention is application number 202110675328.3, and is named as an anti-CD 70 internalizing antibody, an antibody conjugate and a divisional application of the antibody conjugate.

Background

CD70 molecule, i.e. tumor necrosis factor receptor superfamily 7 factor (TNFSF 7), polypeptide chain length 193 a.a., molecular weight 21.1KD, is a type II transmembrane glycoprotein (not yet seen in soluble form), with 155 amino acids at the C-terminal outside the membrane; disulfide bonds are formed among the first, third, second and fourth extracellular cysteines, so that the spatial structure of the extracellular part is maintained; typically it is present on the membrane in the form of homotrimers. In normal tissues, CD70 has a narrow cellular expression profile, and is expressed at low levels in thymic medullary epithelial cells, mainly in germinal center B cells (inducing secretion of antibodies) and in T cells of local lymphoid tissues. But its expression may be greatly increased by antigen induction and reduced with a decrease in immune response, such as high expression of CD70 in antigen-induced activated T, B cells. While its expression is regulated by cytokines such as IL1a, IL12, TNFa, GM-CSF can up-regulate CD70 expression, IL4, IL10 down-regulate CD70 expression. In mature DCs, CD70 expression is mediated by TLR signaling pathways. These phenomena are precisely indicative of CD70 being a co-stimulatory molecule required for complete activation of naive T cells and playing an important role in regulating immune responses. It interacts with ligand CD27 not only to act as a second signal for T cell activation, but also to regulate proliferation and differentiation of immune cells such as B, NK.

CD70, transiently expressed in activated T and B lymphocytes and mature DC cells, is highly expressed in a variety of tumor tissues under pathological conditions. Numerous studies have shown that CD70 is highly expressed in various tumor tissues such as lymphomas, renal cancers, gliomas, breast cancers, hematological malignancies (e.g., hodgkin's lymphoma, multiple myeloma, leukemia, etc.). At the same time, an increase or decrease in CD27 expression in these tissues was observed in synchronization with CD70. Possibly both act as ligand/receptor pairs, forming a feedback loop that promotes lymphocyte proliferation and differentiation, but rather plays a role in mediating disease development. After CD70 interacts with CD27, CD27 activates, TRAF2/5 binds to its intracellular segment, further activating NF-KB and c-Jun kinase signaling pathways, causing cell proliferation, survival, differentiation, etc. In addition, it is also believed that binding of CD70 to CD27 may also cause the intracellular segment of CD27 to bind Siva, thereby causing Caspase-mediated apoptosis.

In view of the above phenomena, specific antibodies are prepared by taking CD70 as a target spot, and great interest is brought to development of tumor immunotherapy. At present, three application modes of CART treatment, antibody medicine and antibody coupling medicine aiming at a CD70 target point are developed, the highest progress is kusakazazumab (developed code ARGX-110) developed by cooperation of prednisone and arGEN-X, the phase I clinic is completed at present, and the result shows that the kusazazumab and the Vidazam can obviously reduce leukemia stem cells in bone marrow of an AML patient, and the reliability of the target point is verified.

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. Current therapies targeting CD70 have CART and naked antibodies with limited clinical efficacy, especially for solid tumors, both of which are almost ineffective. It is widely recognized by developers that antibody drug conjugate ADC will be one of the hopes for future treatment of cancer.

Disclosure of Invention

The invention aims to: in view of the above prior art, the present application provides an anti-CD 70 internalizing antibody, antibody conjugates and uses thereof.

The technical scheme is as follows: a set of anti-CD 70 internalizing antibodies described herein have an amino acid sequence of CDR1 of the heavy chain variable region selected from the group consisting of SEQ ID NOs: 1-16, the amino acid sequence of cdr2 is selected from SEQ ID NO:17-32, the amino acid sequence of cdr3 is selected from SEQ ID NO:33-48.

Further preferred, the anti-CD 70 internalizing antibodies described herein include a heavy chain variable region as set forth in any one of the following:

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:1, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 17, the amino acid sequence of cdr3 is selected from SEQ ID NO:33;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:2, the amino acid sequence of cdr2 is selected from SEQ ID NO:18, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 34;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:3, cdr2 is selected from the amino acid sequence of SEQ ID NO:19, cdr3 is selected from the amino acid sequence of SEQ ID NO:35;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:4, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 20, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 36;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:5, cdr2 is selected from the amino acid sequence of SEQ ID NO:21, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 37, respectively;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:6, the amino acid sequence of cdr2 is selected from SEQ ID NO:22, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 38, a step of carrying out the process;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:7, the amino acid sequence of cdr2 is selected from SEQ ID NO:23, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 39;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:8, the amino acid sequence of cdr2 is selected from SEQ ID NO:24, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 40, a step of performing a;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:9, the amino acid sequence of cdr2 is selected from SEQ ID NO:25, cdr3 is selected from the amino acid sequences of SEQ ID NOs: 41;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:10, 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: 42;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:11, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 27, the amino acid sequence of cdr3 is selected from SEQ ID NO: 43.

The amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:12, 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:44;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:13, the amino acid sequence of cdr2 is selected from SEQ ID NO:29, the amino acid sequence of cdr3 is selected from SEQ ID NO:45;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:14, cdr2 is selected from the amino acid sequences of SEQ ID NOs: 30, the amino acid sequence of cdr3 is selected from SEQ ID NO:46;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:15, 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: 47;

the amino acid sequence of the heavy chain variable region CDR1 is selected from SEQ ID NO:16, 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: 48.

SEQ ID NO:1-48 are shown in the following table:

the present application also discloses DNA molecules encoding the above anti-CD 70 internalizing antibodies, the heavy chain variable region of which SEQ ID NO:1-48, and the nucleotide sequence corresponding to the amino acid is shown as SEQ ID NO: 49-96.

SEQ ID NO:49-96 are shown in the following table:

the application also discloses the application of the anti-CD 70 internalizing antibody for detecting CD70 molecules.

Furthermore, the application also discloses application of the anti-CD 70 internalized antibody in preparing medicines for treating tumors. The tumor is preferably a renal clear cell adenocarcinoma.

Also disclosed is an antibody conjugate comprising the above-described anti-CD 70 internalizing antibody.

The application of the antibody conjugate in preparing a medicine for treating tumors 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 were transfected, the supernatant was harvested after 5 days of shaking culture, and the fusion protein was purified from the supernatant using protein A magnetic bead affinity chromatography and identified for its binding properties to CD70. Target cells were cultured, and antibodies were incubated therewith at 4℃in two groups in parallel. One group was transferred to 37 ℃ after incubation at 4 ℃ for continued incubation, and the other group was continued to be incubated at 4 ℃. After the incubation, adding a fluorescent labeling detection antibody, incubating at 4 ℃ and detecting the fluorescent antibody by using a flow cytometry, namely obtaining the anti-CD 70 antibody of which the cell surface is captured by CD70. Target cells were plated in 96-well plates and cultured overnight to allow the cells to adhere well. Cell culture is performed after addition of antibody and/or toxin reagents and cell growth characteristics are observed. After the completion of the culture, the cell viability was measured.

The technical effects are as follows: the application takes the human CD70 as a target point, successfully develops and obtains the antibody (fusion protein) capable of combining the human CD70 molecules, and the antibody has good specificity and higher affinity, combines the human CD70 expressed on the cell surface, and can be efficiently transported into cells by the cells; because the antibody has an internalization function, toxin molecules can be carried into cells to kill the cells, namely, an antibody-drug conjugate (ADC) approach is utilized, the expression level of CD70 in cancer cells is far higher than that of normal cells, and the ADC mainly targets the cancer cells, so that the effect of treating cancer is achieved. Therefore, the antibody has novel characteristics and a drug development direction, and 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 internalization assay;

FIG. 7 is a comparison of purified antibody internalization performance with ARGX-110;

FIG. 8 is a graph of the detection of the killing efficacy of purified antibody internalization-mediated toxins against cells;

FIG. 9 is a comparison of the killing efficacy of purified antibody internalization-mediated toxins against cells as compared to ARGX-110.

Detailed Description

The present application will be described in detail with reference to specific 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: synchronous with antigen binding to magnetic beads, 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 natamycin 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 natamycin 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 is added, incubated at 37 ℃ for 10min or until blue color is fully developed, 100ul of 1M sulfuric acid is added to stop the reaction, OD450 is read on an enzyme label instrument, the result is shown in figure 1, 50ul of 2-poison particle T medium containing phage virus particles is used for detection during phage panning, and the original data are shown in table 1, wherein the value is the absorbance at 450nm and the ratio thereof.

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

(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), while 70 ug 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 5ml 0.1M 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. With reduction electrophoresis, disulfide bonds in antibody molecules are opened and the molecules undergo electrophoretic migration in the state of extended single peptide chains, as shown in fig. 4.

The purified antibodies obtained from the screening were sequenced to give antibodies numbered 1-16 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:97, the nucleic acid sequence is shown as SEQ ID NO: shown at 98; the full-length amino acid sequence of the variable region of the antibody with the number 2 is shown in SEQ ID NO:99, the nucleic acid sequence is shown in SEQ ID NO: 100; the full-length amino acid sequence of the variable region of the antibody with the number 3 is shown in SEQ ID NO:101, the nucleic acid sequence is shown as SEQ ID NO: 102.

SEQ ID NO：97：

QVQLVESGGGLVQPGGSLRLSCAASGSIFSIYAMGWYRQAPGKQRELVAAITNSGGTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAVLEQASGDPVVYRYLEVWGQGTLVTVSS

SEQ ID NO：98：

CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAGCTATTACTAATAGTGGTGGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCAGTTCTTGAACAGGCTAGCGGCGACCCCGTGGTTTACAGGTATCTCGAAGTTTGGGGCCAGGGCACCCTGGTCACTGTCTCCTCA

SEQ ID NO：99：

QVQLVESGGGLVQPGGSLRLSCAASGSIASIYHMAWYRQASGKQRELVALIISDGKINYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAQNWATRDYWGQGTQVTVSS

SEQ ID NO：100：

CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCGCCAGTATCTATCACATGGCCTGGTACCGCCAGGCTTCAGGGAAGCAGCGCGAGTTGGTCGCACTCATTATTAGTGATGGTAAGATAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCCCAGAATTGGGCCACAAGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

SEQ ID NO：101：

QVQLVESGGGLVQPGGSLRLSCAASGSIFSIYAMGWYRQAPGKQRELVALITNSGGTNYADSVKGRFTISRDNAKNTVYLQMNSLKPVDTAVYYCNAVEEEVVAGTDRYRYLEVWGQGTLVTVSS

SEQ ID NO：102：

CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGTATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCACTTATTACTAATAGTGGTGGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCAGTGGACACGGCCGTCTATTACTGTAATGCGGTTGAGGAGGAGGTAGTAGCTGGTACTGACCGCTACAGGTATCTCGAAGTTTGGGGCCAGGGCACCCTGGTCACTGTCTCCTCA

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 100ul of purified antibody from step (3) 9) 10ug/ml, 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 into single cell suspension, and the suspension was detected by a flow cytometer, wherein the left and right bar graphs of the flow graph are shown in FIG. 2, and the left and right bar graphs represent the binding signals with wild CHO-K1 and CD70 over-expressed CHO-K1 cell lines, respectively, and the original data are shown in Table 2, wherein the values are fluorescence intensity Median (MFI) and the ratio thereof. Purified antibody flow is shown in FIG. 5, wherein ARGX-110 is the positive control and hIgG1 is the negative control, and the raw data is shown in Table 3, wherein the value is the Median Fluorescence Intensity (MFI).

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

Table 3: purified antibodies and cell surface CD70 binding assay

Concentration ug/ml	hIgG1	ARGX-110	1	2	3	4	5	6	7
										5	89.0	12418.8	8609.0	11987.1	13689.4	3432.6	9041	10485.9	10746.8
1.25	64.9	12599.1	9067.2	13122.4	14076.3	2999.1	11379.5	11309.3	9939.8
										0.3125	53.4	10454.3	7496.8	13127.1	11707.1	1580.4	10484.4	11518.5	6865.7
0.0781	140.8	7363.1	4512.0	10307.0	10255.8	686.3	7695.9	9669.2	3319.6
										0.0195	112.4	3930.7	3641.6	6087.3	4576.2	149.1	3786	5428	1109.3
0.0049	100.1	1132.5	697.9	4267.7	2820.6	76.1	957.6	875.3	383.9
										0.0012	119.1	622.5	629.7	1486.6	465.3	66.3	393.3	250.7	144.3
0.0003	96.4	283.5	1263.6	485.2	173.1	343.6	197.4	272	116.5
										Concentration ug/ml	8	9	10	11	12	13	14	15	16
5	17027.1	5318.8	18997.2	18658.2	16308.4	7303.1	9995.4	11034.7	8213.7
										1.25	19287.1	6009.8	18780.9	18599.9	16708.3	7267.5	9000.9	10878.6	8076.9
0.3125	19126.1	6170.7	16032.5	16648.0	17391.7	5319.8	8216.2	9521.7	7723.0
										0.0781	8412.4	3523.3	9042.7	6333.4	17250.2	4247.4	2866.7	3027.9	6113.4
0.0195	2577.6	1156	2964	1942.3	12055.9	1298.6	1653.0	1237.6	3240.5
										0.0049	875.1	306.5	937.5	604.2	4218.5	942.7	1800.8	937.1	1091.9
0.0012	309.2	96.3	347.2	231.5	1590.8	498.2	1216.1	336.8	352.4
										0.0003	137.7	44.8	140.2	110.6	848.1	267.4	399.2	272.7	255.7

(4) Antibody internalization function detection

1) 786-0 cells were digested with 0.25% Typsin-EDTA, the supernatant was discarded by centrifugation and resuspended in complete medium.

2) 75ul of cell suspension was added to a 96-well plate with 1.5 per well and with ⁵ Individual cells.

3) 25ul of antibody EC80 was added at the working concentration (i.e., 80% of the saturation concentration) and mixed well. Each antibody was split into two plates, each plate being plated with one well. Incubation was carried out at 4℃for 1 hour.

4) Adding PBS to a final volume of 250ul, centrifuging at normal temperature for 5min, removing supernatant to retain cell precipitate, sucking residual liquid with water-absorbing paper, tapping the experimental plate to disperse cells, washing with PBS once again, and dispersing cells. This step removes excess unbound antibody.

5) Cells were resuspended in 100ul basal medium, one plate incubated at 37℃for 2 hours and the other plate incubated at 4℃for 2 hours. Antibodies were transferred to cells during incubation at 37℃while internalization was inhibited at 4 ℃.

6) 100ul of the fluorescent secondary antibody diluted with PBS (the concentration of the secondary antibody before addition was 2 times the working concentration) was added and incubated at 4℃for 0.5 hours.

7) Centrifuging at 500g at normal temperature for 5min, removing supernatant, retaining cell precipitate, sucking residual liquid with water absorbing paper, tapping experiment plate to disperse cells, washing with PBS again, and dispersing cells.

8) 100ul PBS was added for resuspension, detection on-machine by flow cytometry, and data were processed by Graphpad Prism. After binding of the antibodies to the cells, the antibodies to be detected bound to the cell surface were detected by fluorescent secondary antibodies as shown in fig. 6, and the raw data of fig. 6 are shown in table 4, where the values are Median Fluorescence Intensity (MFI). FIG. 7 is the relative internalization defined by the MFI measured at 37℃divided by the MFI measured at 4℃for the test sample, seq No. ID herein, as the value A, and by the MFI measured at 37℃divided by the MFI measured at 4℃for ARGX-110 as the value B.

Table 4: purification antibody internalization performance assay

Sample/seq No. ID	37℃	4℃	Sample/seq No. ID	37℃	4℃
						ARGX-110	6335.0	7365.6	9	1296.7	3418.8
1	1149.3	2160.8	10	6910.7	13886.0
						2	1003.9	7450.1	11	8750.5	13581.0
3	1561.3	3420.7	12	6870.6	9654.2
						4	370.3	774.1	13	3774.3	5686.8
5	716.8	2580.4	14	3595.1	4961.0
						6	1884.7	3071.9	15	2505.8	3528.0
7	1492.8	2544.3	16	4025.4	6003.4
						8	6032.4	13007.9

(5) Detection of killing efficacy of antibody internalization-mediated toxins against cells

2) 90ul of cell suspension was added to a 96-well plate and 2500 cells per well were allowed.

3) The antibodies were diluted to 10-fold EC50 values, to which fabf-ZAP human reagent (working concentration of 4.5nM; purchased from Advanced Targeting Systems), 10ul of the mixture was added to the cells in step 2) and mixed well.

4)37℃，5％CO ₂ The incubator was incubated for 72 hours.

5) The CCK8 kit was equilibrated for 15min at room temperature. The 96-well cell culture plate is taken out, CCK8 reagent is added, the culture plate is incubated for 1 to 3 hours in a 37 ℃ incubator in dark place, and an OD450 value is read by an enzyme-labeled instrument. As shown in fig. 8, after incubation of antibody, toxin and cells, the CCK8 method measured lower cell activity than the antibody alone and cell co-incubation. The raw data are shown in Table 5, wherein the value is the absorbance at 450nm (A450). The absorbance at 450nm measured after the simultaneous treatment of the cells with the antibody (ARGX-110 or the antibody indicated by Seq No. ID) and the toxin divided by the absorbance at 450nm measured after the treatment of the cells with the antibody alone (ARGX-110 or the antibody indicated by Seq No. ID) was defined as the value A, and the absorbance at 450nm measured after the treatment of the cells with the toxin divided by the absorbance at 450nm measured with the untreated cells was defined as the value B, and the A/B value was the relative killing. As shown in fig. 9.

Table 5: detection of killing efficacy of purified antibody internalization-mediated toxins against cells

Seq No.ID	Antibody + toxin	Antibodies to	Seq No.ID	Antibody + toxin	Antibodies to
						ARGX-110	2.3615	2.8350	9	1.0498	2.2105
1	1.0227	2.1778	10	1.1626	3.1110
						2	0.8658	2.2844	11	1.0138	3.0691
3	0.7365	2.2092	12	1.2944	2.9997
						4	1.0377	2.2920	13	1.7668	2.5112
5	0.6969	2.2609	14	1.0145	2.1882
						6	0.8546	2.3082	15	1.8259	2.5652
7	0.9212	2.8220	16	1.5359	2.5258
						8	0.9660	2.3672	-	-	-
Toxin+ cells	2.1185	-	Cells	2.4601	-

Claims

1. A panel of anti-CD 70 internalizing antibodies, wherein the amino acid sequence of CDR1 of the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 3, cdr2 is selected from the amino acid sequence of SEQ ID NO:19, cdr3 is selected from the amino acid sequence of SEQ ID NO:35.

2. a DNA molecule encoding an anti-CD 70 internalizing antibody according to claim 1, characterized in that its heavy chain variable region SEQ ID NO: 3. 19, 35 are respectively shown in SEQ ID NO: 51. 67, 83.

3. Use of an anti-CD 70 internalizing antibody according to claim 1 for the preparation of a diagnostic reagent for the detection of CD70 molecules.

4. Use of an anti-CD 70 internalizing antibody according to claim 1 in the manufacture of a medicament for the treatment of a tumor.

5. An antibody conjugate comprising the anti-CD 70 internalizing antibody of claim 1.

6. Use of the antibody conjugate of claim 5 in the preparation of a medicament for treating a tumor.

7. The use according to claim 4 or 6, wherein the tumour is a renal clear cell adenocarcinoma.