NL2031110B1

NL2031110B1 - Recombinant adeno-associated virus vector, recombinant adeno-associated virus aav8-pd1 and use thereof

Info

Publication number: NL2031110B1
Application number: NL2031110A
Authority: NL
Inventors: Lu Yanda; Zhang Xiaodian; Liu Siru; Zheng Shaojiang; Chen Yun; Feng Dongju; Yao Yao; Zhou Zewei
Original assignee: The First Affiliated Hospital Of Hainan Medical Univ
Priority date: 2022-02-28
Filing date: 2022-02-28
Publication date: 2023-09-07

Abstract

The present disclosure belongs to the technical field of biology and relates to a recombinant adeno—associated, virus vector, a recombinant adeno—associated virus and use thereof. The recombinant adeno—associated virus vector is formed by inserting a 5 nucleotide sequence of a PD—l extracellular segment into an adeno— associated virus vector, wherein the nucleotide sequence of a PD—l extracellular segment is as shown in SEQ ID NO: 1. According to the recombinant adeno—associated virus vector provided by the present disclosure, an extracellular segment (a total of 170 amino 10 acids) of PDl is inserted into an adeno—associated virus AAV8 for expression and soluble PDl is secreted. Therefore, the soluble PDl can bind to PDLl on tumor cells, a PD—l/PD—Ll signaling pathway is effectively blocked, and the PDLl on the tumor cells is prevented from binding to the PDl on T cells to avoid exhaustion of the T 15 cells. (+ Fig. 1)

Description

P1149/NLpd

RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR, RECOMBINANT ADENO-

ASSOCIATED VIRUS AAV8-PD1 AND USE THEREOF

TECHNICAL FIELD

The present disclosure belongs to the field of biotechnology and particularly relates to a recombinant adeno-associated virus vector, a recombinant adeno-associated virus and use thereof.

BACKGROUND ART

Hepatocellular carcinoma (HCC) is the sixth most common ma- lignant tumor and the second most common cause of cancer-related death worldwide. The HCC is mainly caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infection and liver cirrhosis.

The GLOBOCAN 2018 data estimated that 841,000 new cases of primary liver cancer and 782,000 deaths would occur each year. Orthotopic liver transplantation (OLT) is an effective method of treating the

HCC and potential liver cirrhosis and considered the best treat- ment option. Early treatment of the HCC includes resection, abla- tion and liver transplantation. Unfortunately, most HCC cases are in an advanced stage or have developed metastases, and thus are amenable to systemic treatment, but are not amenable to OLT. Due to its high metastatic ability and recurrence rate, a five-year survival rate of advanced HCC is very low. Therefore, it is very urgent to seek a high-efficiency and low-toxicity treatment strat- egy for the HCC.

In recent years, some new treatment methods have been contin- uously applied in clinical trials for treating the HCC. For exam- ple, a drug resistance of sorafenib is overcome by inhibiting miR- 222/221 and a PI3K/AKT pathway in a targeted therapy of microRNA; a CDK4/6 inhibitor promotes apoptosis and autophagy of the HCC by inducing a down-regulation of a tumor suppressor AMPK; a trans- forming growth factor-beta (TGF-R) blocker inhibits tumor growth and migration by inhibiting SMAD protein phosphorylation; and a

Wnt signaling pathway antagonist inhibits proliferation and migra-

tion of cancer stem cells (CSCs) and tumor cells. As a new and more effective treatment, an immunotherapy has also been applied to the clinical treatment of the HCC.

The tumor cells have an ability of evading an immune re- sponse. How to effectively activate an anti-tumor activity of T cells in the tumor microenvironment and maintain a persistent ability of the T cells to discover and attack cancer cells are hot spots to be urgently explored by tumor immunity. A recent conver- sion research on use of an immune checkpoint and an immune check- point inhibitor (ICI) in tumor therapy creates new promise for ad- dressing the bottleneck problem. Programmed cell death protein 1 (PDl) is an important immunosuppressive transmembrane protein ex- pressed on surfaces of the T cells. The PDl and its ligand PDL1 (which is mainly expressed in the tumor cells and the tumor micro- environment and exposed to an antigen for a long time, and medi- ates T cell suppression) are combined to inhibit an activation of lymphocytes, thereby inhibiting an immune response of immune cells in the tumor microenvironment. The most common ICIs today include nivolumab and pembrolizumab, which have been approved for market- ing in the United States and the European Union. These antibodies are directed against the receptor PD-1 which is expressed by a number of the immune cells, such as activated T cells, B cells, natural killer cells, monocytes and dendritic cells (DCs). The PD-

Ll is also expressed on various cells, such as the activated T cells and the DCs, as well as in a wide range of non-hematopoietic tissues such as the lungs. The PD-L1 has a physiological function of maintaining immunological homeostasis in the body during pe- ripheral inflammation to prevent autoimmunity. Importantly, the

PD-L1 is highly expressed in some tumor cells and its interaction in the tumor microenvironment can suppress a local anti-tumor T cell response, thereby promoting an immune escape of the cancer cells. Thus, the designed antibodies such as nivolumab can block an interaction between the PD-1 and the PD-L1 to restore T cell activation and prevent immune evasion of the cancer cells. The

ICIs had significant efficacy in clinical trials. The cancer immu- notherapy was considered a breakthrough technique at 2013 and was

FDA approved for treating the HCC. However, the ICIs are expensive and have no targeting property, thus a patient taking the ICIs has systemic reaction after intravenous administration. The treatment is often associated with a number of immune-related adverse events, including colitis, dermatitis, and hepatitis, as well as

Vogt-Koyanagi-Harada disease (VKH). About 7%-12% of patients re- ceiving a single medicine of oa PD-1/a PD-L1 monoclonal antibody have grade 3-4 immune related adverse events. Studies also show that during an experiment, mice obviously lose hair. Therefore, there is a need to explore innovative strategies for tumor- specific delivery of the ICIs. Delivery of therapeutic proteins at a gene level can be accomplished with viral vectors, including vectors derived from adeno-associated virus (AAV).

The AAV is a single-stranded DNA parvovirus and has been widely used in gene therapy of human and animal related diseases due to its advantages of good safety, wide host range, low immuno- genicity, long-term expression of foreign genes, etc. Due to dif- ferences between capsid protein sequences, different AAV serotypes bind to different cell surface receptors, which enable different

AAV serotypes to have different tissue tropisms, e.g. AAV9 has high affinity for the heart and skeletal muscle; and AAV5 has high affinity for the lungs and central nervous system. AAV8 is the most hepatophilic among currently used AAV vectors and the vector carrying a target gene can form a stable liver transfection in an- imals. Recombinant AAV8 can be intrahepatically or intravenously injected into mice and can show significant liver tropism. Be- sides, a target gene expression can last at least 6 months, which is much longer than a clinically used PD-1 antibody.

As a transmembrane protein, the PDl is expressed on a cell membrane and exerts its effect through a binding of its extracel- lular region to the ligand PDL1. Compared with monoclonal antibod- ies, the soluble PD1 molecule can also block a co-stimulatory sig- nal of PDL on the surfaces of the tumor cells through direct re- ceptor/ligand interactions, has a relatively small cleavage vari- ant fragment and is easy to load to achieve exogenous expression and secretion.

Therefore, it is urgent to find a method for increasing the expression of the PDl in the tumor cells.

SUMMARY

The present disclosure aims to provide a recombinant adeno- associated virus vector, a recombinant adeno-associated virus (rAAV) and use thereof to prevent combination of PDL1 on tumor cells with PD1 on T cells, avoid T cell exhaustion and improve an anti-tumor immune activity.

The adeno-associated virus (AAV) is a type of single-stranded linear DNA-deficient virus with a genomic DNA of less than 5kb, has no envelope, and is a naked 20-hedral particle in appearance.

The rAAV is derived from a non-pathogenic wild-type adeno- associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, the rAAV can express foreign genes in vivo for a long time and is re- garded as one of the most promising gene transfer vectors and widely used in gene therapy and vaccine research worldwide. The

AAV vector does not need adenovirus assistance, does not insert into a host genome, but is free from host cell genes, and is sta- bly expressed in a satellite shape. Besides, the AAV has extremely high infection efficiency in vivo.

In order to achieve the above objective, the first aspect of the present disclosure provides a recombinant adeno-associated vi- rus vector. The recombinant adeno-associated virus vector is formed by inserting a nucleotide sequence of a PD-1 extracellular segment into an adeno-associated virus vector, wherein the nucleo- tide sequence of a PD-1 extracellular segment is as shown in SEQ

ID NO: 1.

SEQ ID NO:1 is 5'-

ATGCAGATCCCACAGGCGCCCTGGCCASTCGTCTGGGCGGTGC-

TACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTG-

GAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCAC-

CTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCG-

CATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCG-

CAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGSTCACACAACTGCCTCAAC-

GGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTAC-

CTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGG-

CAGAGCTCAGGSTGACAGAGAGAAGGG-

CAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTG- 3.

Specifically, the PD-1 cells are derived from humans and thus the nucleotide sequence of a PD-1 extracellular segment is a nu- 5 cleotide sequence of a human-derived PD-1 extracellular segment.

A protein sequence of a human PDl extracellular seg- ment>sp/lQ15116/1-170 is as shown in SEQ ID NO: 2.

SEQ ID NO: 2 is MQOIPQAPWPVVWAVLQLGWRPGWELDSPDRPWNPPTF-

SPALLVVTEGDNATETCSEFSNTSESFVLNWYRMSPSNOTDKLAAFPEDRSOPG9OD-

CREFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTER-

RAEVPTAHPSPSPRPAGOFQTLV.

The second aspect of the present disclosure provides a recom- binant adeno-associated virus AAV8-PD1. The recombinant adeno- associated virus belongs to serotype 8.

The third aspect of the present disclosure provides use of the provided recombinant adeno-associated virus vector in prepar- ing a preparation for blocking a PD-1/PD-L1 signaling pathway or promoting growth of a CD8-positive T cell. The recombinant adeno- associated virus vector or the recombinant adeno-associated virus can be intravenously transfected into host cells.

The fourth aspect of the present disclosure provides use of the recombinant adeno-associated virus vector in preparing a prep- aration for treating a tumor.

The fifth aspect of the present disclosure provides use of the recombinant adeno-associated virus vector in preparing a prep- aration for treating liver cancer.

The sixth aspect of the present disclosure provides use of the recombinant adeno-associated virus vector in preparing a prep- aration for blocking a PD-1/PD-L1 signaling pathway or promoting growth of a CD8-positive T cell.

The seventh aspect of the present disclosure provides use of the recombinant adeno-associated virus vector in preparing a prep- aration for treating a tumor.

The eighth aspect of the present disclosure provides use of the recombinant adeno-associated virus in preparing a preparation for treating liver cancer.

According to the recombinant adeno-associated virus vector the first aspect of the present disclosure, an extracellular seg- ment (a total of 170 amino acids) of PD1 is inserted into an adeno-associated virus RAV8 for expression and soluble PDl is se- creted. Therefore, the soluble PDl can bind to PDL1 on tumor cells, a PD-1/PD-L1 signaling pathway is effectively blocked, and the PDL1 on the tumor cells is prevented from binding to the PDI on T cells to avoid exhaustion of the T cells. The recombinant adeno-associated virus vector improves functions of the T cells of a host and a killing effect of immune cells on the tumor cells, almost has no damage to normal cells, tissues and organs, and has extremely high safety and reliability.

The recombinant adeno-associated virus (AAV8-PD1) provided by the second aspect of the present disclosure can target infected liver parts, expresses and secretes a PD-1 extracellular protein in liver cells, thus effectively blocks a PD-1/PD-L1 signaling pathway, improves functions of T cells of a host and a killing ef- fect of immune cells on tumor cells, almost has no damage to nor- mal cells, tissues and organs, and has extremely high safety and reliability.

The recombinant adeno-associated virus AAV8-PD1l provided by the present disclosure can stably infect liver tissues and express a PD1 extracellular segment protein.

The recombinant adeno-associated virus AAV8-PD1 provided by the present disclosure obviously inhibits growth of tumors of mice.

The recombinant adeno-associated virus AAV8-PD1 provided by the present disclosure increases the number of cytotoxic T lympho- cytes after treatment.

The recombinant adeno-associated virus AAV8-PD1 provided by the present disclosure increases the number of CD8-positive memory

T cells in the spleen after treatment.

Other features and advantages of the present disclosure are described in detail in the following DETAILED DESCRIPTION part.

BRIEF DESCRIPTION OF THE DRAWINGS

Through the accompanying drawings, the embodiments of the present disclosure are described in more detail and the above and other objectives, features and advantages of the present disclo- sure will become more apparent.

FIG. 1 shows a flow chart of construction of AAV, vGEP and

AAV8-PD1;

FIG. 2A shows fluorescence micrographs of Hepal-6 cells in- fected with different titers of AAV9-PD1 after 72 hours;

FIG. 2B shows a fluorescence statistical result of Hepal-6 cells infected with different titers of AAV9-PD1 after 72 hours;

FIG. 3 shows an expression of a PDl protein after Hepal-6 cells are cultured for 96 hours;

FIG. 4 shows expressions of AAV8-PD1 and AAV8-GFP in the liv- er in example 2;

FIG. 5A shows a visual observation of mouse liver after 10 days of treatment with a recombinant adeno-associated virus;

FIG. 5B shows a result of a statistical analysis of mouse liver after 10 days of treatment with a recombinant adeno- associated virus; where P > 0.05; * indicates P < 0.05; ** indi- cates P < 0.01; *** indicates P < 0.001; and **** indicates P < 0.0001;

FIG. 6 shows a flow cytometric analysis result of the number of CD4+ and CD8+ CTL cells in tumor tissues treated with a recom- binant adeno-associated virus for 7 days;

FIG. 7 shows a statistical result of a percentage of the num- ber of CD8+ CTL cells to the total viable cells; where P > 0.05; * indicates P < 0.05; ** indicates P < 0.01; and *** indicates P < 0.001;

FIG. 8 shows that the number of CD8+, CD44+, cCD122+ and CD4+,

CD44+, CD122+ memory T cells in mouse splenocytes increases sig- nificantly after AAV8-PD1l treatment; and

FIG. 9 shows a statistical result of a percentage of the num- ber of CD8-positive memory T cells to the total viable cells; where P > 0.05; * indicates P < 0.05; ** indicates P < 0.01; and *** indicates P < 0.005.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The preferred embodiments of the present disclosure will be described in more detail below. Although the preferred embodiments of the present disclosure are described below, it should be under- stood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth here- in.

Materials used in the following examples

Mouse hepatocarcinoma cells Hepal-6 are from the laboratory of Professor Chen Yun of Nanjing Medical University; 6-8-week old male C57BL/6 mice are provided by Hunan SJA Laboratory Animal Co.,

LTD (Experimental animal production license: SCXK(Xiang)201lé- 0002); and an escherichia coli strain DH5o is from Invitrogen; a re- striction enzyme is from Thermo Fisher Scientific; an HB-infusion™ seamless cloning kit is from Hanbio Biotechnology Co., Ltd.; plas- mid DNA is small and a large-scale extraction kit is from Beijing

ComWin Biotech; a gel recovery kit is from Shanghai Generay Bio- tech; agarose and agar powder are from Sangon Biotech; a DNA lad- der is from Thermo Fisher Scientific; and a KOD-Plus kit is from

TOYOBO CO., LTD.

Consumables such as cell culture flasks, cryopreservation tubes, pipettes, centrifuge tubes, pipette tips, cryopreservation boxes, and cell culture plates are purchased from Corning; a DMEM medium and fetal calf serum (FCS) are purchased from Gibico BRL,

USA; and rabbit anti-mouse PCNA antibody is purchased from Santa

Cruz Company. A PDl monoclonal antibody is purchased from Abcam; a p-actin antibody is purchased from CST; and CD45, CD3, CD4, CDS,

CD25, CD44, CD69, CD122, FOXP3 and other flow cytometry antibodies are from BD.

A nucleotide sequence of a PD-1 extracellular segment is shown in SEQ ID NO: 1. 5'-

ATGCAGATCCCACAGGCGCCCTGGCCAGTCETCTGGGCGETGC—

TACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTG-

GAACCCCCCCACCTTCTCCCCAGCCCTSCTCGTGSTGACCGAAGGGGACAACGCCAC-

CTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCG-

CATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCG-

CAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAAC-

GGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTAC-

CTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGG-

CAGAGCTCAGGGTGACAGAGAGAAGGG-

CAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTG- 3°.

A protein sequence of a human PDl extracellular seg- ment>splQ15116/1-170 is as shown in SEQ ID NO: 2. 51

MQIPQAPWPVVWAVLOLGWRPGWEFLDSPDRPWNPPTEFSPALLVVTEGD-

NATFTCSESNTSESEVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRERVTQLPNGRD—

FHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV- 37

An adeno-associated virus serotype 8 (AAV8) vector is from

Hanbio Biotechnology and has a nucleotide sequence as shown in SEQ

ID NO: 3. 5'-

ATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGSGCGTGGATAGCGGTTTGACTCAC-

GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCAC-

CAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGG-

TAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTG-

GAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGSGATCCGCCACCGGTAC-

CTTAATTAACGACTACAAGGATGACGATGACAAGGATTACAAAGACGACGATGATAAGGAC-

TATAAGGATGATGACGACAAATTAATTAACGAGGGCAGAG-

GAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTACCGGTGCCAC-

CATGGCCCAGTCCAAGCACGGCCTGACCAAAGAGATGACCATGAAGTACCG-

CATGGAGGGCTGCGTGGACGGCCACAAGTTCGTGATCACCGGCGAGGGCATCGGC-

TACCCCTTCAAGGGCAAGCACGCCATCAACCTGTGCGTGGTGGAGGGCGGCCCCTT-

GCCCTTCGCCGAGGACATCTTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAGTACC- 3.

Statistical analysis

The mean value of an experimental result in the following ex- amples is represented by, measurement data is analyzed by a Stu- dent's T test, a comparison of mouse survival rates is analyzed by a Long rank test; and SPSS 17.0 is used for statistical analysis.

Example 1

I. Cell cultivation 1 Cell resuscitation culture

Mouse hepatocellular carcinoma Hepal-6 cells preserved in liquid nitrogen were taken out and quickly put in a 37°C water bath pot to rewarm and thaw, cryopreserved liquid was transferred to a sterile 15-ml centrifuge tube in a super clean bench, 3 ml of

DMEM containing 10% of fetus calf serum, 100 U/ml of penicillin and 100 mg/ml of streptomycin was gently rinsed several times, centrifugation was conducted at 500 rpm for 5 min, a supernatant was removed, 2 ml of a medium was added to resuspend, and the cells were transferred to a 6-well plate to be cultured at 37°C in a 54%CO: incubator. 2 Cell subculture

Growth adhering to wall and cell passage: after the cells grew about 90% under a microscope, an old medium was discarded, the cells were washed twice with PBS, 0.25% trypsin was added, the cells were shrunk and round under the microscope, a complete medi- um was added to terminate digestion, the cells were gently pipet- ted until the cells were fully dispersed and fell off, liquid was collected in a sterile 15-ml centrifuge tube, centrifugation was conducted at 800 rpm for 5 min, a supernatant was discarded, and a cell precipitate was resuspended with a complete medium, pipetted evenly and divided into dishes for culture.

II. Construction of recombinant adeno-associated virus sero- type 8 (AAV8-PD1) expression vector

In order to construct a AAV8-PD1l vector, a human PDl extra- cellular sequence (derived from UniProtKB-Q15116 (PDCDl HUMAN); https://www.uniprot.org/uniprot/Q15116) was amplified with pri- mers; the vector was digested at 37°C and a gel was recovered; af- ter a fragment PCR, a treated target fragment was ligated with the vector; the ligated product was transformed into competent cells

DH5o; the cells were cultured overnight at resistance of Amp, 37°C and 230 rpm; and the bacteria were picked from a transformed plate and shaken at 37°C and 230 rpm for 14 hours, the bacteria liquid was used for PCR identification, and positive cloned bacteria liq- uid was sent to a sequencing company for sequencing. The primers included an upstream primer and a downstream primer, and had nu- cleotide sequences as shown below: the upstream primer h-PD1-F: ttttgacctccatagaagacaccgg- gatccgccaccatgcagatccca (SEQ ID NO: 4); and the downstream primer h-PD1-R: tcatccttgtag- tcgttaattaaggtacccaccagggtttggaactgg (SEQ ID NO: 5).

III. Construction of AAV8-PD1l expression vector

As a gene delivery vector, AAV can introduce a target gene into host cells and stably express the target gene. Besides, when the AAV infects human host cells, a site is specifically integrat- ed into a specific region of human chromosome 19. An extracellular domain of a human PDl protein (amino acids 1-170) and a marker gene GFP were inserted into an open reading frame of AAV8. A con- struction mode was shown in FIG. 1.

IV. Construction of AAV8-PD1 expression vector

In order to test whether the virus was constructed success- fully, Hepal-6 cells were infected with different titers of the

AAV8-PD1 separately. An expression of a green fluorescent protein (GFP) of the AAV8-PD1l in the cells was observed after 72 hours.

The results were shown in FIG. 2A and FIG. ZB. The above results showed that as a virus infection titer increased, the expression of the GFP increased and the virus infection efficiency increased; where the 5x10° vg viruses had a best infection effect. The Hepal-6 cells were infected with the viruses of a MOI value of 5x10° and cultured in a 37° C in a 5% CO: incubator for 96 hours. A protein was extracted and subjected to a western blot analysis. A result was shown in FIG. 3. It can be seen from FIG. 3 that the Hepal-6 cells can secrete a PDl protein after infection with the AAV8-PD1, while the cells in a control group did not secrete the PD1 pro- tein.

Example 2

The example is used to determine that a recombinant adeno- associated virus (AAV8-PD1) has a relatively strong affinity for the liver.

Mice in an AAV8-PD1 treatment group (AAV8-PD1), an AAV8-GFP treatment group (vGFP) and a control group (Mock) were injected with 1x10 vg virus/mouse in a tail vein manner. After 10 days, visible light irradiation of small animal imaging showed that GFP fluorescence was obvious in the liver area, indicating that the

AAV8-PD1 had a targeting effect on liver tissues (FIG. 4).

Example 3

A mouse hepatocellular carcinoma model was constructed in the example. 6-8 week-old male C57BL/6 mice were subcutaneously injected with 1x10’ Hepal-6 cells in 100 pl of sterile PBS to a right arm- pit. The experimental mice were randomly divided into four groups: a recombinant adeno-associated virus AAV8-PD1 treatment group (vPD1l), an AAV8-GFP treatment group (vGFP), a PDl monoclonal anti- body treatment group (aPDl) and a control group (Mock). When a size of a subcutaneous tumor was about 150 mm’ after infection of

Hepal-6 cells, 2.5x10%* vg viruses were diluted with PBS to 100 ul and injected once into the tumor, and the tumor of the mice in the control group was only injected with PBS. Body condition and weight of the mice were monitored every day. The diameter of the tumor was measured with a vernier caliper. Besides, when the tumor reached 20 mm in any direction, the mice were euthanized. The re- sults were shown in FIG. 5A and FIG. 5B. The tumor volume was cal- culated according to the formula: tumor volume (mm?) = 0.52xlengthxwidth?®.

It can be seen from FIG. 5A and FIG. 5B that compared with the control group, the tumor volume of the mice in the AAV8-PD1 treatment group was significantly reduced and the tumor weight was significantly reduced (FIG. 5A). The AAV8 and PDl monoclonal anti- body treatment groups had different degrees of anti-tumor effects, but the AAV8-PD1 treatment group had a more significant effect (P < 0.0001, FIG. 5B).

Example 4

In the example, tumor tissues were collected for a flow cy- tometric detection on mice treated with a recombinant adeno- associated virus AAV8-PD1l for 7 days. The experimental mice were randomly divided into four groups: a recombinant adeno-associated virus AAV8-PDl treatment group (vPDl), an AAV8-GFP treatment group (VGFP), a PD1l monoclonal antibody treatment group {aPDl) and a control group (Mock).

Fresh tumor tissues were put in a petri dish containing a small amount of PBS and cut into a homogenate shape with scissors; 10 ml of PBS was added, a tissue homogenate was sucked with a pi- pette and filtered into a test tube with a 300-mesh nylon mesh;

centrifugation was conducted at 1,000 rpm for 5 min, the cells were washed with PBS for 3 times, and cell debris was removed at 800 rpm for 5 min each time; the cells were fixed with 70% ice ethanol at 4°C overnight; centrifugation was conducted at 1,000 rpm for 3 min and a supernatant was removed, CD4, CD8, CD44 and CD122 flow cytometry antibodies were added respectively for an incuba- tion for 30 min; centrifugation was conducted at 1,000 rpm for 3 min and a supernatant was removed; the cells were washed with PBS for 3 times, centrifugation was conducted at 1,000 rpm for 3 min each time and a supernatant was removed; and 200 ul of PBS was added to resuspend the cells after the last wash and detection was conducted on a flow cytometer. Results were as shown in FIG. © and

FIG. 7.

As shown in FIG. 6, the number of cytotoxic T cells (CD8- positive T cells) in the vPDl treatment group was significantly higher than that in the control group. As shown in FIG. 7, the number of the cytotoxic T cells was 0.16% in the control group, 0.33% in the vGFP treatment group, 1.22% in the aPDl treatment group, and 6.08% in the vPDl treatment group. There were signifi- cant differences between the groups (all P < 0.001).

In a process of performing the above-mentioned flow cytometry detection, changes of the number of the CD8-positive memory T cells (TCM) in the spleen by different treatments were detected.

As shown in FIG. 8 and FIG. 9, the number of the CD8-positive memory T cells was 13.4% in the Mock control group, 20.4% in the

VGFP treatment group, 19.7% in the aPDl treatment group, and 25.7% in the vPDl treatment group. There were significant differences between the groups (all 2 < 0.001).

It can be seen from the above examples that compared with di- rect use of antibodies as proteins, the recombinant adeno- associated virus vector or recombinant adeno-associated virus of the present disclosure was used, a genetic delivery of therapeutic antibodies has many advantages. For example, the use of the recom- binant adeno-associated virus vector for delivery can lead to long-term antibody production after a single injection, thus re- ducing a manufacturing cost and a treatment cycle. In this case, the naturally available serotype AAV delivers an antibody gene to the liver or muscle tissue and immune checkpoint inhibitors (ICIs) are released into blood, resulting in a persistent and high anti- body serum level.

Before that, oncolytic measles viruses and adenoviruses have been modified to encode scFv-Fc of oPD-L1 and scFv-Fc of oCLTA-4 or oCTLA-4 antibody. Both oncolytic viruses are particles with a replication ability, can lyse human tumor cells and must be pro- cessed under biosafety level 2. Although this property may enhance an invasion of tumor infiltrating lymphocytes (TILs), they cannot or can only disappear during infection and replication. The AAV vector used here can be processed under safety level 1. In addi- tion, there is now enough in vivo clinical experience for use of the AAV vectors and two products have been approved for marketing in the European Union and the United States. This makes the AAV vectors an ideal tool for genetic delivery because they are easier to apply to research and treatment than the oncolytic viruses.

In the present disclosure, the AAV8-PD1 recombinant adenovi- rus significantly inhibited tumor growth and significantly reduced the tumor volume and the tumor weight. The AAV8 and PD1 monoclonal antibody treatment groups had different degrees of anti-tumor ef- fects, but the AAV8-PD1 treatment group had a more significant ef- fect. In the present disclosure the anti-tumor effect of the AAV8 was related to an expressed Rep protein and a special ITR struc- ture of a genome. The Rep mainly affects activities of these genes by combining with abnormal promoters of proto-oncogenes or viral genes, thereby exerting the anti-tumor effects and having cell specificity. The ITR mainly activates a DNA damage response, thereby causing cell apoptosis, death or cell cycle arrest. At the same time, the AAV can also increase sensitivity of tumor cells to various anti-tumor factors. In addition, after an AAV infection gene is integrated into the cells, it may stimulate antiviral pro- tein immunity of body and at the same time kill the tumor cells infected by the AAV.

In the present disclosure, the recombinant adenc-associated virus AAV8-PD1 can stably infect liver tissues and enable the ex- pression of the ICIs in orthotopic implanted tumors to be better than other sites. Therefore, AAV8-PD1 produced by infected tumor cells is continuously released into the tumor microenvironment and specifically binds to PDL1 on surfaces of tumor cells to activate

T cell immune response and exert an anti-tumor immune effect.

It can be seen from the experimental results of the above ex- amples that the number of cytotoxic T cells (CD8 positive T cells) in the AAV8-PD1 treatment group was significantly higher than that in the control group, which indicated that after the viruses in- fected the tumor cells, soluble PDl was released and bound to PDL1 on the tumor cells, thus binding of the T cells to PD1-PDL1 of the tumor cells was blocked.

From the experimental results of the above examples, it can be seen that the number of the CD8-positive memory T cells in the spleen of the AAV8-PD1 treatment group was significantly higher than that of the control group, indicating that there was a sys- temic immune effect, which may have important significance for tu- mor metastasis and recurrence.

The above descriptions of the examples of the present disclo- sure are illustrative, not exhaustive, and not limited to the dis- closed examples. It is apparent to those skilled in the art that many modifications and changes may be made without departing from the scope and spirit of the described embodiments.

SEQUENCE LISTING

<110> The First Affiliated Hospital of Hainan Medical University <120> RECOMBINANT ADENG-ASSOCIATED VIRUS VECTOR, RECOMBINANT

ADENO-ASSOCIATED VIRUS AAVE-PD1 AND USE THEREOF <130> HKJP202110572 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 510 <212> DNA <213> Homo sapiens <400> 1 atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60 ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120 ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctec caacacatcg 180 gagagctteg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240 gccttecccg aggaccgcag ccagcccggc caggactgcc gcttecgtgt cacacaactg 300 cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360 tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420 gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480 aggccagccg gccagttcca aaccctggtg 510

<210> 2 <211> 170 <212> PRT <213> Homo sapiens <400> 2

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 g0 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val 165 170 <210> 3 <211> 681 <212> DNA <213> adeno-associated virus 8 <400> 3 attaccatgg tgatgeggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 60 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 120 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 180 cgtgtacggt gggaggtcta tataagcaga gctegtttag tgaaccgtca gatcgcctgg 240 agacgccatc cacgctgttt tgacctccat agaagacacc gggatcegcc accggtacct 300 taattaacga ctacaaggat gacgatgaca aggattacaa agacgacgat gataaggact 360 ataaggatga tgacgacaaa ttaattaacg agggcagagg aagtcttcta acatgcggtg 420 acgtggagga gaatcccggc cctaccggtg ccaccatgge ccagtccaag cacggcctga 480 ccaaagagat gaccatgaag taccgcatgg agggctgegt ggacggccac aagttcgtga 540 tcaccggcga gggcatceggc taccccttca agggcaagca cgccatcaac ctgtgcgtgg 600 tggagggegg ccccettgcce ttegcegagg acatcttgte cgecgectte atgtacggca 060 accgcgtgtt caccgagtac c 681

<210> 4 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PCR amplication of PDl extracellular domain <400> 4 ttttgacctc catagaagac accgggatcc gccaccatgc agatccca 48 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PCR amplication of PDl extracellular domain <400> 5 tcatcettgt agtcgttaat taaggtaccc accagggttt ggaactgg 48

SEQUENCE LISTING

<110> The First Affiliated Hospital of Hainan Medical University <120> RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR, RECOMBINANT

ADENO-ASSOCIATED VIRUS AAV8-PD1 AND USE THEREOF <130> HKJIP202110572 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 510 <212> DNA <213> Homo sapiens <400> 1 atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60 ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120 ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180 gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240 gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300 cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360 tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420 gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480 aggccagccg gccagttcca aaccctggtg 510 <210> 2 <211> 170 <212> PRT <213> Homo sapiens <400> 2

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val 165 170 <210> 3 <211> 681 <212> DNA <213> adeno-associated virus 8 <400> 3 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 60 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 120 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 180 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg 240 agacgccatc cacgctgttt tgacctccat agaagacacc gggatccgcc accggtacct 300 taattaacga ctacaaggat gacgatgaca aggattacaa agacgacgat gataaggact 360 ataaggatga tgacgacaaa ttaattaacg agggcagagg aagtcttcta acatgcggtg 420 acgtggagga gaatcccggc cctaccggtg ccaccatggc ccagtccaag cacggcctga 480 ccaaagagat gaccatgaag taccgcatgg agggctgcgt ggacggccac aagttcgtga 540 tcaccggcga gggcatcggc taccccttca agggcaagca cgccatcaac ctgtgcgtgg 600 tggagggcgg ccccttgccc ttcgccgagg acatcttgtc cgccgccttc atgtacggca 660 accgcgtgtt caccgagtac c 681 <210> 4

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward primer for PCR amplication of PD1 extracellular domain <400> 4 ttttgacctc catagaagac accgggatcc gccaccatgc agatccca 48 <210> 5

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Reverse primer for PCR amplication of PD1 extracellular domain <400> 5 tcatccttgt agtcgttaat taaggtaccc accagggttt ggaactgg 48

Claims

CONCLUSIONS

1. Recombinant adeno-associated virus vector formed by inserting a nucleotide sequence of an extracellular PD-1 segment into an adeno-associated virus vector, wherein the nucleotide sequence of an extracellular PD-1 segment is as shown in SEQ ID NO: 1 .

The recombinant adeno-associated virus vector of claim 1, wherein a PD-1 cell is of human origin.

The recombinant adeno-associated virus vector according to claim 1 or 2, wherein the recombinant adeno-associated virus vector belongs to serotype 8.

A recombinant adeno-associated virus AAV8-PD1, wherein the recombinant adeno-associated virus contains the recombinant adeno-associated virus vector according to any one of claims 1 to 3.

The recombinant adeno-associated virus according to claim 4, wherein the recombinant adeno-associated virus belongs to serotype 8.

Use of the recombinant adeno-associated virus vector according to any one of claims 1 to 3 in the preparation of a preparation for blocking a PD-1/PD-L1 signaling pathway or promoting the growth of a CD8-positive T-cell cell.

Use of the recombinant adeno-associated virus vector according to any one of claims 1 to 3 in the preparation of a composition for treating a tumor.

Use of the recombinant adeno-associated virus vector according to any one of claims 1 to 3 in the preparation of a preparation for treating liver cancer.

Use of the recombinant adeno-associated virus according to claim 4 or 5 in the preparation of a composition for blocking a PD-1/PD-L1 signaling pathway or promoting the growth of a CD8-positive T cell .

Use of the recombinant adeno-associated virus according to claim 4 or 5 in the preparation of a composition for the treatment of liver cancer.