阅读说明：本技术 Ox40抗体、其编码基因、及抗体的制备方法和在增强人体免疫功能中的应用 (OX40 antibody, encoding gene thereof, preparation method of antibody and application of antibody in enhancing human immune function ) 是由 刘明录 王立新 韩庆梅 刘敏 万磊 张传鹏 强邦明 金海锋 韩国英 于 2020-08-20 设计创作，主要内容包括：OX40抗体、其编码基因、及抗体的制备方法和在增强人体免疫功能中的应用,该抗体包含VH域,该VH域包含氨基酸序列的SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19中的任一个；还包含VL域,该VL域包含氨基酸序列的SEQ ID NO.12、SEQ ID NO.14、SEQ ID NO.16、SEQ ID NO.18、SEQ ID NO.20中的任一个。OX40即可增加人体免疫功能,又可以解除免疫抑制功能,从而提高制剂的增强人体免疫功能的效果。(OX40 antibody, its coding gene, a preparation method of the antibody and application of the antibody in enhancing human immunity function, wherein the antibody comprises a VH domain, and the VH domain comprises any one of SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17 and SEQ ID NO.19 of amino acid sequence; also comprises a VL domain comprising any of SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20 of the amino acid sequence. OX40 can increase human body immunity and relieve immunosuppression, thereby improving the effect of the preparation in enhancing human body immunity.)

An OX40 antibody characterized by: the antibody comprises a VH domain comprising any one of SEQ ID No.11, SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.19 of the amino acid sequence;

also comprises a VL domain comprising any of SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20 of the amino acid sequence.

2. The OX40 antibody of claim 1, wherein: the antibody is a humanized OX40 antibody.

3. The OX40 antibody of claim 2, wherein: the combination of the amino acid sequences of the VH domain and the VL domain of the antibody is preferably any one of the following:

the VH region of OX40-1 is SEQ ID NO.11, and the VL region is SEQ ID NO. 12; or

The VH region of OX40-2 is SEQ ID NO.13, and the VL region is SEQ ID NO. 14; or

The VH region of OX40-3 is SEQ ID NO.15, and the VL region is SEQ ID NO. 16; or

The VH region of OX40-4 is SEQ ID NO.17, and the VL region is SEQ ID NO. 18; or

The VH region of OX40-5 is SEQ ID NO.19, and the VL region is SEQ ID NO. 20.

4. A gene encoding an OX40 antibody as claimed in any one of claims 1 to 3, characterized in that: comprising a nucleotide sequence encoding an OX40 antibody as described above.

5. The coding gene of claim 4, wherein: the nucleic acid sequence of the encoding gene for encoding the VH domain is any one of SEQ ID NO.1, 3, 5, 7 and 9; the nucleic acid sequence encoding the VL domain is any of SEQ ID NO.2, 4, 6, 8, 10.

6. The coding gene of claim 4, wherein: preferred combinations of the encoding genes are:

the nucleotide sequence of the nucleotide sequence encoding OX40-1 VH region is SEQ ID NO.1, and the nucleotide sequence of the VL region is SEQ ID NO. 2; or

The nucleotide sequence of the nucleotide sequence encoding OX40-2 VH domain is SEQ ID NO.3, and the nucleotide sequence of the VL domain is SEQ ID NO. 4; or

The nucleotide sequence of the nucleotide sequence encoding OX40-3 VH domain is SEQ ID NO.5, and the nucleotide sequence of the VL domain is SEQ ID NO. 6; or

The nucleotide sequence of the nucleotide sequence encoding OX40-4 VH domain is SEQ ID NO.7, and the nucleotide sequence of the VL domain is SEQ ID NO. 8; or

The nucleic acid sequence encoding the VH domain of OX40-5 is SEQ ID NO.9, and the VL domain is SEQ ID NO. 10.

7. A method of making an OX40 antibody as recited in any one of claims 1-3, characterized by: the method comprises the following steps:

respectively artificially synthesizing a nucleic acid sequence for coding a VH domain and a nucleic acid sequence for coding a VL domain, carrying out double digestion, connecting the nucleic acid sequence for coding the VH domain to a pAZ-V5-hCH-IgG1 vector subjected to double digestion, and connecting the nucleic acid sequence for coding the VL domain to a pAZ-V5-hCL-IgG kappa vector subjected to double digestion to obtain a recombinant expression vector of a heavy chain and a light chain; then the antibody is transfected into HEK293T cells, and the antibody can be obtained in the supernatant obtained by culturing the cells.

8. The method of claim 7, wherein: further comprises a purification step:

mixing the cell culture supernatant with a binding buffer solution at a ratio of 1:1, and filtering; passing the ProteinA column with 5-10 volumes of binding buffer; loading the prepared supernatant sample; washing the column with binding buffer until the binding solution is free of protein; passing the eluent through a column, and collecting a leakage liquid at the same time until the leakage liquid does not contain protein; collecting the protein in each collecting pipe, and combining the protein pipes; the collected antibody was dialyzed against PBS.

Use of an OX40 antibody for the manufacture of a medicament for enhancing immune function in a human.

10. The use of claim 9, wherein: the medicament comprises a kit, which comprises:

(1) an OX40 antibody;

(2) antibody dilutions.

Technical Field

The invention relates to the technical field of biology, in particular to an OX40 antibody, a coding gene thereof, a preparation method of the antibody and application of the antibody in enhancing human immune function.

Background

Immunity is the body's own defense mechanism, and is the body's ability to recognize and destroy any foreign body (virus, bacteria, etc.) that invades from the outside. The ability to treat senescent, damaged, dead, degenerating self-cells, and to recognize and treat mutant and virally infected cells in vivo. Improving immunity is the physiological response of human body to recognize and eliminate "abnormal self". The immune system performs this function in the human body. The primary role played in the immune system is the T cell.

Killing among T cells is by CD4+ and CD8+ T cells, and when humans encounter antigen challenge, a proportion of the naive CD4+ T cells form Treg 1-type cells during the T cell response. Treg 1-type cells play a crucial role in peripheral tolerance. It is particularly important in limiting tissue damage to the host in response to inflammatory immunity. In the tumor microenvironment, Treg type 1 cells have important immune functions and can allow tumor cells to escape immune attack. Treg 1-type cells possess the ability to suppress the development of autoimmune diseases and can limit the magnitude of immune responses to microbial pathogens. In order to improve the immune function of the human body, the generation of Treg1 type cells needs to be reduced.

The research finds that OX40 receptors exist on Treg1 type cells, and the activation of OX40 receptors blocks the generation of Treg1 type cells by primary or memory CD4+ T cells, and simultaneously blocks the immunosuppressive functions of IL-10 and Treg1 type cells by Treg1 type cells.

OX40, also known as CD134, TNFRSF4, is a member of the tumor necrosis factor superfamily. The ligand of OX40, OX40L, is a member of the TNF family, and is expressed primarily on activated antigen presenting cells, including B cells, macrophages, endothelial cells, and DC cells. OX40 is a membrane-bound receptor that is not expressed on resting T cells, but is transiently expressed on activated T cells. OX40 is a major co-stimulatory receptor that, in conjunction with CD28, promotes T cell proliferation. Stimulation of activated T cells by OX40 results in cytokine production and promotes proliferation of CD4+ and CD8+ T cells, while co-stimulation of OX40 prolongs T cell survival and increases the number of memory T cells by inhibiting death of effector T cells.

OX40 protein is present predominantly on antigen-exposed memory T cells. When an OX40 antibody binds to an OX40 protein receptor, it triggers a costimulatory signal that increases T cell and inflammatory cytokine production. Activation of the OX40 receptor by OX40 antibodies can prevent the formation of Treg 1-type cells and FOXp3+ Treg cells, and can also prevent the production of IL-10 while preventing their immunosuppressive function. This mechanism is thought to activate the dormant immune system, which may help to enhance the immune function of the cells against cancer. Although there are patents on OX40, these prior patents are directed to monoclonal antibodies, and antibodies obtained by hybridoma cells are of murine origin, and the immune function-enhancing effect on humans is not ideal.

Therefore, the development of a novel OX40 antibody, a coding gene thereof, a preparation method of the antibody and application of the antibody in enhancing human immune function not only have urgent research values, but also have good economic benefits and industrial application potentials, which are the basis and the motivation for the completion of the invention.

Disclosure of Invention

The present inventors have conducted intensive studies to overcome the above-identified drawbacks of the prior art, and as a result, have completed the present invention after having made a great deal of creative efforts.

Specifically, the technical problems to be solved by the present invention are: provides OX40 antibody, its coding gene, the preparation method of the antibody and the application of the antibody in enhancing human body immunity function, so as to improve the effect of the preparation in enhancing human body immunity function.

In order to solve the technical problems, the technical scheme of the invention is as follows:

in a first aspect, the invention provides an OX40 antibody comprising a VH domain comprising any one of SEQ ID No.11, SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.19 of the amino acid sequence;

also comprises a VL domain comprising any of SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20 of the amino acid sequence.

In the present invention, as an improvement, the antibody is a humanized OX40 antibody.

In the present invention, as an improvement, the combination of the amino acid sequences of the VH domain and the VL domain of the antibody is preferably any one of the following:

the VH region of OX40-1 is SEQ ID NO.11, and the VL region is SEQ ID NO. 12; or

The VH region of OX40-2 is SEQ ID NO.13, and the VL region is SEQ ID NO. 14; or

The VH region of OX40-3 is SEQ ID NO.15, and the VL region is SEQ ID NO. 16; or

The VH region of OX40-4 is SEQ ID NO.17, and the VL region is SEQ ID NO. 18; or

The VH region of OX40-5 is SEQ ID NO.19, and the VL region is SEQ ID NO. 20.

The numerals following OX40-1, OX40-2, etc., are self-numbering for ease of distinction, and have no actual meaning.

In a second aspect, the invention provides a coding gene comprising a nucleotide sequence encoding an OX40 antibody as described above.

In the invention, as an improvement, the nucleic acid sequence of the encoding gene for encoding the VH domain is any one of SEQ ID No.1, 3, 5, 7 and 9; the nucleic acid sequence encoding the VL domain is any of SEQ ID NO.2, 4, 6, 8, 10.

In the present invention, as an improvement, the preferred combinations of the encoding genes are:

the nucleotide sequence of the nucleotide sequence encoding OX40-1 VH region is SEQ ID NO.1, and the nucleotide sequence of the VL region is SEQ ID NO. 2; or

The nucleotide sequence of the nucleotide sequence encoding OX40-2 VH domain is SEQ ID NO.3, and the nucleotide sequence of the VL domain is SEQ ID NO. 4; or

The nucleotide sequence of the nucleotide sequence encoding OX40-3 VH domain is SEQ ID NO.5, and the nucleotide sequence of the VL domain is SEQ ID NO. 6; or

The nucleotide sequence of the nucleotide sequence encoding OX40-4 VH domain is SEQ ID NO.7, and the nucleotide sequence of the VL domain is SEQ ID NO. 8; or

The nucleic acid sequence encoding the VH domain of OX40-5 is SEQ ID NO.9, and the VL domain is SEQ ID NO. 10.

In a third aspect, the present invention provides a method for producing an antibody, comprising the steps of:

respectively artificially synthesizing a nucleic acid sequence for coding a VH domain and a nucleic acid sequence for coding a VL domain, carrying out double digestion, connecting the nucleic acid sequence for coding the VH domain to a pAZ-V5-hCH-IgG1 vector subjected to double digestion, and connecting the nucleic acid sequence for coding the VL domain to a pAZ-V5-hCL-IgG kappa vector subjected to double digestion to obtain a recombinant expression vector of a heavy chain and a light chain; then the antibody is transfected into HEK293T cells, and the obtained supernatant is cultured to obtain the antibody.

In the present invention, as a preferred embodiment, the method for preparing the antibody further comprises a purification step:

mixing the cell culture supernatant with a binding buffer solution at a ratio of 1:1, and filtering; passing the ProteinA column with 5-10 volumes of binding buffer; loading the prepared supernatant sample; washing the column with binding buffer until the binding solution is free of protein; passing the eluent through a column, and collecting a leakage liquid at the same time until the leakage liquid does not contain protein; collecting the protein in each collecting pipe, and combining the protein pipes; the collected antibody was dialyzed against PBS.

In a fourth aspect, the invention provides an application of an OX40 antibody in enhancing human immune function, which means that the OX40 antibody can be effectively applied in preparing a medicament for enhancing human immune function.

In the present invention, as a preferred technical scheme, the medicament comprises a kit, which comprises:

(1) an OX40 antibody;

(2) antibody dilutions.

After the technical scheme is adopted, the invention has the beneficial effects that:

the preparation for enhancing human immune function, which is disclosed by the invention, comprises OX40 antibody, and OX40 antibody can trigger a costimulatory signal when being combined with OX40 protein receptor, so that the generation of T cells can be increased. Activation of the OX40 receptor by OX40 antibodies can prevent the formation of Treg 1-type cells and FOXp3+ Treg cells, and can also prevent the production of IL-10 while preventing their immunosuppressive function. OX40 can increase immunity and relieve immunosuppression.

Drawings

FIG. 1 is an electrophoretogram of OX40-VH-1DNA fragment (right) and OX40-VL-1DNA fragment (left) according to the invention.

FIG. 2 is a schematic diagram of the recombinant expression vectors pAZ-V5-hCH-IgG1 and pAZ-V5-hCL-IgG kappa according to the present invention.

FIG. 3 shows HEK293T cells used for transfection in the present invention.

FIG. 4 shows PBMC cells isolated and cultured according to the present invention.

FIG. 5 is a graph of the number of CD8+ T cells detected by flow cytometry in accordance with the present invention without (upper) or with (lower) addition of OX40 antibody.

Detailed Description

The invention is further illustrated by the following specific examples. The use and purpose of these exemplary embodiments are to illustrate the present invention, not to limit the actual scope of the present invention in any way, and not to limit the scope of the present invention in any way.

Example 1

OX40 antibody examples

The OX40 antibody comprises a VH domain comprising any one of SEQ ID No.11, SEQ ID No.13, SEQ ID No.15, SEQ ID No.17, SEQ ID No.19 of the amino acid sequence; also comprises a VL domain comprising any of SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, SEQ ID No.20 of the amino acid sequence. Wherein the antibody is a humanized OX40 antibody.

This example illustrates the antibody OX40-1 of SEQ ID NO.11 for the VH domain amino acid sequence and SEQ ID NO.12 for the VL domain amino acid sequence.

When any one of OX40-2, OX40-3, OX40-4 and OX40-5 is adopted, the preparation method and other technical parameters are basically the same, and redundant description is not repeated.

Example 2

A coding gene comprising a nucleotide sequence encoding an OX40 antibody as described above. Correspondingly, the nucleic acid sequence of the encoding gene for encoding the VH domain is any one of SEQ ID NO.1, 3, 5, 7 and 9; the nucleic acid sequence encoding the VL domain is any of SEQ ID NO.2, 4, 6, 8, 10.

The present example selects as a specific example the gene encoding the OX40 antibody described in example 1, wherein the nucleic acid sequence encoding the VH domain of OX40-1 is SEQ ID NO.1 and the VL domain is SEQ ID NO. 2.

The preparation method of the antibody comprises the following steps: respectively artificially synthesizing a nucleic acid sequence for coding a VH domain and a nucleic acid sequence for coding a VL domain, carrying out double digestion, connecting the nucleic acid sequence for coding the VH domain to a pAZ-V5-hCH-IgG1 vector subjected to double digestion, and connecting the nucleic acid sequence for coding the VL domain to a pAZ-V5-hCL-IgG kappa vector subjected to double digestion to obtain a recombinant expression vector of a heavy chain and a light chain; then the antibody is transfected into HEK293T cells, and the obtained supernatant is cultured to obtain the antibody.

The present invention is described in detail by the following examples.

Example 3

Construction of pAZ-V5-hCH-IgG1 and pAZ-V5-hCL-IgG kappa expression vectors

A method for producing OX40-VH-1 and OX40-VL-1 genes, comprising the steps of:

(1) synthesizing artificial sequences of OX40-VH-1 and OX40-VL-1 regions respectively, and inserting the artificial sequences into a cloning vector pUC57 to obtain pUC-OX40-VH-1 and pUC-OX 40-VL-1;

(2) performing double enzyme digestion on pUC-OX40-VH-1 and pUC-OX40-VL-1, utilizing agar electrophoresis to cut off the agar part containing OX40-VH-1 and OX40-VL-1DNA fragments, utilizing DNA extraction kit to treat, passing through a DF column to discard filtrate, rinsing the DF column, separating, eluting the DF column, and collecting centrifugate to obtain purified OX40-VH-1 and OX40-VL-1DNA fragments, namely the heavy chain variable region and the light chain variable region of the OX40 antibody.

In more detail, the preparation method of pAZ-V5-hCH-IgG1 and pAZ-V5-hCL-IgG kappa expression vectors provided in this example comprises the following steps:

the nucleic acid artificial sequences of OX40-VH-1 and OX40-VL-1 regions, respectively, were synthesized by the firm of Committee Biotechnology engineering (Shanghai) and inserted into the cloning vector pUC57, and were thus designated pUC-OX40-VH-1 and pUC-OX 40-VL-1.

pUC-OX40-VH-1 and pUC-OX40-VL-1 were subjected to a double digestion with Fast Digest BamHI (from NEB) and Fast Digest XbaI (from NEB), at 37 ℃ for 4 h. The 100. mu.l enzyme system is: 10 × buffer: 10 mu l of the mixture; 6 mu g of DNA; BamHI enzyme: 3 mu l of the solution; XbaI enzyme: 3 mu l of the solution; deionized water to make up the volume.

The agar sites containing the OX40-VH-1 and OX40-VL-1DNA fragments were excised by agarose electrophoresis and placed in two centrifuge tubes. The DNA was dissolved from the agar using a DNA extraction kit (available from ThermoFisher Co.) and concentrated by first adding 500. mu.l DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. Then 500. mu.l of Wash Buffer was added and centrifuged at 8000rpm for 1 minute, and the filtrate was decanted off. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally, transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of Elution Buffer, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min, wherein the liquid in the micro-centrifuge tube is the purified OX40-VH-1 and OX40-VL-1DNA fragments (see figure 1).

Both vectors pAZ-V5-hCH-IgG1 and pAZ-V5-hCL-IgG kappa (see FIG. 2) were subjected to a double digestion with Fast Digest BamHI (from NEB) and Fast Digest XbaI (from NEB), at 37 ℃ for 4 h. The gel was cut and recovered in the same manner as above to obtain linearized pAZ-V5-hCH-IgG1 and pAZ-V5-hCL-IgG kappa vectors.

The purified OX40-VH-1DNA fragment and the pAZ-V5-hCH-IgG1 linearized vector were ligated overnight at 16 ℃ to form the pAZ-hCH-VH-1 plasmid, while OX40-VL-1 and pAZ-V5-hCL-IgG kappa were ligated to form the pAZ-hCL-VL-1 plasmid. The connecting system is as follows: 10 × buffer: 1 mul; t4 ligase: 1 mul; OX40-VH-1 DNA: 4 mu l of the solution; linearized pAZ-V5-hCH-IgG 1: 4 μ l, light chain ligation system identical.

Example 4

Examples of the purification of both plasmids pAZ-hCH-VH-1 and pAZ-hCL-VL-1.

Coli (TOP10) was transformed with both the above pAZ-hCH-VH-1 and pAZ-hCL-VL-1 plasmids. The method comprises the following specific steps: the plasmid and the competent cells are evenly mixed and incubated on ice for half an hour, then heat shock is carried out at 42 ℃ for 90 seconds, then the mixture is placed on ice for 2min, finally liquid LB culture medium is added and slowly shaken for about 1 hour, then centrifugation is carried out at 3000rpm for 5min, and 100 mul of bacterial liquid is coated on a solid plate containing ampicillin LB.

A single colony is picked for overnight culture the next day, and two plasmids, namely pAZ-hCH-VH-1 and pAZ-hCL-VL-1, are extracted by a plasmid extraction and purification kit (purchased from Qiagen company), and the specific steps are as follows: (1) 1.5ml of the bacterial solution was centrifuged at room temperature at 10000 Xg for 1 min. (2) The supernatant was removed, 250. mu.l of solution I (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended. (3) Adding 250 mu l of solution II, and gently inverting the centrifuge tube for 4-6 times to obtain a clear lysate. Preferably, the incubation is carried out at room temperature for 2 min. (4) Add 350. mu.l of solution III, mix gently by inversion several times until white flocculent precipitate appears, centrifuge at room temperature 10000 Xg for 10 min. (5) The supernatant was aspirated with special care and transferred to a clean adsorption column equipped with 2ml centrifuge tubes. It is ensured that there are no aspiration deposits and cell debris. Centrifugation was carried out at room temperature at 10000 Xg for 1min until the lysate was completely passed through the column. (6) The filtrate was discarded, 500. mu.l Buffer HBC was added, 10000 Xg was centrifuged for 1min, and the column was washed to remove residual protein to ensure the purity of DNA. (7) The filtrate was discarded, and the column was washed with 750. mu.l of Wash Buffer diluted with 100% ethanol and centrifuged at 10000 Xg for 1 min. (8) The column was washed with 750. mu.l of Wash Buffer. (9) The column must be centrifuged at 10000 Xg for 2min to ensure that the ethanol is removed. (10) The column was placed into a clean 1.5ml centrifuge tube, 50-100. mu.l (depending on the desired final concentration) sterile deionized water or TE buffer was added to the filter, and the plasmid DNA was collected by centrifugation at 10000 Xg for 2 min.

The above-mentioned two plasmids, pAZ-hCH-VH-1 and pAZ-hCL-VL-1, were sequenced by committee of Biotechnology engineering (Shanghai) Co., Ltd. Sequencing is carried out correctly for later use.

Example 5

Examples of HEK293T cells were co-transfected with both pAZ-hCH-VH-1 and pAZ-hCL-VL-1 plasmids.

And taking out the frozen HEK293T cells from the liquid nitrogen tank, quickly throwing the cells into a water bath kettle at 37 ℃, quickly shaking, and completely dissolving the cell solution within 1-2 min to the greatest extent. The cell solution was transferred to a 50mL centrifuge tube and 5mL of fresh complete medium was added to the tube, mixed well and centrifuged at 1500rpm for 5 min. The supernatant was removed and 1mL of fresh complete medium was added to resuspend the cell pellet and transferred to six well plates, each well being replenished to 3mL of medium. The six-well plate was incubated in an incubator at 37 ℃ with 5% CO2 and 95% relative humidity. Cell viability was observed the next day and the medium was changed. The growth of the cells was observed daily thereafter, and the cells were used in the experiment when the state was good (see FIG. 3).

One day before transfection, HEK293T cells with good morphology and vigorous growth in logarithmic growth phase were digested with trypsin and diluted to a density of 4X 105/mL, plated into 6-well cell culture plates, gently shaken to uniformly spread the cells in the wells of the cell plate at 2mL per well, and cultured and passaged in DMEM high-sugar medium. The cells are kept flat in a 5% carbon dioxide incubator at 37 ℃ and transfection operation can be started when the cells grow to 80-90% confluency.

3ug of each of the pAZ-hCH-VH-1 and pAZ-hCL-VL-1 plasmids was added to 200ul of Optipro SFM, mixed well, added to 8ul of transfection reagent, mixed well again and incubated at room temperature for 20 min. The DNA-transfection reagent complex is dripped into a 6-well plate, mixed evenly and cultured in an incubator at 37 ℃ for 48 h. The supernatant was collected and contained OX40 antibody. And (5) obtaining a cell line for stably expressing the antibody through antibiotic screening after the verification is correct, and carrying out expanded culture.

Example 6

Example of purification of recombinant antibody.

The antibody was purified using a ProteinA column. The method comprises the following specific steps: (1) sample preparation: the cell supernatant was mixed with binding buffer 1:1 and filtered (to prevent clogging of the column). (2) Column equilibration: the ProteinA column was passed over 5-10 volumes of binding buffer. (3) Loading: the prepared supernatant sample is loaded, taking into account the volume of the loading volume according to the binding capacity of the column. (4) And (3) eluting the hybrid protein: the column was washed with binding buffer until the binding solution was free of protein. (5) Collecting the antibody: and (4) passing the eluent through the column, and collecting the leaked liquid at the same time until the leaked liquid does not contain protein. And (4) determining the protein content in each collection tube, and combining the protein tubes. (6) The collected antibody was dialyzed against PBS.

Example 7

Preparation example of PBMC cells.

100ml of fresh peripheral blood from healthy donors were taken and used with TBD sample density separation (purchased from Tianjin primary ocean organisms) to isolate PBMC from peripheral blood mononuclear cells as follows:

(1) peripheral blood 100ml was diluted with physiological saline at a ratio of 1: 1. Carefully adding diluted blood to lymphocyte separation solution with the same volume to form obvious layering, horizontally centrifuging at room temperature for 900g/min, and 25 min. At the moment, 4 layers are formed in the centrifugal tube from top to bottom; plasma, a buffy coat layer composed of PBMCs, a lymphocyte separation liquid layer, and a lowest erythrocyte sedimentation layer.

(2) The buffy coat was carefully aspirated with a pipette, and the PBMC aspirated as completely as possible. Adding 2 times of physiological saline, washing cells for 2 times, mixing uniformly, centrifuging at 1500rpm/min for 5min, centrifuging, discarding supernatant, collecting PBMC cells, and culturing (see figure 4).

Example 8

OX40 antibody can promote proliferation of T cells and inhibit production of IL-10.

The collected PBMCs were cultured in two equal portions, one portion was cultured in the presence of OX40 antibody, the other portion was cultured normally, and after 15 days of culture, the content of CD8+ T cells was measured by a flow cytometer (see FIG. 5), and the content of CD8+ T cells was 27.4% in PBMC cultured with OX40 antibody added and the content of CD3+ CD8+ T cells was 6.3% in PBMC cultured without OX40 antibody added, indicating that OX40 antibody can promote the proliferation of CD8+ T cells. Meanwhile, the concentration of IL-10 is detected by an ELISA method, and the result shows that the concentration of IL-10 in the T cells after the OX40 antibody is added is far lower than that of the T cells without the OX40 antibody, which indicates that the OX40 antibody can inhibit the secretion of IL-10.

Example 9

Preparation for enhancing immunity

(1) An OX40 antibody;

(2) antibody diluent;

(3) instructions for use.

Wherein the instructions for use comprise the steps as described in examples 7, 8.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.

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<400> 3

gaggtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc cctgagactc 60

tcctgtgcag cctctgaata cgagttccct tcccatgaca tgtcttgggt ccgccaggct 120

ccggggaagg ggctggagtt ggtcgcagcc attaatagtg atggtggtag cacctactat 180

ccagacacca tggagagacg attcaccatc tccagagaca atgccaagaa ctcactgtac 240

ctgcaaatga acagtctgag ggccgaggac acagccgtgt attactgtgc aagacactat 300

gatgattact acgcctggtt tgcttactgg ggccaaggga ctatggtcac tgtctcttca 360

<210> 4

<211> 333

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 4

gaaattgtgc tgacacagtc tcctgctacc ttatctttgt ctccagggga aagggccacc 60

ctctcatgca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggtac 120

caacagaaac caggacaggc tcccagactc ctcatctatc ttgcatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caccatcagc 240

agcctagagc ctgaggattt tgcagtttat tactgtcagc acagtaggga gcttccgctc 300

acgttcggcg gagggaccaa ggtcgagatc aaa 333

<210> 5

<211> 357

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 5

gaagtgaagc tggaggagtc tggaggaggc ttggtgcaac ctggaggatc catgaaactc 60

tcttgtgctg cctctggatt cacttttagt gacgcctgga tggactgggt ccgccagtct 120

ccagagaagg ggcttgagtg ggttgctgaa attagaagca aagctaataa tcatgcaaca 180

tactatgctg agtctgtgaa tgggaggttc accatctcaa gagatgattc caaaagtagt 240

gtctacctgc aaatgaacag cttaagagct gaagacactg gcatttatta ctgtacgtgg 300

ggggaagtgt tctactttga ctactggggc caaggcacca ctctcacagt ctcctca 357

<210> 6

<211> 318

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 6

gacatccaga tgacacagtc tccatcctca ctgtctgcat ctctgggagg caaagtcacc 60

atcacttgca agtcaagcca agacattaac aagtatatag cttggtacca acacaagcct 120

ggaaaaggtc ctaggctgct catacattac acatctacat tacagccagg catcccatca 180

aggttcagtg gaagtgggtc tgggagagat tattccttca gcatcagcaa cctggagcct 240

gaagatattg caacttatta ttgtctacag tatgataatc ttctcacgtt cggtgctggg 300

accaagctgg agctgaaa 318

<210> 7

<211> 372

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 7

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180

gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagactat 300

tatgattctt ctggatattc tgattatgga atggatgttt ggggccaagg caccactgtc 360

accgtctcga gc 372

<210> 8

<211> 324

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 8

gacatcgtta tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caatttacta ctgtcaacag aattacaata cccgacaagt tactttcggc 300

caagggacac gactggagat taaa 324

<210> 9

<211> 372

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 9

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180

gcacagaagt tccagggcag agtcaccatg accactgaca cgtccacgag cacagcttac 240

atggagctga gaagcctgag atctgatgac acggccgtgt attactgtgc tagagatcct 300

tattctagta gttggtatgg agctgaatac tttcaacatt ggggccaggg aaccctggtc 360

accgtctcat ca 372

<210> 10

<211> 333

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 10

cagtctgttc tgactcagcc tagatccgtg tctgggtctc ctggacagtc ggttaccatc 60

tcctgcactg gaaccagcag tgacggaggt gattataact atgtctcctg gtaccaacag 120

cacccaggcc aagcccccaa actccttatt tatgaggtca gtaatcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc agctcatata caagcagcag cactctcgtg 300

gtattcggcg gagggaccaa gctgaccgtc cta 333

<210> 11

<211> 122

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 11

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

1 5 10 15

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

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 12

<211> 107

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 12

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 120

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 13

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Glu Phe Pro Ser His

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 14

<211> 111

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 14

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

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

50 55 60

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

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 15

<211> 119

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 15

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

1 5 10 15

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

20 25 30

Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

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

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Thr Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 16

<211> 106

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 16

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

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ser Ser Gln Asp Ile Asn Lys Tyr

20 25 30

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

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 17

<211> 124

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Asp Ser Ser Gly Tyr Ser Asp Tyr Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 18

<211> 108

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 18

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Asn Tyr Asn Thr Arg Gln

85 90 95

Val Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 19

<211> 124

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Pro Tyr Ser Ser Ser Trp Tyr Gly Ala Glu Tyr Phe Gln

100 105 110

His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 111

<212> PRT

<213> ethnic species (Homo sapiens)

<400> 20

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

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Gly Gly Asp Tyr

20 25 30

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

35 40 45

Leu Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110