CN111363040A

CN111363040A - anti-OX 40 monoclonal antibodies and uses thereof

Info

Publication number: CN111363040A
Application number: CN201811593852.0A
Authority: CN
Inventors: 黄莺; 张发明; 席甘
Original assignee: Hangzhou Hansi Biomedical Co ltd
Current assignee: Hangzhou Hansi Biomedical Co ltd
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2020-07-03
Anticipated expiration: 2038-12-25
Also published as: CN111363040B

Abstract

The invention provides anti-OX 40 antibodies or antigen-binding fragments thereof, as well as corresponding isolated polynucleotides, expression vectors, recombinant cells, pharmaceutical compositions, and uses thereof. The antibody can be used for treating cancer or autoimmune diseases.

Description

anti-OX 40 monoclonal antibodies and uses thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an anti-OX 40 monoclonal antibody and application thereof.

Background

OX40, also known as CD134 and TNFRSF4, belongs to the Tumor Necrosis Factor Receptor (TNFR) superfamily, and OX40 is expressed only on the surface of activated CD4+ T, CD8+ T cells. Human OX40 is an I-type transmembrane glycoprotein with the molecular mass of 47-51 kDa, and consists of 249 amino acids, wherein the extracellular region, the transmembrane region and the intracellular region respectively consist of 188, 24 and 37 amino acids, and the corresponding genes are positioned on a human chromosome 1p 36. OX40 signals can activate the downstream NF-kB, PI3K and PKB pathways, and the sustained activation of these pathways ultimately can prolong the survival time of T cells, expand T cell memory, and promote the cell killing ability of T cells; in addition, OX40 can also improve immunosuppressive effects in the tumor microenvironment by inhibiting the differentiation and activity of regulatory T cells (tregs), further enhancing the function of effector T cells.

Treatment with OX40 as a target has yet to be further improved.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. To this end, it is an object of the present invention to propose a monoclonal antibody against OX 40. The OX40 antibodies provided by the invention can be used to enhance immune responses to treat cancer and infectious diseases, or as adjuvants for cancer vaccines.

According to a first aspect of the invention, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof, comprising at least one of: (1) a heavy chain variable region having the amino acid sequences GFTFSDYY, ISDGGSNT and ARRGTGTGFGY, and a light chain variable region having the amino acid sequences ENIYST, AAT and QHFWGIPWT; (2) heavy chain variable region with amino acid sequences of GYTFTNYD, IYPEDGST and ARDTRGYFDY, and light chain variable region with amino acid sequences of SSVNY, YTS and QQFTSSPWT; (3) a heavy chain variable region having the amino acid sequence GDSITSGY, ISFSGNT and ARYPYSYSNWDYAMDY; and a light chain variable region having amino acid sequences QFLLYSSSQKNY, WAS and HQYYSYPLT; (4) a heavy chain variable region having amino acid sequences gdsiigf, inhsgs and ARSGTDLDY, and a light chain variable region having amino acid sequences QSLLDSDGKTY, LVS and WQGTHFPRT; (5) an amino acid sequence having one or more conservative amino acid mutations as compared with (1) to (4). According to an embodiment of the present invention, the amino acid sequence having one or more conservative amino acid mutations compared to (1) to (4) is preferably an amino acid sequence having one conservative amino acid mutation, an amino acid sequence having two conservative amino acid mutations, or an amino acid sequence having three conservative amino acid mutations.

According to embodiments of the invention, the antibody or antigen-binding fragment thereof may further comprise: (a) a heavy chain having an amino acid sequence shown in SEQ ID NO. 1 and a light chain having an amino acid sequence shown in SEQ ID NO. 2; (b) a heavy chain having an amino acid sequence shown in SEQ ID No. 3 and a light chain having an amino acid sequence shown in SEQ ID No. 4; (c) a heavy chain having an amino acid sequence shown in SEQ ID NO. 5 and a light chain having an amino acid sequence shown in SEQ ID NO. 6; (d) a heavy chain having the amino acid sequence shown in SEQ ID NO. 7 and a light chain having the amino acid sequence shown in SEQ ID NO. 8; (e) has one or more conservative amino acid mutations compared with the amino acid sequences of (a) to (d) above except for the heavy chain variable region and the light chain variable region. According to an embodiment of the present invention, the amino acid sequence having one or more conservative amino acid mutations compared to the amino acid sequences of (a) to (d) other than the heavy chain variable region and the light chain variable region may have one conservative amino acid mutation, two conservative amino acid mutations, or three conservative amino acid mutations.

According to an embodiment of the invention, the antibody is a monoclonal antibody.

According to a second aspect of the invention, there is provided an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of the first aspect of the invention.

According to an embodiment of the invention, the polynucleotide has a nucleotide sequence of one of: the nucleotide sequence shown in SEQ ID No. 9 and the nucleotide sequence shown in SEQ ID No. 10; the nucleotide sequence shown in SEQ ID NO. 11 and the nucleotide sequence shown in SEQ ID NO. 12; the nucleotide sequence shown in SEQ ID NO. 13 and the nucleotide sequence shown in SEQ ID NO. 14; the nucleotide sequence shown in SEQ ID NO. 15 and the nucleotide sequence shown in SEQ ID NO. 16;

according to a third aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the second aspect of the invention. The expression vector comprising the polynucleotide may be a plasmid, for example, a double stranded DNA loop capable of ligating the polynucleotide into a loop, for example, therein; the expression vector may also be a viral vector, and the polynucleotide may be ligated into the viral genome. Certain expression vectors are capable of autonomous replication when introduced into a host cell, and certain expression vectors may be integrated into the genome of a host cell upon introduction into the host cell and thereby replicate in conjunction with the host genome.

According to an embodiment of the present invention, the above-mentioned expression vector may be further added with the following technical features:

in some embodiments of the invention, the expression vector further comprises: a control element operably linked to the polynucleotide for controlling expression of the polynucleotide in a host cell. "operably linked" refers to the attachment of a foreign gene to a vector such that control elements, e.g., transcriptional and translational control sequences, within the vector are capable of performing their intended function of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the light and heavy chains of the antibody may be inserted into separate vectors, more typically into the same vector.

In some embodiments of the invention, the control element comprises at least one of: promoters, enhancers and terminators.

In some embodiments of the invention, the host cell is a mammalian cell.

According to a fourth aspect of the present invention there is provided a recombinant cell comprising an expression vector according to any one of the embodiments of the third aspect of the present invention. Herein, the recombinant cell refers to a cell into which an expression vector is introduced. It is understood that the recombinant cell refers not only to the cell of a particular subject, but also to the progeny of such a cell. Such progeny cells may not in fact be identical to the parent cell, but are still included within the scope of the recombinant cells referred to herein, since some variation may occur in subsequent passages due to mutation or environmental influences.

According to a fifth aspect of the invention there is provided a method of preparing an antibody or antigen-binding fragment thereof according to any one of the embodiments of the first aspect of the invention. The method comprises culturing a recombinant cell according to the fourth aspect of the invention.

According to a sixth aspect of the invention, there is provided the use of a polynucleotide, expression vector, or recombinant cell in the preparation of an antibody, or antigen-binding fragment thereof, which antibody specifically binds to OX 40. According to an embodiment of the invention, the polynucleotide is a polynucleotide according to the second aspect of the invention, the expression vector is an expression vector according to the third aspect of the invention, and the recombinant cell is a recombinant cell according to the fourth aspect of the invention.

According to a seventh aspect of the present invention, there is provided a hybridoma cell selected from at least one of: the hybridoma HX011-9C12 with the preservation number of C2018197 is preserved in the China center for type culture Collection with the preservation time of 09 and 27 days in 2018; the hybridoma cell HX011-1D9 with the preservation number of C2018198 is preserved in the China center for type culture Collection with the preservation time of 09 and 27 days in 2018.

According to an eighth aspect of the invention, there is provided use of a hybridoma cell according to the seventh aspect of the invention for the preparation of a monoclonal antibody to OX 40.

According to a ninth aspect of the invention, there is provided the use of an antibody or antigen binding fragment, polynucleotide, expression vector, recombinant cell or hybridoma cell in the manufacture of a medicament for the treatment of an autoimmune disease or cancer. The drug can be used to inhibit the growth of tumor cells, including but not limited to cells of breast cancer, prostate cancer, colorectal cancer, or B-cell lymphoma. Furthermore, the medicament can be used for treating autoimmune diseases, including but not limited to rheumatoid arthritis, multiple sclerosis, diabetes, Crohn's disease, inflammatory bowel syndrome, ulcerative colitis, celiac disease, psoriasis, proliferative lupus nephritis, granulomatous myopathy, polymyositis, and the like.

According to an embodiment of the invention, the antibody or antigen-binding fragment is an antibody or antigen-binding fragment according to the first aspect of the invention, the polynucleotide is a polynucleotide according to the second aspect of the invention, the expression vector is an expression vector according to the third aspect of the invention, the recombinant cell is a recombinant cell according to the fourth aspect of the invention, and the hybridoma cell is a hybridoma cell according to the seventh aspect of the invention.

According to a tenth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment, polynucleotide, expression vector, recombinant cell or hybridoma cell. According to an embodiment of the invention, the antibody or antigen-binding fragment is an antibody or antigen-binding fragment according to the first aspect of the invention, the polynucleotide is a polynucleotide according to the second aspect of the invention, the expression vector is an expression vector according to the third aspect of the invention, the recombinant cell is a recombinant cell according to the fourth aspect of the invention, and the hybridoma cell is a hybridoma cell according to the seventh aspect of the invention.

The anti-OX 40 antibodies provided herein can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, these pharmaceutical compositions include an anti-OX 40 antibody provided herein and a pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, and the like, and combinations thereof. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the pharmaceutical composition. Of course, the pharmaceutically acceptable carrier may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, to prolong the shelf life or effectiveness of the antibody.

For example, the antibodies of the invention can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms. Such as liquid, semi-solid, and solid dosage forms, and the like, including, but not limited to, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions. The antibody may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

According to an eleventh aspect of the invention, there is provided a method for identifying an agent capable of binding to OX40, comprising: contacting the antibody or antigen-binding fragment thereof according to any embodiment of the first aspect of the invention with an antigen in the presence of a candidate agent and confirming a first amount of binding of the antibody or antigen-binding fragment thereof to the antigen; and contacting the antibody or antigen-binding fragment thereof according to any one of the embodiments of the first aspect of the invention with an antigen in the absence of the drug candidate and determining a second amount of binding of the antibody or antigen-binding fragment thereof to the antigen; wherein the antigen is OX40 or a fragment thereof, and the second amount of binding being greater than the first amount of binding is indicative that the candidate drug is capable of binding to OX 40. A drug capable of binding to OX40 can be identified by determining the amount of antibody bound to antigen by detection in the absence of the drug candidate, then adding the drug candidate, determining the amount of antibody bound to antigen by detection, comparing the difference between the two determined amounts of binding, and if the amount of antibody bound to antigen decreases when the drug candidate is added, indicating that the drug candidate is capable of competitively binding to OX40 antigen.

According to a twelfth aspect of the invention, there is provided a pharmaceutical combination comprising: (1) an antibody or antigen binding fragment, a polynucleotide, an expression vector, a recombinant cell, a hybridoma cell; and (2) an immunopotentiating agent different from (1), wherein the antibody or antigen-binding fragment is the antibody or antigen-binding fragment according to the first aspect of the present invention, the polynucleotide is the polynucleotide according to the second aspect of the present invention, the expression vector is the expression vector according to the third aspect of the present invention, the recombinant cell is the recombinant cell according to the fourth aspect of the present invention, and the hybridoma cell is the hybridoma cell according to the seventh aspect of the present invention.

According to an embodiment of the present invention, the immunopotentiating drug different from (1) is selected from at least one of: an anti-PD 1 antibody, a LAG-3 antibody, a CTLA-4 antibody, a Tim3 antibody, or a PD-L1 antibody. These immunopotentiating drugs can bind to specific targets, and combination therapy can exert therapeutic effects at lower doses by combining or combining these immunopotentiating drugs with anti-OX 40 antibodies.

According to a thirteenth aspect of the invention, there is provided a method for treating cancer, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof according to any one of the first aspects of the invention.

According to an embodiment of the invention, in the method for treating cancer described above, the cancer is selected from the group consisting of: breast cancer, prostate cancer, ovarian cancer, colorectal cancer, or B-cell lymphoma.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

fig. 1 is a graph of the results of the kinetic parameters of the 9C12 antibody provided in accordance with an embodiment of the present invention.

Figure 2 is a graph of the results of the kinetic parameters of the 8a7 antibody provided in accordance with an embodiment of the present invention.

FIG. 3 is a graph of species-specific binding ELISA results provided in accordance with an embodiment of the present invention.

FIG. 4 is a graph showing the results of FACS methods provided according to the present invention to detect the binding activity of 9C12 to OX 40.

FIG. 5 is a graph of the results of OX40 antibody stimulating IFN- γ secretion by primary CD4+ T cells, according to an embodiment of the invention.

FIG. 6 is a graph showing the results of OX40 antibody activity assay by Jurkat-NF kappa B-OX40 reporter gene method of OX40 provided in accordance with an example of the present invention.

FIG. 7 is a graph showing the change in tumor-bearing volume of experimental animals according to an embodiment of the present invention.

Biological preservation

The hybridoma HX011-9C12 with the preservation number of C2018197 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation time of 09 months and 27 days in 2018 and the addresses of: wuhan university in Wuhan City, China.

The hybridoma cell HX011-1D9 with the preservation number of C2018198 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation time of 09 months and 27 days in 2018 and the addresses of: wuhan university in Wuhan City, China.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

To enhance the therapeutic effect of OX40 as a target, the invention provides an anti-OX 40 antibody and the use of the antibody in drug therapy. Autoimmune diseases or cancer can be treated by the anti-OX 40 antibodies provided by the invention. Herein, an anti-OX 40 antibody may also be expressed as an OX40 antibody or may also be expressed as an isolated anti-OX 40 antibody, as desired.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, including, but not limited to, monoclonal antibodies produced by hybridoma or recombinant DNA methods. An "anti-OX 40 antibody" is also sometimes referred to herein as an "OX 40 antibody".

As used herein, the term "antibody" is an immunoglobulin molecule consisting of two heavy chains and two light chains, which are linked internally by four polypeptide chains via disulfide bonds. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region. Each light chain is composed of a light chain variable region and a light chain constant region. The heavy chain variable region and the light chain variable region may further include complementarity determining regions, also known as hypervariable regions or hypervariable regions (CDRs).

The term "antigen-binding fragment" of an antibody includes antibody fragments that retain the ability to specifically bind to antigen (hOX 40).

In particular, the cancer described herein may be lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, hematologic cancer, or any other disease or disorder characterized by uncontrolled cell growth.

Herein, the autoimmune disease may be rheumatoid arthritis, multiple sclerosis, diabetes, crohn's disease, inflammatory bowel disease, ulcerative colitis, celiac disease, psoriasis, proliferative lupus nephritis, granulomatous myopathy, multiple myositis, and the like.

According to one aspect of the invention, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising at least one of: (1) a heavy chain variable region having the amino acid sequences GFTFSDYY, ISDGGSNT and ARRGTGTGFGY, and a light chain variable region having the amino acid sequences ENIYST, AAT and QHFWGIPWT; (2) heavy chain variable region with amino acid sequences of GYTFTNYD, IYPEDGST and ARDTRGYFDY, and light chain variable region with amino acid sequences of SSVNY, YTS and QQFTSSPWT; (3) a heavy chain variable region having the amino acid sequence GDSITSGY, ISFSGNT and ARYPYSYSNWDYAMDY; and a light chain variable region having amino acid sequences QFLLYSSSQKNY, WAS and HQYYSYPLT; (4) a heavy chain variable region having amino acid sequences gdsiigf, inhsgs and ARSGTDLDY, and a light chain variable region having amino acid sequences QSLLDSDGKTY, LVS and WQGTHFPRT; (5) an amino acid sequence having one or more conservative amino acid substitutions as compared with (1) to (4).

Where "conservative amino acid substitution" is one in which the amino acid is substituted with another amino acid residue that is biologically, chemically or structurally similar. Biologically similar means that the substitution does not destroy the biological activity of OX 40. Structurally similar refers to amino acids having side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity refers to amino acids that are identically charged or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine. Or substitution of polar amino acids such as lysine with arginine, aspartic acid with glutamic acid, asparagine with glutamine, threonine with serine, and the like.

According to an embodiment of the invention, the invention provides an anti-OX 40 antibody having a heavy chain variable region with three hypervariable regions comprising the amino acid sequences: CDRH1(GFTFSDYY), CDRH2 (isddgnt), CDRH3 (ARRGTGTGFGY); the light chain variable region has three hypervariable regions which comprise the amino acid sequences: CDRL1(ENIYST), CDRL2(AAT), CDRL3 (QHFWGIPWT).

In a specific embodiment of the invention, the antibody is 9C12, and the heavy chain amino acid sequence (SEQ ID NO:1) is: q V Q L Q Q S G G G L V K P G G S L K L S C A A SG F T F S D Y YM YW V R Q T P E K R L E W V A TI S D G G S N TY Y P D S V K G R F T I S R D NA K N N L Y L Q M S S L K S E D T A T Y Y CA R R G T G T G F G YW G Q G T LV T V S A, respectively; its light chain amino acid sequence (SEQ ID NO: 2): d I V L T Q T T AS L S V S V G E T VT I T C R A SE N I Y S TL A W Y Q Q K Q G K S P Q L L V YA A TN L V AG VP S R F S G S G S G T Q Y S L K I N S L Q S E D F G S Y Y CQ H F W G I P W TF G G G T K L E I K are provided. The hypervariable region sequences on the antibody heavy and light chains are underlined.

According to a particular embodiment of the invention, the nucleotide sequence encoding the amino acid sequence of the heavy chain shown in SEQ ID NO:1 (SEQ ID NO:9) is:

CAAGTTCAGCTGCAGCAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAATGGGTCGCAACCATTAGTGATGGTGGAAGTAACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCCGAGGACACAGCCACATATTACTGTGCAAGACGAGGGACTGGGACGGGGTTTGGTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA；

according to a specific embodiment of the present invention, the nucleotide sequence encoding the light chain amino acid sequence shown in SEQ ID NO. 2 (SEQ ID NO:10) is:

GACATTGTGCTGACCCAGACTACAGCCTCCCTATCTGTTTCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTACTTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCTGCAACAAACTTAGTAGCTGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTGCAGTCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGGGTATTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

according to an embodiment of the invention, the invention provides a further anti-OX 40 antibody having a heavy chain variable region with three hypervariable regions comprising the amino acid sequence: CDRH1(GYTFTNYD), CDRH2(IYPEDGST), CDRH3(ARDTRGYFDY), the light chain variable region of which has three hypervariable regions comprising the amino acid sequence: CDRL1(SSVNY), CDRL2(YTS), CDRL3 (QQFTSSPWT).

In one embodiment of the invention, the antibody is 1D9, having a heavy chain amino acid sequence (SEQ ID NO: 3): e V Q L Q Q S G P E L V K P G A L V K I S C K A SG Y T F T N Y DI N WV K Q R P G Q G L E W I G WI Y P E D G S TK Y N E K F K G K A T L T A D K SS S T A Y M Q L S S L T S E N S A V Y F CA R D T R G Y F D YW G Q G T T L TV S S, respectively; the light chain amino acid sequence (SEQ ID NO:4) is: d I V M T Q T T A I M S A S L G E K V TM S C R A SS S V N YI Y W Y Q Q K S D A S P K L W I YY T SN L A P G V P AR F S G S G S G N S Y S L T I S S M E G E D A A T Y Y CQ Q F T S S P W TF GG G T K L E F K R are provided. The hypervariable region sequences on the antibody heavy and light chains are underlined.

According to an embodiment of the present invention, the nucleotide sequence encoding the heavy chain amino acid sequence shown in SEQ ID NO. 3 (SEQ ID NO:11) is:

GAGGTTCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAACCTGGGGCTTTAGTGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACAAACTACGATATAAACTGGGTGAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGAAGATGGTAGTACTAAGTACAATGAGAAATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACTTCTGAGAACTCTGCAGTCTATTTCTGTGCAAGAGACACACGTGGCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA；

according to an embodiment of the present invention, the nucleotide sequence encoding the light chain amino acid sequence shown in SEQ ID NO. 4 (SEQ ID NO:12) is:

GACATTGTGCTCACACAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTAAATTACATATACTGGTACCAGCAGAAGTCAGATGCCTCCCCCAAACTCTGGATTTATTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCAGTTTACTAGTTCCCCATGGACGTTCGGTGGAGGCACCAAGCTGGAATTCAAACGG。

according to an embodiment of the invention, there is provided another anti-OX 40 antibody having a heavy chain variable region with three hypervariable regions comprising the amino acid sequences: CDRH1(GDSITSGY), CDRH2(ISFSGNT), CDRH3(ARYPYSYSNWDYAMDY), the light chain variable region of which has three hypervariable regions comprising the amino acid sequence: CDRL1(QFLLYSSSQKNY), CDRL2(WAS), CDRL3 (HQYYSYPLT).

In one embodiment of the invention, the antibody is 8A7, and the heavy chain amino acid sequence (SEQ ID NO:5) is: q V Q L Q E S G P S H V K P S Q T L S L T C S V TG D S I T S G YW NW I R K F P G N K L E Y L G YI S F S G N TY Y N P S L K S R I S I I R D T SK N Q Y Y L Q L N S V T T E D T A T Y Y CA R Y P Y S Y S N W D Y A M D YW GQ G T S V T V S S, respectively; the light chain amino acid sequence (SEQ ID NO:6) is: d I V M T Q S P S S L V V SV G E K V T M S C K S SQ F L L Y S S S Q K N YL A W Y Q Q K P G Q S P Q L LI YW A ST R E S G V P D R F T G S G S G T D F T L T I S S V K A E D L A V YY CH Q Y Y S Y P L TF G A G T K L E L K are provided. The hypervariable region sequences on the antibody heavy and light chains are underlined.

According to a specific embodiment of the present invention, the nucleotide sequence capable of encoding the heavy chain amino acid sequence shown in SEQ ID NO. 5 (SEQ ID NO:13) is:

CAGGTTCAGCTGCAAGAGTCAGGACCTAGCCACGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGAAATAAACTTGAGTATTTGGGGTACATAAGCTTCAGTGGTAACACTTACTACAATCCATCTCTCAAAAGTCGAATCTCCATCATTCGAGACACATCCAAGAACCAGTATTATTTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATATCCTTACTCCTATAGTAACTGGGACTATGCTATGGACTACTGGGGTCAAGGAACTTCAGTCACCGTCTCCTCA；

according to a specific embodiment of the present invention, the nucleotide sequence capable of encoding the light chain amino acid sequence shown in SEQ ID NO. 6 (SEQ ID NO:14) is:

GATATTGTGATGACACAATCTCCATCCTCCCTAGTTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGTTCCTTTTATATAGTAGCAGTCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTCAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCACCAATATTATAGCTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA。

according to an embodiment of the invention, the invention provides a further anti-OX 40 antibody having a heavy chain variable region with three hypervariable regions comprising the amino acid sequence: CDRH1 (gdsiigf), CDRH2 (inhsgs), CDRH3(ARSGTDLDY), the light chain variable region of which has three hypervariable regions comprising the amino acid sequence: CDRL1(QSLLDSDGKTY), CDRL2(LVS), CDRL3 (WQGTHFPRT).

In one embodiment of the invention, the antibody is 9D7, having a heavy chain amino acid sequence (SEQ ID NO: 7): e V K L E E S G P S L V K P S Q T L S L T C S V SG D S I T I G FW N WI R K F P G N K L E Y M G YI N Y S G S SY Y N P S L K S R I S I T R D T S KN Q Y Y L Q L N S V T P E D T A T Y Y CA R S G T D L D YW G Q G T T L T V SS, respectively; its light chain amino acid sequence (SEQ ID NO: 8): d I V L T Q S P L T L S V T I G Q P A S I S CK S SQ S L L D S D G K T YL N W L L Q R P G Q S P K R L I YL V SK L D S GV P D R F T G S G S G T D F T L K I S R V E A E D L G V Y Y CW Q G T H F P R TF G G G T K L E I K R are provided. The hypervariable region sequences on the antibody heavy and light chains are underlined.

According to an embodiment of the invention, the nucleotide sequence encoding the heavy chain amino acid sequence shown in SEQ ID NO. 7 (SEQ ID NO:15) is:

GAGGTGAAGCTGGAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCTCTGGCGACTCCATCACCATTGGTTTCTGGAACTGGATCCGGAAATTCCCAGGAAATAAACTTGAGTACATGGGATACATAAACTACAGTGGTAGCAGTTACTACAATCCATCTCTCAAAAGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTATTACCTGCAGTTGAATTCTGTGACTCCTGAGGACACAGCCACATATTACTGTGCAAGATCTGGGACGGACCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA；

according to an embodiment of the present invention, the nucleotide sequence encoding the light chain amino acid sequence shown in SEQ ID NO. 8 (SEQ ID NO:16) is:

GATATTGTGCTCACACAGTCTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG。

according to an embodiment of the present invention, the anti-OX 40 antibody may also be a sequence having 90% or more homology, preferably 95% or more homology, more preferably 98% or more homology, and more preferably 99% or more homology to the amino acid sequences of antibodies 9C12, 1D9, 8a7, and 9D 7. For example, may exhibit one conservative amino acid substitution, two conservative amino acid substitutions, or three conservative amino acid substitutions, or even more, as compared to the amino acid sequence of antibodies 9C12, 1D9, 8a7, and 9D 7.

The anti-OX 40 antibodies herein can also be made part of a kit or other diagnostic reagent as desired. The kit may comprise any one or more of the following: an antagonist, an anti-OX 40 antibody, or a drug reference material; a protein purification column; an immunoglobulin affinity purification buffer; an assay diluent for the cells; the specification or literature, and the like. anti-OX 40 antibodies can be used in different types of diagnostic tests, e.g., to detect a wide variety of diseases or the presence of drugs, toxins, or other proteins, etc., in vitro or in vivo. For example, the test may be performed by testing the serum or blood of the subject for the relevant disease. Such related diseases may include OX40 related diseases, such as various cancers, inflammatory conditions, or autoimmune diseases, among others. Of course the antibodies provided herein may also be used for radioimmunoassay and radioimmunotherapy of cancer and the like.

The following embodiments of the present invention are described in detail, and it should be noted that the following embodiments are exemplary only, and are not to be construed as limiting the present invention. In addition, all reagents used in the following examples are commercially available or can be synthesized according to texts or known methods, and are readily available to those skilled in the art for reaction conditions not listed, if not explicitly stated.

EXAMPLE 1 establishment of OX40 hybridoma cell line

According to the full-length amino acid sequence of hOX40, a human fusion protein hOX40-HIS having the following amino acid sequence was prepared according to a molecular biological method:

LSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAHHHHHH(SEQ ID NO:17)。

then, the prepared hOX40-HIS fusion protein is used as an antigen, an adjuvant is added, and a mouse is immunized, so that a monoclonal antibody capable of being specifically combined with the hOX40 protein and a hybridoma cell line are obtained. The method comprises the following steps:

1. mouse immunization and cell fusion

The prepared hOX40-HIS fusion protein was used as an antigen, and dissolved in PBS to obtain a protein solution. Taking a proper amount of protein solution, uniformly mixing and emulsifying by using Freund's complete adjuvant (Sigma), taking a BALB/c female mouse with the age of 4 weeks, and injecting antigen subcutaneously at multiple points. After 2 weeks, an appropriate amount of the protein solution was mixed with Freund's incomplete adjuvant (Sigma) and emulsified, and then a second immunization was performed subcutaneously on the mice. The third and fourth immunizations were performed every 3 weeks thereafter. 3 days after the fourth immunization, spleens were taken for cell fusion.

When the fused cells are cultured to the 5 th to 10 th days, the culture supernatant with the cloned cell clusters appearing in the holes of the 96-hole culture plate is sucked to detect the content of the antibody by an enzyme-linked immunosorbent assay method, and cell strains with high titer and high specificity are screened according to the secretion condition of the antibody.

The cell lines obtained by screening were subcloned to screen for monoclonal stable cell lines secreting a protein that specifically binds to hOX 40.

After obtaining the stable cell strain, culturing the stable cell strain by using 10% low IgG fetal calf serum, collecting cell culture supernatant after culturing for 7-10 days, and purifying the antibody by using a Hi-Trap protein A HP (GE Healthcare) affinity chromatography column to obtain the corresponding hybridoma antibody. The purified hybridoma antibodies were used directly in subsequent experiments.

The hybridoma antibody comprises the following four types of antibodies through amino acid sequence determination, wherein the four types of antibodies are respectively as follows:

the first one is: antibody 9C12, whose heavy chain amino acid sequence is SEQ ID NO. 1 and light chain amino acid sequence is SEQ ID NO. 2;

the second method is as follows: antibody 1D9, whose heavy chain amino acid sequence is SEQ ID NO. 3 and light chain amino acid sequence is SEQ ID NO. 4;

the third is: antibody 8A7, whose heavy chain amino acid sequence is SEQ ID NO. 5 and light chain amino acid sequence is SEQ ID NO. 6;

the fourth method is as follows: antibody 9D7, whose heavy chain amino acid sequence is SEQ ID NO. 7 and light chain amino acid sequence is SEQ ID NO. 8.

EXAMPLE 2 obtaining cDNA sequence of hOX40 hybridoma cell line

Total RNA from hybridoma cell lines was extracted by Trizol (Invitrogen) method, and then Reverse transcribed into cDNA using oligo-dT primer and SuperScript II Reverse Transcriptase (lnvirogen, cat # 18064-014). DNA fragments of the heavy and light chains of the different antibodies of example 1 were obtained by PCR amplification, ligated to cloning vectors, and clones were picked for sequencing. And comparing the sequencing results to obtain the correct sequence.

Wherein, the heavy chain nucleotide sequence of the antibody 9C12 obtained by PCR amplification is SEQ ID NO. 9; the light chain nucleotide sequence is SEQ ID NO. 10;

the heavy chain nucleotide sequence of the antibody 1D9 obtained by PCR amplification is SEQ ID NO. 11, and the light chain nucleotide sequence is SEQ ID NO. 12;

the heavy chain nucleotide sequence of the antibody 8A7 obtained by PCR amplification is SEQ ID NO. 13; the light chain nucleotide sequence is SEQ ID NO. 14;

the heavy chain nucleotide sequence of the antibody 9D7 obtained by PCR amplification is SEQ ID NO. 15; the light chain nucleotide sequence is SEQ ID NO 16.

Example 3 detection of hOX 40-specific antibodies by ELISA method

The specific binding of different antibodies to hOX40 was detected for the hybridoma antibodies prepared in example 1 by the following steps:

1) coating antigen: preparing hOX40-HIS antigen with concentration of 0.6 μ g/ml and 100 μ l/well, and coating overnight at 4 deg.C;

2) blocking with 1% BSA (PBS dilution) for 2 hours at 37 deg.C, washing 3 times with 1 × PBST (Tween-20, 1%), patting gently dry;

3) adding a primary antibody: respectively preparing the concentrations of the four antibodies into 2 mu g/ml, taking 2 mu g/ml as an initial concentration, diluting the four antibodies according to a 1:5 gradient to 7 gradient concentrations (namely the concentrations are respectively 2 mu g/ml, 400ng/ml, 80ng/ml, 16ng/ml, 3.2ng/ml, 0.64ng/ml and 0.128ng/ml), taking a blank control group as PBS, respectively adding the prepared primary antibodies, and incubating the mixture for 1 hour at 37 ℃;

4) adding a secondary antibody: PBST is washed for 3 times, gently patted dry, added with HRP enzyme-labeled goat anti-mouse IgG (H + L) secondary antibody diluted by 1:5000, each well is 100 mul, incubated for 1 hour at 37 ℃;

5) color development: PBST was washed 3 times, patted dry gently; adding a TMB color developing agent, reacting for 5-15 min at room temperature, wherein each hole is 100 mu l;

6) and (3) color development termination: 2M H was added at 50. mu.l/well₂SO₄Terminating the color development reaction by the solution;

7) reading: the absorbance of each well was measured on a microplate reader using absorbance 450 nm.

The ELISA test results are shown in Table 1. Wherein, EC₅₀The values represent the half maximal effect concentrations, i.e. the concentrations that are capable of causing 50% of the maximal effect. As can be seen from Table 1, all 4 monoclonal antibodies specifically bound to hOX40, binding force ranged from 10.8nM to 0.017 nM. And the effect of specific binding of the 9C12 antibody, the 8a7 antibody and the 9D7 antibody was better compared to the 1D9 antibody.

TABLE 1 monoclonal antibody binding to hOX40 ELISA results

Example 4 determination of OX40 antibody kinetic parameters using a ProteOn XPR molecular interactor

The kinetics and affinity constants of the interaction between different drugs and various target proteins can be detected by using a ProteOn XPR molecular interaction instrument. 250nM of hOX40-HIS antigen (histone modified hOX40 antigen) was immobilized on the surface of the GLM chip, after equilibration in PBST, bound to antibody 9C12, 9C12 antibody was diluted twice with PBST at concentrations of 50, 25, 12.5, 6.25, 3.125, 0nM, respectively, and dissociated in PBST. The 8A7 antibody was detected by the same detection method, and the concentrations of the 8A7 antibody were 308, 154, 77, 38.5, 19.25, and 0nM, respectively.

The kinetic parameters of 9C12 and 8A7 are shown in Table 2, and the results are shown in FIG. 1 and FIG. 2.

TABLE 2 kinetic parameters of different antibodies

Wherein ka (1/Ms) in Table 2 represents an association rate constant, KD represents an dissociation rate constant, KD represents an equilibrium constant, Rmax represents a maximum binding capacity of a chip surface, Chi²Representative is the chi-squared check value. Wherein the association rate constant and the dissociation rate constant represent the ease of binding of the antigen to the antibody.

From the above values, it can be seen that the 9C12 antibody binds more readily to the hOX40-HIS antigen than the 8a7 antibody.

Example 5 species-specific study of OX40 antibodies

Respectively coating human origin OX40 antigen, monkey origin OX40 antigen and murine origin OX40 antigen, and detecting whether OX40 antibody cross reacts with OX40 protein of different species or not, the specific steps are as follows:

1) coating antigen: respectively preparing human OX40 antigen, monkey OX40 antigen and murine OX40 antigen, wherein the concentrations of the human OX40 antigen, the monkey OX40 antigen and the murine OX40 antigen are 0.25 mu g/ml and 100 mu l/hole, and coating the mixture overnight at 4 ℃;

2) blocking with 1% BSA (PBS dilution) for 2 hours at 37 deg.C, washing 3 times with 1 × PBST (Tween-20, 1%), tapping dry;

3) adding a primary antibody: preparing the concentration of the antibody 9C12 to be 2 mu g/ml, then diluting the solution by taking 2 mu g/ml as an initial concentration according to a 1:5 gradient to be 7 gradient concentrations (namely, the concentrations are respectively 2 mu g/ml, 400ng/ml, 80ng/ml, 16ng/ml, 3.2ng/ml, 0.64ng/ml and 0.128ng/ml), adding the prepared primary antibody into a blank control group which is PBS, and then respectively adding the prepared primary antibody to incubate for 1 hour at 37 ℃;

The results are shown in table 3 and fig. 3, and 9C12 antibody binds to human OX40 antigen, monkey OX40 antigen with comparable affinity and does not cross-react with murine OX40 antigen.

Table 3: species specific binding ELISA results plot

Example 6 detection of binding Activity of 9C12 with hOX40 Using FACS (flow cytofluorimetric sorting) method

In the experiment, 293T-OX40 stable transfected cell line cells are used as experimental cells, and the combination condition of 9C12 and hOX40 protein on the surface of a cell membrane is detected by a FACS method. The specific method comprises the following steps:

293T cells expressing hOX40 were digested to a final concentration of 10⁶Cells/ml. 100. mu.l of each cell group was mixedThe suspension was placed in a 1.5ml EP tube. The prepared 9C12 antibody (10000ng/ml, 4000ng/ml, 2000ng/ml, 1000ng/ml, 500ng/ml, 200ng/ml, 20ng/ml, 2ng/ml) was added at each concentration and incubated on ice for 1 hour. After 1 hour, wash once with HBSS. Adding prepared APC goat anti-human IgG secondary antibody into each group, incubating for 20min on ice in dark place, washing with HBSS once, finally suspending with 200 μ l HBSS, and detecting on cell machine.

The binding activity of 9C12 to hOX40 on the cell membrane surface was measured by FACS method, and the results are shown in FIG. 4 and Table 4 below.

TABLE 4 results of FACS method for measuring binding activity of 9C12 to hOX40

As shown in FIG. 4 and the statistics in the table above, the binding EC of 9C12 antibody to hOX40 on the cell membrane surface is shown₅₀It was 2.84 nM.

Example 7 study of OX40 antibodies to costimulate human primary CD4+ T cells in vitro

40ml of fresh human blood was collected, human PBMC was separated using lymphocyte separation medium, CD14 monocytes were first positive-selected using CD14 magnetic beads (Miltenyi, cat # 130-. CD4+ T cells were supplemented with 2. mu.g/ml PHA-L (Sigma) and 200IU/ml human recombinant IL-2 (R)&D) Complete culture medium with PBMC (90% RPMI 1640(Hyclone) + 10% FBS (Sijiqing)), 1X10⁶The cells/ml were plated in six well plates, charged with 5% CO at 37 ℃₂The culture was carried out in an incubator for 48 hours.

The next day, 100. mu.l of PBS solution (Hyclone) containing 2. mu.g/ml of goat anti-mouse Fc γ -specific IgG (Jackson) (or 2. mu.g/ml of goat anti-mouse Fc γ -specific IgG plus 2. mu.g/ml of goat anti-human Fc γ -specific IgG (Jackson)) was added to each well of the 96-well plate. Placing in refrigerator, and coating at 4 deg.C overnight. The following day the supernatant was removed and washed with PBS. Mu.l of PBS containing 1% BSA was added to each well, and the mixture was incubated at 37 ℃ with 5% CO₂The incubator is sealed for 90 min. After completion, the supernatant was aspirated from each well again, and 200. mu.l of PBS solution was added for washing.

CD4+ T cells were recovered again and centrifuged at 1800rpm for 5min before resuspension in PBMC complete medium, adjusting the cell concentration to 1X10⁶Individual cells/ml. Mu.l per well were added to the coated wells and 50. mu.l per well of CD3 antibody OKT3(2ng/ml, Biolegend) and 50. mu.l of different concentrations (10-fold dilution from 1. mu.g/ml to 1ng/ml) of OX40 antibody or 50. mu.l of complete PBMC medium were mixed well. Put in 5% CO at 37 DEG C₂The culture was carried out in an incubator for 72 hours.

The supernatant was harvested after 72 hours, and the expression of IFN-. gamma.in the supernatant was detected using an IFN-. gamma.ELISA detection kit (Dayou, cat # DKW 12-1000-096).

The results are shown in FIG. 5, in which OX40 antibody has the ability to stimulate human primary CD4+ T cells, promoting IFN-. gamma.expression.

Example 8 detection of OX40 antibody bioactivity based on 2-cell luciferase reporter assay

In this example, a set of 2-cell reporter genes were used to perform a biological activity assay to assess the ability of OX40 antibodies to signal through human OX 40. The amount of T cell activation is measured by the overexpression of luciferase that can be produced in response to stimulation of the NF κ B signaling pathway. That is, Jurkat-NF κ B luciferase reporting occurs by stimulation of the NF κ B signaling pathway by the OX40 antibody. Nfkb signaling occurs downstream of OX40 and can be correlated with other measures of T cell activation (e.g., proliferation and cytokine release). The amount of T cell activation can be measured by detecting the amount of luciferase.

The method specifically comprises the following steps:

respectively constructing 293T cells (293T-CD32A) capable of stably expressing CD32A and Jurkat cells (Jurkat-NF kappa B-OX40) capable of stably expressing human OX40 and containing firefly luciferase (firefly luciferase) gene regulated by NF kappa B signal pathway by using lentiviruses, re-suspending the 293T-CD32A cells by using a complete culture medium after trypsinization, and regulating the cell concentration to be 2 × 10⁶Jurkat-NF kappa B-OX40 cells were resuspended in complete medium after centrifugation at 1800rpm for 5min and adjusted to a cell concentration of 2 × 10⁶Individual cells/ml.

In 96 wells, divided in each wellSeparately, 50. mu.l of 293T-CD32A cell and Jurkat-NF kappa B-OX40 cell suspensions were added and mixed. Mu.l of different concentrations of OX40 antibodies (9C12 antibody and 1D9 antibody at concentrations of 10. mu.g/ml, 1. mu.g/ml, 0.1. mu.g/ml, 0.01. mu.g/ml, 0.001. mu.g/ml, respectively) or 100. mu.l of Jurkat cell complete medium containing 1. mu.g/ml puromycin (puromycin) were added and mixed well. Put in 5% CO at 37 DEG C₂The culture was carried out overnight in an incubator for 20 hours.

Centrifuging at 1800rpm the next day for 5min, discarding supernatant, adding 100 μ l of firefly luciferase assay kit (manufacturer: Biyunyan, cat # RG005) lysate into each well, and shaking in a shaker at normal temperature for 30min to release luciferase from cell lysis. After centrifugation at 2000rpm for 5min again, 90. mu.l of supernatant per well was removed and added to a white opaque 96-well plate (Costar). After 100. mu.l of a developing solution was added to each well supernatant, the resulting mixture was placed in a full-function microplate detector (Perkinelmer, model: Envison) to detect the self-luminous intensity.

The results are shown in FIG. 6, and indicate that both the 9C12 antibody and the 1D9 antibody have significant stimulatory effects on Jurkat-NF κ B-OX40 reporter cells expressing human OX 40.

Example 9 anti-tumor Effect of OX40 antibody in the MC38 mouse B-hOX40 humanized mouse model of colorectal cancer

The method comprises the following specific steps:

MC38 cells were murine colon cancer cells derived from C57BL/6 mice, purchased from N.N. Shanghai Biotech, Inc. The B-hOX40 humanized mouse is a mouse model obtained by partially replacing a murine OX40 protein molecule interacted with an OX40L protein molecule in a C57BL/6 mouse body by a human protein through a genetic engineering technology, and is purchased from Jiangsu gene biotechnology limited of the Pogostemon baiyaoshima.

Resuspended MC38 cells in PBS at 5 × 10⁵The cells were inoculated subcutaneously on the right side of B-h OX40 humanized mice at a concentration of 0.1mL, at a volume of 0.1 mL. When the average tumor volume reaches 100-³In the meantime, mice with moderate tumor volume and body weight were selected and distributed into 4 experimental groups on average, 5 mice in each group, and administration was started on the day of grouping, and the specific administration schedule is shown in the following table, wherein PBS was given to G1 group as a control group.

Table 5: design of pharmacodynamics experiment

Note: a: the administration volume is calculated according to the body weight of the experimental animal by 10 mu L/g;

b: BIW refers to 2 administrations per week.

The tumor growth inhibition in tumor-bearing mice of each treatment group is shown in Table 6.

Table 6: tumor growth inhibitory effect

TGI (the tumor growth inhibition value) in Table 6 represents the tumor volume inhibition rate and is used to evaluate the inhibition of tumor growth by drugs in animals. The TGI value is calculated as follows:

TGI value (%) ═ 100% (1-experimental relative tumor volume/control relative tumor volume)%

Wherein the relative tumor volume is the change value of the tumor volume after a period of drug treatment.

Taking group G2 as an example, the TGI value is calculated as follows: the change in tumor volume of the G2 group on day 23 was divided by the change in tumor volume of the control group on day 23 to obtain a difference, and the difference was subtracted from 1 to obtain the TGI value.

As can be seen from table 6, the 8a7 antibody, the 1D9 antibody, or the 9C12 antibody showed good tumor suppression rate. Especially, the 9C12 antibody shows higher tumor inhibition rate and has better treatment effect.

The tumor volumes of tumor-bearing mice, i.e., tumor growth curves of mice treated with different drugs, measured at different times are shown in fig. 7. As can be seen from fig. 7, the treatment with 8a7 antibody administration, or the treatment with 1D9 antibody administration, or the treatment with 9C12 antibody administration all showed significant inhibition of tumor volume in mice compared to the PBS control group. Furthermore, the 1D9 antibody and the 9C12 antibody showed higher tumor suppression effects compared to the 8a7 antibody.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Hangzhou Hansi biomedical Co., Ltd

<120> anti-OX 40 monoclonal antibody and use thereof

<130>PIDC3183947

<160>17

<170>PatentIn version 3.5

<210>1

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> heavy chain amino acid

<400>1

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

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

20 25 30

Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Gly Thr Gly Thr Gly Phe Gly Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210>2

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> light chain amino acids

<400>2

Asp Ile Val Leu Thr Gln Thr Thr Ala Ser Leu Ser Val Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Thr

20 25 30

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

35 40 45

Tyr Ala Ala Thr Asn Leu Val Ala Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>3

<211>117

<212>PRT

<213> Artificial sequence

<220>

<223> heavy chain amino acid

<400>3

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

1 5 10 15

Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Glu Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asp Thr Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210>4

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> light chain amino acids

<400>4

Asp Ile Val Met Thr Gln Thr Thr Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Tyr Thr Ser Asn Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Phe Lys Arg

100 105

<210>5

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> heavy chain amino acid

<400>5

Gln Val Gln Leu Gln Glu Ser Gly Pro Ser His Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Leu

35 40 45

Gly Tyr Ile Ser Phe Ser Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Ile Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Tyr Pro Tyr Ser Tyr Ser Asn Trp Asp Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>6

<211>113

<212>PRT

<213> Artificial sequence

<220>

<223> light chain amino acids

<400>6

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Val Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Phe Leu Leu Tyr Ser

20 25 30

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Gln Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210>7

<211>115

<212>PRT

<213> Artificial sequence

<220>

<223> heavy chain amino acid

<400>7

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

1 510 15

Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Asp Ser Ile Thr Ile Gly

20 25 30

Phe Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Asn Tyr Ser Gly Ser Ser Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu

65 70 75 80

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

85 90 95

Arg Ser Gly Thr Asp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210>8

<211>113

<212>PRT

<213> Artificial sequence

<220>

<223> light chain amino acids

<400>8

Asp Ile Val Leu Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

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

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210>9

<211>354

<212>DNA

<213> Artificial sequence

<220>

<223> heavy chain coding sequence

<400>9

caagttcagc tgcagcagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt gactattaca tgtattgggt tcgccagact 120

ccggaaaaga ggctggaatg ggtcgcaacc attagtgatg gtggaagtaa cacctactat 180

ccagacagtg tgaaggggcg attcaccatc tccagagacaatgccaagaa caacctgtac 240

ctgcaaatga gcagtctgaa gtccgaggac acagccacat attactgtgc aagacgaggg 300

actgggacgg ggtttggtta ctggggccaa gggactctgg tcactgtctc tgca 354

<210>10

<211>321

<212>DNA

<213> Artificial sequence

<220>

<223> light chain coding sequence

<400>10

gacattgtgc tgacccagac tacagcctcc ctatctgttt ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtga gaatatttac agtactttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctatgct gcaacaaact tagtagctgg tgtgccatca 180

aggttcagtg gcagtggatc aggcacacag tattccctca agatcaacag cctgcagtct 240

gaagattttg ggagttatta ctgtcaacat ttttggggta ttccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210>11

<211>351

<212>DNA

<213> Artificial sequence

<220>

<223> heavy chain coding sequence

<400>11

gaggttcagc tgcagcagtc tggacctgag ttggtgaaac ctggggcttt agtgaagata 60

tcctgcaagg cttctggtta caccttcaca aactacgata taaactgggt gaaacagagg 120

cctggacagg gacttgagtg gattggatgg atttatcctg aagatggtag tactaagtac 180

aatgagaaat tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240

atgcagctca gcagcctgac ttctgagaac tctgcagtct atttctgtgc aagagacaca 300

cgtggctact ttgactactg gggccaaggc accactctca cagtctcctc a 351

<210>12

<211>321

<212>DNA

<213> Artificial sequence

<220>

<223> light chain coding sequence

<400>12

gacattgtgc tcacacagtc tccagcaatc atgtctgcat ctctagggga gaaggtcacc 60

atgagctgca gggccagctc aagtgtaaat tacatatact ggtaccagca gaagtcagat 120

gcctccccca aactctggat ttattacaca tccaacctgg ctcctggagt cccagctcgc 180

ttcagtggca gtgggtctgg gaactcttat tctctcacaa tcagcagcat ggagggtgaa 240

gatgctgcca cttattactg ccagcagttt actagttccc catggacgtt cggtggaggc 300

accaagctgg aattcaaacg g 321

<210>13

<211>366

<212>DNA

<213> Artificial sequence

<220>

<223> heavy chain coding sequence

<400>13

caggttcagc tgcaagagtc aggacctagc cacgtgaaac cttctcagac tctgtccctc 60

acctgttctg tcactggcga ctccatcacc agtggttact ggaactggat ccggaaattc 120

ccaggaaata aacttgagta tttggggtac ataagcttca gtggtaacac ttactacaat 180

ccatctctca aaagtcgaat ctccatcatt cgagacacat ccaagaacca gtattatttg 240

cagttgaatt ctgtgactac tgaggacaca gccacatatt actgtgcaag atatccttac 300

tcctatagta actgggacta tgctatggac tactggggtc aaggaacttc agtcaccgtc 360

tcctca 366

<210>14

<211>339

<212>DNA

<213> Artificial sequence

<220>

<223> light chain coding sequence

<400>14

gatattgtga tgacacaatc tccatcctcc ctagttgtgt cagttggaga gaaggttact 60

atgagctgca agtccagtca gttcctttta tatagtagca gtcaaaagaa ctacttggcc 120

tggtaccagc agaaaccagg gcagtctcct caactgctga tttactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgaaggctga agacctggca gtttattact gtcaccaata ttatagctat 300

ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339

<210>15

<211>345

<212>DNA

<213> Artificial sequence

<220>

<223> heavy chain coding sequence

<400>15

gaggtgaagc tggaggagtcaggacctagc ctcgtgaaac cttctcagac tctgtccctc 60

acctgttctg tctctggcga ctccatcacc attggtttct ggaactggat ccggaaattc 120

ccaggaaata aacttgagta catgggatac ataaactaca gtggtagcag ttactacaat 180

ccatctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca gtattacctg 240

cagttgaatt ctgtgactcc tgaggacaca gccacatatt actgtgcaag atctgggacg 300

gaccttgact actggggcca aggcaccact ctcacagtct cctca 345

<210>16

<211>339

<212>DNA

<213> Artificial sequence

<220>

<223> light chain coding sequence

<400>16

gatattgtgc tcacacagtc tccactcact ttgtcggtta ccattggaca accagcctcc 60

atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120

ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180

tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240

agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttcct 300

cggacgttcg gtggaggcac caagctggaa atcaaacgg 339

<210>17

<211>200

<212>PRT

<213> Artificial sequence

<220>

<223> fusion protein

<400>17

Leu Ser Thr Val Thr Gly Leu His Cys Val Gly Asp Thr Tyr Pro Ser

1 5 10 15

Asn Asp Arg Cys Cys His Glu Cys Arg Pro Gly Asn Gly Met Val Ser

20 25 30

Arg Cys Ser Arg Ser Gln Asn Thr Val Cys Arg Pro Cys Gly Pro Gly

35 40 45

Phe Tyr Asn Asp Val Val Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp

50 55 60

Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr

65 70 75 80

Gln Asp Thr Val Cys Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser

85 90 95

Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys Pro Pro Gly His Phe Ser

100 105 110

Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala

115 120 125

Gly Lys His Thr Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys

130 135 140

Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro

145 150 155160

Pro Ala Arg Pro Ile Thr Val Gln Pro Thr Glu Ala Trp Pro Arg Thr

165 170 175

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

180 185 190

Val Ala His His His His His His

195 200

Claims

1. An antibody or antigen-binding fragment thereof against OX40 comprising at least one of:

(1) a heavy chain variable region having amino acid sequences of GFTFSDYY, isddgnt and ARRGTGTGFGY, and a light chain variable region having amino acid sequences of ENIYST, AAT and QHFWGIPWT;

(2) heavy chain variable region with amino acid sequences of GYTFTNYD, IYPEDGST and ARDTRGYFDY, and light chain variable region with amino acid sequences of SSVNY, YTS and QQFTSSPWT;

(3) a heavy chain variable region having the amino acid sequence GDSITSGY, ISFSGNT and ARYPYSYSNWDYAMDY; and a light chain variable region having amino acid sequences QFLLYSSSQKNY, WAS and HQYYSYPLT;

(4) a heavy chain variable region having amino acid sequences gdsiigf, inhsgs and ARSGTDLDY, and a light chain variable region having amino acid sequences QSLLDSDGKTY, LVS and WQGTHFPRT;

(5) an amino acid sequence having one or more conservative amino acid substitutions as compared with (1) to (4);

optionally, at least one of the following is included:

(a) a heavy chain having an amino acid sequence shown in SEQ ID NO. 1 and a light chain having an amino acid sequence shown in SEQ ID NO. 2;

(b) a heavy chain having an amino acid sequence shown in SEQ ID NO. 3 and a light chain having an amino acid sequence shown in SEQ ID NO. 4;

(c) a heavy chain having an amino acid sequence shown in SEQ ID NO. 5 and a light chain having an amino acid sequence shown in SEQ ID NO. 6;

(d) a heavy chain having the amino acid sequence shown in SEQ ID NO. 7 and a light chain having the amino acid sequence shown in SEQ ID NO. 8;

(e) (ii) has one or more conservative amino acid substitutions as compared to the amino acid sequences of (a) - (d) above except for the heavy chain variable region and the light chain variable region;

optionally, the antibody is a monoclonal antibody;

optionally, the antibody or antigen-binding fragment thereof is a sequence having a homology of 90% or more with (a) to (d), optionally a sequence having a homology of 95% or more, more preferably a sequence having a homology of 98% or more, and still more preferably a sequence having a homology of 99% or more.

2. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1;

optionally, the polynucleotide has a nucleotide sequence of one of:

the nucleotide sequence shown in SEQ ID NO. 9 and the nucleotide sequence shown in SEQ ID NO. 10;

the nucleotide sequence shown in SEQ ID NO. 11 and the nucleotide sequence shown in SEQ ID NO. 12;

the nucleotide sequence shown in SEQ ID NO. 13 and the nucleotide sequence shown in SEQ ID NO. 14;

the nucleotide sequence shown in SEQ ID NO. 15 and the nucleotide sequence shown in SEQ ID NO. 16.

3. An expression vector comprising the polynucleotide of claim 2;

optionally, further comprising:

a control element operably linked to the polynucleotide for controlling expression of the polynucleotide in a host cell;

optionally, the control element comprises at least one of: promoters, enhancers and terminators;

optionally, the host cell is a mammalian cell.

4. A recombinant cell comprising the expression vector of claim 3.

5. A method of making the antibody or antigen-binding fragment thereof of claim 1, comprising culturing the recombinant cell of claim 4.

6. A hybridoma cell selected from at least one of the following:

the hybridoma HX011-9C12 with the preservation number of C2018197 is preserved in the China center for type culture Collection with the preservation time of 09 and 27 days in 2018;

the hybridoma cell HX011-1D9 with the preservation number of C2018198 is preserved in the China center for type culture Collection with the preservation time of 09 and 27 days in 2018.

7. Use of the antibody or antigen-binding fragment of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, or the recombinant cell of claim 4 or the hybridoma cell of claim 6 in the preparation of a medicament for treating an autoimmune disease or cancer.

8. A pharmaceutical composition, comprising: the antibody or antigen-binding fragment of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, the recombinant cell of claim 4, or the hybridoma cell of claim 6.

9. A method for identifying an agent capable of binding to OX40, comprising:

contacting the antibody or antigen-binding fragment thereof of claim 1 with an antigen in the presence of a candidate drug and confirming a first amount of binding of the antibody or antigen-binding fragment thereof to the antigen; and

contacting the antibody or antigen-binding fragment thereof of claim 1 with an antigen in the absence of the drug candidate and determining a second amount of binding of the antibody or antigen-binding fragment thereof to the antigen;

wherein the antigen is OX40 or a fragment thereof, and the second amount of binding being greater than the first amount of binding is indicative that the candidate drug is capable of binding to OX 40.

10. A pharmaceutical combination, comprising:

(1) the antibody or antigen-binding fragment thereof of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, the recombinant cell of claim 4, or the hybridoma cell of claim 6; and

(2) an immunopotentiating agent different from (1);

optionally, the immunopotentiating drug different from (1) is selected from at least one of:

an anti-PD 1 antibody, a LAG-3 antibody, a CTLA-4 antibody, a Tim3 antibody, or a PD-L1 antibody.