CN111793134A - Medicine, tumor vaccine and inhibitor for cancer treatment - Google Patents
Medicine, tumor vaccine and inhibitor for cancer treatment Download PDFInfo
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- CN111793134A CN111793134A CN201910276548.1A CN201910276548A CN111793134A CN 111793134 A CN111793134 A CN 111793134A CN 201910276548 A CN201910276548 A CN 201910276548A CN 111793134 A CN111793134 A CN 111793134A
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Abstract
The invention belongs to the technical field of biological medicines, and discloses a medicament, a tumor vaccine and an inhibitor for cancer treatment, wherein the medicament for cancer treatment is a BTNL2 monoclonal therapeutic antibody or a composition containing the BTNL2 monoclonal therapeutic antibody; the BTNL2 monoclonal therapeutic antibody is human BTNL2 or mouse BTNL 2; the amino acid sequence of human BTNL2 is SEQ ID NO.1, and the coding nucleotide sequence is SEQ ID NO. 2; mouse BTNL2 has the amino acid sequence of SEQ ID NO.3 and the coding nucleotide sequence of SEQ ID NO. 4. The monoclonal antibody produced by the hybridoma (BTN-1) obtained by the invention can be applied to detection and screening of Immunohistochemistry (IHC), immunoblotting (Western blot), indirect ELISA, antibody chip preparation and the like, and has high specificity and sensitivity.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a medicine, a tumor vaccine and an inhibitor for treating cancers.
Background
Currently, the current state of the art commonly used in the industry is such that: the immune checkpoint is blocked by a humanized or fully human antibody of the immune checkpoint, thereby achieving the effects of activating immune response and killing tumors. Common immune checkpoints are PD-1, PD-L1, CTLA 4.
Tumor immunotherapy is a method of activating human immunity through different mechanisms, and finally eliminating tumors through specific immune cells in a collective body. Currently, tumor immunotherapy is divided into active immunotherapy and passive immunotherapy, depending on the method of activating the immune system. Active immunotherapy mainly comprises tumor vaccines, and passive immunotherapy mainly comprises antibody drug therapy, immune checkpoint inhibitor therapy, adoptive immune cell therapy, cytokine therapy and the like.
Currently the most interesting is immune checkpoint inhibitor therapy. The immune check point is a molecule playing a protective role in the immune system of a human body, plays a role similar to braking, and prevents inflammatory injury and the like caused by over-activation of T cells. The tumor cells utilize the characteristics of the human immune system, inhibit the reaction of the human immune system by over-expressing immune check point molecules, escape the immune monitoring and killing of the human body, and promote the growth of the tumor cells. The most widely studied and used immune checkpoint inhibitors at present include inhibitors of CTLA-4, PD-1 and its ligand PD-L1. Immune checkpoint inhibitor therapy achieves anti-tumor effects by inhibiting immune checkpoint activity, releasing immune brakes in the tumor microenvironment, reactivating the immune response effects of T cells against tumors, which also makes it a new weapon against tumors. After clinical application of the therapy, some patients can obtain lasting clinical effect and no clinical symptoms related to tumors exist in several years.
The major CTLA-4 inhibitors currently include Iplilimumab, Tremelimumab, and others, with Iplilimumab being the earliest FDA approved and clinically used immune checkpoint inhibitor. The FDA in the united states finally approved Ipilimumab for the treatment of metastatic melanoma in 2011 based on survival results obtained in phase III randomized controlled trials. Currently FDA approved PD-1 antibodies are Nivolumab and Pembrolizumab, used to treat melanoma, non-small cell lung cancer and renal cancer, respectively. PD-L1 antibody atezolizumab (trade name tecentiq) of the roquel gene taxol, approved four months in advance by the FDA in 2016 as a second-line drug for the treatment of one of the most common advanced bladder cancers called urothelial carcinoma. The PD-L1 antibody bavenciio (avelumab), co-developed by merck, gmelin, germany, was approved by FDA in 2017 with an accelerated approval for the treatment of adults suffering from metastatic Merck Cell Carcinoma (MCC) and children over 12 years old, including those patients who had not previously been treated with chemotherapy. The "PD-1 antibody, Keytruda, from saxidong was approved by the FDA in 2017 for the treatment of any solid tumor that carries a specific genetic signature. This is the U.S. FDA approved top line of anti-tumor therapy that does not rely on tumor origin, but rather on biomarkers for differentiation. In particular, Keytruda is approved for the treatment of adult and pediatric solid tumor patients who carry high microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR). At present, hundreds of clinical tests aiming at different tumors are carried out on antibodies of immune checkpoint inhibitors PD-1, PD-L1 and CTLA-4, and the antibodies are expected to make breakthrough on the treatment of various tumors. Despite the tremendous advances made by immune checkpoint inhibitors in the treatment of numerous tumors, there are also some deficiencies with this therapy, such as lower overall response rates, development of drug resistance, etc. Therefore, screening for new tumor immune checkpoints is essential.
BTNL2(Butyrophin-like2) is one of BTNL family members and has high homology with B7 family proteins. In recent years, some genetic studies find that the single nucleotide polymorphism of the gene is closely related to a plurality of diseases, such as autoimmune diseases, tumors and the like. The study finds that the BTNL2 protein can inhibit the activation of T cells, and the BTNL2 plays an important role in immune regulation and tolerance.
In summary, the problems of the prior art are as follows:
in the prior art, the overall response rate of the immune checkpoint inhibitor is low, and drug resistance occurs.
The humanized level of the antibody is low, and immunological rejection reaction is easy to occur;
the humanized antibody has poor affinity, resulting in poor blocking activity.
The difficulty and significance for solving the technical problems are as follows:
blockade against other checkpoint proteins is likely to increase the overall response rate of treatment;
the optimized humanization can reduce rejection reaction and enable the antibody to play a better role;
increasing the affinity promotes better blocking activity and better drug efficacy.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a medicament, a tumor vaccine and an inhibitor for treating cancer.
The invention is realized in that a drug for use in cancer therapy, which is a BTNL2 monoclonal therapeutic antibody, can be used to treat a variety of human cancers.
The BTNL2 monoclonal therapeutic antibody is human BTNL2 or mouse BTNL 2; the amino acid sequence of human BTNL2 is SEQ ID NO.1, and the coding nucleotide sequence is SEQ ID NO. 2; mouse BTNL2 has the amino acid sequence of SEQ ID NO.3 and the coding nucleotide sequence of SEQ ID NO. 4.
Further, the BTNL2 extracellular domain polypeptide used to purify the antibody is an immunogen; the BTNL2 extracellular region polypeptide is from leucine 24 to serine 455 of human BTNL2 and from lysine 24 to serine 454 of mouse Ephrin-B1, and the coding nucleotides of the BTNL2 extracellular region polypeptide are respectively from nucleotide 69 to nucleotide 1365 of the sequence shown in SEQ ID NO.2 and from nucleotide 69 to nucleotide 1362 of the sequence shown in SEQ ID NO. 4.
Further, a fusion polypeptide consisting of the extracellular region of BTNL2 and a 6XHis tag was used for purification of the immunogen.
Another objective of the invention is to provide a vector for expressing a fusion antigen polypeptide, which is constructed by BTNL2 monoclonal therapeutic antibody, wherein the vector is pINFUSE-hIgG2-Fc2 (the vector is purchased from INVIVOGEN company) and comprises nucleotides coding for BTNL2 extracellular region, and the vector is used for treating cancers.
Another object of the present invention is to provide a method for constructing the vector, the method comprising: cloning and purifying a gene sequence encoding leucine 24 to serine 455 of BTNL2 by expression using mammalian cells, and immunizing a rat as an immunogen; obtaining a positive hybridoma cell line efficiently secreting the monoclonal antibody by fusing with mouse hybridoma cells and screening and cloning by recombinant CP4 EPSPS;
collecting culture supernatant by culturing the hybridoma cell line, and purifying the culture supernatant by Protein G column affinity chromatography to obtain a rat monoclonal antibody; an immunoblotting Western blot experiment is carried out, and rat monoclonal antibodies are analyzed to specifically recognize BTNL2 proteins endogenous to tumor cells.
Another object of the present invention is to provide an animal model construction method for verifying the efficacy of the drug for cancer treatment, the animal model construction method comprising:
firstly, selecting 8-week-old female mice to perform animal experiments; mice were randomly divided into 2-3 groups of 10 mice each, one group injected with control antibody, one group injected with anti-BTNL 2 antibody, and the other group injected with anti-PD-L1 antibody as positive control;
pINFUSE-hIgG2-His-BTNL2 vector construction:
through synthesizing a mouse BTNL2 full cDNA sequence, the coding nucleotide sequence is shown as SEQ ID NO.4, and the mouse BTNL2 extracellular region expression vector is constructed by taking the coding nucleotide sequence as a template, wherein the amino acids of the extracellular region are from 24 th lysine to 454 th serine; the expression vector is pINFUSE-hIgG2-Fc 2;
the PCR reaction system is as follows:
10XPCR buffer:5μl,
dNTP:1μl,
upstream/downstream primers: 1ul (10. mu.M) each,
template DNA: 1ng of the feed water is added into the feed water,
PFU:1μl
the total reaction system is as follows: 50 μ l;
the PCR reaction program is:
5 minutes at 95 ℃, 30 times of circulation, 10 minutes at 72 ℃ and 4 ℃;
running nucleic acid for electrophoresis in a PCR reaction system, cutting gel and recovering DNA fragments; and (3) carrying out enzyme digestion on the DNA fragment after gel cutting recovery and the pINFUSE-hIgG2-Fc2 empty vector at 37 ℃ for 2 hours, and recovering the DNA fragment. Connecting the recovered DNA fragment with the vector at 16 ℃ overnight; mu.l of the ligation product was used to transform DH 5. alpha. competent cells. Putting the transformation plate into a 37 ℃ bacterial incubator overnight, taking a monoclonal colony the next day, carrying out colony PCR identification, extracting plasmid from the colony which is correctly identified, and sending the colony to a company for sequencing; carrying out plasmid extraction on the plasmid with correct sequencing to prepare transfected cells; DNA gel cutting recovery kit and DNA solution recovery kit;
preparation of recombinant protein His-BTNL 2:
the constructed expression vector pINFUSE-hIgG2-His-BTNL2 is transfected into 293F cells, 1L cultured cells and 1x 106Per mL, 5% CO at 37 ℃2Shaking at 130rpm for 6 days; collecting cell culture supernatant on 6 days after transfection, centrifuging to remove cells, filtering with 0.45 μm filter membrane at 4 deg.C and 1500rpm for 5 min; purifying the filtered supernatant with His-tag purification kit to obtain recombinant protein, ultrafiltering to obtain protein, dissolving in 1 × PBS, filtering with 0.22 μm filter membrane, measuring concentration, and storing at-80 deg.C;
secondly, establishing a hybridoma cell line:
e) immunization
Emulsifying the cross-linked polypeptide in the first step, immunizing female rats with the age of 6-8 weeks, injecting 6 points per rat by abdominal subcutaneous injection, wherein the dosage is 60 ug/rat, boosting immunity once in 14 days, emulsifying the antigen, and the dosage is 30 ug/rat; detecting the multi-antibody titer of the anti-immunogen in the rat serum by indirect ELISA 7 days after 3 rd boosting immunization, injecting the rat with the highest titer into tail vein for impact immunization, and uniformly mixing the antigen with normal saline, wherein the dosage is 50 ug/mouse;
the antigen used for immunizing rats is recombinant His-BTNL2(24-454) antigen protein, and the recombinant His-BTNL2(24-454) antigen protein is obtained by encoding a nucleotide sequence of SEQ ID No.1 in a sequence table and recombining and expressing the encoded protein by an animal cell 293F;
f) cell fusion:
aseptically preparing spleen cell suspension of rat with immunity reaching standard, mixing with mouse myeloma cell sp2/0(ATCC) at ratio of 5:1, centrifuging at 1500rpm for 5 min; discarding the supernatant, placing the centrifuge tube into a 37 ℃ water bath, slowly adding Iml PEG1500 in 1 minute, and stirring the cells; standing in warm water for 1min, adding 10ml serum-free top DM, mixing, and centrifuging at 1000rpm for 5 min; discarding the supernatant, adding 10ml serum to blow the cells, adding 5ml thymocytes mixed with 10XHAT, and mixing; then adding 25ml of semisolid culture medium containing 2.1% of nitrocellulose, fully mixing, and then uniformly pouring into 20 cell culture dishes; putting the cell culture dish into a wet box, and putting the cell culture dish into a 5% CO2 incubator at 37 ℃ for culture;
g) selecting and cloning:
the size and density of the clone cell mass are moderate 7 days after fusion, round, solid and large clone masses are sucked and injected into a 96-hole culture plate of a culture medium, and the culture is carried out in a 5% CO2 incubator at 37 ℃;
h) ELISA screening positive hybridoma cells:
after 3 days, the cell mass was about 2/3 basal areas, and IOOyl supernatants were screened by ELISA using the immunogen and the synthetic polypeptide, respectively; positive clones were completely changed and 200y1 complete medium containing feeder cells and 1% HT was added; performing second ELISA screening after two days, and transferring the positive clone into a 24-pore plate prepared with a culture medium in advance for culture; taking 100y1 supernatant after five days, carrying out third ELISA screening, and gradually transferring the positive clones into a 6-hole plate and a cell culture bottle for amplification culture and freezing; the obtained mouse hybridoma cell line BTN-1;
e) purification of antibody secreted by mouse hybridoma cell line BTN-1:
the mouse hybridoma cell line BTN-1 was cultured using serum replacement. Mixing 5X106Cells were transferred to 10cm dishes at a cell density of about 50% the following day. After three days, the supernatant was removed and replaced with fresh medium, and the culture was continued for 3 days, and the supernatant was collected. The cells were removed by centrifugation and filtered through a 0.45 μm filter. Purifying the filtered supernatant by using a protein G prepacked column to obtain recombinant protein; washing after purificationUltrafiltering the antibody-removed antibody with ultrafiltration strain of Milipore, fusing the antibody in 1 × PBS, filtering with 0.22 μm filter membrane, measuring concentration, and storing at-80 deg.C;
f) t cell activation assay screening blocking/therapeutic BTNL2 antibodies:
coating a 24-well plate with anti-CD3ePBS and anti-CD28T cell activators, and standing at 4 ℃ overnight; the following day the 24-well plate was washed twice with 1XPBS and 20ug/ml of BTNL2-His protein was added for two hours at room temperature. 1XPBS washes 24 hole plate twice; mouse CD4+ T lymphocytes were isolated and CD4+ T cells were plated in coated 24-well plates, 5X105Per well/1 ML; culturing the cells for 3 days, collecting cell supernatant, and detecting IL-2 secretion by ELISA;
g) establishment of mouse subcutaneous tumor and antibody:
c57BL/6 mice or Balb/C mice 8 weeks old were randomly divided into 2 groups; c57BL/6 mice were inoculated subcutaneously in the right dorsal side with 5X105100ul PBSde mouse intestinal cancer cell MC38, or 3X105100ul PBSde mouse lung cancer cell LLC, or 5X105100ul PBS mouse liver cancer cell Hepa 1-6; balb/c mice were inoculated subcutaneously on the right dorsal side with 5X105Mouse breast cancer cell 4T1 in 100ul PBS; intraperitoneal injection of 200ug of control antibody or anti-BTNL 2 antibody was performed 4, 7, 10, 12 days after inoculation; tumor start measurement, tumor measurement formula is: tumor volume (length X width)2) 2; and the survival time of the mice is plotted as a survival curve.
Another object of the present invention is to provide a tumor vaccine prepared using the drug for cancer treatment.
It is another object of the present invention to provide a tumor immune checkpoint inhibitor prepared using the drug for use in the treatment of cancer.
Another object of the present invention is to provide an adoptive cancer cell therapy inhibitor for tumors prepared using the drug for cancer therapy.
Another object of the present invention is to provide a tumor cytokine therapy inhibitor prepared using the drug for cancer therapy.
In summary, the advantages and positive effects of the invention are:
because BTNL2 can inhibit the activation of T cells, the invention prepares the blocking monoclonal antibody aiming at BTNL2, and the blocking antibody can reverse the inhibition effect of BTNL2 protein on the T cells through in vitro experiments. Through a mouse tumor-bearing experiment, the invention discovers that the injection of the antibody can obviously inhibit the growth of various tumors and can obviously prolong the survival time of a mouse. BTNL2 is an important immune checkpoint protein, and a blocked monoclonal antibody against BTNL2 can be used as a drug to treat tumors.
The invention has the following advantages:
the monoclonal antibody secreted by the hybridoma (BTN-1) can identify a recombinant protein BTNL2 molecule through western blot (western blot), and can detect endogenous BTNL2 proteins expressed by various tumor tissues such as colon cancer, melanoma, lung cancer, breast cancer, prostate cancer, liver cancer and the like through the western blot.
The monoclonal antibody produced by the hybridoma (BTN-1) obtained by the invention can be applied to detection and screening of Immunohistochemistry (IHC), immunoblotting (Western blot), indirect ELISA, antibody chip preparation and the like, and has high specificity and sensitivity.
The antibody secreted by the hybridoma (BTN-1) obtained by the invention has extremely strong specificity and sensitivity in combination with the recombinant and endogenously expressed BTNL2 protein, and can block, neutralize the activity of the recombinant and endogenous BTNL2 protein.
Fourthly, the monoclonal antibody injection produced by the hybridoma (BTN-1) obtained by the invention can greatly prolong the survival period of tumor-bearing mice and can inhibit the growth of tumors, and the monoclonal antibody can be used as a medicament for treating cancers to treat various tumors.
The invention finds that therapeutic antibodies against BTNL2 can be used for the treatment of a variety of cancers, providing new targets and drugs for cancer therapy.
Drawings
FIG. 1 is a photograph of the running gel, Coomassie Brilliant blue staining after purification of the recombinant protein His-BTNL2(24-454) provided in the examples of the present invention. His-BTNL2(24-454) was seen to be of good purity and was mostly secreted in the culture supernatant.
Figure 2 is a screen for blocking/therapeutic BTNL2 antibodies using the T cell activation assay provided in the examples of the invention. It can be seen that antibodies secreted by hybridoma No.2 significantly reversed the inhibitory effect of BTNL2 on T cells. Hybridoma No.2 was named BTN-1.
FIG. 3 shows the detection of endogenous BTNL2 expression in different tumors using Western blot of purified antibody secreted from BTN-1 hybridoma according to the present invention. The arrow indicates endogenous BTNL2 with a molecular weight of about 72 kD.
FIG. 4 is a graph of the therapeutic effect of an anti-BTNL 2 antibody provided in an example of the invention on the treatment of tumors vaccinated with mouse lung cancer LLC. As can be seen, the anti-BTNL 2 antibody significantly inhibited the growth of mouse lung cancer relative to the control antibody.
FIG. 5 is a graph of the tumor treatment effect of anti-BTNL 2 antibody treatment on MC38 cell vaccination of mouse colon cancer provided by the examples of the present invention. It can be seen that the anti-BTNL 2 antibody significantly prolonged the survival of the mice relative to the control antibody.
FIG. 6 shows the effect of anti-BTNL 2 antibody therapy on tumor treatment by mouse liver cancer Hepa1-6 cell vaccination. It can be seen that the anti-BTNL 2 antibody significantly prolonged the survival of the mice relative to the control antibody.
FIG. 7 is the tumor therapeutic effect of anti-BTNL 2 antibody treatment provided by the present invention on vaccination of mouse breast cancer 4T1 cells. It can be seen that the anti-BTNL 2 antibody significantly prolonged the survival of the mice relative to the control antibody.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the prior art, the effect of being able to reverse the inhibitory effect of the BTNL2 protein on T cells was poor. The medicine for inhibiting the growth of various tumors has poor effect. In the prior art, the overall response rate of the immune checkpoint inhibitor is low, and drug resistance occurs.
In order to solve the above technical problems, the present invention will be described in detail with reference to specific embodiments.
The invention constructs the coding sequence of BTNL2 extracellular region on an expression vector pINFUSE-hIgG2-Fc2, expresses recombinant protein His-BTNL2 through mammalian cells, immunizes rats with the recombinant protein His-BTNL2, and prepares a monoclonal antibody aiming at BTNL 2. Therapeutic monoclonal antibodies capable of blocking the BTNL2 protein were selected by in vitro T cell activation experiments and antibody purified. Through establishing different subcutaneous tumor models of mice, the anti-BTNL 2 antibody is found to be capable of inhibiting the growth of tumors and prolonging the survival period of the mice in various tumor models, so that the BTNL2 is suggested to be a new tumor immunotherapy target, and the anti-BTNL 2 antibody has the potential of becoming a new anti-tumor drug.
The medicine for treating cancer provided by the embodiment of the invention comprises:
a BTNL2 antibody or a composition comprising a BTNL2 antibody.
The BTNL2 is human BTNL2 or mouse BTNL 2. Wherein, the amino acid sequence of human BTNL2 is shown as SEQ ID NO.1, and the coding nucleotide sequence is shown as SEQ ID NO. 2; the amino acid sequence of mouse BTNL2 is shown in SEQ ID NO.3, and the coding nucleotide sequence is shown in SEQ ID NO. 4.
In embodiments of the invention, the BTNL2 extracellular domain polypeptide used to purify the antibody is an immunogen. The BTNL2 extracellular region polypeptide is respectively leucine at position 24 to serine at position 455 of human BTNL2 and serine at position 24 to serine at position 454 of mouse Ephrin-B1, and the coding nucleotides are respectively nucleotide 69-1365 of the sequence shown in SEQ ID NO.2 and nucleotide 69-1362 of the sequence shown in SEQ ID NO. 4.
In embodiments of the invention, the BTNL2 extracellular domain polypeptide used to purify the antibody is an immunogen. A fusion polypeptide consisting of the BTNL2 extracellular region and a 6XHis tag was used for purification of the immunogen.
The embodiment of the invention provides a vector for expressing the fusion antigen polypeptide, wherein the framework vector of the vector is preferably pINFUSE-hIgG2-Fc2 and comprises nucleotides for coding the BTNL2 extracellular region.
In embodiments of the invention, host vector systems for producing the above-described fusion antigen polypeptides, host cells comprising the above-described vectors, are provided. The host cell is a bacterial cell, a yeast cell, an insect cell or a mammalian cell, preferably 293F, COS7, a CHO cell.
In an embodiment of the present invention, a method for constructing the vector of the fusion antigen polypeptide comprises: fusion antigen polypeptides are polypeptides produced by the above-described host vector system under conditions that permit production of the fusion polypeptide. The invention clones the gene sequence coding the leucine 24 to the serine 455 of BTNL2 and uses the expression and purification of mammalian cells as immunogen to immunize rats. The positive hybridoma cell line capable of efficiently secreting the monoclonal antibody is obtained by fusion with mouse hybridoma cells and screening and cloning of recombinant CP4 EPSPS.
Collecting culture supernatant by culturing the hybridoma cell line, and purifying the culture supernatant by Protein G column affinity chromatography to obtain the rat monoclonal antibody. Immunoblotting (Western blot) experiments showed that the antibody specifically recognized BTNL2 protein endogenous to tumor cells.
Through T cell activation experiments, a therapeutic antibody with a blocked activity of BTNL2 protein was identified.
Through mouse tumor-bearing experiments, the BTNL2 therapeutic antibody can greatly prolong the survival time of tumor-bearing mice and can inhibit the growth of tumors. The tested tumor types include intestinal cancer, lung cancer, liver cancer and breast cancer.
In the embodiment of the invention, the BTNL2 antibody is used for preparing a medicament for treating various cancers, wherein the cancer types include, but are not limited to, lung cancer, intestinal cancer, liver cancer, breast cancer, stomach cancer, esophageal cancer, cervical cancer, prostate cancer, nasopharyngeal cancer and the like; the administration route of the drug for treating cancer is oral administration or intravenous administration.
The invention is further described with reference to specific examples.
Example 1
(1) Laboratory mouse
C57BL/6 mice, purchased from the drug safety evaluation center of Hubei province and housed in the SPF-level animal laboratory at the drug safety evaluation center of Hubei province. And selecting female mice with the age of 8 weeks for animal experiments. Mice were randomized into 2-3 groups of 10 mice each, one group injected with control antibody, one group injected with anti-BTNL 2 antibody, and the other group injected with anti-PD-L1 antibody as a positive control.
(2) pINFUSE-hIgG2-His-BTNL2 vector construction:
a mouse BTNL2 complete cDNA sequence (coding nucleotide sequence is shown in SEQ ID NO. 4) was synthesized by the company, and using this as a template, a mouse BTNL2 extracellular region expression vector (amino acids in the extracellular region were from lysine at position 24 to serine at position 454, constructed by the company clone) was constructed. The expression vector was pINFUSE-hIgG2-Fc2, purchased from Invivogen.
The PCR reaction system is as follows:
10XPCR buffer:5μl,
dNTP:1μl,
upstream/downstream primers: 1ul (10. mu.M) each,
template DNA: 1ng of the feed water is added into the feed water,
PFU:1μl
the total reaction system is as follows: 50 μ l.
The PCR reaction procedure was as follows:
5 minutes at 95 deg.C, (1 minute at 95 deg.C, 30 seconds at 58 deg.C, 2 minutes at 72 deg.C) 30 cycles, 10 minutes at 72 deg.C, 4 deg.C.
And (4) running nucleic acid electrophoresis on the PCR reaction system, and cutting gel to recover DNA fragments. The DNA fragment after gel cutting recovery and pINFUSE-hIgG2-Fc2 empty vector were digested at 37 ℃ for 2 hours (NCoI and BglII digestion), and the DNA fragment was recovered. The recovered DNA fragment and the vector were ligated at 16 ℃ overnight. Mu.l of the ligation product was used to transform DH 5. alpha. competent cells. And (3) putting the transformation plate into a 37 ℃ bacterial incubator overnight, taking a monoclonal colony the next day, carrying out colony PCR identification, extracting a plasmid from the correctly identified colony, and sending the colony to a company for sequencing. Sequencing the correct plasmid will be extracted from the plasmid and prepared to transfect cells. NCoI and BglII enzymes were purchased from NEB. The DNA gel cutting recovery kit and the DNA solution recovery kit were purchased from Omega, and the article numbers were D2500-01 and D2500-02, and the recovery methods were referred to the product manual. DNA T4 ligase was purchased from THERMO FISHER and assigned the accession number EL0014, and the ligation protocol was referred to the product description. DH 5. alpha. competent cells were purchased from Tiangen Biochemical technology Inc., having the designation CB 101.
(3) Preparation of recombinant protein His-BTNL2 (24-454):
the constructed expression vector pINFUSE-hIgG2-His-BTNL2(24-454) was transfected into 293F cells (1L cultured cells, 1X 10)6/mL), 5% CO at 37 deg.C2Shaking at 130rpm for 6 days. The transfection reagent is PolyPlus transfection reagent from PolyPlus transfection company, the product number is FECTO PRO, and the specific loading method is shown in the product description. Cell culture supernatants were harvested at day 6 post-transfection, centrifuged to remove cells (4 ℃, 1500rpm, 5 min), and filtered through 0.45 μm filters. And purifying the filtered supernatant by using a His-tag purification kit to obtain recombinant protein (His-tag protein purification kit produced in Biyunsian, product number P2226), wherein the purification method is shown in the product specification. The purified eluted protein was ultrafiltered by Millipore's ultrafiltration strain (stock number UFC501096), the protein was dissolved in 1XPBS, and the solution was passed through a 0.22 μm filter and stored at-80 ℃.
(4) The second step is that: establishment of hybridoma cell lines:
i) immunization
The polypeptide crosslinked in the first step was emulsified with Freund's complete adjuvant (Sigma Co.), female rats of 6-8 weeks of age (purchased from Beijing Wintolite laboratories Inc.), injected subcutaneously at 6 spots per rat in the abdominal region, at a dose of 60 ug/mouse, boosted once for 14 days, and the antigen emulsified with Freund's incomplete adjuvant (Sigma Co.) at a dose of 30 ug/mouse. 7 days after 3 times of booster immunization, indirect ELISA (wavelength of 450nm) is used for detecting the multi-antibody titer of the anti-immunogen in the rat serum, the rat with the highest titer is injected with tail vein for impact immunization, and the antigen is uniformly mixed with normal saline, and the dosage is 50 ug/mouse.
The antigen for immunizing rats is recombinant His-BTNL2(24-454) antigen protein, and the recombinant His-BTNL2(24-454) antigen protein is obtained by encoding a nucleotide sequence of SEQ ID No.1 in a sequence table and recombining and expressing the encoded nucleotide sequence by a mammalian cell 293F.
j) Cell fusion
Aseptically preparing spleen cell suspension of rat with immunity reaching standard, mixing with mouse myeloma cell sp2/0(ATCC) at ratio of 5:1, centrifuging at 1500rpm for 5 min; the supernatant was discarded and the tube was placed in a 37 ℃ water bath, Iml of PEG1500(Roche Co.) was added slowly over 1 minute, and the cells were agitated; after standing in warm water for 1min, 10ml serum-free top DM (Sigma) was added, mixed with a bar and centrifuged at 1000rpm for 5 min; discarding the supernatant, adding 10ml serum (PAA company) to blow the cells carefully, adding 5ml thymocytes mixed with 10XHAT (Sigma company), and mixing well; then 25ml of semisolid culture medium containing 2.1% of nitrocellulose (Sigma company) is added and mixed evenly, and then the mixture is poured into 20 cell culture dishes evenly; the cell culture dish was placed in a wet box and incubated in a 5% CO2) incubator at 37 ℃.
k) Picking clone
The size and density of the clone cell mass are moderate 7 days after fusion, and the round, solid and large clone mass is sucked under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture is carried out in a 5% CO2 incubator at 37 ℃.
l) ELISA screening for Positive hybridoma cells
After 3 days, the cell mass was about 2/3 basal areas, and IOOyl supernatants were screened by ELISA using the immunogen and the synthetic polypeptide, respectively; positive clones were completely changed and 200y1 complete medium containing feeder cells and 1% HT (Sigma) was added; two days later, performing second ELISA screening, and transferring the positive clone into a 24-well plate prepared with a culture medium (containing feeder cells and HT) in advance for culture; after five days, 100y1 supernatant is taken for the third ELISA screening, and positive clones are transferred into a 6-well plate and a cell culture bottle successively for amplification culture and frozen storage. The obtained mouse hybridoma cell line BTN-1.
(5) Purification of antibody secreted by mouse hybridoma cell line BTN-1
Using serum replacement (KNOCKOUTTMSerum replacement, GIBCO, cat # 10828-. Mixing 5X106Cells were transferred to 10cm dishes at a cell density of about 50% the following day. After three days, the supernatant was removed and replaced with fresh medium, and the culture was continued for 3 days, and the supernatant was collected. The cells were removed by centrifugation (4 ℃, 1500rpm, 5 minutes) and filtered through a 0.45 μm filter. The filtered supernatant was purified of recombinant protein using protein G pre-packed column (pre-packed column purchased from Wuhan Hui research, Inc., cat #)HZ1012-1), and the purification method is described in the product specification. The purified eluted antibody was ultrafiltered by Millipore (stock No. UFC501096), the antibody was fused in 1XPBS, and the antibody was passed through a 0.22 μm filter and stored at-80 ℃.
(6) T cell activation assay screening for blocking/therapeutic BTNL2 antibodies
24-well plates were coated with anti-CD3e (1ug/ml PBS) and anti-CD28(1ug/ml PBS) T cell activator (T cell activator from biolenged, cat. 423304) overnight at 4 degrees. The following day the 24-well plate was washed twice with 1XPBS and 20ug/ml of BTNL2-His protein was added for two hours at room temperature. 1XPBS washed 24 well plates twice. Mouse CD4+ T lymphocytes are separated (by using a cell separation kit of Tian-whirly mouse CD4+ in Germany, the cargo number is 130-104-454), and the specific method is described in the product specification. Planting CD4+ T into coated 24-well plate, 5X105Per well/1 ML (cell culture medium RPMI1640+ 10% FBS). Cells were cultured for 3 days, and cell supernatants were collected and assayed for IL-2 secretion by ELISA.
(7) Establishment of mouse subcutaneous tumors and antibody treatment:
the 8-week-old C57BL/6 mice or Balb/C mice were randomly divided into 2 groups. C57BL/6 mice were inoculated subcutaneously in the right dorsal side with mouse intestinal cancer cells MC38(5X 10)5100ul PBS), or mouse lung cancer cell LLC (3X 10)5100ul PBS), or mouse hepatoma cell Hepa1-6(5X 10)5100ul PBS). Balb/c mice were inoculated subcutaneously on the right dorsal side with mouse breast cancer cells 4T1(5X 10)5100ul PBS). 200ug of control antibody or anti-BTNL 2 antibody were intraperitoneally injected 4, 7, 10, and 12 days after inoculation (some experiments injected 200ug of anti-PD-L1 antibody as a positive control, anti-PD-L1 antibody purchased from BioXcell, Inc., Cat. No. BE 0101). The measurement is started when the tumor is visible by naked eyes, and the tumor measurement formula is as follows: tumor volume (length X width)2)/2。
And the survival time of the mice is plotted as a survival curve.
The present invention is further described below in conjunction with the experimental results.
The results are shown in FIGS. 1 to 7. FIG. 1 is a photograph of the running gel, Coomassie Brilliant blue staining after purification of the recombinant protein His-BTNL2 (24-454). His-BTNL2(24-454) was found to be of good purity and was mostly secreted in the culture supernatant. Figure 2 is a screening of blocking/therapeutic BTNL2 antibodies using a T cell activation assay. It can be seen that antibodies secreted by hybridoma No.2 significantly reversed the inhibitory effect of BTNL2 on T cells. We named hybridoma No.2 as BTN-1. FIG. 3 shows the detection of endogenous BTNL2 expression in different tumors using protein imprinting (western blot) with purified antibodies secreted from BTN-1 hybridomas. The arrow indicates endogenous BTNL2 with a molecular weight of about 72 kD. FIG. 4 is the therapeutic effect of anti-BTNL 2 antibody on mouse lung cancer LLC inoculated tumors. As can be seen, the anti-BTNL 2 antibody significantly inhibited the growth of mouse lung cancer relative to the control antibody. FIG. 5 is the tumor therapeutic effect of anti-BTNL 2 antibody treatment on vaccination of mouse colon cancer MC38 cells. It can be seen that the anti-BTNL 2 antibody significantly prolonged the survival of the mice relative to the control antibody. FIG. 6 shows the effect of anti-BTNL 2 antibody treatment on tumor therapy by mouse hepatoma Hepa1-6 cell vaccination. It can be seen that the anti-BTNL 2 antibody significantly prolonged the survival of the mice relative to the control antibody. Figure 7 is the tumor therapeutic effect of anti-BTNL 2 antibody treatment on vaccination of mouse breast cancer 4T1 cells. It can be seen that the anti-BTNL 2 antibody significantly prolonged the survival of the mice relative to the control antibody.
In the present invention, SEQ ID NO.1
MVDFPGYNLSGAVASFLFILLTMKQSEDFRVIGPAHPILAGVGEDALLTCQLLPKRTTMHVEVRWYRSEPSTPVFVHRDGVEVTEMQMEEYRGWVEWIENGIAKGNVALKIHNIQPSDNGQYWCHFQDGNYCGETSLLLKVAGLGSAPSIHMEGPGESGVQLVCTARGWFPEPQVYWEDIRGEKLLAVSEHRIQDKDGLFYAEATLVVRNASAESVSCLVHNPVLTEEKGSVISLPEKLQTELASLKVNGPSQPILVRVGEDIQLTCYLSPKANAQSMEVRWDRSHRYPAVHVYMDGDHVAGEQMAEYRGRTVLVSDAIDEGRLTLQILSARPSDDGQYRCLFEKDDVYQEASLDLKVVSLGSSPLITVEGQEDGEMQPMCSSDGWFPQPHVPWRDMEGKTIPSSSQALTQGSHGLFHVQTLLRVTNISAVDVTCSISIPFLGEEKIATFSLSESRMTFLWKTLLVWGLLLAVAVGLPRKRS。
SEQ ID NO.2
SEQ ID NO.3
MVDCPRYSLSGVAASFLFVLLTIKHPDDFRVVGPNLPILAKVGEDALLTCQLLPKRTTAHMEVRWYRSDPDMPVIMYRDGAEVTGLPMEGYGGRAEWMEDSTEEGSVALKIRQVQPSDDGQYWCRFQEGDYWRETSVLLQVAALGSSPNIHVEGLGEGEV
QLVCTSRGWFPEPEVHWEGIWGEKLMSFSENHVPGEDGLFYVEDTLMVRNDSVETISCFIYSHGLRETQEATIALSERLQTELASVSVIGHSQPSPVQVGENIELTCHLSPQTDAQNLEVRWLRSRYYPAVHVYANGTHVAGEQMVEYKGRTSLVTDAIHEGKLTLQIHNARTSDEGQYRCLFGKDGVYQEARVDVQVMAVGSTPRITREVLKDGGMQLRCTSDGWFPRPHVQWRDRDGKTMPSFSEAFQQGSQELFQVETLLLVTNGSMVNVTCSISLPLGQEKTARFPLSDSKIALLWMTLPVVVLPLAMAIDLIKVKRWRRTNEQTHSSNQENNKNDENHRRRLPSDERLR。
SEQ ID NO.4
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> university of science and technology in Huazhong
<120> a drug, a tumor vaccine and an inhibitor for cancer treatment
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>482
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Met Val Asp Phe Pro Gly Tyr Asn Leu Ser Gly Ala Val Ala Ser Phe
1 5 10 15
Leu Phe Ile Leu Leu Thr Met Lys Gln Ser Glu Asp Phe Arg Val Ile
20 25 30
Gly Pro Ala His Pro Ile Leu Ala Gly Val Gly Glu Asp Ala Leu Leu
35 40 45
Thr Cys Gln Leu Leu Pro Lys Arg Thr Thr Met His Val Glu Val Arg
50 55 60
Trp Tyr Arg Ser Glu Pro Ser Thr Pro Val Phe Val His Arg Asp Gly
65 70 75 80
Val Glu Val Thr Glu Met Gln Met Glu Glu Tyr Arg Gly Trp Val Glu
85 90 95
Trp Ile Glu Asn Gly Ile Ala Lys Gly Asn Val Ala Leu Lys Ile His
100 105 110
Asn Ile Gln Pro Ser Asp Asn Gly Gln Tyr Trp Cys His Phe Gln Asp
115 120 125
Gly Asn Tyr Cys Gly Glu Thr Ser Leu Leu Leu Lys Val Ala Gly Leu
130 135 140
Gly Ser Ala Pro Ser Ile His Met Glu Gly Pro Gly Glu Ser Gly Val
145 150 155 160
Gln Leu Val Cys Thr Ala Arg Gly Trp Phe Pro Glu Pro Gln Val Tyr
165 170 175
Trp Glu Asp Ile Arg Gly Glu Lys Leu Leu Ala Val Ser Glu His Arg
180 185 190
Ile Gln Asp Lys Asp Gly Leu Phe Tyr Ala Glu Ala Thr Leu Val Val
195 200 205
Arg Asn Ala Ser Ala Glu Ser Val Ser Cys Leu Val His Asn Pro Val
210 215 220
Leu Thr Glu Glu Lys Gly Ser Val Ile Ser Leu Pro Glu Lys Leu Gln
225 230 235 240
Thr Glu Leu Ala Ser Leu Lys Val Asn Gly Pro Ser Gln Pro Ile Leu
245 250 255
Val Arg Val Gly Glu Asp Ile Gln Leu Thr Cys Tyr Leu Ser Pro Lys
260 265 270
Ala Asn Ala Gln Ser Met Glu Val Arg Trp Asp Arg Ser His Arg Tyr
275 280 285
Pro Ala Val His Val Tyr Met Asp Gly Asp His Val Ala Gly Glu Gln
290 295 300
Met Ala Glu Tyr Arg Gly Arg Thr Val Leu Val Ser Asp Ala Ile Asp
305 310 315 320
Glu Gly Arg Leu Thr Leu Gln Ile Leu Ser Ala Arg Pro Ser Asp Asp
325 330 335
Gly Gln Tyr Arg Cys Leu Phe Glu Lys Asp Asp Val Tyr Gln Glu Ala
340 345 350
Ser Leu Asp Leu Lys Val Val Ser Leu Gly Ser Ser Pro Leu Ile Thr
355 360 365
Val Glu Gly Gln Glu Asp Gly Glu Met Gln Pro Met Cys Ser Ser Asp
370 375 380
Gly Trp Phe Pro Gln Pro His Val Pro Trp Arg Asp Met Glu Gly Lys
385 390 395 400
Thr Ile Pro Ser Ser Ser Gln Ala Leu Thr Gln Gly Ser His Gly Leu
405 410 415
Phe His Val Gln Thr Leu Leu Arg Val Thr Asn Ile Ser Ala Val Asp
420 425 430
Val Thr Cys Ser Ile Ser Ile Pro Phe Leu Gly Glu Glu Lys Ile Ala
435 440 445
Thr Phe Ser Leu Ser Glu Ser Arg Met Thr Phe Leu Trp Lys Thr Leu
450 455 460
Leu Val Trp Gly Leu Leu Leu Ala Val Ala Val Gly Leu Pro Arg Lys
465 470 475 480
Arg Ser
<210>2
<211>1449
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
atggtggatt ttccaggcta caatctgtct ggtgcagtcg cctccttcct attcatcctg 60
ctgacaatga agcagtcaga agactttaga gtcattggcc ctgctcatcc tatcctggcc 120
ggggttgggg aagatgccct gttaacctgc cagctactcc ccaagaggac cacaatgcac 180
gtggaggtga ggtggtaccg ctcagagccc agcacacctg tgtttgtgca cagggatgga 240
gtggaggtga ctgagatgca gatggaggag tacagaggct gggtagagtg gatagagaat 300
ggcattgcaa agggaaatgt ggcactgaag atacacaaca tccagccctc cgacaatgga 360
caatactggt gccatttcca ggatgggaac tactgtggag aaacaagctt gctgctcaaa 420
gtagcaggtc tggggtctgc ccctagcatc cacatggagg gacctgggga gagtggagtc 480
cagcttgtgt gcactgcaag gggctggttc ccagagcccc aggtgtattg ggaagacatc 540
cggggagaga agctgctggc cgtgtctgag catcgcatcc aagataaaga tggcctgttc 600
tatgcggaag ccaccctggt ggtcaggaac gcctctgcag agtctgtgtc ctgcttggtc 660
cacaaccccg tcctcactga ggagaagggg tcggtcatca gcctcccaga gaaactccag 720
actgagctgg cttctttaaa agtgaatgga ccttcccagc ccatcctcgt cagagtggga 780
gaagatatac agctaacctg ttacctgtcc cccaaggcga atgcacagag catggaggtg 840
aggtgggacc gatcccaccg ttaccctgct gtgcatgtgt atatggatgg ggaccatgtg 900
gctggagagc agatggcaga gtacagaggg aggactgtac tggtgagtga cgccattgac 960
gagggcagac tgaccctgca gatactcagt gccagacctt cggacgacgg gcagtaccgc 1020
tgcctttttg aaaaagatga tgtctaccag gaggccagtt tggatctgaa ggtggtaagt 1080
ctgggttctt ccccactgat cactgtggag gggcaagaag atggagaaat gcagccgatg 1140
tgctcttcag atgggtggtt cccacagccc cacgtgccat ggagggacat ggaaggaaag 1200
acgataccat catcttccca ggccctgact caaggcagcc acgggctgtt ccacgtgcag 1260
acattgctaa gggtcacaaa catctccgct gtggacgtca cttgttccat cagcatcccc 1320
tttttgggcg aggagaaaat cgcaactttt tctctctcag agtccaggat gacgtttttg 1380
tggaaaacac tgcttgtttg gggattgctt cttgctgtgg ctgtaggcct gcccaggaag 1440
aggagctga 1449
<210>3
<211>514
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Met Val Asp Cys Pro Arg Tyr Ser Leu Ser Gly Val Ala Ala Ser Phe
1 5 10 15
Leu Phe Val Leu Leu Thr Ile Lys His Pro Asp Asp Phe Arg Val Val
20 25 30
Gly Pro Asn Leu Pro Ile Leu Ala Lys Val Gly Glu Asp Ala Leu Leu
35 40 45
Thr Cys Gln Leu Leu Pro Lys Arg Thr Thr Ala His Met Glu Val Arg
50 55 60
Trp Tyr Arg Ser Asp Pro Asp Met Pro Val Ile Met Tyr Arg Asp Gly
65 70 75 80
Ala Glu Val Thr Gly Leu Pro Met Glu Gly Tyr Gly Gly Arg Ala Glu
85 90 95
Trp Met Glu Asp Ser Thr Glu Glu Gly Ser Val Ala Leu Lys Ile Arg
100 105 110
Gln Val Gln Pro Ser Asp Asp Gly Gln Tyr Trp Cys Arg Phe Gln Glu
115 120 125
Gly Asp Tyr Trp Arg Glu Thr Ser Val Leu Leu Gln Val Ala Ala Leu
130 135 140
Gly Ser Ser Pro Asn Ile His Val Glu Gly Leu Gly Glu Gly Glu Val
145 150 155 160
Gln LeuVal Cys Thr Ser Arg Gly Trp Phe Pro Glu Pro Glu Val His
165 170 175
Trp Glu Gly Ile Trp Gly Glu Lys Leu Met Ser Phe Ser Glu Asn His
180 185 190
Val Pro Gly Glu Asp Gly Leu Phe Tyr Val Glu Asp Thr Leu Met Val
195 200 205
Arg Asn Asp Ser Val Glu Thr Ile Ser Cys Phe Ile Tyr Ser His Gly
210 215 220
Leu Arg Glu Thr Gln Glu Ala Thr Ile Ala Leu Ser Glu Arg Leu Gln
225 230 235 240
Thr Glu Leu Ala Ser Val Ser Val Ile Gly His Ser Gln Pro Ser Pro
245 250 255
Val Gln Val Gly Glu Asn Ile Glu Leu Thr Cys His Leu Ser Pro Gln
260 265 270
Thr Asp Ala Gln Asn Leu Glu Val Arg Trp Leu Arg Ser Arg Tyr Tyr
275 280 285
Pro Ala Val His Val Tyr Ala Asn Gly Thr His Val Ala Gly Glu Gln
290 295 300
Met Val Glu Tyr Lys Gly Arg Thr Ser Leu Val Thr Asp Ala Ile His
305 310 315 320
Glu Gly Lys LeuThr Leu Gln Ile His Asn Ala Arg Thr Ser Asp Glu
325 330 335
Gly Gln Tyr Arg Cys Leu Phe Gly Lys Asp Gly Val Tyr Gln Glu Ala
340 345 350
Arg Val Asp Val Gln Val Met Ala Val Gly Ser Thr Pro Arg Ile Thr
355 360 365
Arg Glu Val Leu Lys Asp Gly Gly Met Gln Leu Arg Cys Thr Ser Asp
370 375 380
Gly Trp Phe Pro Arg Pro His Val Gln Trp Arg Asp Arg Asp Gly Lys
385 390 395 400
Thr Met Pro Ser Phe Ser Glu Ala Phe Gln Gln Gly Ser Gln Glu Leu
405 410 415
Phe Gln Val Glu Thr Leu Leu Leu Val Thr Asn Gly Ser Met Val Asn
420 425 430
Val Thr Cys Ser Ile Ser Leu Pro Leu Gly Gln Glu Lys Thr Ala Arg
435 440 445
Phe Pro Leu Ser Asp Ser Lys Ile Ala Leu Leu Trp Met Thr Leu Pro
450 455 460
Val Val Val Leu Pro Leu Ala Met Ala Ile Asp Leu Ile Lys Val Lys
465 470 475 480
Arg Trp Arg Arg Thr AsnGlu Gln Thr His Ser Ser Asn Gln Glu Asn
485 490 495
Asn Lys Asn Asp Glu Asn His Arg Arg Arg Leu Pro Ser Asp Glu Arg
500 505 510
Leu Arg
<210>4
<211>1545
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
atggtggatt gcccacggta tagtctatct ggcgtggctg cctccttcct cttcgtcctg 60
ctgactataa agcacccaga tgacttcaga gtggtcggtc ctaacctccc aatcttggct 120
aaagtcgggg aagatgccct gctaacgtgt cagctcctcc ccaagaggac cacggcacac 180
atggaggtga ggtggtaccg ctccgaccct gacatgccag tgattatgta ccgggatgga 240
gctgaggtga ctgggctacc gatggagggg tacggaggcc gggcagagtg gatggaggac 300
agcactgaag agggcagtgt ggctctgaag attcgccagg tccagccaag tgacgatggc 360
cagtactggt gccgcttcca ggagggggac tactggagag agacaagcgt gctactccaa 420
gtggctgctc taggatcttc cccaaatatc catgtggagg gactcggaga aggagaggtc 480
cagcttgtat gcacgtcccg aggctggttc cctgagcctg aggtgcactg ggaaggcatc 540
tggggagaaa agttgatgag tttctctgag aatcatgtgc caggtgaaga tgggctattc 600
tatgtggaag acacactgat ggtcaggaat gacagtgtag agaccatttc ctgcttcatc 660
tacagccatg gcctcagaga gacccaggag gccaccatcg ctctgtcaga gaggctccag 720
accgaactgg cttccgttag cgtaatcgga cattcccagc ccagccctgt tcaagtcgga 780
gagaacatag aattaacttg tcacctctca cctcaaacgg atgctcagaa cttagaggtg 840
aggtggctcc gatcccgcta ttaccctgca gtccacgtgt atgcaaatgg cacccacgtg 900
gctggagagc agatggtaga atacaaaggg aggacttcat tggtgactga tgccatccac 960
gagggaaaac tgaccctgca aattcacaat gccagaactt cggatgaagg gcagtaccgg 1020
tgcctttttg gaaaagatgg tgtctaccag gaggcccgtg tggatgtgca ggtgatggcg 1080
gtgggttcca ccccacggat caccagggag gtcttgaaag atggaggcat gcagctgagg 1140
tgtacgtctg atgggtggtt cccacggccc catgtgcagt ggagggacag agatggaaag 1200
acaatgccat cgttttccga ggcctttcag caagggagcc aggagctgtt ccaggtggag 1260
acacttctgc tggtcacaaa cggctccatg gtgaatgtga cctgctccat cagcctccct 1320
ctgggccagg agaaaacagc ccgtttccct ctctcagact ccaagatagc tttgctatgg 1380
atgaccctgc ctgttgtggt gctgcctctg gccatggcta tcgacctgat caaggtgaaa 1440
cggtggcggc ggaccaatga acaaacacac agcagcaatc aggaaaataa caagaatgac 1500
gaaaaccaca ggcggcgact tccttctgat gagaggctca gatga 1545
Claims (10)
1. A medicament for use in the treatment of cancer, wherein the medicament for use in the treatment of cancer is a BTNL2 monoclonal therapeutic antibody;
the amino acid sequence of the BTNL2 monoclonal therapeutic antibody is SEQ ID NO.1 or SEQ ID NO. 3; the coding nucleotide sequence of SEQ ID NO.1 is SEQ ID NO. 2; the coding nucleotide sequence of SEQ ID NO.3 is SEQ ID NO. 4.
2. The medicament for use in the treatment of cancer according to claim 1, wherein the BTNL2 extracellular domain polypeptide used for purifying the antibody is an immunogen; the BTNL2 extracellular domain polypeptide is from leucine 24 to serine 455 of amino acid sequence SEQ ID NO.1 or from lysine 24 to serine 454 of amino acid sequence SEQ ID NO.3, and the coding nucleotide of the BTNL2 extracellular domain polypeptide is nucleotide 69-1365 of the sequence shown in SEQ ID NO.2 or nucleotide 69-1362 of the sequence shown in SEQ ID NO. 4.
3. The medicament for use in the treatment of cancer according to claim 1, wherein a fusion polypeptide consisting of the extracellular region of BTNL2 and a 6XHis tag is used for purification of the immunogen.
4. A vector expressing a fusion antigen polypeptide constructed from a BTNL2 monoclonal therapeutic antibody for use in the treatment of cancer according to claim 1, wherein the vector is pINFUSE-hIgG2-Fc2 comprising nucleotides encoding the extracellular domain of BTNL 2.
5. A method for constructing the vector of claim 4, comprising: cloning and purifying a gene sequence encoding leucine 24 to serine 455 of BTNL2 by expression in mammalian cells, and immunizing a mouse with the cloned gene sequence as an immunogen; obtaining a positive hybridoma cell line efficiently secreting the monoclonal antibody by fusing with mouse hybridoma cells and screening and cloning by recombinant CP4 EPSPS;
collecting culture supernatant by culturing the hybridoma cell line, and purifying the culture supernatant by Protein G column affinity chromatography to obtain a rat monoclonal antibody; western blot experiments were performed to analyze the specific recognition of BTNL2 protein endogenous to tumor cells by mouse monoclonal antibodies.
6. An animal model construction method for verifying the efficacy of a drug for use in cancer therapy according to claim 1, comprising:
firstly, selecting 8-week-old female mice to perform animal experiments; mice were randomly divided into 2-3 groups of 10 mice each, one group injected with control antibody, one group injected with anti-BTNL 2 antibody, and the other group injected with anti-PD-L1 antibody as positive control;
pINFUSE-hIgG2-His-BTNL2 vector construction:
through synthesizing a mouse BTNL2 full cDNA sequence, the coding nucleotide sequence is shown as SEQ ID NO.4, and the mouse BTNL2 extracellular region expression vector is constructed by taking the coding nucleotide sequence as a template, wherein the amino acids of the extracellular region are from 24 th lysine to 454 th serine; the expression vector is pINFUSE-hIgG2-Fc 2;
the PCR reaction system is as follows:
10XPCR buffer:5μl,
dNTP:1μl,
upstream/downstream primers: 1ul (10. mu.M) each,
template DNA: 1ng of the feed water is added into the feed water,
PFU:1μl
the total reaction system is as follows: 50 μ l;
the PCR reaction program is:
5 minutes at 95 ℃, 30 times of circulation, 10 minutes at 72 ℃ and 4 ℃;
running nucleic acid for electrophoresis in a PCR reaction system, cutting gel and recovering DNA fragments; and (3) carrying out enzyme digestion on the DNA fragment after gel cutting recovery and the pINFUSE-hIgG2-Fc2 empty vector at 37 ℃ for 2 hours, and recovering the DNA fragment. Connecting the recovered DNA fragment with the vector at 16 ℃ overnight; mu.l of the ligation product was used to transform DH 5. alpha. competent cells. Putting the transformation plate into a 37 ℃ bacterial incubator overnight, taking a monoclonal colony the next day, carrying out colony PCR identification, extracting plasmid from the colony which is correctly identified, and sending the colony to a company for sequencing; carrying out plasmid extraction on the plasmid with correct sequencing to prepare transfected cells; DNA gel cutting recovery kit and DNA solution recovery kit;
preparation of recombinant protein His-BTNL 2:
the constructed expression vector pINFUSE-hIgG2-His-BTNL2 is transfected into 293F cells, 1L cultured cells and 1x 106Per mL, 5% CO at 37 ℃2Shaking at 130rpm for 6 days; collecting cell culture supernatant on 6 days after transfection, centrifuging to remove cells, filtering with 0.45 μm filter membrane at 4 deg.C and 1500rpm for 5 min; purifying the filtered supernatant with His-tag purification kit to obtain recombinant protein, ultrafiltering to obtain protein, dissolving in 1 × PBS, filtering with 0.22 μm filter membrane, measuring concentration, and storing at-80 deg.C;
secondly, establishing a hybridoma cell line:
a) immunization
Emulsifying the cross-linked polypeptide in the first step, immunizing female rats with the age of 6-8 weeks, injecting 6 points per rat by abdominal subcutaneous injection, wherein the dosage is 60 ug/rat, boosting immunity once in 14 days, emulsifying the antigen, and the dosage is 30 ug/rat; detecting the multi-antibody titer of the anti-immunogen in the rat serum by indirect ELISA 7 days after 3 rd boosting immunization, injecting the rat with the highest titer into tail vein for impact immunization, and uniformly mixing the antigen with normal saline, wherein the dosage is 50 ug/mouse;
the antigen used for immunizing rats is recombinant His-BTNL2(24-454) antigen protein, and the recombinant His-BTNL2(24-454) antigen protein is obtained by encoding a nucleotide sequence of SEQ ID No.1 in a sequence table and recombining and expressing the encoded protein by an animal cell 293F;
b) cell fusion:
aseptically preparing spleen cell suspension of rat with immunity reaching standard, mixing with mouse myeloma cell sp2/0(ATCC) at ratio of 5:1, centrifuging at 1500rpm for 5 min; discarding the supernatant, placing the centrifuge tube into a 37 ℃ water bath, slowly adding Iml PEG1500 in 1 minute, and stirring the cells; standing in warm water for 1min, adding 10ml serum-free top DM, mixing, and centrifuging at 1000rpm for 5 min; discarding the supernatant, adding 10ml serum to blow the cells, adding 5ml thymocytes mixed with 10XHAT, and mixing; then adding 25ml of semisolid culture medium containing 2.1% of nitrocellulose, fully mixing, and then uniformly pouring into 20 cell culture dishes; placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C2Culturing in an incubator;
c) selecting and cloning:
the cell mass is moderate in size and density 7 days after fusion, the round, solid and large cell masses are sucked and injected into a 96-hole culture plate of a culture medium, and 5% CO at 37℃ is added2Culturing in an incubator;
d) ELISA screening positive hybridoma cells:
after 3 days, the cell mass was about 2/3 basal areas, and IOOyl supernatants were screened by ELISA using the immunogen and the synthetic polypeptide, respectively; positive clones were completely changed and 200y1 complete medium containing feeder cells and 1% HT was added; performing second ELISA screening after two days, and transferring the positive clone into a 24-pore plate prepared with a culture medium in advance for culture; taking 100y1 supernatant after five days, carrying out third ELISA screening, and gradually transferring the positive clones into a 6-hole plate and a cell culture bottle for amplification culture and freezing; the obtained mouse hybridoma cell line BTN-1;
e) purification of antibody secreted by mouse hybridoma cell line BTN-1:
the mouse hybridoma cell line BTN-1 was cultured using serum replacement. Mixing 5X106Cells were transferred to 10cm dishes at a cell density of about 50% the following day. After three days, goThe supernatant was replaced with fresh medium, and the culture was continued for 3 days, and the supernatant was collected. The cells were removed by centrifugation and filtered through a 0.45 μm filter. Purifying the filtered supernatant by using a protein G prepacked column to obtain recombinant protein; ultrafiltering the purified eluted antibody with ultrafiltration strain of Milipore, fusing the ultrafiltered antibody in 1 × PBS, filtering with 0.22 μm filter membrane, measuring concentration, and storing at-80 deg.C;
f) t cell activation assay screening blocking/therapeutic BTNL2 antibodies:
coating a 24-well plate with anti-CD3ePBS and anti-CD28T cell activators, and standing at 4 ℃ overnight; the following day the 24-well plate was washed twice with 1XPBS and 20ug/ml of BTNL2-His protein was added for two hours at room temperature. 1XPBS washes 24 hole plate twice; mouse CD4+ T lymphocytes were isolated and CD4+ T cells were plated in coated 24-well plates, 5X105Per well/1 ML; culturing the cells for 3 days, collecting cell supernatant, and detecting IL-2 secretion by ELISA;
g) establishment of mouse subcutaneous tumor and antibody:
c57BL/6 mice or Balb/C mice 8 weeks old were randomly divided into 2 groups; c57BL/6 mice were inoculated subcutaneously in the right dorsal side with 5X105100ul PBSde mouse intestinal cancer cell MC38, or 3X105100ul PBSde mouse lung cancer cell LLC, or 5X105100ul PBS mouse liver cancer cell Hepa 1-6; balb/c mice were inoculated subcutaneously on the right dorsal side with 5X105Mouse breast cancer cell 4T1 in 100ul PBS; intraperitoneal injection of 200ug of control antibody or anti-BTNL 2 antibody was performed 4, 7, 10, 12 days after inoculation; tumor start measurement, tumor measurement formula is: tumor volume (length X width)2) 2; and the survival time of the mice is plotted as a survival curve.
7. A tumor vaccine prepared using the medicament of claim 1 for use in the treatment of cancer.
8. A tumor immune checkpoint inhibitor prepared using the medicament of claim 1 for use in the treatment of cancer.
9. An adoptive cancer cell therapy inhibitor for tumor prepared by using the pharmaceutical composition for cancer therapy according to claim 1.
10. A tumor cytokine therapy inhibitor prepared using the medicament for cancer therapy according to claim 1.
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CN114009399A (en) * | 2021-10-28 | 2022-02-08 | 复旦大学附属华山医院 | Preparation and application of drug-resistant mouse and cell strain of liver cancer immune check point antibody |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112735513A (en) * | 2021-01-04 | 2021-04-30 | 江苏先声医疗器械有限公司 | Construction method of tumor immune checkpoint inhibitor treatment effectiveness evaluation model based on DNA methylation spectrum |
CN112735513B (en) * | 2021-01-04 | 2021-11-19 | 江苏先声医疗器械有限公司 | Construction method of tumor immune checkpoint inhibitor treatment effectiveness evaluation model based on DNA methylation spectrum |
CN114009399A (en) * | 2021-10-28 | 2022-02-08 | 复旦大学附属华山医院 | Preparation and application of drug-resistant mouse and cell strain of liver cancer immune check point antibody |
CN114437234A (en) * | 2021-12-28 | 2022-05-06 | 四川省医学科学院·四川省人民医院 | BTNL2 recombinant protein, preparation method thereof and application thereof in preparing medicines for treating inflammatory bowel diseases |
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