WO2009009935A1 - Replication-deficient recombinant virus, pharmaceutical composition comprising the same and the uses thereof - Google Patents

Abstract

A replication-deficient recombinant virus, comprising human fusion gene LFA3-Fc under the control of promoter. The human fusion gene LFA3-Fc is fused by soluble extracellular domain of human LFA3 and Fc fragment of human IgG. Pharmaceutical composition against psoriasis, consisting of the replication-deficient virus and pharmaceutical acceptable dressing. Injection of pharmaceutical composition against psoriasis produced by the method according to the present invention can make the target protein be expressed in vivo for long time, therefore administration times can be reduced.

Description

 Recombinant replication-deficient virus, pharmaceutical composition containing the same and application thereof

 The present invention relates to the field of genetic engineering, and in particular to a recombinant replication-defective virus, a pharmaceutical composition containing the same, and an application thereof, particularly a replication-defective virus containing the human fusion gene LFA3-FC, and an anti- A pharmaceutical composition of psoriasis. Background technique

 Psoriasis is a common relapsing, inflammatory skin disease characterized by hyperplasia of keratinocytes, inflammatory cell infiltration, and neovascularization as the three elements of histopathology. In addition, psoriasis is a genetically related immune disease. Among the many factors associated with the pathogenesis of psoriasis, immune factors are the primary factors, especially the activation of tau cells (thymus-dependent lymphocytes), which are the main components of lymphocytes and their cells. There are a large number of CD2 receptor expression on the surface, and examples thereof include CD4+CD45RO+ and CD8+CD45RO+.

 According to the American Psoriasis Association, the activation of T cells that induce psoriasis must meet two conditions: First, antigen presenting cells (APCs, examples of macrophages, Langerhans cells and Dendritic cells, etc.) must present to the T cells an antigenic component that can be recognized and matched by the cell surface receptor (but the specific components of these antigens have not yet been elucidated); Second, the combined APC and T cells T cells must be activated by one or more stimulation channels. Once T cells are activated, they release a large number of cytokines that act on the keratinocytes located at the outermost layer of the epidermis, prompting the keratinocyte to divide faster. This phenomenon is called "hyperproliferation". . When T cells are activated, they can also stimulate a cascade of cascades, including activation of helper T cells and accumulation of intradermal skin. This ultimately results in a large amount of APC in the lesions, which in turn further activates more T cells, thereby creating a vicious circle.

LFA3 is Lymphocyte function-associated antigen-3 (LFA-3; aka CD58). LPA3 molecule on the surface of APC and CD2 of T cells The combination is one of the co-stimulatory signals. This binding of LFA3 to CD2 not only activates T cells, but further promotes the secretion of some cytokines such as γ-interferon, and the occurrence of psoriasis is also closely related to the excessive secretion of γ-interferon.

 It can be seen that an effective psoriasis treatment is a drug that must be able to block this vicious cycle. Currently, many biopharmaceutical companies also use biopharmaceuticals (LFA3-FC) that block this process before the start of the cycle to treat psoriasis. In other words, the LFA3-FC directly interferes with the activation of sputum cells. The LFA3-FC is a novel molecule formed by fusing an extracellular soluble fraction of human LFA3 with an Fc fragment of human IgG1. The LFA3-FC is a bifunctional molecule whose LFA3 moiety specifically binds to the CD2 receptor on the surface of T cells, inhibits the activation of T cells and changes the progression of inflammatory processes; the Fc fragment of IgG1 can interact with CD16 on the surface of NK cells ( Fey receptor III) binds, induces apoptosis of T cells, and ultimately reduces and eliminates T cells with memory effects. Since the LFA3/CD2 costimulatory channel on a large number of activated T cells present in the lesions of psoriasis is blocked by LFA3-Fc, the effect of T cells is inhibited, and the purpose of relieving and curing psoriasis is achieved.

 However, due to the high clinical dose of LFA3-FC (15 mg intramuscularly or 7.5 mg intravenously, 12 weeks is a course of treatment), Chinese hamster ovary (CHO) expression system is low in yield and difficult to scale. Production makes the current LFA3-FC difficult to meet clinical needs. The present invention adopts a gene therapy protocol to introduce the LFA3-FC gene into an adenovirus to construct a recombinant adenovirus (Ad-LF), thereby continuously expressing LFA3-FC in vivo. The method of the invention can overcome the problems of low protein expression in vitro, cumbersome expression and purification process, and short half-life of recombinant protein, so that the target protein can be continuously expressed in vivo for a long time, and the number of administrations can be reduced. Summary of the invention

 A first aspect of the invention relates to a replication-deficient virus comprising a human fusion gene LFA3-Fc under the control of a promoter, the human fusion gene LFA3-FC being an extracellular soluble fraction of human LFA3 It is formed by fusion with the Fc fragment of human IgG.

According to the first aspect of the present invention, with regard to a viral vector, a replication-deficient virus such as a replication-deficient adenovirus, a replication-deficient herpes simplex virus, and a replication defect must be used. Newcastle disease virus. The reason why the replication-defective virus is preferably used in the present invention is that the viral vector cannot replicate in cells other than the packaging cell line (293, 911, PERC6, etc.), and its common features are: deletion of some replication in the viral genome The genes necessary for proliferation. Among them, the present invention preferably uses a first generation type 5 adenovirus or a second generation, third generation type 5 adenovirus.

 A second aspect of the invention relates to a host cell comprising the replication-defective virus of the first aspect of the invention. For example, a host cell, such as 293 cell, can be directly transformed with a recombinant replication-deficient virus of the present invention, or a host cell can be co-transfected with a liposome with a cloning vector carrying the fusion gene LFA3-FC.

 With respect to host cells, host cells useful in the practice of the invention may be eukaryotic or prokaryotic cells, such as bacteria, yeast, or animal cells. Furthermore, the host cell is preferably a eukaryotic cell. Further, the host cell is a 293 cell. The 293 cell is used in the present invention because the cell line is transfected with human adenovirus E1A gene in human renal epithelial cells, and the recombinant replication-deficient adenovirus can be replicated and packaged in the cell; and three large-scale culture methods ( Adherent culture, microcarrier culture, serum-free suspension culture can be used for the culture of 293 cells.

 A third aspect of the invention provides a pharmaceutical composition against psoriasis comprising the following components:

 a recombinant replication-deficient virus comprising a human fusion gene LFA3-Fc under the control of a promoter, the human fusion gene LFA3-FC being derived from the extracellular soluble portion of human LFA3 and human IgG Fc fragment fusion formed; and

 b. Pharmaceutically acceptable excipients.

 In a fourth aspect of the invention, a method of preparing a pharmaceutical composition resistant to psoriasis is provided. In order to prepare a pharmaceutical composition against psoriasis, a replication-defective virus comprising a human fusion gene LFA3-Fc under the control of a promoter, and a pharmaceutically acceptable excipient, The human fusion gene LFA3-FC is formed by fusing an extracellular soluble portion of human LFA3 with an Fc fragment of human IgG; and mixing the replication-defective virus with the pharmaceutically acceptable excipient.

The pharmaceutical composition against psoriasis of the present invention is preferably in the form of a liquid preparation, more preferably an injection preparation. A liquid carrier generally used for formulating a viral preparation, such as purified water, physiological salt Water, a glucose solution or the like is used as the pharmaceutically acceptable adjuvant. For injections, a sterile, non-pyrogenic liquid carrier should be used.

 The pharmaceutical composition of the present invention should generally be stored at a low temperature, for example, at a temperature of -20 °C.

 According to the present invention, it is possible to realize a human recombinant LFA3-FC polypeptide which expresses a highly active host cell (which can infect various cell types including lung cancer, liver cancer, endometrial cancer, ovarian cancer, etc.). Tests have shown that these secreted human recombinant LFA3-FC polypeptides are effective in inhibiting lymphocyte proliferation in vitro. At the same time, it is also proved that the anti-psoriatic injection prepared by the recombinant adenovirus of the invention can continuously express the high level of human LFA3-FC and maintain the complete biological activity for a long time after transfecting the cells in vivo, and can effectively treat the silver crumb. disease.

The invention also provides a method of treating psoriasis comprising administering to a patient an effective amount of a recombinant replication-defective virion of the invention. For the injection as a preferred pharmaceutical composition of the present invention, it can be administered by intramuscular injection or intravenous injection, and the dose can be administered according to some known factors such as the severity of psoriasis, the patient's weight, sex, and age. The administration method and the like are easily determined by a physician. For example, the virion content per ml of the injection may generally be in the range of 3 x 10 ⁹ -3 x l0", preferably in the range of 3 χ 10 ¹⁰ -3 χ 10 ¹³ .

 The anti-psoriatic injection of the present invention can compensate for defects such as poor stability of the drug in direct protein infusion, can increase the interval of administration time, greatly reduce the dose, reduce the economic burden on the patient, and improve the quality of life of the patient. Detailed description of the invention

 For the human fusion gene LFA3-Fc used, the fusion LFA3-Fc available in the prior art can be used, or it can be prepared by the following procedure:

 a. The following PCR primers were synthesized to amplify the extracellular gene sequence of human LFA3 and the gene sequence of the Fc fragment of human IgG1:

 PI (SEQ ID No. 2): 5' CTC AAG CTT GCT AGC ATG GTT GCT

 GGG AGC GAC GC 3'

P2 (SEQ ID No. 3): 5' ACG CGT CGA CAT AAA GAA AGA ACT TCA TGG 3'

 P3 (SEQ ID No. 4): 5' ACG CGT CGA CAA AAC TCA CAC ATG

 CC 3'

 P4 (SEQ ID No. 5): 5' CGG GAT CCT CAT TTA CCC GGA GAC

 AGG 3'

 b. constructing the gene sequence of the extracellular domain of human LFA3 into a cloning vector;

 c The gene sequence of the Fc fragment of human IgG1 was constructed into a cloning vector to obtain an LFA3-Fc fusion gene.

 A specific example of the above process includes the PCR primer (having a restriction enzyme sequence) of the synthetic sequence of SEQ ID NO. 2 and SEQ ID NO. 3, and the amplification of the human LFA3 extracellular segment cDNA by PCR; The vector pUC18 was constructed as pUC18-L; the PCR primers (having a restriction enzyme sequence) of the synthetic sequences SEQ ID NO. 4 and SEQ ID NO. 5 were synthesized, and human IgG1 was amplified from human lymphocyte total RNA by RT-PCR. cDNA of Fc fragment; cDNA of Fc fragment was inserted into pUC18-L to construct pUC18-LF containing the human LFA3-FC fusion gene. The human LFA3-Fc fusion gene obtained at this time has a nucleotide sequence as shown in SEQ ID NO.

 The cloning vector used in the above process is the most commonly used vector in recombinant DNA technology, such as plasmid, phage lambda, cosmid and phage sputum 13, and the recipient cells of these vectors are Escherichia coli. The cloning vector used in the present invention is not particularly limited as long as it can replicate autonomously in Escherichia coli, can replicate together with the foreign DNA to be carried, and can be easily separated from the bacterial DNA and purified. In a preferred embodiment, the cloning vector used is pUC18.

 Then, the cloned human fusion gene LFA3-FC is constructed into a shuttle vector, and a host cell such as Escherichia coli cells is co-transformed with a plasmid containing the viral genome, and the recombinant replication-deficient virus pAd-LF of the present invention is selected.

With respect to host cells, host cells useful in the practice of the invention may be eukaryotic or prokaryotic cells, such as bacteria, yeast, or animal cells. Preferably, the host cell is a eukaryotic cell. Further, the host cell is a 293 cell. The 293 cells are used in the present invention because the cell line is easy to transfect, and three large-scale culture methods (adherent culture, microcarrier culture, serum-free suspension culture) can be used for 293 cell culture. Raise.

 The methods for co-transformation can be divided into two types, one is direct gene transfer technology, such as gene gun method, protoplast method, liposome method, pollen tube channel method, electro-optic transformation method, PEG-mediated transformation method, etc. The gene gun transformation method is representative; the other is bio-mediated transformation methods such as Agrobacterium-mediated and virus-mediated transformation methods. In the present invention, the method of co-transformation is not particularly limited. In a preferred embodiment, a method of electroporation can be employed.

After the infected host cells are cultured, the replication-defective virus is obtained by centrifugation. The injection of the anti-psoriatic composition of the third aspect of the present invention is obtained by mixing the replication-deficient virus with, for example, sterile water for injection and adjusting the pH. In a preferred embodiment, the injection contains 3 χ 10 ^{Ι () -} 3 χ 10 ^ί3 virions (VP) of virus particles per ml.

 A fourth aspect of the invention provides a method of preparing a pharmaceutical composition against psoriasis. According to a specific embodiment of the present invention, there is provided a method of preparing an injection comprising the steps of:

a. Take water for injection under aseptic conditions, add replication-defective virus at 3x l0 ⁽⁾ -3>< 10 ¹³ VP per ml;

 b. Under the aseptic conditions, the raw materials in step a are mixed and adjusted to adjust the pH to 7.5-8.5;

 c The step b product is sterile filtered and dispensed into an injection. DRAWINGS

 Figure 1 is a flow chart showing the construction of recombinant adenovirus (Ad-LF).

 Figure 2 is a 1% agarose gel electrophoresis map of the PCR product. Wherein: M is lkb + lOObp DNA ladder (GBICO), and 1 is a PCR product.

 Figure 3 is a 1% agarose gel electrophoresis pattern of the RT-PCR product. Among them: M is lOObp DNA ladder (GBICO), 1 is RT-PCR product.

 Figure 4 is a 1% agarose gel electrophoresis map of the digestion result of pAd-LF (Pac l). Wherein: M is the lkb DNA ladder (GIBCO); 1 is the number of the selected monoclonal.

Figure 5 is a 1% agarose electrophoresis map of recombinant adenovirus (PL1+PL2) identified by PCR amplification. Spectrum. Wherein: M is a lOObp DNA ladder (GBICO), C is a positive control (pAd-LF), and 1-6 represents a plaque of No. 1-6, respectively.

 Figure 6 is a 1% agarose electrophoresis pattern of recombinant adenovirus (PE1+PE2) identified by PCR amplification. Among them: M is lOObp DNA ladder (GBICO), C is positive control (pAd-LF), and 1-6 indicates 1-6 plaque.

 Figure 7 shows the results of Western Blot identification of Hela cells, where R is the reducing condition, NR is the non-reducing condition, and the protein molecular weight standard is Rainbow Marker (GE Healthcare).

 Fig. 8 is an experiment for inhibiting proliferation of mixed lymphocytes by virus-infected supernatant, wherein Ad-Null is an infection supernatant of an empty adenovirus, and Ad-LF is an infection supernatant of recombinant adenovirus Ad-LF. detailed description

 The present invention will be further described with reference to the accompanying drawings, which are not to be construed as a limitation of the invention. The basic idea of the invention is within the scope of the invention. Example 1 Preparation of human LFA3 gene

 In this example, the human LFA3 extracellular domain (aa.1-120) cDNA fragment was amplified by PCR from the template plasmid pORF5-LFA-3 (purchased from Invivogen).

 PCR primers were designed and synthesized based on the human LFA3 gene sequence. In order to insert the PCR product directly into the cloning vector-pUC 18 (purchased from Invitrogen) and fused with the IgGl Fc fragment, the Hind III and Nhe l were designed in the 5' primer. The restriction enzyme site, the 3' primer was designed into the Sai l restriction site, and the primer sequence was as follows:

 PI (SEQ ID NO. 2): 5' CTC AAG CTT GCT AGC ATG GTT GCT GGG AGC GAC GC 3'

 P2 (SEQ ID NO. 3): 5' ACG CGT CGA CAT AAA GAA AGA ACT TCA TGG 3'

The PCR conditions are as follows: After the PCR reaction mixture was denatured at 94 ° C for 5 minutes, the reaction was carried out under the following conditions: denaturation at 94 ° C for 30 seconds; annealing at 55 ° C for 30 seconds; 72 Ό extension for 30 seconds. The reaction was carried out for 30 cycles. Then extend at 72 ° C for another 12 minutes.

 After the reaction was completed, the PCR product was detected by 1% agarose gel electrophoresis, and it was revealed that a DNA fragment of about 380 bp which is identical in size to the expected fragment was amplified (see Fig. 2). The pUC18 was constructed and pUC18-L was obtained. The sequencing results showed that the target gene in the obtained recombinant plasmid was identical to the published extracellular domain of human LFA3. Example 2 Preparation of human LFA3-FC fusion gene

 In this example, a cDNA fragment of human IgG1 Fc (aa, 247-473) was amplified from human lymphocyte total RNA by RT-PCR.

 The RT-PCR primer was designed and synthesized based on the human IgG1 gene sequence. In order to insert the PCR product into the LFA3 fragment, the inventors designed the Sal I site in the 5' primer and the BamH I site in the 3 primer. The sequence is as follows:

 P3 (SEQ ID NO. 4): 5' ACG CGT CGA CAA AAC TCA CAC ATG CC 3'

 P4 (SEQ ID NO. 5): 5, CGG GAT CCT CAT TTA CCC GGA GAC AGG 3'

 The RT-PCR conditions are as follows:

 After the RT-PCR reaction mixture was denatured at 50 ° C for 30 minutes, the reaction was carried out under the following conditions:

 Reverse transcription reaction: 94 Ό denaturation 30 sec; 50 ° C annealing 30 sec; 68 ° C extension 1 min. The reaction was carried out for 10 cycles.

 PCR reaction: denaturation at 94 ° C for 30 seconds; annealing at 52 30 for 30 seconds; extension at 68 ° C for 1 minute. The reaction was carried out for 25 cycles. Then extend at 68 ° C for another 7 minutes.

After the completion of the reaction, the RT-PCR product was detected by 1% agarose gel electrophoresis, and it was revealed that a DNA fragment of about 720 bp which is identical in size to the expected fragment was amplified (see Fig. 3). Construction of pUC18-L to obtain pUC18-LF, sequencing indicated that the target gene in the resulting recombinant plasmid and the predicted human LFA3 extracellular domain sequence (aa.1-120) and human IgG1 Fc (aa.247-473) fusion gene The sequence is exactly the same. Example 3 Construction and screening of recombinant adenovirus Ad-LF

 The cloned human LFA3-FC fusion gene was first constructed into the pAdenoVator-CMV5 shuttle vector, and co-transformed into E. coli BJ5183 with the plasmid pAdenoVatorAElE3 containing the adenoviral genome, and the recombinant plasmid was selected and then transfected into 293 cells to screen the recombinant adenovirus.

1. The cloned human LFA3-FC fusion gene was constructed into the pAdenoVator-CMV5 shuttle vector, and then subjected to conventional electroporation with the circular plasmid _P AdenoVatorAElE3 (purchased from Qbiogene) containing the adenoviral genome (El, E3 deletion). Methods Escherichia coli BJ5183 (purchased from Qbiogene) was co-transformed and the recombinant p-Ad-LF was screened for identification. The results indicated that the recombinant pAd-LF (see Figure 4) with the recombination site between the replicon and the right arm was screened, and a 4.5 kb fragment was excised.

 2. The linearized recombinant plasmid pAd-LF and liposome Lipofectamine (purchased from Invitrogen) were co-transfected into 293 cells (purchased from ATCC) using a conventional co-transfection method, and the recombinant adenovirus Ad-LF was screened. After 14 days, a total of 6 plaques appeared in the cultured dishes, and each plaque was extracted and expanded.

 3. Extract the adenoviral DNA, and identify the recombinant adenovirus by PCR amplification of the human LFA3-F cDNA fragment, and identify the E2B region fragment characteristic of the adenovirus.

 The primers for amplifying the human LF A3 -Fc DNA fragment are:

 5, Primer P1 : 5' CTC AAG CTT GCT AGC ATG GTT GCT GGG AGC GAC GC 3'

 3, Primer P4: 5' CGG GAT CCT CAT TTA CCC GGA GAC AGG

3,

 The primers for amplifying the adenovirus-specific E2B region are:

 5, Primer PE1 : 5' TCG TTT CTC AGC AGC TGT TG 3'

 3, Primer PE2: 5' CAT CTG AAC TCA AAG GGT GG 3' was identified, the selected plaques were inserted into the human LFA3-Fc DNA fragment (a band around 1060 bp, see Figure 5); The adenovirus E2B positive band was amplified (around 860 bp, see Figure 6).

4. Infect the Hda cells with the selected virus, and adopt the conventional ELISA method to identify the expression of the LFA3-FC fusion protein. The expression results are shown in Table 1. Expression of LFA3-FC fusion protein in each plaque

从测定结果看来， 6个空斑均能表达融合蛋白 LFA3-Fc， 2#空斑 表达最高。 我们选择表达最高的空斑 Ad-LF-2#， 命名为 Ad-LF。 实施例 4 重组腺病毒的扩增和纯化

From the results of the assay, the six plaques were able to express the fusion protein LFA3-Fc, and the 2# plaque expression was the highest. We chose the highest-profile plaque Ad-LF-2#, named Ad-LF. Example 4 Amplification and Purification of Recombinant Adenovirus

The Ad-LF virus seed solution was expanded step by step, and finally 5 χ 10 ^δ 293 cells were infected, cultured for 48-72 hours until complete cytopathic effect (CPE), and the cells were collected by centrifugation, and the cell pellet was resuspended in 100 ml. % FBS (fetal calf serum) in DMEM medium, - 20 °C / 37 °C repeated freeze-thaw three times, centrifuge to remove cell debris, the supernatant is the virus lysate, spare.

 The Ad-LF was purified by conventional two-step CsCl density gradient ultracentrifugation. The first step was to remove most of the cell debris and defective virus particles (ie, virus particles without infectious activity) using a discontinuous CsCl gradient. In the second step, the infectious virus particles were completely separated from the defective virus particles by continuous CsCl density gradient. Finally, the salt was removed by dialysis to remove CsCl to obtain recombinant human LFA3-FC adenovirus-Ad-LF. Example 5 Infectious activity of recombinant adenovirus Ad-LF on different cells and identification by Western Blot

We infect Ad-LF with different MOI values (multiplicity of infection), including human lung cancer cell line A549, human cervical cancer. The cell line HeLa, human hepatoma cell line HepG2, and human ovarian cancer cell line SKOV3 were used to detect the expression of human LFA3-FC fusion protein by ELISA and Western Blot, respectively.

 Human lung cancer cell line A549, human cervical cancer cell line HeLa, human liver cancer cell line HepG2, and human ovarian cancer cell line SKOV3 (purchased from ATCC) were inoculated into a 6-well plate, and the recombinant adenovirus Ad- obtained in Example 4 was used. LF was infected with different MOI values: 0, 5, 10, 50. After 48 hours of culture, the supernatant was assayed for the expression of LFA3-FC using the kit (purchased from Chemicon). The results are shown in Table 2.

结果表明， 重组腺病毒 Ad-LF 能感染包括肺癌、 肝癌、 子宫内 膜癌、卵巢癌在内的多种细胞类型，表达并分泌人 LFA3-FC融合蛋白， 说明 Ad-LF具有较广谱的感染范围及较高的表达活性。

The results showed that the recombinant adenovirus Ad-LF can infect many cell types including lung cancer, liver cancer, endometrial cancer and ovarian cancer, and express and secrete human LFA3-FC fusion protein, indicating that Ad-LF has a broad spectrum. Infection range and high expression activity.

 At the same time, rabbit anti-human LFA3 antibody (1:500) (purchased from Chemicon) was used as primary antibody, goat anti-rabbit IgG-HRP (purchased from DAKO) as secondary antibody (1:3000), using conventional Western

The Blot method further identified whether the expression product of Hela cells was the same as expected.

The results are shown in Figure 7. The results indicate that Hela cells specifically express the human LFA3-Fc fusion protein, which are subjected to electrophoresis under reducing and non-reducing conditions, respectively, and the hybridization conditions are consistent with the expected bands (about 60 kDa), non-reducing hybridization bands. It is a dimeric form (about 119 kDa:), which is very diffuse and presents a typical glycoprotein electropherogram. Example 6 Activity of recombinant adenovirus Ad-LF to inhibit lymphocyte proliferation In vitro, we tested recombinant adenocarcinoma by a mixed lymphocyte proliferation inhibition assay. The activity of the fusion protein expressed by toxic Ad-LF.

 First, Ad-LF infected Hela cells (MOI = 10) to prepare a viral infection supernatant for use.

Human lymphocytes isolated by lymphocyte separation (Ficoll) were divided into two groups. One group was washed twice with RPMI1640 and resuspended in RPMI1640 containing 10% FBS to obtain a cell suspension with a final concentration of 3×10 ⁶ /ml. As a responder cell; the other group was treated with 2000 rads-ray to obtain a cell fluid having a final concentration of 3×10 ⁶ /ml as a stimulator cell.

The reaction cells and the stimulator cells were added to a round-bottom 96-well plate (1.5×10 ⁵ /well cells) in a ratio of 1:1, and 50 μl of the virus-infected supernatant was added to each well (diluted twice), and placed at 37 °C.

Incubate for 5 days in a 5% CO ₂ incubator.

On the fifth day, 1 μ of ³ Η-labeled thymidine was added to each well. After incubation for 15 hours, the cells were collected using a multi-head cell harvester, and the relative radioactivity (expressed in CPM) of each well was measured by a liquid scintillation meter.

 The results are shown in Fig. 8. The results showed that there was dose compliance between the concentration of LFA3-FC fusion protein and the inhibition of lymphocyte proliferation, and the inhibition of lymphocyte cells reached 50% at a dilution of 1:4; Ad-Null infection supernatant has no inhibitory effect. This indicates that the LFA3-FC fusion protein expressed by Ad-LF can significantly inhibit lymphocyte proliferation in vitro. Example 7 Preparation of recombinant adenovirus Ad-LF injection

500 ml of water for injection was taken under aseptic conditions, and the recombinant adenovirus Ad-LF (3 χ 10 ¹⁰ -3 χ 10 ¹³ VP ) obtained in Example 4, Tris 0.484 g, MgCl ₂ 0.076, and 16 g of sucrose were added and mixed uniformly. Water for injection was added to a total volume of 1000 ml while adjusting the pH to 8.0 with HCl. After sterile filtration, the recombinant adenovirus Ad-LF injection was dispensed into 1 ml/sp., and stored at - 20 °C.

In conclusion, the recombinant adenovirus Ad-LF constructed by the human LFA3-FC fusion gene of the present invention can infect various types of cells and stably secrete a highly active recombinant human LFA3-FC polypeptide in a large amount. The anti-psoriatic injection prepared by the recombinant adenovirus of the present invention enables the body to express a high level of recombinant human LFA3-FC polypeptide having intact biological activity. This compensates for the poor stability of the protein-infused human LFA3-FC drug and the short duration of the effective concentration, which can greatly reduce the dosage, reduce the cost of medication, reduce the burden on patients, and improve the quality of life of patients. Good market prospects.

 The above preferred embodiments are further exemplified by the present invention, but are not intended to limit the scope of the present invention, and those skilled in the art can make various modifications or improvements according to the basic idea of the present invention without departing from the invention. The basic idea is within the scope of the spirit of the invention and the scope of the appended claims.

Claims

Rights request: A recombinant replication-deficient virus, characterized in that the replication-defective virus comprises a human fusion gene LFA3-Fc under the control of a promoter. The replication-defective virus according to claim 1, wherein the human fusion gene LFA3-FC is formed by fusing an extracellular soluble portion of human LFA3 with an Fc fragment of human IgG. The replication-defective virus according to claim 2, wherein the nucleotide sequence of the human fusion gene LFA3-FC is as shown in SEQ ID NO. The replication-defective virus according to any one of claims 1 to 3, wherein the replication-deficient virus is a replication-deficient adenovirus, a replication-deficient herpes simplex virus or a replication-defective Newcastle disease virus. Any of them. The replication-defective virus according to claim 4, wherein the replication-deficient virus is an adenovirus type 5. A host cell comprising the replication-defective virus according to any one of claims 1 to 6. 7. The host cell according to claim 6, wherein the host cell is a 293 cell. A method of producing a recombinant replication-defective virus according to any one of claims 1 to 6, the method comprising: The human fusion gene LFA3-FC was inserted into the replication-defective virus genome and placed under the control of a promoter. A pharmaceutical composition comprising the replication-defective virus according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is an injection solution containing 3 x 10 ^{1Q - 3} x 10 ¹³ virions per ml of the injection. The use of the replication-defective virus according to any one of claims 1 to 6 for the preparation of a medicament against psoriasis.