WO2024148521A1 - Recombinant human 2ig-b7-h3 protein-coding gene, recombinant vector, host cell, pharmaceutical composition and use thereof - Google Patents

Abstract

A recombinant human 2Ig-B7-H3 protein-coding gene, a recombinant vector, a host cell, a pharmaceutical composition and a use thereof. The recombinant human 2Ig-B7-H3 protein-coding gene comprises a nucleotide sequence having a length of 3095 bp, the base C at the 1722th site being replaced with T at a fixed point. The recombinant human 2Ig-B7-H3 protein-coding gene packaged by a lentiviral vector and wild type human 2Ig-B7-H3 protein-coding gene packaged by a lentiviral vector are combined in a set ratio to prepare a drug, and unexpected effects are achieved in the treatment or prevention of cancers.

Description

Recombinant human 2Ig-B7-H3 protein encoding gene, recombinant vector, host cell, pharmaceutical composition and application thereof Technical Field

The present invention relates to the field of genetic engineering, and in particular to a human 2Ig-B7-H3 protein encoding gene, a recombinant vector comprising the gene, a host cell, a pharmaceutical composition and applications thereof.

Background technique

The activation of T cells requires two different signals. The first signal comes from the interaction between TCR and antigen peptide-MHC complex, and the second signal comes from the co-stimulatory signal generated by the combination of B7 family molecules on APC and their ligand CD28 family molecules on T cells, such as B7-1B7-2 combined with CD28 and CTLA-4. This pathway is called the classical B7 pathway.

The human B7-H3 gene was first discovered by Chapoval et al. in a cDNA library of human dendritic cells. Because its structure is similar to that of the B7 family genes, it was named B7Homolog 3, or B7-H3 for short. It is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily, and has 20-27% homology with other members of the B7 family in the extracellular amino acid sequence.

B7-H3 is widely expressed: at the transcriptional level, B7-H3 is expressed in most tissues, but at the protein level, it is only expressed in a few tissues such as human liver, lung, bladder, testis, prostate, breast, placenta and lymphoid organs; the difference in the expression of B7-H3 at the gene (mRNA) level and protein level may be related to the post-transcriptional regulation of the molecule.

In addition to being able to regulate lymphocyte proliferation in the process of antigen-specific humoral immunity, B7-H3 is an immune regulatory molecule. In recent years, it has been found that it also has important clinical significance in many tumor cells: that is, it may be a regulatory factor of tumor resistance.

The human B7-H3 gene is located on chromosome 15. The protein has two different splice forms in the body: 2IgB7-H3 and 4IgB7-H3. The extracellular segment of 2IgB7-H3 is composed of two immunoglobulin domains, IgV and IgC.

The prior art has not reported the correlation between site-directed replacement of the 2IgB7-H3 gene and tumor resistance. This application aims to study the correlation between site-directed replacement of the 2IgB7-H3 gene and tumor resistance, thereby providing a new approach for gene therapy of cancer.

Summary of the invention

The technical problem to be solved by the present invention is to provide a human 2Ig-B7-H3 protein encoding gene, a recombinant vector, a host cell containing the same, a pharmaceutical composition and an application thereof. The human 2Ig-B7-H3 protein encoding gene provides a new approach to gene therapy of cancer.

On the one hand, the present invention provides a recombinant human 2Ig-B7-H3 protein encoding gene, which comprises a nucleotide sequence of 3095 bp in length, wherein the base C at position 1722 is site-specifically replaced by T.

Preferably, the recombinant human 2Ig-B7-H3 protein encoding gene has the nucleotide sequence shown in SEQ ID NO: 2.

On the other hand, the present invention provides a recombinant vector, comprising a vector and the above-mentioned recombinant human 2Ig-B7-H3 protein encoding gene packaged by the vector.

Preferably, when the target gene is DNA, the vector is selected from the group consisting of a lentiviral vector and an adeno-associated viral vector, and when the target gene is mRNA, the vector is a nanoparticle vector.

In still another aspect, the present invention provides a host cell comprising the recombinant vector as described above.

Preferably, the host cell is selected from one or both of 293T cells and SHG44 cells.

On the other hand, the present invention provides a pharmaceutical composition comprising the recombinant human 2Ig-B7-H3 protein encoding gene as described above packaged by a lentiviral vector and the wild-type human 2Ig-B7-H3 protein encoding gene packaged by a lentiviral vector, and a pharmaceutically acceptable excipient.

Preferably, in the pharmaceutical composition, the ratio of the recombinant human 2Ig-B7-H3 protein encoding gene packaged by the lentiviral vector to the wild-type human 2Ig-B7-H3 protein encoding gene is 1:2 to 2.0:1, preferably 1:1.5 to 1.5:1, more preferably 1:1.25 to 1.25:1, and most preferably 1:1.

Preferably, the pharmaceutical composition is an injection.

The present invention also provides use of the above-mentioned pharmaceutical composition in preparing a drug for preventing or treating cancer.

Preferably, the cancer is liver cancer, lung cancer, prostate cancer, pancreatic cancer, small intestine cancer, colon cancer and cervical cancer.

Compared with the prior art, the present invention provides a recombinant human 2Ig-B7H3 protein encoding gene medicine, which plays an extremely important role in the tumor immune response process and provides a new and advantageous approach for the prevention and treatment of cancer. Compared with the wild-type human 2Ig-B7-H3 protein encoding gene alone, the recombinant human 2Ig-B7-H3 protein encoding gene packaged by the lentiviral vector of the present invention is mixed with the wild-type human 2Ig-B7-H3 protein encoding gene in a certain ratio to prepare a medicine, which has achieved unexpected technical effects in the treatment and prevention of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the synthesis process of the DNA sequence of the recombinant human 2Ig-B7-H3 protein encoding gene of the present application;

FIG2 shows a recombinant 2IgB7H3 plasmid vector carrying a recombinant human 2Ig-B7-H3 protein encoding gene of the present invention;

Figure 3A shows a picture of APC cells before infection;

FIG3B shows a picture of APC cells after infection;

Figure 3C shows a picture of cells before co-culture;

FIG3D shows a picture of cells after co-culture (24 h);

FIG3E shows a picture after co-cultivation (72 h); and

Figures 4A-4C respectively show the secretion levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and gamma-interferon (IFN-γ) by antigen presenting cells 293 cells and SHG44 cells after the introduction of the plasmid vector carrying the recombinant human 2Ig-B7-H3 protein encoding gene of the present invention.

Detailed ways

The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

In one embodiment, the present invention provides a recombinant human 2Ig-B7-H3 protein encoding gene, which is a 3095 bp long nucleotide sequence, wherein the base C at position 1722 is site-specifically replaced by T.

Compared with the wild-type human 2Ig-B7-H3 protein coding gene, four consecutive T bases at positions 121 to 124 at the 5’ end of the human 2Ig-B7-H3 protein coding gene are deleted, and/or four consecutive C bases at positions 3036-3040 at the 3’ end are deleted.

Preferably, the recombinant human 2Ig-B7-H3 protein encoding gene has the nucleotide sequence shown in SEQ ID NO: 2.

In one embodiment, the present invention provides a recombinant vector, comprising a vector and a target gene packaged therein, wherein the target gene is the recombinant human 2Ig-B7-H3 protein encoding gene described in the above technical solution.

Preferably, the target gene may also include regulatory sequences, such as promoters, terminators and enhancers for the expression of the one or more target genes. The target gene may also include marker genes (e.g., genes encoding β-galactosidase, green fluorescent protein or other fluorescent proteins) or genes whose products regulate the expression of other genes.

The target gene may be not only DNA, but also mRNA, tRNA or rRNA, and may also include related transcription regulatory sequences usually associated with transcription sequences, such as transcription termination signals, polyadenylation sites and downstream enhancer elements.

The carrier can be any carrier commonly used in the art that can package the target gene, and any available carrier that can carry the target gene that has been improved by technological development, such as plasmid (naked DNA), liposome, molecular coupling body, polymer, nanoparticle carrier and virus.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide containing an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), nanoparticle vectors (in the case of a target gene being mRNA), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) introduced into the recombinant polynucleotide.

In one embodiment, when the target gene is DNA, the vector is selected from the group consisting of a lentiviral vector and an adeno-associated viral vector, and when the target gene is mRNA, the vector is a nanoparticle vector.

The term "lentivirus" belongs to the family of retroviruses. Lentiviruses can infect both dividing and non-dividing cells. After infection, lentiviruses can deliver a large amount of genetic information to host cells and express it stably for a long time, while being able to be stably inherited as cells divide. Therefore, lentiviruses are one of the most effective tools for introducing foreign genes. Examples of lentiviruses include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious anemia (EIA), and feline immunodeficiency virus (FIV).

Lentiviral vectors can effectively integrate foreign genes into host chromosomes, thereby achieving persistent expression. In terms of infection ability, they can effectively infect various types of cells such as neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc., thereby achieving good gene therapy effects.

Preferably, the present invention uses a lentiviral vector.

The present invention also provides a host cell, wherein the host contains the recombinant vector of the present invention. The recombinant vector containing the recombinant human 2Ig-B7-H3 protein encoding gene of the present invention is transformed into the host, which can be used to study its relationship with tumor cell expression. Preferably, the host is selected from one or more of Escherichia coli, 293 cells and SHG44 cells.

An embodiment of the present invention discloses a pharmaceutical composition, which includes the recombinant human 2Ig-B7-H3 protein encoding gene as described above packaged by a lentiviral vector and the wild-type human 2Ig-B7-H3 protein encoding gene packaged by a lentiviral vector, and a pharmaceutically acceptable excipient.

In the pharmaceutical composition of the present invention, the ratio of the recombinant human 2Ig-B7-H3 protein encoding gene packaged by the lentiviral vector to the wild-type human 2Ig-B7-H3 protein encoding gene is 1:2 to 2.0:1, preferably 1:1.5 to 1.5:1, more preferably 1:1.25 to 1.25:1, and most preferably 1:1.

The pharmaceutically acceptable excipients refer to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulation materials or other formulation excipients, for example, including but not limited to saline, buffered saline, glucose, water, glycerol, ethanol and mixtures thereof. The pharmaceutical composition is suitable for parenteral, sublingual, intracisternal, intravaginal, intraperitoneal, rectal, buccal or epidermal administration.

Parenteral administration includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intraarticular injection and infusion. Pharmaceutical compositions suitable for parenteral administration include sterile aqueous solutions or non-aqueous solutions, dispersions, suspensions or emulsions, and powders prepared in sterile injectable solutions or dispersions before use. Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, glycerol, propylene glycol, polyethylene glycol, carboxymethyl cellulose, vegetable oils and injectable organic esters such as ethyl oleate. These compositions can also contain preservatives, wetting agents, emulsifiers, protective agents and dispersant adjuvants, such as inositol, sorbitol and sucrose. Osmotic pressure regulators such as sugars, sodium chloride, potassium chloride are preferably added.

Epidermal administration includes administration on the skin, mucous membranes, and on the lungs and ocular surfaces. Such pharmaceutical compositions include powders, ointments, drops, transdermal patches, iontophoresis devices, and inhalants, etc. Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the recombinant vector of the present invention with suitable non-irritating excipients such as cocoa butter, polyethylene glycol, or suppository wax, wherein the excipient or carrier is solid at room temperature and liquid at body temperature, so it melts and releases the active compound in the rectum or vaginal cavity.

Preferably, the pharmaceutical composition is an injection, which comprises a pharmaceutically acceptable excipient and one or more selected from the recombinant human 2Ig-B7H3 protein encoding gene described in the present invention and the recombinant vector described in the present invention.

The embodiments of the present invention also provide the use of the pharmaceutical composition described in the above technical solution in the preparation of drugs for preventing or treating cancer.

After ectopic expression in several tumor cell lines of mice, it can induce the activation of tumor-specific cytotoxic T lymphocytes, thereby delaying the growth of cancer cells or even completely eliminating the tumor. After the transfected cancer cell lines are implanted into mice, the survival time of the mice can be significantly prolonged.

The technical scheme of the present invention is further illustrated by the following examples. It should be understood by those skilled in the art that the examples are only used to help understand the present disclosure and should not be regarded as specific limitations of the present disclosure.

In order to enable persons with ordinary knowledge in the art to understand the characteristics and effects of the present disclosure, the following is a general description and definition of the terms and expressions mentioned in the specification and the scope of the patent application.

Unless otherwise specified, all technical and scientific terms used herein have the common meanings understood by those skilled in the art for the present disclosure. In case of conflict, the definitions in this specification shall prevail.

Example

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example 1

Preparation of recombinant human 2Ig-B7-H3 protein encoding gene

According to the nucleotide sequence of the recombinant human 2Ig-B7-H3 protein encoding gene shown in SEQ ID NO: 2, restriction endonuclease map analysis was performed to obtain three sequences for step-by-step synthesis. The sequence was entered at https://hpcwebapps.cit.nih.gov/dnaworks/ to obtain the recommended oligonucleotide fragment synthesis plan. The nucleotide fragments were chemically synthesized by a commercial synthesis company, and the fragments were spliced to obtain the complete gene sequence, which was then sequenced and verified for construction of a plasmid vector.

Technologies used for gene sequence synthesis:

PCR, or polymerase chain reaction, is a technology that can replicate a small amount of DNA in large quantities. By adding target DNA, primers, enzymes, buffers, and raw materials for DNA synthesis (dNTPs) into a container (usually a PCR tube) and controlling its temperature, the target DNA can be replicated in large quantities, from a small amount to a large amount.

The basic principle of "bridge" PCR is the same as that of ordinary PCR, but the target DNA does not need to be added. This technology is used to splice the pre-designed primers into the target DNA. After this experiment is completed, "ordinary" PCR is performed to copy the target DNA in large quantities, from a small amount to a large amount.

As shown in Figure 1, "bridge" PCR and "normal" PCR were used together to synthesize multiple recombinant 2IgB7H3 gene fragments into a sequence. In this plasmid construction process, "bridge" PCR was used as the first step of the two-step method to synthesize the gene sequence, and "normal" PCR was used as the second step of the two-step method to synthesize the gene sequence. The two steps are collectively referred to as "gene synthesis".

On this basis, PCR amplification and site-directed recombination were performed to obtain the recombinant 2IgB7H3 gene fragment, which was used to construct a plasmid vector after sequencing verification.

The oligonucleotide fragments used to synthesize the recombinant human 2Ig-B7-H3 protein encoding gene (hereinafter referred to as "muB7H3 gene") of the present invention are as follows:

Overlapping oligonucleotide PCR

1. Step 1 PCR

Oligonucleotide fragments with complementary ends are used to form overlapping chains of PCR products, so that template-free amplification can be achieved by extending the overlapping chains in the subsequent amplification reaction.

PCR reaction system

PCR reaction procedure

2. Step 2 PCR

The reaction system was prepared using the products of oligonucleotide fragment fusion PCR reaction (muB7H3-I, muB7H3-II, muB7H3-III) as templates, and PCR was performed after sufficient mixing and instant centrifugation to obtain the target fragment 3095. The reaction system and procedure are detailed in the table below.

PCR reaction system

PCR reaction procedure

Example 2 Construction of plasmid vector

The nucleotide sequence of the recombinant human 2Ig-B7-H3 protein encoding gene shown in SEQ ID NO: 2 prepared in Example 1 was inserted between the AgeI+EcoRII restriction sites of the pLV.CBh.WPRE vector to obtain the recombinant plasmid pLV.CBh.T1912(MUT).WPRE, as shown in Figure 2.

2.1 Use LipofectamineTM2000 cationic liposome transfection kit and operate according to the kit instructions to introduce the recombinant plasmid pLV.CBh.T1912(MUT).WPRE into 293T cells to obtain recombinant cells.

2.2 The recombinant cells obtained in step 1 were inoculated into DMEM/F12 medium containing 5% (volume ratio) newborn calf serum, and then cultured in an incubator at 37° C. and 5% CO 2 for 48 hours, and then the supernatant was collected.

2.3 Take the supernatant obtained in step 2, filter it with a 0.45 μm filter membrane and collect the filtrate, then adjust the pH to 7.4.

2.4 The filtrate obtained in step 3 is purified by affinity chromatography.

Equilibration buffer: pH 7.4, 0.5 M Tris-HCl buffer containing 0.5 M NaCl;

Eluent: pH 3.0, 0.1 M Gly-HCl buffer.

First, wash 3 column volumes with the equilibration buffer, and then wash the target product with the eluent, both at a flow rate of 5 mL/min.

A280nm detects the ultraviolet absorption peak.

The target peak was collected using a collection tube, and the solution in the collection tube was then transferred to a dialysis bag and dialyzed in pH 7.4, 0.01 M PBS buffer to obtain the Lenti-reB7H3 lentivirus stock solution.

Example 3 Drug Preparation

According to the method of Example 1, the wild-type human 2IgB7-H3 gene sequence was obtained, and the vector was constructed according to the method of Example 2, and the lentiviral particles carrying the wild-type human 2IgB7-H3 gene sequence were packaged to obtain the Lenti-wtB7H3 lentiviral stock solution.

3.3 The above two stock solutions were calibrated and adjusted to a concentration of 1E+8TU/mL, and mixed into a combined Lenti-combiB7H3 stock solution in the particle number ratio shown in Table 1 below. The final concentration of each configured stock solution was 1E+8TU/mL.

Table 1

Human liver cancer SMMC7721 cells frozen in liquid nitrogen were quickly thawed in a 37.0℃ water bath, and the cell density was adjusted to 1X 10 ⁷ /mL. Two BALB/c nude mice were subcutaneously inoculated with 0.2mL each. When the nude mice had tumors, the minimum diameter of the tumor was not less than 1.0cm. Under aseptic operation, the tumor was removed and divided into clumps with a maximum diameter of no more than 3mm, and subcutaneously inoculated into 21 nude mice.

Group 1, 3 mice, received no treatment;

Group 2, 3 mice, gemcitabine drug control, 120 mg/Kg body weight;

Group 3, 3 mice, circulatory system administration, tail vein injection of configuration 1, 0.2 mL, 5 times every other day;

Group 4, 3 mice, circulatory system administration, tail vein injection of configuration 2, 0.2 mL, 5 times every other day;

Group 5, 3 mice, circulatory system administration, tail vein injection of configuration 3, 0.2 mL, 5 times every other day;

Group 6, 3 mice, circulatory system administration, tail vein injection of configuration 4, 0.2 mL, 5 times every other day;

Group 7, 3 mice, circulatory system administration, tail vein injection of configuration 5, 0.2 mL, 5 times every other day.

From the 4th day, the tumor growth was observed every day, the tumor size was recorded, and the tumor volume was calculated according to the following formula: V=ab ² /2 (V-volume, a-tumor long diameter, b-tumor short diameter). The changes in tumor volume of each group are shown in Table 2 below (mean).

Table 2

The results in Table 2 show that:

In group 1, in nude mice without drug intervention, the tumor volume increased 170 times after 27 days;

In group 2, under the intervention of the positive drug gemcitabine, the tumor volume increased 105 times after 27 days, indicating the therapeutic effect of the positive drug;

Group 3, configuration 1 (recombinant: wild type = 1:2), after 27 days, the tumor volume increased 86 times, which was better than the positive drug;

Group 4, configuration 2 (recombinant: wild type = 1:2.5), after 27 days, the tumor volume increased 121 times, and the effect was poor;

Group 5, configuration 3 (recombinant: wild type = 2:1), after 27 days, the tumor volume increased 80 times, which was better than the positive drug;

In group 6, configuration 4 (recombinant: wild type = 2.5:1), after 27 days, the tumor volume increased by 137 times, and the effect was poor.

In group 7, configuration 5 (recombinant: wild type = 1:1), after 27 days, the tumor volume increased 18 times, which was superior to all the drugs in the above groups.

It can be concluded that the optimal configuration ratio of the recombinant B7H3 and wild-type B7H3 combination is 1:1. The ratio between 2:1 and 1:2 can show good therapeutic effects. When the ratio exceeds the above ratio, the therapeutic effect of the combination is poor.

Example 4 Cell experiment: ELSIA detection was completed after virus-infected antigen presenting cells (APC) were co-cultured with CD3 or CD11B+ monocytes)

After virus-infected APC cells were co-cultured with CD3 or CD11B+ monocytes, cytokines in the supernatant were detected by ELISA.

4.1 Experimental Materials

1) Target cells: antigen presenting cells 293T cells, SHG44 cells; culture medium: DMEM + 10% FBS + 1% P/S + 1% Gln. Culture medium manufacturer: Jiangsu Keyi, product number KGM12800-500; CD11B+ monocyte culture medium: 1640 + 10% FBS + 1% P/S + 1% Gln. Culture medium manufacturer: TRANSGEN BIOTECH, product number: M20301

2) FBS manufacturer: Gemini, product number: 9001-108

3) Drug name: CD3 antibody abcam catalog number: ab5690, stock solution concentration: 0.2 mg/ml; CD28 antibody abcam catalog number: ab213043, stock solution concentration: 1 mg/ml; LPS, stock solution concentration 1 mg/ml

4) Virus stock solution:

Lenti-wtB7H3

Lenti-reB7H3

Lenti-combiB7H3 with a particle number ratio of 1:1 between Lenti-wtB7H3 and Lenti-reB7H3

5) Detection factors: IL-2, IFN-γ (CD3+ and APC cell incubation group); IL-6, TNF-α (CD11b+ and APC incubation group)

4.2 Experimental procedures

4.2.1 Cell culture:

The cells were cultured in a 5% CO ₂ , 37°C carbon dioxide incubator.

4.2.2 Drugs act on target cells:

1) APC cells were digested with trypsin, counted, and seeded into 6-well culture plates, with 2×10 ⁵ cells per well. Cultured overnight in a 37°C 5% CO ₂ incubator.

2) On the second day, according to the MOI value (100-200) of APC cells found in the preliminary experiment, add the corresponding number of virus particles, culture for 8 hours, replace with fresh complete medium, and continue to culture for 48 hours.

3) APC cell (293T and SHG44) suspension was inoculated into a 48-well culture plate (10,000 cells/well), cultured in a 37°C 5% CO ₂ incubator, photographed and recorded the next day, and then antibodies and CD3 or CD11b cells were added to the cell culture plate, respectively, and grouped as shown in Table 3:

table 3

i) Virus-infected cells were co-incubated with CD3+ T cells for 72 hours, and the levels of cytokines IL-2 and IFN-γ were detected by ELISA.

Note: Two cell lines, 8 groups in total, the cell ratio was 1:30, and the concentrations of CD3 and CD28 were 1ug/ml.

ii) Virus-infected APC cells were co-incubated with CD11b+ monocytes (ratio of 1:30) for 24 hours, and the changes in the levels of cytokines IL-6 and TNF-α were detected by Elisa.

Note: Two cell lines, 8 groups in total, cell ratio is 1:30, LPS concentration is 2ug/ml.

5) After co-culture of CD11b+ cells and APC cells for 24 hours, take photos and collect the supernatant; after co-culture of CD3+ cells and APC cells for 72 hours, take photos and collect the supernatant.

6) The collected supernatant was centrifuged at 1000 rpm, the precipitate was removed, and the supernatant was collected.

4.2.3 Preparation of reagents and collection of cell culture supernatant

1) Collect specimens;

2) For the preparation of standard solution, please refer to the instruction manual.

3) Dilute the 10× specimen diluent 1:10 with distilled water (for example: 1 ml concentrated diluent + 9 ml distilled water).

4) Washing solution: dilute with deionized water 1:20 (Example: add 19 ml of deionized water to 1 ml of concentrated washing solution).

4.2.4 Testing procedures

1) Sample addition: Add 50ul of standard or sample to be tested to each well, and 50ul of enzyme-labeled antibody working solution. Mix the reaction plate thoroughly and place it at 37℃ for 120 minutes.

2) Washing the plate: Wash the reaction plate thoroughly 4-6 times with washing solution and blot dry on filter paper.

3) Add 100ul of substrate working solution to each well and incubate in the dark at 37℃ for 15 minutes.

4) Add 100ul of stop solution to each well and mix well.

5) Measure the absorbance at 450 nm using an enzyme reader within 30 minutes.

4.3 Result calculation and judgment

1) Use the standard product as the horizontal axis and the OD value as the vertical axis to draw a standard curve on the coordinate paper.

2) Find the protein content of the factor to be tested in the corresponding sample on the curve according to the sample OD value. (All OD values should be calculated after subtracting the blank value).

Figure 3A shows a picture before APC cell infection; Figure 3B shows a picture after APC cell infection; Figure 3C shows a picture before cell co-culture; Figure 3D shows a picture after cell co-culture (24h); Figure 3E shows a picture after co-culture (72h).

Tables 4a-4c to 6a-6c and Figures 4A-4C show the secretion amounts of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) by antigen presenting cells 293 cells and SHG44 cells, respectively.

Table 4a-4b TNF-α secretion of antigen presenting cells 293 cells and SHG44 cells

Table 5a-5b IL-6 secretion of antigen presenting cells 293 cells and SHG44 cells

Table 6a-6b IFN-γ secretion of antigen presenting cells 293 cells and SHG44 cells

4.4 Conclusion

From the above results, we can see

After 293T cells were infected with Lenti-wtB7H3, Lenti-mtB7H3, and Lenti-combiB7H3 (1:1), and co-cultured with CD3+T cells and CD11b+monocytes:

Compared with the control group, the tumor necrosis factor (TNF-α) in the Lenti-wtB7H3 group increased by 8%; compared with the control group, the tumor necrosis factor (TNF-α) in the Lenti-mtB7H3 group increased by 3%; compared with the control group, the tumor necrosis factor (TNF-α) in the Lenti-combiB7H3 group increased by 11%.

Compared with the control group, interleukin 6 (IL-6) in the Lenti-wtB7H3 group increased by 199%; compared with the control group, interleukin 6 (IL-6) in the Lenti-mtB7H3 group increased by 194%; compared with the control group, interleukin 6 (IL-6) in the Lenti-combiB7H3 group increased by 255%.

Compared with the control group, the gamma interferon (IFN-γ) level in the Lenti-wtB7H3 group increased by 2%; compared with the control group, the gamma interferon (IFN-γ) level in the Lenti-mtB7H3 group increased by 44%; compared with the control group, the gamma interferon (IFN-γ) level in the Lenti-combiB7H3 group increased by 130%.

After SHG44 cells were infected with Lenti-wtB7H3, Lenti-mtB7H3, and Lenti-combiB7H3 (1:1), and co-cultured with CD3+T cells and CD11b+monocytes:

Compared with the control group, the tumor necrosis factor (TNF-α) in the Lenti-wtB7H3 group decreased by 2%; compared with the control group, the tumor necrosis factor (TNF-α) in the Lenti-mtB7H3 group decreased by 4%; compared with the control group, the tumor necrosis factor (TNF-α) in the Lenti-combiB7H3 group increased by 5%.

Compared with the control group, interleukin 6 (IL-6) in the Lenti-wtB7H3 group increased by 250%; compared with the control group, interleukin 6 (IL-6) in the Lenti-mtB7H3 group increased by 181%; compared with the control group, interleukin 6 (IL-6) in the Lenti-combiB7H3 group increased by 261%.

Compared with the control group, the gamma interferon (IFN-γ) level in the Lenti-wtB7H3 group increased by 13%; compared with the control group, the gamma interferon (IFN-γ) level in the Lenti-mtB7H3 group increased by 62%; compared with the control group, the gamma interferon (IFN-γ) level in the Lenti-combiB7H3 group increased by 112%.

Because the cell experiment used the method of adding antibodies to make antigen-presenting cells output signals to immune cells, there were only antigen-presenting cells (293T and SHG44) and immune cells in the experimental system, and there were no target cells attacked by immune cells (tumor cells), and there was a lack of synergistic stimulation formed after the immune system attacked the target cells.

Therefore, the intensity of immune factors secreted by immune cells is far from the level detected in tumor-bearing animals and volunteers (cancer patients). This is because in in vivo experiments, the immune system can locate and attack actual tumor cells, synergistically stimulating the secretion of immune factors. In practice, the levels of immune factors measured in animals and volunteers can usually reach 5-10 times the basal value.

It is particularly noteworthy that the increase in tumor necrosis factor in cell experiments is not obvious. This is because there are no tumor cells in the cell experiment system. In order to protect their own survival, the experimental cells will inhibit the secretion of tumor necrosis factor to avoid being affected by tumor necrosis factor. Therefore, in cell experiments, the secretion-promoting effect of tumor necrosis factor is affected.

From the above results, it can be seen that after 293T and SHG44 cells were infected with WT and MU viruses of 2lgB7-H3I, they were co-incubated with CD11b+ monocytes. Compared with NC, WT and MU, the expression of IL-6 in the MU+WT group was increased, among which the MU+WT group in 293T cells was higher than the WT and MU groups, and the WT and WT+MU groups in SHG44 cells were higher than the MU group; compared with NC, WT and MU, the expression of TNF-α in the MU+WT group did not change significantly; co-incubated with CD3+ T cells, compared with NC, WT and MU, the expression of IFN-γ in the MU+WT group was increased, among which the MU+WT group was higher than the WT and MU groups.

Example 5 Animal Experiment

Human liver cancer SMMC7721 cells frozen in liquid nitrogen were quickly thawed in a 37.0℃ water bath, the cell density was adjusted to 1X 10 ⁷ /mL, and two BALB/c nude mice were subcutaneously inoculated with 0.2mL each. When the nude mice had tumors, the minimum diameter of the tumor was not less than 1.0cm. Under aseptic operation, the tumor was removed and divided into clumps with a maximum diameter of no more than 3mm, and subcutaneously inoculated into 25 nude mice.

The first group, 5 mice, received no treatment;

The second group, 5 mice, gemcitabine drug control, 120 mg/Kg body weight;

Group 3, 5 mice, circulatory system administration, tail vein injection of wild-type Lenti-wtB7H3, 0.2mL, 1E+8TU/mL, 5 times every other day;

Group 4, 5 mice, circulatory system administration, tail vein injection of recombinant Lenti-reB7H3, 0.2mL, 1E+8TU/mL, 5 times every other day;

Group 5, 5 mice, circulatory system administration, tail vein injection of combined Lenti-combiB7H3 (1:1), 0.2mL, 1E+8TU/mL, 5 times every other day;

From the 4th day, the tumor growth was observed every day, the tumor size was recorded, and the tumor volume was calculated according to the following formula: V = ab ² /2 (V-volume, a-tumor long diameter, b-tumor short diameter). The changes in tumor volume of each group are shown in Table 7 below (mean ± standard deviation).

From the results in Table 7, it can be seen that after nude mice were tumor-bearing, the tumor volume of the non-intervention group increased by 350 times after 38 days; the tumor volume of the positive drug gemcitabine group increased by 184 times after 38 days, showing the therapeutic effect of the positive drug. The wild-type Lenti-wtB7H3 group increased the tumor volume by 223 times after 38 days, showing the effect of inhibiting tumor growth compared with the non-intervention group. The recombinant Lenti-reB7H3 group increased the tumor volume by 201 times after 38 days, showing the effect of inhibiting tumor growth compared with the non-intervention group. At the same time, the tumor inhibition effect of recombinant Lenti-reB7H3 is similar to that of the chemical drug gemcitabine. After 38 days, the tumor volume of the combined Lenti-combiB7H3 group increased by only 38 times, which is much better than the chemical drug group, the wild-type group and the recombinant group. The results of this animal experiment are far better than the previous cell experiment results, and the reasons are as described in the previous cell experiment.

Example 6 Clinical Trial

The test subject is a 57-year-old female patient with cholangiocarcinoma, stage II, no family history. She is intolerant to chemotherapy and develops severe myocarditis after treatment with Teplizumab. She is treated with albumin + paclitaxel combined with gemcitabine. After 22 weeks of treatment, tumor markers are elevated and the reaction is severe. The CT report shows that liver lesions have progressed significantly and there is ascites. Therefore, the gene drug of the present invention is used for treatment.

The Lenti-combi B7H3 (1:1) preparation of the present invention was intravenously administered 3 times, 3.0 mL each time, 1E+8TU/mL intravenous retention needle push injection, and the push injection time was 15 seconds. Two biochemical indicators, tumor necrosis factor-α and gamma-interferon, were tested before the patient received the Lenti-combi B7H3 preparation. 12 days after receiving the preparation, the two indicators were retested, and the results are shown in Table 8 below.

Table 8 Levels of tumor necrosis factor-α and interferon-γ before and 12 days after injection of Lenti-combi B7H3 preparation

Biochemical Indicators Before injection 12 days after injection Increase multiple TNF-α 0.87pg/mL 4.19 pg/mL 4.8 Interferon-gamma 0.27pg/mL 3.44pg/mL 12.7

The CT scan results of the patient at 1 month and 3 months after Lenti-combi B7H3 (1:1) injection were as follows:

One month after receiving the Lenti-combiB7H3 (1:1) preparation, the CT report showed no obvious progression of the lesion site compared with the previous CT report without receiving the Lenti-combiB7H3 (1:1) preparation.

Three months after receiving the Lenti-combiB7H3 (1:1) preparation, the CT report showed that the ascites was absorbed and there was no obvious progression of the lesion.

It can be seen from the above Example 6 that after the patient received the Lenti-combiB7H3 (1:1) preparation injection, the tumor necrosis factor and gamma interferon increased significantly, indicating that the preparation improved the patient's immune level, and the CT report showed no obvious progression of the tumor lesion site, proving that under the action of the Lenti-combiB7H3 preparation, tumor growth was inhibited.

It can be seen that the preparation of the present invention made of recombinant Lenti-B7H3 and wild-type Lenti-B7H3 (1:1) has a much better effect in inhibiting tumor growth than existing chemical drugs and the use of recombinant Lenti-B7H3 or wild-type Lenti-B7H3 alone.

The above embodiments are only used to help understand the method and core idea of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

A recombinant human 2Ig-B7H3 protein encoding gene comprises a nucleotide sequence of 3095 bp in length, wherein the base C at position 1722 is site-specifically replaced by T. The recombinant human 2Ig-B7-H3 protein encoding gene according to claim 1, wherein the recombinant human 2Ig-B7-H3 protein encoding gene has the nucleotide sequence shown in SEQ ID NO: 2. A recombinant vector comprises a vector and the recombinant human 2Ig-B7-H3 protein encoding gene according to claim 1 or 2 packaged by the vector. The recombinant vector according to claim 3, wherein, when the target gene is DNA, the vector is selected from the group consisting of a lentiviral vector and an adeno-associated viral vector, and when the target gene is mRNA, the vector is a nanoparticle vector. A host cell comprising the recombinant vector according to claim 1 or 2. The host cell according to claim 5, characterized in that the host cell is selected from one or both of 293T cells and SHG44 cells. A pharmaceutical composition comprising the recombinant human 2Ig-B7-H3 protein encoding gene according to claim 1 or 2 packaged by a lentiviral vector and the wild-type human 2Ig-B7-H3 protein encoding gene packaged by a lentiviral vector, and a pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 7, characterized in that, in the pharmaceutical composition, the ratio of the recombinant human 2Ig-B7-H3 protein encoding gene packaged by the lentiviral vector to the wild-type human 2Ig-B7-H3 protein encoding gene is 1:2 to 2.0:1, preferably 1:1.5 to 1.5:1, more preferably 1:1.25 to 1.25:1, and most preferably 1:1. Use of the pharmaceutical composition according to claim 7 or 8 in the preparation of a drug for preventing or treating cancer. The use according to claim 9 is characterized in that the cancer is liver cancer, lung cancer, prostate cancer, pancreatic cancer, small intestine cancer, colon cancer and cervical cancer.

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