CN110590951A

CN110590951A - ROR1 monoclonal antibody, preparation method and application thereof

Info

Publication number: CN110590951A
Application number: CN201910929922.3A
Authority: CN
Inventors: 范国煌; 丁成立
Original assignee: Anmengde Pharmaceutical Technology Shanghai Co Ltd
Current assignee: Anmengde Pharmaceutical Technology Shanghai Co Ltd
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2019-12-20

Abstract

The invention provides a ROR1 monoclonal antibody, which comprises a heavy chain and a light chain structure of the ROR1 monoclonal antibody, wherein a heavy chain variable region of the heavy chain and a light chain variable region of the light chain respectively have 3 CDRs, and a secretion-optimized signal peptide is added, so that the ROR1 monoclonal antibody can be efficiently expressed, is specifically combined with a Frizzled domain of a ROR1 receptor, inhibits the transmission of related channels, and effectively treats cancer, particularly has remarkable application in the aspect of treating non-small cell lung adenocarcinoma.

Description

ROR1 monoclonal antibody, preparation method and application thereof

Technical Field

The invention belongs to the field of biotechnology and antibodies, and particularly relates to a ROR1 monoclonal antibody, a preparation method and application thereof.

Background

Receptor tyrosine kinase-like orphan receptor (ROR 1) is a transmembrane protein with a molecular weight of about 104kDa and a size of about 130kDa after glycosylation modification. The extracellular domain of the ROR1 receptor consists of three distinct domains: an immunoglobulin-like (Ig-like) domain distal to the membrane; a Frizzled (Frizzled) domain; and a Kringle (Kringle) domain proximal to the membrane.

There are studies that show that ROR1 is expressed at high levels during embryonic development and plays an important role in the regulation of embryonic, muscle, skeletal, pulmonary and nervous system development. After developmental maturation, the expression of ROR1 protein is restricted, with only a small amount being expressed in some organs and tissues, including lungs, pancreas, adipocytes, etc. However, ROR1 is expressed in a variety of tumor cells, including chronic lymphocytic leukemia, mantle cell lymphoma, lung, breast, and ovarian cancer, among others.

At present, a ROR1 monoclonal antibody drug (UC-961) is in clinical experiments and is used for treating chronic lymphocytic leukemia and mantle cell lymphoma. UC-961 specifically binds to the immunoglobulin-like (Ig-like) domain of ROR1 protein. No ROR1 monoclonal antibody directed against the other domain of ROR1 has been discovered.

Disclosure of Invention

In view of the above technical problems, the present invention provides a ROR1 monoclonal antibody, the ROR1 monoclonal antibody comprises heavy chain and light chain structures, the variable region of the heavy chain and the variable region of the light chain each have 3 complementarity determining region CDRs, wherein: the amino acid sequence of CDR1 of the heavy chain variable region is SEQ ID NO.3, the amino acid sequence of CDR2 of the heavy chain variable region is SEQ ID NO.4, the amino acid sequence of CDR3 of the heavy chain variable region is SEQ ID NO.5, the amino acid sequence of CDR1 of the light chain variable region is SEQ ID NO.6, the amino acid sequence of CDR2 of the light chain variable region is SEQ ID NO.7, and the amino acid sequence of CDR3 of the light chain variable region is SEQ ID NO. 8.

Namely heavy chain CDR 1: GGSFSG (SEQ ID NO: 3);

heavy chain CDR 1: NHSGSTS (SEQ ID NO: 4);

heavy chain CDR 1: GHSSGWYRRYFDL (SEQ ID NO: 5);

light chain CDR 1: RASQSVSSYLA (SEQ ID NO: 6);

light chain CDR 2: DASNRAT (SEQ ID NO: 7);

light chain CDR 3: QQRSNWPPT (SEQ ID NO: 8).

The invention further provides the amino acid sequence of the heavy chain variable region of the ROR1 monoclonal antibody shown in SEQ ID NO. 1.

The invention further provides the amino acid sequence of the variable region of the light chain of the ROR1 monoclonal antibody shown in SEQ ID NO. 2.

In the present invention, this monoclonal antibody was designated as G3 monoclonal antibody.

Further, the present invention provides a ROR1 monoclonal antibody, wherein based on the amino acid sequences of the heavy chain variable region and the light chain variable region, a signal peptide sequence (for example, the sequence is ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCA, as shown in SEQ ID NO. 11; the protein sequence is MGWSCIILFLVATATGVHS, as shown in SEQ ID NO. 12) optimized for heavy chain secretory expression is added to the N-terminal of the heavy chain variable region (VH), and a human IgG1 constant region sequence CH1-CH3 (for example, the nucleotide sequence is:

GCTTCCACCAAGGGCCCCTCCGTGTTCCCCCTGGCTCCCTCTTCCAAGAGCACCAGCGGCGGCACCGCTGCTCTGGGATGTCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAATTCCGGCGCCCTGACCTCCGGCGTGCACACATTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTATAGCCTGTCCTCCGTGGTGACAGTGCCTAGCTCCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTAGCAATACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAATGATAA, as shown in SEQ ID NO. 13; amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, shown as SEQ ID NO. 14), constructing the full-length amino acid sequence of the heavy chain.

Further, the present invention provides ROR1 monoclonal antibody, wherein a signal peptide sequence optimized for light chain secretory expression (for example, the nucleotide sequence is:

ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCA, as shown in SEQ ID NO. 15; the amino acid sequence is: MGWSCIILFLVATATGVHS, as shown in SEQ ID NO. 16), and a human Kappa constant region sequence (for example, the nucleotide sequence is:

AGGACCGTGGCTGCCCCCAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGTCTGCTGAATAACTTCTATCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAACTCCCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCTCCACCCTGACCCTGTCCAAGGCTGATTATGAGAAGCACAAGGTGTATGCTTGCGAGGTGACACACCAGGGCCTGTCCAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGCTGATAA, as shown in SEQ ID NO. 17; amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, as shown in SEQ ID NO. 18), constructing the full-length amino acid sequence of the light chain.

That is, according to a specific embodiment of the present invention, the heavy chain constant region of the monoclonal antibody of the present invention is a human IgG1-Fc constant region and the light chain constant region is a human kappa chain constant region.

According to a specific embodiment of the present invention, the amino acid sequences of the heavy chain and the light chain of the monoclonal antibody of the present invention are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively; the antibody is a specific example of the fully human monoclonal antibody G3 against ROR1 of the present invention. (SEQ ID NO.1 and 2 do not contain a signal peptide, but contain a constant region)

In another aspect, the invention also provides a ROR1 monoclonal antibody, wherein the nucleotide sequences of the heavy chain and the light chain of the monoclonal antibody are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10; the antibody is a specific example of the fully human monoclonal antibody G3 against ROR1 of the present invention.

The invention further provides a preparation method of the ROR1 monoclonal antibody, which comprises the following steps:

1) synthesizing sequences according to the nucleotide sequences of the heavy chain and the light chain, and constructing the sequences into an expression antibody;

2) eukaryotic cell transfection is carried out according to the proportion of the heavy chain and the light chain, and the corresponding ROR1 monoclonal antibody is obtained after protein purification is carried out on the expressed antibody.

In the above preparation method, the expressed antibody is pcDNA3.4.

In the preparation method, the plasmid weight molar ratio of the heavy chain to the light chain is 1: 1-5. Preferably 1: 2. preventing the production of Fc dimers without light chains.

Further, in the above preparation method, the heavy chain and the light chain include a secretion-optimizing signal peptide.

An application of the ROR1 monoclonal antibody in preparing a medicament for treating cancer.

In the above applications, the cancer includes: chronic granulocytic lymphoma, mantle cell lymphoma, non-small cell lung cancer and breast cancer

In the above application, the cancer is non-small cell lung cancer adenocarcinoma.

In conclusion, the invention constructs and prepares the full-length monoclonal antibody of full human source by screening VH and VL sequences of a monoclonal antibody from a natural phage display library. The monoclonal antibody G3 of the invention has specificity combined with the Frizzled (Frizzled) domain of human ROR1, the effect is better than the treatment effect combined with other domains of human ROR1, the monoclonal antibody has prominent effect for the first time, and the monoclonal antibody has important application value in the clinical diagnosis and treatment of clinical tumor.

Drawings

FIG. 1 binding (ELISA) of anti-human ROR1 antibody G3 to human ROR 1;

FIG. 2 binding (ELISA) of anti-human ROR1 antibody G3 to murine ROR 1;

FIG. 3 binding (FACS) of anti-human ROR1 monoclonal antibody G3 to JeKo-1 cell ROR1 protein;

FIG. 4 binding (FACS) of anti-human ROR1 monoclonal antibody G3 to ROR1 on the cell surface of CHO-K1-hROR 1;

FIG. 5 binding (ELISA) of monoclonal antibody G3 against human ROR1 to the Frizzled (Frizzled) domain of human ROR1 protein;

FIG. 6G 3 antibody inhibited the proliferation of NCI-H1975 cells.

Detailed Description

The following examples are conducted by methods commonly used in the art or according to commercial kit instructions.

Example 1

Preparation method of anti-ROR 1 antibody

The ROR1 antibody VH and VL sequences of the present invention were derived from a natural phage display library (ProMab Biotechnologies, Inc).

The sequences of the heavy chain and the light chain of the antibody are obtained by means of gene synthesis (namely SEQ ID NO.9 and 10, and corresponding secretion signal peptides SEQ ID NO.11 and 15 are added), and are respectively constructed on a pcDNA3.4 vector. After expanding the Expi-CHO-S cells to a certain density, the plasmid DNA was transfected into Expi-CHO-S by transient Transfection (Gibco, Expifeacamine CHO Transfection Kit 1L) with a molar ratio of heavy chain to light chain of 1:2, fed separately on day 1 and day 5 after Transfection according to the protocol of the Transfection Kit, and purified on day 7 after Transfection.

After the expression was completed, the antibody was purified by Protein A (GE mabselect Sure) to obtain a purified antibody. Finally, the antibodies were mass analyzed by SDS-PAGE and SEC-HPLC.

The molecular weight of the antibody is 150kDa, and the purity of the antibody is 93.9%.

Example 2

Activity characterization of ROR1 antibody

I enzyme-linked immunosorbent assay (ELISA)

Coating solution (3.56g Na)₂CO₃And 8.4g NaHCO₃Ph9.5) diluted recombinant human ROR1 protein (available from beijing percent pocersus biotechnology limited, product No.: RO1-H522y) to 1. mu.g/mL coated ELISA 96-well plates, 100. mu.L per well, and incubating overnight at 4 ℃; blocking the 96-well plate by PBS (phosphate buffer solution) containing 2% BSA, and incubating for 1h at room temperature; after 3 washes of PBST (0.05% Tween 20 in PBS), each well was washedTo this was added 100. mu.L of ROR1 antibody molecule G3 (0.6. mu.g/ml starting concentration, diluted 5-fold into 8 concentration gradients) and incubated for 1h at room temperature; diluting horseradish peroxidase-conjugated goat anti-human IgG Fc secondary antibody (purchased from Abcam company, product number: ab97225) at a ratio of 1:10000, adding into the wells at a ratio of 100. mu.L/well, and incubating at room temperature for 1 h; horse radish peroxidase substrate developing solution (purchased from Shanghai biological engineering Co., Ltd., product number: C520026) is prepared, added into the hole according to 100 μ L/hole, incubated at room temperature for 1-2min, stopped reacting with 2mol sulfuric acid, and read OD450 value with enzyme labeling instrument.

Coating solution (3.56g Na)₂CO₃And 8.4g NaHCO₃Ph9.5) diluted recombinant murine ROR1 protein (purchased from beijing percent pocersus biotechnology limited, product No.: RO1-M5221) to 1. mu.g/mL coated ELISA 96-well plates, 100. mu.L per well, incubated overnight at 4 ℃; blocking the 96-well plate by PBS (phosphate buffer solution) containing 2% BSA, and incubating for 1h at room temperature; after 3 PBST (PBS containing 0.05% Tween 20) washes, 100. mu.L of ROR1 antibody molecule G3 (15. mu.g/ml starting concentration, diluted 5-fold into 8 concentration gradients) was added to each well and incubated for 1h at room temperature; after PBST was washed 3 times, a goat anti-human IgG Fc secondary antibody (purchased from Abcam, product number: ab97225) conjugated with horseradish peroxidase was diluted at a ratio of 1:10000, added to the wells at a rate of 100. mu.L/well, and incubated at room temperature for 1 hour; horse radish peroxidase substrate developing solution (purchased from Shanghai biological engineering Co., Ltd., product number: C520026) is prepared, added into the hole according to 100 μ L/hole, incubated at room temperature for 1-2min, stopped reacting with 2mol sulfuric acid, and read OD450 value with enzyme labeling instrument.

The above results show that anti-human ROR1 antibody G3 binds to both human ROR1 and murine ROR1 (fig. 1 and 2). The antibody was demonstrated to be able to stably bind to the receptor.

II flow cytometry (FACS)

The binding activity of human ROR1 antibody to endogenous ROR1 antigen was analyzed using flow cytometry (FACS).

The specific method comprises the following steps: JeKo-1 cells were collected and evenly divided into 1.5mL EP tubes, 5X 10 cells per tube⁵JeKo-1 cells. Wash 1 time with PBS, 1000rpm, 5 min. PBS blocking solution containing 1% BSA was added to each tube, incubated at 4 ℃ for 1h, washed 1 time with PBS, and run at 1000rpm for 5 min. The 2 tubes of cells were all treated with 100. mu.L PBSResuspend, resuspend the cells of the remaining tubes with 100. mu.L of ROR1 antibody G3 (20. mu.g/mL starting concentration, 4-fold dilution to 8 concentration gradients), incubate at 4 ℃ for 1h, wash 2 times with PBS, 1000rpm, 5 min.

The cells to which ROR1 antibody had been added were resuspended in 100. mu.L of PE-labeled goat anti-human IgG-Fc secondary antibody (purchased from Abcam, product No.: ab98596), and the remaining 2 tubes of cells were resuspended in 100. mu.L of PBS and PE-labeled goat anti-human IgG-Fc secondary antibody, respectively, and incubated at 4 ℃ with exclusion of light for 30 min. After 1 PBS wash, each tube was resuspended in 200. mu.L PBS and immunofluorescence was performed on FACS.

The binding activity of human ROR1 antibody to exogenous ROR1 antigen was analyzed using flow cytometry (FACS). The specific method comprises the following steps: CHO-K1 cells were infected with lentivirus packaging a plasmid expressing human ROR1 protein to obtain a stable transgenic cell strain CHO-K1-hROR1 overexpressing ROR 1. The cell line CHO-K1-hROR1 stably expressing human ROR1 was collected and divided into 1.5mL EP tubes with 5X 10 cells per tube⁵CHO-K1-human-ROR1 cells. PBS wash 1 time, 300g, 5 min. PBS blocking solution containing 1% BSA was added to each tube, incubated at 4 ℃ for 1h, washed 1 time with PBS, 300g, 5 min. 2 tubes of the cells were resuspended in 100. mu.L PBS, and the cells in the remaining tubes were resuspended in 100. mu.L of ROR1 antibody G3 (starting concentration 30. mu.g/mL, 3-fold dilution to 8 concentration gradients), incubated at 4 ℃ for 1h, washed 2 times in PBS, 300G, 5 min. The cells to which ROR1 antibody had been added were resuspended in 100. mu.L of PE-labeled goat anti-human IgG-Fc secondary antibody (purchased from Abcam, product No.: ab98596), and the remaining 2 tubes of cells were resuspended in 100. mu.L of PBS and PE-labeled goat anti-human IgG-Fc secondary antibody, respectively, and incubated at 4 ℃ with exclusion of light for 30 min. After 1 PBS wash, each tube was resuspended in 200. mu.L PBS and immunofluorescence was performed on FACS.

The above results show that anti-human ROR1 antibody G3 can bind to ROR1 on the cell surface of JeKo-1 and CHO-K1-hROR1 (FIGS. 3 and 4).

Example 3

Identification of antigen binding epitopes of ROR1 antibody

The recombinant ROR1 protein comprises 3 extracellular domains: ig domain, frizzled domain, Kringg domain (available from Bethes Biotech, Inc., Beijing under the product numbers RO1-H5221, RO1-H5222, and RO1-H5223, respectively). The extracellular domain of 3 ROR1 proteins was diluted to 1. mu.g/mL with coating solution (3.56g Na2CO3 and 8.4g NaHCO3, pH9.5), coated on ELISA 96-well plates, added at 100. mu.L/well, and incubated overnight at 4 ℃; blocking the 96-well plate by PBS (phosphate buffer solution) containing 2% BSA, and incubating for 1h at room temperature; after 3 PBST (PBS containing 0.05% Tween 20) washes, 100. mu.L of ROR1 antibody molecules G3 and C3 (20. mu.g/ml starting concentration, diluted 5-fold into 8 concentration gradients) were added to each well and incubated for 1h at room temperature; after PBST was washed 3 times, a goat anti-human IgG Fc secondary antibody (purchased from Abcam, product number: ab97225) conjugated with horseradish peroxidase was diluted at a ratio of 1:10000, added to the wells at a rate of 100. mu.L/well, and incubated at room temperature for 1 hour; horse radish peroxidase substrate developing solution (purchased from Shanghai Biotechnology engineering Co., Ltd., product number: C520026) is prepared, added into the hole according to 100 μ L/hole, incubated at room temperature for 10min, stopped reacting with 2mol sulfuric acid, and read OD450 value with enzyme-labeling instrument.

The above results show that anti-human ROR1 antibody G3 binds to the Frizzled (Frizzled) domain of human ROR1 (fig. 5).

Example 4

The G3 antibody can inhibit the proliferation of NCL-H1975 cells

Selecting 96-well plate, and laying 3 × 10 plates in each well³NCL-H1975 cells (human non-small cell lung carcinoma adenocarcinoma cells, ATCC, CRL-5908), cultured overnight at 37 ℃ in a 5% CO2 incubator. The following day ROR1 antibodies G3 and UC-961 (made according to US20160097776A 1) (starting concentration 24. mu.g/ml, diluted 2-fold into 10 gradients), 5% CO at 37 ℃ were added₂The incubator is used for 3 days.

Add 50. mu.l to each wellLuminessent reagent (available from Promega, product No: G7572) was lysed at room temperature for 10 minutes. The values were read in a microplate detector (from Tecan, model: Infinite 200 pro).

The experimental results show that the proliferation inhibition effect of G3 on NCL-H1975 cells is obviously better than that of UC-961 (FIG. 6).

Sequence listing

<110> Anmengde pharmaceutical technology (Shanghai) Co., Ltd

<120> ROR1 monoclonal antibody, preparation method and application thereof

<141> 2019-09-27

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 451

<212> PRT

<213> Artificial Sequence

<400> 1

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly His Ser Ser Gly Trp Tyr Arg Arg Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

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

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 2

<211> 213

<212> PRT

<213> Artificial Sequence

<400> 2

Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu

1 5 10 15

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Lys Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 3

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 3

Gly Gly Ser Phe Ser Gly

1 5

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 4

Asn His Ser Gly Ser Thr Ser

1 5

<210> 5

<211> 13

<212> PRT

<213> Artificial Sequence

<400> 5

Gly His Ser Ser Gly Trp Tyr Arg Arg Tyr Phe Asp Leu

1 5 10

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 6

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 7

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 8

Gln Gln Arg Ser Asn Trp Pro Pro Thr

1 5

<210> 9

<211> 1359

<212> DNA/RNA

<213> Artificial Sequence

<400> 9

caggtgcagc tgcagcagtg gggcgccgga ctgctgaagc cttccgagac cctgtccctg 60

acctgtgccg tgtacggcgg ctccttcagc ggctattact ggtcctggat ccggcagcct 120

cctggcaagg gcctggagtg gatcggcgag atcaatcaca gcggctccac ctcctataac 180

cccagcctga agagccgggt gaccatcagc gtggatacca gcaagaatca gttcagcctg 240

aagctgagct ccgtgaccgc cgctgacacc gccgtgtatt attgtgccag gggccacagc 300

agcggctggt atcggcggta cttcgatctg tggggcaggg gcaccctggt gaccgtgagc 360

tccgcttcca ccaagggccc ctccgtgttc cccctggctc cctcttccaa gagcaccagc 420

ggcggcaccg ctgctctggg atgtctggtg aaggactact tccctgagcc tgtgaccgtg 480

tcctggaatt ccggcgccct gacctccggc gtgcacacat tccctgctgt gctgcagtcc 540

tccggcctgt atagcctgtc ctccgtggtg acagtgccta gctccagcct gggcacccag 600

acctatatct gcaacgtgaa ccacaagcct agcaatacca aggtggacaa gaaggtggag 660

cctaagagct gcgacaagac ccacacctgt cctccatgtc ctgctccaga actgctcggc 720

ggaccttccg tgttcctgtt tcctccaaag cctaaggaca ccctgatgat cagcagaacc 780

cctgaagtga cctgcgtggt ggtggatgtg tcccacgagg atcccgaagt gaagttcaat 840

tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900

aacagcacct acagagtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 960

aaagagtaca agtgcaaggt gtccaacaag gccctgcctg ctcctatcga gaaaaccatc 1020

agcaaggcca agggccagcc tagggaaccc caggtttaca cactgcctcc aagcagggac 1080

gagctgacca agaatcaggt gtccctgacc tgcctggtca agggcttcta cccttccgat 1140

atcgccgtgg aatgggagag caatggccag cctgagaaca actacaagac aacccctcct 1200

gtgctggaca gcgacggctc attcttcctg tacagcaagc tgacagtgga caagagcaga 1260

tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320

acccagaagt ccctgagcct gtctcctggc aaatgataa 1359

<210> 10

<211> 648

<212> DNA/RNA

<213> Artificial Sequence

<400> 10

gagatcgtgc tgacccagtc ccctgccacc ctgagcctgt cccccggaga gagagctacc 60

ctgagctgcc gggctagcca gagcgtgagc agctatctgg cctggtatca gcagaagccc 120

ggccaggccc cccggctgct gatctatgac gctagcaacc gggccaccgg catccctgct 180

aagttcagcg gctccggcag cggcaccgac ttcaccctga ccatctccag cctggagccc 240

gaggacttcg ctgtgtacta ctgtcagcag aggtccaact ggccccctac cttcggcggc 300

ggcaccaagg tggagatcaa gaggaccgtg gctgccccca gcgtgttcat cttccctcct 360

agcgacgagc agctgaagag cggcaccgct agcgtggtgt gtctgctgaa taacttctat 420

cccagggagg ccaaggtgca gtggaaggtg gataacgccc tgcagagcgg caactcccag 480

gagtccgtga ccgagcagga ctccaaggac agcacctact ccctgagctc caccctgacc 540

ctgtccaagg ctgattatga gaagcacaag gtgtatgctt gcgaggtgac acaccagggc 600

ctgtccagcc ctgtgaccaa gagcttcaac cggggcgagt gctgataa 648

<210> 11

<211> 57

<212> DNA/RNA

<213> Artificial Sequence

<400> 11

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattca 57

<210> 12

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 12

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 13

<211> 996

<212> DNA/RNA

<213> Artificial Sequence

<400> 13

gcttccacca agggcccctc cgtgttcccc ctggctccct cttccaagag caccagcggc 60

ggcaccgctg ctctgggatg tctggtgaag gactacttcc ctgagcctgt gaccgtgtcc 120

tggaattccg gcgccctgac ctccggcgtg cacacattcc ctgctgtgct gcagtcctcc 180

ggcctgtata gcctgtcctc cgtggtgaca gtgcctagct ccagcctggg cacccagacc 240

tatatctgca acgtgaacca caagcctagc aataccaagg tggacaagaa ggtggagcct 300

aagagctgcg acaagaccca cacctgtcct ccatgtcctg ctccagaact gctcggcgga 360

ccttccgtgt tcctgtttcc tccaaagcct aaggacaccc tgatgatcag cagaacccct 420

gaagtgacct gcgtggtggt ggatgtgtcc cacgaggatc ccgaagtgaa gttcaattgg 480

tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ctagagagga acagtacaac 540

agcacctaca gagtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaaa 600

gagtacaagt gcaaggtgtc caacaaggcc ctgcctgctc ctatcgagaa aaccatcagc 660

aaggccaagg gccagcctag ggaaccccag gtttacacac tgcctccaag cagggacgag 720

ctgaccaaga atcaggtgtc cctgacctgc ctggtcaagg gcttctaccc ttccgatatc 780

gccgtggaat gggagagcaa tggccagcct gagaacaact acaagacaac ccctcctgtg 840

ctggacagcg acggctcatt cttcctgtac agcaagctga cagtggacaa gagcagatgg 900

cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 960

cagaagtccc tgagcctgtc tcctggcaaa tgataa 996

<210> 14

<211> 330

<212> PRT

<213> Artificial Sequence

<400> 14

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 15

<211> 57

<212> DNA/RNA

<213> Artificial Sequence

<400> 15

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattca 57

<210> 16

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 16

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 17

<211> 327

<212> DNA/RNA

<213> Artificial Sequence

<400> 17

aggaccgtgg ctgcccccag cgtgttcatc ttccctccta gcgacgagca gctgaagagc 60

ggcaccgcta gcgtggtgtg tctgctgaat aacttctatc ccagggaggc caaggtgcag 120

tggaaggtgg ataacgccct gcagagcggc aactcccagg agtccgtgac cgagcaggac 180

tccaaggaca gcacctactc cctgagctcc accctgaccc tgtccaaggc tgattatgag 240

aagcacaagg tgtatgcttg cgaggtgaca caccagggcc tgtccagccc tgtgaccaag 300

agcttcaacc ggggcgagtg ctgataa 327

<210> 18

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 18

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims

1. A ROR1 monoclonal antibody, wherein the ROR1 monoclonal antibody comprises heavy and light chain structures, the heavy chain variable region of the heavy chain and the light chain variable region of the light chain each having 3 complementarity determining region CDRs, wherein: the amino acid sequence of CDR1 of the heavy chain variable region is SEQ ID NO.3, the amino acid sequence of CDR2 of the heavy chain variable region is SEQ ID NO.4, the amino acid sequence of CDR3 of the heavy chain variable region is SEQ ID NO.5, the amino acid sequence of CDR1 of the light chain variable region is SEQ ID NO.6, the amino acid sequence of CDR2 of the light chain variable region is SEQ ID NO.7, and the amino acid sequence of CDR3 of the light chain variable region is SEQ ID NO. 8.

2. The ROR1 monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1.

3. The ROR1 monoclonal antibody of claim 1, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

4. The ROR1 monoclonal antibody of claim 1, wherein the N-terminal of VH of the heavy chain variable region comprises a signal peptide sequence optimized for heavy chain secretory expression, and the C-terminal of VH comprises the human IgG1 constant region sequence CH1-CH 3.

5. The ROR1 monoclonal antibody of claim 1, wherein the N-terminal of the light chain variable region VL is added with a signal peptide sequence optimized for light chain secretory expression, and the C-terminal of VL is added with a human Kappa constant region sequence.

6. The ROR1 monoclonal antibody of claim 1, wherein the amino acid sequences of the heavy chain and the light chain of the monoclonal antibody are shown as SEQ ID No.1 and SEQ ID No.2, respectively.

7. The ROR1 monoclonal antibody of claim 1, wherein the nucleotide sequences of the heavy chain and the light chain of the monoclonal antibody are shown as SEQ ID No.9 and SEQ ID No.10, respectively.

8. The ROR1 monoclonal antibody of claim 1, consisting of the steps of:

9. Use of the ROR1 monoclonal antibody of claim 1 in the preparation of a medicament for the treatment of cancer.

10. The use of claim 9, wherein the cancer comprises chronic granulocytic lymphoma, mantle cell lymphoma, non-small cell lung cancer and breast cancer.