Replication-type oncolytic adenovirus for inhibiting tumor progression and prolonging lifetime of tumor-bearing individual and application of replication-type oncolytic adenovirus

Abstract

The invention discloses a recombinant adenovirus or an adenovirus vector for expressing a glucagon-like peptide-1 (GLP-1) receptor agonist, the recombinant adenovirus or the adenovirus vector shows an expected anti-tumor effect in a tumor-bearing subject, the lifetime of the tumor-bearing subject is prolonged, and a pharmaceutical composition containing the recombinant adenovirus or the adenovirus vector is also disclosed.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment using recombinant adenovirus or adenovirus vector. Background Art

[0002] Cancer has become the leading disease that threatens human life and health. In my country, there are about 4 million new cancer patients and nearly 3 million cancer deaths each year. Surgery is the most effective treatment for early-stage cancer, but it has limited effect on most patients with advanced and metastatic cancer. Although adjuvant therapy such as radiotherapy and chemotherapy can inhibit tumor progression to a certain extent, the overall survival rate of cancer patients has not been significantly improved. Finding new safe and effective therapeutic agents is a hot topic in global pharmaceutical research.

[0003] Tumor immunotherapy has attracted much attention in recent years due to its significant efficacy. This therapy stimulates the body's immune system to kill and monitor cancer cells by activating and / or regulating immune-related pathways. Oncolytic viruses are an emerging direction in the field of tumor immunotherapy. With the FDA's approval of the oncolytic virus T-Vec at the end of 2015, oncolytic virus-mediated anti-tumor immunotherapy has received increasing attention. Oncolytic viruses are a type of virus that can replicate and proliferate in large quantities in tumor cells. They have multiple unique anti-tumor effects: they can induce immunogenic death of tumor cells; activate the body's immune surveillance; and express recombinant proteins to regulate the tumor microenvironment. At present, clinical trials related to oncolytic viruses such as adenovirus, herpes simplex virus, measles virus, poxvirus, and vesicular stomatitis virus have been carried out, and good therapeutic effects have been achieved.

[0004] However, solid tumors escape the body's immune surveillance and promote invasion and metastasis through multiple mechanisms such as metabolic abnormalities and pro-inflammatory microenvironment. Studies have found that oncolytic viruses can upregulate tumor-promoting inflammation in the tumor microenvironment and change the metabolic microenvironment while activating immunity, thereby adversely affecting its anti-tumor effect. The present invention discloses a novel oncolytic virus construct and its use in anti-tumor therapy. Summary of the invention

[0005] On one hand, the present invention relates to a recombinant adenovirus or an adenoviral vector, which comprises: a first promoter, an E1A early activation replication element, an optional second promoter, and a target nucleotide (such as DNA) sequence encoding a target protein, wherein the target protein comprises a glucagon-like peptide-1 (GLP-1) receptor agonist; wherein the first promoter is a constitutive promoter, and the optional second promoter is a constitutive promoter that is the same as or different from the first promoter; when the target protein is GLP-1 (7-36), the Ad5 adenovirus or adenoviral vector needs to contain two promoters.

[0006] Another aspect relates to a pharmaceutical composition comprising the above-mentioned recombinant adenovirus or adenovirus vector and a pharmaceutically acceptable excipient.

[0007] Another aspect relates to the use of the recombinant adenovirus or adenoviral vector or a pharmaceutical composition thereof for anti-tumor treatment and / or prevention in a subject, including inducing CD8+T cell-mediated anti-tumor effects.

[0008] Another aspect relates to a method for preparing the recombinant adenovirus or adenovirus vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1A shows an example of a single promoter shuttle vector used in the preparation of a recombinant adenovirus or adenoviral vector expressing GLP-1 according to the present invention.

[0010] FIG1B shows an example of a single promoter shuttle vector DNA construct as described in Example 1, which is used to prepare a recombinant adenovirus or adenoviral vector expressing a GLP-1 analog (such as AdV-GLP-1 (7-37 / A8G)-CMV); wherein: IL-2SS: a DNA sequence encoding an IL-2 signal peptide, the amino acid sequence of which is MDWTWRILFLVAAATGAHS; GLP-1 analogs: DNA sequences encoding GLP-1 (7-37 / A8G), as shown in Table 1.

[0011] FIG. 1C shows another example of a single promoter shuttle vector used in the preparation of a recombinant adenovirus or adenoviral vector expressing GLP-1 according to the present invention.

[0012] FIG. 2A shows an example of constructing a dual-promoter shuttle plasmid.

[0013] FIG2B shows an example of the DNA sequence of the dual-promoter shuttle vector AdV-GLP-1(7-36)-CMV / EF1a, which contains CMV and EF1a promoters, and displays an example of the DNA sequence encoding GLP-1(7-36).

[0014] FIG2C shows an example of the DNA sequence of the dual-promoter shuttle vector AdV-GLP-1(7-36)-PLGF2-CMV / EF1a, which contains the CMV and EF1a promoters.

[0015] FIG2D shows an example of the DNA sequence of the dual-promoter shuttle vector AdV-PLGF2-GLP1(7-36)-CMV / EF1a, which contains the CMV and EF1a promoters.

[0016] FIG. 2E shows another construction example of a dual-promoter shuttle plasmid.

[0017] FIG3A shows the expression and secretion function of the recombinant adenovirus expressing GLP1(7-36) described in Example II evaluated by ELISA.

[0018] FIG. 3B shows that the viral replication ability of the recombinant adenovirus expressing GLP1 (Ad5_G01) in the example in pancreatic cancer cells is significantly higher than that of the control adenovirus not expressing GLP1 (Ad5_con).

[0019] FIG3C shows that the viral replication ability of the recombinant adenovirus expressing GLP1 (Ad5_G01) in the example is significantly higher than that of the control adenovirus not expressing GLP1 (Ad5_con) in colorectal cancer cells.

[0020] FIG. 4 shows the biologically active form of GLP1 produced by the recombinant adenovirus expressing the GLP1 analog (ADV GLP1(7-37 / A8G)) in Example II.

[0021] FIG. 5 shows the biologically active form of GLP1 produced by the recombinant adenovirus expressing GLP1(7-36) in Example II.

[0022] FIG. 6 shows the experimental design for evaluating the recombinant adenovirus expressing GLP1(7-36) in a pancreatic cancer mouse model in Example III(A).

[0023] FIG. 7A shows the tumor growth data of the PBS negative control group in the pancreatic cancer mouse model shown in FIG. 6 (see Example III(A)).

[0024] FIG. 7B shows the tumor growth data of the group treated with a control adenovirus (AdV-Ctrl) that does not express the target peptide in the pancreatic cancer mouse model (as shown in FIG. 6 , see Example III(A)).

[0025] FIG. 7C shows the tumor growth data of the group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) in the pancreatic cancer mouse model (as shown in FIG. 6 , see Example III(A)).

[0026] Figure 8 shows that in the pancreatic cancer mouse model (as shown in Figure 6, see Example III (A)), the survival rate of the group treated with recombinant adenovirus expressing GLP1 (7-36) (AdV-GLP1 (7-36)) was significantly improved (p < 0.05) compared with the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) and the negative control group treated only with phosphate buffered saline (PBS).

[0027] Figure 9 shows that in the pancreatic cancer mouse model (as shown in Figure 6, see Example III (A)), there was no significant difference in body weight between the recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)), the control adenovirus not expressing the target peptide (AdV-Ctrl), and the negative control group treated only with phosphate buffered saline (PBS).

[0028] FIG. 10 shows the experimental design for evaluating recombinant adenovirus expressing GLP1(7-36) (see Example III(A)) in an orthotopic pancreatic cancer mouse model.

[0029] Figure 11A shows the total luciferin counts between the recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)), the control adenovirus not expressing the target peptide (AdV-Ctrl), and the negative control group treated only with phosphate buffered saline (PBS) in the orthotopic Panc02-car mouse model (as shown in Figure 10, see Example III(A)).

[0030] Figure 11B shows the tumor volume measured by luminescence in the orthotopic Panc02-car mouse model (as shown in Figure 10, see Example III (A)), comparing the use of recombinant adenovirus expressing GLP1 (7-36) (AdV-GLP1 (7-36)), a control adenovirus not expressing the target peptide (AdV-Ctrl), and a negative control group treated only with phosphate buffered saline (PBS).

[0031] Figure 12 shows that in the in situ Panc02-car mouse model (as shown in Figure 10, see Example III (A)), the survival rate of the group treated with recombinant adenovirus expressing GLP1 (7-36) (AdV-GLP1 (7-36)) was significantly improved (p<0.05) compared with the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) and the negative control group treated only with phosphate buffered saline (PBS).

[0032] FIG. 13 shows the experimental design for evaluating recombinant adenovirus expressing GLP1(7-36) (see Example III(B)) in a mouse model of colorectal cancer.

[0033] FIG. 14A shows the tumor growth data of the negative control group treated with phosphate buffered saline (PBS) only in the colorectal cancer mouse model (as shown in FIG. 13 , see Example III(B)).

[0034] FIG. 14B shows the tumor growth data of the group treated with a control adenovirus (AdV-Ctrl) that does not express the target peptide in the colorectal cancer mouse model (as shown in FIG. 13 , see Example III(B)).

[0035] FIG. 14C shows the tumor growth data of the group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) in the colorectal cancer mouse model (as shown in FIG. 13 , see Example III(B)).

[0036] Figure 15 shows that in the colorectal cancer mouse model (as shown in Figure 13, see Example III (B)), the survival rate of the group treated with recombinant adenovirus expressing GLP1 (7-36) (AdV-GLP1 (7-36)) was significantly improved compared with the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) (p<0.01) and the negative control group treated only with phosphate buffered saline (PBS) (p<0.05).

[0037] Figure 16 shows that in the colorectal cancer mouse model (as shown in Figure 13, see Example III (B)), there was no significant difference in body weight between the recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)), the control adenovirus not expressing the target peptide (AdV-Ctrl), and the negative control group treated only with phosphate buffered saline (PBS).

[0038] Figure 17 shows the experimental design for evaluating recombinant adenovirus expressing GLP1(7-36) (see Example III(C)) in a melanoma mouse model.

[0039] Figure 18 shows that in the melanoma mouse model (as shown in Figure 17, see Example III (C)), the survival rate of the group treated with recombinant adenovirus expressing GLP1 (7-36) (AdV-GLP1 (7-36)) was significantly improved compared with the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) (P = 0.0014) and the negative control group treated only with phosphate buffered saline (PBS) (P = 0.0015).

[0040] FIG. 19 shows the experimental design for evaluating recombinant adenovirus expressing GLP1(7-36) (see Example III(D)) in a glioma mouse model.

[0041] Figure 20 shows that in the glioma mouse model (as shown in Figure 19), the tumor growth rate of the group treated with recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) was significantly slowed compared with the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) (p=0.0001) and the negative control group treated only with phosphate buffered saline (PBS) (p=0.0001).

[0042] Figure 21 shows that in the glioma mouse model (as shown in Figure 19, see Example III (D)), the survival rate of the group treated with recombinant adenovirus expressing GLP1 (7-36) (AdV-GLP1 (7-36)) was significantly improved compared with the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) (p = 0.0027) and the negative control group treated only with phosphate buffered saline (PBS) (p = 0.0082).

[0043] FIG. 22 shows the experimental design for evaluating recombinant adenovirus expressing GLP1(7-36) (eg, I(E)) in a mouse model of renal cancer.

[0044] FIG. 23A shows the tumor growth data of the negative control group treated with phosphate buffered saline (PBS) only in the renal cancer mouse model (as shown in FIG. 22 , see Example III(E)).

[0045] FIG. 23B shows the tumor growth data of the group treated with a control adenovirus (AdV-Ctrl) that does not express the target peptide in the renal cancer mouse model (as shown in FIG. 22 , see Example III(C)).

[0046] FIG. 23C shows the tumor growth data of the group treated with a recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) in a renal cancer mouse model (as shown in FIG. 22 , see Example III(C)).

[0047] Figure 23D shows that in the renal cancer mouse model (as shown in Figure 22), the tumor growth rate of the group treated with recombinant adenovirus expressing GLP1(7-36) (AdV-GLP1(7-36)) was significantly slowed compared with the group treated with a control adenovirus not expressing the target peptide (AdV-Ctrl) (p<0.0001) and the negative control group treated only with phosphate buffered saline (PBS) (p<0.05).

[0048] Figure 24 shows that in a pancreatic cancer mouse model (see Example IV (A)), the group treated with AdV-GLP-1 (7-36) after treatment with anti-CD8 antibody (GLP1 (7-36) + anti-CD8) showed a slower tumor growth rate compared to the negative control group treated with phosphate buffered saline (PBS) only, the group treated with control adenovirus (AdV-Ctrl), and the group treated with anti-CD4 antibody (GLP1 (7-36) + anti-CD4) or anti-NK antibody (GLP1 (7-36) + anti-NK) before treatment with AdV-GLP-1 (7-36)-CMV / EFla.

[0049] Figure 25 shows that in the pancreatic cancer mouse model (see Example IV (A)), the group treated with anti-CD8 antibody (GLP1 (7-36) + anti-CD8) before AdV-GLP-1 (7-36)-CMV / EFla treatment did not show statistical significance in survival rate compared with the group treated with control adenovirus only (AdV-Ctrl); however, its survival rate was significantly reduced compared with the group treated with AdV-GLP-1 (7-36)-CMV / EFla only (AdV-GLP1 (7-36)). The survival rates of the groups treated with AdV-GLP-1(7-36)-CMV / EFla alone (AdV-GLP1(7-36)) and the groups treated with anti-CD4 antibody (GLP1(7-36) + anti-CD4) or anti-NK antibody (GLP1(7-36) + anti-NK) before AdV-GLP-1(7-36)-CMV / EFla treatment were significantly longer than those of the group treated with control adenovirus (AdV-Ctrl) alone (*: p<0.05). There was no significant difference in survival rate between the groups treated with anti-CD4 antibody (GLP1(7-36) + anti-CD4) or anti-NK antibody (GLP1(7-36) + anti-NK) before AdV-GLP-1(7-36)-CMV / EFla treatment and the group treated with AdV-GLP-1(7-36)-CMV / EFla alone (AdV-GLP1(7-36)).

[0050] FIG26A shows the expression of interferon-γ positive CD8 positive T cells (IFN-γ) in the pancreatic cancer mouse model (see Example IV (B)) in the AdV-GLP1 (7-36) group, the AdV-Ctrl group, and the PBS group. + CD8 + The proportion of T cells.

[0051] FIG26B shows the expression of granzyme B-positive CD8-positive T cells (GZMB) in the AdV-GLP1(7-36) group, the AdV-Ctrl group, and the PBS group in the pancreatic cancer mouse model (see Example IV(B)). + CD8 + The proportion of T cells.

[0052] FIG26C shows the expression of PD1-positive CD8-positive T cells (PD1 + CD8 + The proportion of T cells.

[0053] FIG27A shows the expression of CD8 positive T cells (CD8 + The expression level of LCN2 in T cells.

[0054] FIG27B shows the expression of CD4 positive T cells (CD4 + The expression level of LCN2 in T cells.

[0055] FIG. 28A shows the expression level of LCN2 when cultured CD8-positive T cells were co-cultured with Panc02 cells in the presence or absence of GLP-1(7-36) in a pancreatic cancer mouse model (see Example IV(C)).

[0056] FIG. 28B shows the expression level of LCN2 when cultured CD8-positive T cells were co-cultured with Panc02 cells in the presence or absence of GLP-1(7-36) and liraglutide in a pancreatic cancer mouse model (see Example IV(C)).

[0057] Figure 29 shows that AdV-GLP-1(7-36)-CMV / EFla (CD8-Panc02-AdV-GLP1(7-36)) significantly inhibited CD8-positive T cell-mediated neutrophil recruitment, showing significant differences in in vitro experiments compared with the control adenovirus (AdV-Ctrl) treatment group (CD8-Panc02-AdV-Ctrl) (see Example IV (C)).

[0058] FIG. 30 shows that the recombinant adenovirus expressing GLP1 (7-37 / A8G) has an oncolytic effect on non-small cell lung cancer cells.

[0059] Figure 31 shows the experimental design for evaluating recombinant adenovirus expressing GLP1 (7-37 / A8G) in a pancreatic cancer mouse model (see Example V(B)).

[0060] Figure 32 shows that in the pancreatic cancer mouse model (as shown in Figure 31), the tumor growth rate of the group treated with recombinant adenovirus expressing GLP1 (7-36 / A8G) (AdV-GLP-1 (7-36 / A8G)) was significantly slowed compared with the group treated with the control adenovirus (AdV-Ctrl) that did not express the target peptide (P<0.0001) and the negative control group treated only with phosphate buffered saline (PBS).

[0061] Figure 33 shows that in the pancreatic cancer mouse model (as shown in Figure 31, see Example V (B)), the survival rate of the group treated with recombinant adenovirus expressing GLP1 (7-36 / A8G) (AdV-GLP1 (7-36 / A8G)) was significantly improved (p<0.05) compared with the group treated with the control adenovirus (AdV-Ctrl) that did not express the target peptide.

[0062] Figure 34 shows that in the pancreatic cancer mouse model (as shown in Figure 31, see Example V (B)), there was no significant difference in body weight between the recombinant adenovirus expressing GLP1 (7-36 / A8G) (AdV-GLP1 (7-36 / A8G)), the control adenovirus not expressing the target peptide (AdV-Ctrl), and the negative control group treated only with phosphate buffered saline (PBS).

[0063] Figure 35 shows the experimental design for evaluating recombinant adenovirus expressing GLP1(7-36)-PLGF2 or PLGF2-GLP1(7-36) in a pancreatic cancer mouse model (see Example VI).

[0064] FIG. 36A shows tumor growth data of a negative control group treated with phosphate buffered saline (PBS) alone in a pancreatic cancer mouse model (as shown in FIG. 35 , see Example VI).

[0065] FIG. 36B shows the tumor growth data of the group treated with a recombinant adenovirus expressing GLP1(7-36)-PLGF2 (AdV-GLP1) in a pancreatic cancer mouse model (as shown in FIG. 35 , see Example VI).

[0066] FIG. 36C shows the tumor growth data of the group treated with a recombinant adenovirus expressing PLGF2-GLP1(7-36) (AdV-GLP1) in a pancreatic cancer mouse model (as shown in FIG. 35 , see Example VI). Detailed Description

[0067] Glucagon-like peptide-1 (GLP-1) is a polypeptide composed of 30 amino acid residues and is an agonist of the glucagon-like peptide-1 receptor. It can not only lower blood sugar, but also significantly inhibit the activation of multiple tumor-promoting inflammatory pathways, including the activation of the NF-κB / IL-6 / STAT3 pathway and the formation of the NLRP3 / IL-1β inflammasome.

[0068] The present invention provides a recombinant adenovirus (e.g., adenovirus C subtype Ad5) or an adenovirus vector, which comprises a first promoter, an E1A early activation replication element, an optional second promoter, and a nucleotide (e.g., DNA) sequence encoding a target protein. The target protein comprises a glucagon-like peptide-1 (GLP-1) receptor agonist. The first promoter is a constitutive promoter, and the optional second promoter is also a constitutive promoter, which may be the same as or different from the first promoter. The present invention also provides a method for preparing the above-mentioned recombinant adenovirus or adenovirus vector and its use.

[0069] As shown in the Examples, the present invention prepares a variety of Ad5 adenoviruses or adenovirus vectors by encoding a target protein containing a GLP-1 receptor agonist through one or two nucleotide (e.g., DNA) sequences upstream of the promoter. These viruses can express the target protein both in vivo and in vitro, and show in vivo and in vitro tumor growth inhibition effects on a variety of tumors (e.g., non-small cell lung cancer, pancreatic cancer, colon cancer, melanoma, glioma, and renal cancer), and even in some cases, the effect of in vivo tumor regression was observed, and no significant side effects were observed.

[0070] In addition, in at least one example of an Ad5 adenovirus or adenoviral vector, which comprises a nucleotide (e.g., DNA) sequence encoding GLP-1 (7-37), it induces CD8+ T cell-mediated anti-tumor effects in pancreatic cancer, rather than natural killer (NK) cell-mediated anti-tumor effects induced by liraglutide (a GLP-1 analog) in hepatocellular carcinoma by intratumoral injection (reference: Xian Lu, Chun Xu, Jie Dong, Shuguang Zuo, Hailin Zhang, Chunping Jiang, Junhua Wu, Jiwu Wei, Liraglutide activates natural killer cell-mediated antitumor responses by inhibiting IL-6 / STAT3 signaling in hepatocellular carcinoma, Translational Oncology, Volume 14, Issue 1, 2021, 100872). I. Recombinant adenovirus or adenovirus vector and its pharmaceutical composition

[0071] One aspect of the present invention relates to a recombinant adenovirus or adenoviral vector, comprising a first promoter, an E1A early activating replication element, an optional second promoter, and a nucleotide (e.g., DNA) sequence encoding a target protein. The target protein comprises a glucagon-like peptide-1 (GLP-1) receptor agonist. The first promoter is a constitutive promoter, and the optional second promoter is also a constitutive promoter, which may be the same as or different from the first promoter. In certain embodiments, the nucleotide (e.g., DNA) sequence further comprises a DNA sequence encoding an interleukin-2 (IL-2) signal peptide to promote extracellular secretion of the target peptide. In certain embodiments, when the target protein is GLP-1 (7-36), the recombinant adenovirus or adenoviral vector comprises two promoters. In certain embodiments, the E1A early activating replication element may be located upstream or downstream of the nucleotide (e.g., DNA) sequence, as long as the E1A early activating replication element and the nucleotide (e.g., DNA) sequence are both located between the first promoter and the poly (A) tail. For example, see Figures 1A, 1C, 2A and 2E, in which the first promoter is the CMV promoter as an example.

[0072] Examples of the above-mentioned recombinant adenovirus include but are not limited to adenovirus subtype C Ad5 and the like.

[0073] Certain recombinant adenoviruses (e.g., Ad5) or adenoviral vectors disclosed herein can be used to treat or prevent one or more tumors in a subject. In certain embodiments, the recombinant adenovirus (e.g., Ad5) or adenoviral vector can induce CD8+T cell-mediated anti-tumor effects in a subject. In certain embodiments, the recombinant adenovirus (e.g., Ad5) or adenoviral vector can cause tumor regression in a subject.

[0074] Certain recombinant adenoviruses (e.g., Ad5) or adenoviral vectors disclosed herein comprise one or more constitutive promoters selected from CMV promoters and EF1a promoters. Multiple constitutive promoters may be the same or different. For example, a recombinant adenovirus (e.g., Ad5) or adenoviral vector may use the CMV promoter as both the first and second promoters; a recombinant adenovirus (e.g., Ad5) or adenoviral vector may use the EF1a promoter as both the first and second promoters; a recombinant adenovirus (e.g., Ad5) or adenoviral vector may use the CMV promoter as the first promoter and the EF1a promoter as the second promoter; or conversely, a recombinant adenovirus (e.g., Ad5) or adenoviral vector may use the EF1a promoter as the first promoter and the CMV promoter as the second promoter.

[0075] Certain recombinant adenoviruses (eg, Ad5) or adenovirus vectors disclosed in the present invention comprise two constitutive promoters and a DNA sequence encoding GLP-1 (7-36), wherein the amino acid sequence of GLP-1 (7-36) is SEQ ID NO: 1.

[0076] Examples of glucagon-like peptide-1 receptor agonists include, but are not limited to, amino acid sequences having 80% or more homology with GLP-1, active fragments of GLP-1 (e.g., GLP-1(7-36) (SEQ ID NO: 1), GLP-1(7-37)), or GLP-1 analogs (e.g., GLP-1(7-37 / A8G) (SEQ ID NO: 2) having one or more mutations). Certain recombinant adenoviruses (e.g., Ad5) or adenovirus vectors disclosed herein contain secretory glucagon-like peptide-1 protein as a GLP-1 receptor agonist. Examples of GLP-1 analogs include, but are not limited to, GLP-1 fragments having one or more substituted amino acids, deleted amino acids, and / or added amino acids, and these GLP-1 analogs are all GLP-1 receptor agonists.

[0077] In certain embodiments, the target protein comprises a fusion protein of a tumor homing protein (e.g., PLGF2) and a GLP-1 receptor agonist. In certain embodiments, the fusion protein further comprises a connecting peptide (e.g., a 4GS linker, such as (GGGGS)n, n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) connecting the tumor homing protein (e.g., PLGF2) and the GLP-1 receptor agonist. In certain embodiments, the GLP-1 receptor agonist is close to the N-terminus in the fusion protein, while the tumor homing protein (e.g., PLGF2) is close to the C-terminus. In certain embodiments, the tumor homing protein (e.g., PLGF2) is close to the N-terminus in the fusion protein, while the GLP-1 receptor agonist is close to the C-terminus.

[0078] Examples of fusion proteins include, but are not limited to, GLP-1(7-36)-4GS-PLGF2 (e.g., SEQ ID NO: 3, wherein n=3 is used for the 4GS linker, Table 1), PLGF2-4GS-GLP-1(7-36) (e.g., SEQ ID NO: 4, wherein n=3 is used for the 4GS linker, Table 1), GLP-1(7-37)-4GS-PLGF2, PLGF2-4GS-GLP-1(7-37, GLP-1(7-37 / A8G)-4GS-PLGF2, and PLGF2-4GS-GLP-1(7-37 / A8G).

[0079] Certain recombinant adenoviruses (eg, Ad5) or adenovirus vectors disclosed in the present invention comprise one or two constitutive promoters and a DNA sequence encoding a target protein, the details of which are summarized in Table 1 ( FIGS. 1A-2D ). Table 1. Examples of target proteins and their encoding DNA sequences

[0080] Another aspect of the present invention relates to a pharmaceutical composition comprising the recombinant adenovirus (eg, Ad5) or adenovirus vector disclosed in the present invention, and one or more pharmaceutically acceptable carriers, adjuvants, excipients or a combination thereof.

[0081] Examples of pharmaceutically acceptable carriers include physiologically acceptable solutions, such as sterile saline and sterile buffered saline.

[0082] The pharmaceutically acceptable carrier may contain trace amounts of excipients, such as substances that enhance isotonicity and chemical stability. These excipients should be non-toxic at the dosages and concentrations used for human or other mammalian subjects. Examples of such excipients include buffers (such as phosphoric acid, citric acid, succinic acid, acetic acid and other organic acids and their salts), antioxidants (such as ascorbic acid), low molecular weight (e.g., less than about 10 residues) polypeptides (e.g., polyarginine and tripeptides), proteins (e.g., serum albumin, gelatin and immunoglobulins), amino acids (e.g., glycine, glutamic acid, aspartic acid and arginine), monosaccharides, disaccharides and other carbohydrates (e.g., cellulose and its derivatives, glucose, mannose and dextrin), chelating agents (e.g., EDTA), sugar alcohols (e.g., mannitol and sorbitol), counterions (e.g., sodium), nonionic surfactants (e.g., polysorbates and poloxamers), antibiotics and polyethylene glycol (PEG).

[0083] The recombinant adenovirus or adenoviral vector disclosed in the present invention, or the pharmaceutical composition comprising the recombinant adenovirus or adenoviral vector, can be stored as an aqueous solution or a lyophilized product in a single-dose or multi-dose container, such as a sealed ampoule or vial. II. Use of recombinant adenovirus (eg, Ad5) or adenovirus vector and pharmaceutical composition thereof

[0084] Another aspect of the present invention relates to the use of the recombinant adenovirus (e.g., Ad5) or adenoviral vector and pharmaceutical compositions thereof disclosed in the present invention for anti-tumor treatment and / or prevention in a subject. In certain embodiments, the anti-tumor treatment and / or prevention comprises inducing CD8+T cell-mediated anti-tumor effects in the subject.

[0085] In certain embodiments, the method of inducing CD8+ T cell-mediated anti-tumor effects in a subject comprises administering to the subject an effective therapeutic amount of a recombinant adenovirus (eg, Ad5) or an adenoviral vector, or a pharmaceutical composition comprising the recombinant adenovirus or adenoviral vector.

[0086] In certain embodiments, a method of treating one or more cancers in a subject comprises administering to the subject a therapeutically effective amount of a recombinant adenovirus (eg, Ad5) or an adenoviral vector, or a pharmaceutical composition comprising the recombinant adenovirus or adenoviral vector.

[0087] Examples of the one or more cancers include, but are not limited to, liver cancer, pancreatic cancer, colon cancer, glioma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, head and neck squamous cell carcinoma, melanoma, multiple myeloma, and lymphoma. In certain embodiments, the lung cancer is non-small cell lung cancer.

[0088] In certain embodiments, the cancer is a metastatic cancer.

[0089] Certain anti-tumor methods disclosed herein further comprise inhibiting tumor invasion and / or metastasis.

[0090] Certain anti-tumor methods disclosed in the present invention further include one or more effects selected from the group consisting of: reducing the expression level of LCN2 in CD8+T cells, and reducing the infiltration of neutrophils in tumors.

[0091] Certain anti-tumor methods disclosed herein further comprise delaying the progression of cachexia in the subject.

[0092] Certain anti-tumor methods disclosed herein further comprise extending the life span of the subject.

[0093] Herein, "effective amount" refers to the amount of a compound that can produce the desired effect. For example, the in vitro (e.g., cell culture) effect can be studied by contacting a group of cells with the compound, or the expected therapeutic effect can be produced by in vitro or in vivo experiments. The effective amount of the compound can also be used to produce a therapeutic effect in a subject, such as preventing or treating a target disease (e.g., one or more tumors), alleviating symptoms associated with the disease, or producing an expected physiological effect. In this case, the effective amount of the compound is referred to as a "therapeutically effective amount," "therapeutically effective concentration," or "therapeutically effective dose." The exact effective amount or therapeutically effective amount is the amount of the compound that can achieve the best therapeutic effect in a given subject or cell population. The amount varies depending on a variety of factors, including but not limited to the properties of the compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, overall health, response to a specific dose, and the type of drug used), and the nature of the pharmaceutically acceptable carrier or carrier used in the formulation, as well as the route of administration. In addition, the effective amount or therapeutically effective amount may also vary depending on whether the compound is administered alone or in combination with other compounds, drugs, therapies, or other treatment modalities. Those skilled in the art of clinical and pharmacology can determine the effective amount or therapeutically effective amount by routine experimentation, i.e., by monitoring the response of cells or subjects to the compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st edition, University of the Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, 2005, which is hereby incorporated by reference in its entirety.

[0094] "Treating" or "treating" a disease may mean preventing the disease, slowing the onset or development of the disease, reducing the risk of developing the disease, preventing or delaying the development of symptoms associated with the disease, alleviating or eliminating symptoms associated with the disease, achieving complete or partial regression of the disease, or some combination of the above. Treatment may also refer to prophylactic or preventative treatment of a disease.

[0095] In certain embodiments, the recombinant adenovirus (e.g., Ad5) or adenoviral vector and its pharmaceutical composition disclosed in the present invention can be used in combination with other known pharmaceutical products, such as peptides that promote immune response and antibacterial agents (synthetic antibacterial agents). The recombinant adenovirus (e.g., Ad5) or adenoviral vector and its pharmaceutical composition disclosed in the present invention may also contain other drugs and excipients. Examples of drugs or excipients that can be used in combination with the recombinant adenovirus (e.g., Ad5) or adenoviral vector and its pharmaceutical composition disclosed in the present invention include drugs that help the recombinant adenovirus (e.g., Ad5) or adenoviral vector and its pharmaceutical composition enter cells, liposomes and other drugs and / or excipients that help transfection (e.g., fluorocarbon emulsifiers, cochleates, nanotubes, gold particles, biodegradable microspheres and cationic polymers).

[0096] In certain embodiments, the amount of active ingredient contained in the recombinant adenovirus (e.g., Ad5) or adenovirus vector and its pharmaceutical composition can be selected from a wide range of concentrations, such as expressed in infectious units (ifu), weight volume percentage (w / v%) or other quantitative indicators of the amount of active ingredient, as long as it is a therapeutically or pharmaceutically effective amount. The dosage range of the recombinant adenovirus (e.g., Ad5) or adenovirus vector and its pharmaceutical composition disclosed in the present invention can be appropriately selected according to factors such as the expected therapeutic effect, administration method (administration route), treatment cycle, patient age, gender and other conditions.

[0097] In certain aspects, when a recombinant adenovirus (e.g., Ad5) or an adenoviral vector is administered to a human subject as an active ingredient, the dose of the recombinant adenovirus (e.g., Ad5) or adenoviral vector may be approximately corresponding to about 5×10 5 About 5×10 10 Infectious units (ifu), about 5×10 6 About 5×10 9 ifu, about 1×10 7 About 1×10 9 ifu, about 5×10 7 About 5×10 8 ifu, about 1×10 8 About 1×10 10 ifu, about 5×10 8 About 5×10 10 ifu, about 5×10 7 to about 5×1011 ifu, about 1×10 8 About 1×10 10 ifu, or about 1×10 8 About 1×10 11 An ifu.

[0098] In a further aspect, when a recombinant adenovirus (e.g., Ad5) or an adenoviral vector is administered to a subject as an active ingredient, the dosage can be selected within a wide range depending on the amount of expressible DNA introduced into the recombinant adenovirus (e.g., Ad5) or adenoviral vector. The dosage also depends on the strength of the transcription and translation promoter used in any transfer vector.

[0099] In certain embodiments, the recombinant adenovirus (e.g., Ad5) or adenoviral vector and pharmaceutical compositions thereof disclosed herein can be administered by directly injecting a suspension of the recombinant adenovirus (e.g., Ad5) or adenoviral vector into a target site (e.g., one or more cancer sites). The suspension can be prepared by suspending the recombinant adenovirus (e.g., Ad5) or adenoviral vector in phosphate buffered saline (PBS) or normal saline. The recombinant adenovirus (e.g., Ad5) or adenoviral vector and pharmaceutical compositions thereof disclosed herein can be administered more than once. Specifically, after the initial administration, one or more additional recombinant adenoviruses (e.g., Ad5) or adenoviral vectors can be administered.

[0100] In certain anti-tumor methods disclosed in the present invention, the recombinant adenovirus (eg, Ad5) or adenoviral vector and pharmaceutical compositions thereof can be administered as a single drug therapy, adjuvant therapy, or combined therapy.

[0101] In certain anti-tumor methods disclosed in the present invention, one or more therapies selected from the following group are further included: one or more chemotherapies and one or more radiotherapies.

[0102] In certain embodiments, the therapeutically effective amount of the recombinant adenovirus (eg, Ad5) or adenoviral vector and pharmaceutical composition thereof can be administered once a day.

[0103] In certain embodiments, the therapeutically effective amount of the recombinant adenovirus (eg, Ad5) or adenoviral vector and pharmaceutical composition thereof can be administered once every other day.

[0104] In certain embodiments, the therapeutically effective amount of the recombinant adenovirus (e.g., Ad5) or adenoviral vector and its pharmaceutical composition can be administered for at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, at least about twelve months, at least about one year, at least about two years, at least about three years, at least about four years, or at least about five years. In certain embodiments, the therapeutically effective amount of the recombinant adenovirus (e.g., Ad5) or adenoviral vector and its pharmaceutical composition can be administered to one or more cancers in a subject until the one or more cancers are significantly regressed or disappear. III. Preparation of recombinant adenovirus (eg Ad5) or adenovirus vector and pharmaceutical composition thereof

[0105] Another aspect of the present invention relates to the preparation of recombinant adenovirus (e.g., Ad5) or adenoviral vector. One embodiment of preparing recombinant adenovirus (e.g., Ad5) or adenoviral vector includes the following steps: i) constructing a shuttle plasmid; ii) virus rescue; iii) virus amplification, as disclosed in the present invention.

[0106] In one embodiment, the step of constructing a shuttle plasmid includes preparing a shuttle plasmid comprising nucleotides (e.g., DNA) encoding a target protein, wherein the target protein comprises a GLP-1 receptor agonist as disclosed in the present invention. For example, but not limited to, the target protein may be GLP-1, a GLP-1 fragment (e.g., GLP-1(7-36), GLP-1(7-37)), or a GLP-1 analog (e.g., a GLP-1 fragment having one or more substituted amino acids, one or more deleted amino acids, and / or one or more additionally added amino acids). In certain embodiments, the target protein may be a fusion protein of a GLP-1 receptor agonist and another protein (e.g., PLGF2).

[0107] The following examples are intended to illustrate various embodiments of the present invention. Therefore, the specific embodiments discussed should not be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that various equivalent substitutions, changes and modifications may be made without departing from the scope of the present invention. Furthermore, it is understood that these equivalent embodiments should be included in the present invention. In addition, all references cited in the present invention are hereby incorporated herein by reference in their entirety, as if fully set forth herein. IV. Examples

[0108] The following examples illustrate the Ad5 examples summarized below. Table 2. Examples of recombinant Ad5 adenovirus IV. Examples Example I. Preparation of recombinant Ad5 adenovirus or adenoviral vector with a single promoter or dual promoters

[0109] An embodiment of a recombinant Ad5 adenovirus or adenoviral vector having a single promoter is prepared by the following three steps: i) Ad5 shuttle plasmid construction; ii) Ad5 virus rescue; iii) virus amplification, and the specific method is described as follows. I(A). Construction of Ad5 shuttle plasmid

[0110] A DNA sequence encoding the desired element was synthesized in vitro (e.g., a recombinant adenovirus example with CMV as a single promoter: HomologousA-CMV-E1A-P2A-GLP1(7-36)-Homologous B, see Figures 1A-1B; a recombinant adenovirus example with CMV as the first promoter and EFla as the second promoter: HomologousA-CMV-E1A-EFla-GLP1(7-36)-Homologous B, see Figures 2A-2D). A shuttle plasmid (e.g., pDC316AdMax shuttle plasmid) was cut using two restriction endonucleases (e.g., XbaI and HindIII), and the synthesized DNA sequence was inserted into the plasmid in the presence of the In-Fusion enzyme. The shuttle plasmid containing the DNA sequence encoding the target protein, i.e., the target DNA (see the example in Table 1), was confirmed by DNA sequencing. I(B). Ad5 virus rescue

[0111] 293T cells were inoculated into T25 culture flasks, pBHG-lox-(Δ)E1E3-Cre and Ad5 shuttle plasmid containing target DNA were added, and cultured at 37°C and 5% CO2. After 7-10 days, cells were collected into 15mL centrifuge tubes (using 1mL of culture medium to collect by pipetting), frozen and thawed twice, and centrifuged at 3,000rpm / min for 15 minutes. Viral supernatant was collected and stored at -80°C as virus stock. I(C). Ad5 virus amplification

[0112] Add 50 μL of the virus stock solution to a 10 cm dish containing 60% 293T cells and culture at 37°C, 5% CO2. Harvest the cells when they show cytopathic effect. Collect the virus as described above and purify the virus by cesium chloride density gradient centrifugation. Determine the virus titer using the TCID50 method. Example II. Viral Function Evaluation of Recombinant Ad5 Adenovirus Examples II(A). Expression and secretion function of GLP1

[0114] A549 cells were seeded in six-well plates (8×10 5 / well), and cultured overnight in DMEM medium containing 10% FBS. Subsequently, the medium was replaced with DMEM medium containing 2% FBS (1 mL / well), and recombinant Ad5 adenovirus example (Example I, MOI=100, 10, 1, 0.1, 0.01 or 0) containing target DNA (GLP-1 (7-36)) was added, and the supernatant was collected 48 hours and 72 hours after virus infection. The expression and secretion function of GLP1 were detected by ELISA (Figure 3A, the left bar graph of each group of MOI represents the concentration of GLP-1 (7-36) in the sample 48 hours after virus infection; the right bar graph represents the concentration of GLP-1 (7-36) in the sample 72 hours after virus infection).

[0115] Pancreatic cancer cells (Panc02) and colorectal cancer cells (MC38) were infected with recombinant Ad5 virus (containing target DNA, Example I) at the same MOI and a control virus (Ad5 con) containing only the E1A sequence as a viral replication element but not the target DNA sequence. Cells were collected at different time points. The virus suspension was obtained by repeated freezing and thawing and centrifugation. The virus titer was determined using 293T cells, and the changes in viral replication ability were analyzed (Figures 3B-3C). The results showed that the viral replication ability of the recombinant adenovirus expressing GLP1 (Ad5_G01, Figures 3B-3C) in pancreatic cancer and colorectal cancer cells was higher than that of the control adenovirus (Ad5_con, Figures 3B-3C) that did not express GLP1. II(B). After the A11A cell line was infected with recombinant Ad5 adenovirus, the biological activity of the target protein was equivalent to that of liraglutide

[0116] A11A is a cell line that stably expresses a fusion reporter gene and is used to monitor the biological activity of GLP-1. HEK293 cells (4.7×10 5 ~5.2×10 5 / well) are inoculated in a six-well plate and cultured overnight at 37°C and 5% CO2. A recombinant Ad5 adenovirus example containing the target DNA encoding the target protein (e.g., AdV-GLP-1 (7-37 / A8G) and AdV-GLP-1 (7-36)-CMV / EFla prepared according to Example 1) is added to infect the cells at the desired MOI (e.g., MOI = 15). 48-72 hours after viral infection, the cells and the liquid in the wells are collected respectively and stored at -70°C as GLP-1 receptor agonist samples for A11A cell treatment. The negative control sample is prepared in the same way, but the control virus Ad5_con is added instead of the recombinant Ad5 adenovirus AdV-GLP-1 (7-37 / A8G) and AdV-GLP-1 (7-36)-CMV / EFla. A11A cells are inoculated in a 96-well plate (5×10 3cells / well) and cultured overnight at 37°C and 5% CO2. 100 μL of GLP-1 receptor agonist sample, negative control sample or liraglutide (1,000 nM, 100 μL) was added to A11A cells, and the fluorescence intensity was measured after 4 hours of incubation to show the biological activity of the GLP-1 agonist in the GLP-1 receptor agonist sample (Table 3 and Figures 4-5). Table 3. AdV-GLP-1(7-37 / A8G)-CMVA11A data Example III. In vivo antitumor effect of recombinant Ad5 adenovirus AdV-GLP-1(7-36)-CMV / EFla

[0117] AdV-GLP-1(7-36)-CMV / EF1a was prepared according to the method of Example I (B), wherein the target protein sequence was SEQ ID NO: 1 and the target DNA sequence was SEQ ID NO: 5. Viral replication, expression of GLP-1 receptor agonist and its biological activity were verified according to the method of Example II (see Figure 5). Ⅲ(A). In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla on pancreatic cancer animal model Ⅲ(A)(i) Panc02-car subcutaneous animal model

[0118] C57BL / 6 mice aged 6-8 weeks were selected and inoculated with 1×10 Panc02-car cells (suspended in 10% Matrigel in PBS, with each mouse inoculated with 1×10 6 18 mice (see Figure 6) were used to establish a subcutaneous tumor model. When the tumor volume reached about 100 mm 3 The mice were randomly divided into three groups: negative control group: injected with PBS (0.1 mL); control Ad5 group: injected with Ad5 control virus (AdV-Ctrl, 5 × 10 8 ifu / dose); treatment group: injected with Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EF1a, 5×10 8 Ifu / dose). All treatments were intratumorally injected once every other day for a total of three doses (see Figure 6). The tumor volume, body weight changes and survival period of the mice after natural death were recorded.

[0119] Compared with the control Ad5 group (p<0.0001, see Figure 7B) and the negative control group injected with PBS only (p<0.0001, see Figure 7A), the AdV-GLP-1 (7-36) -CMV / EF1a treatment group showed a slower tumor growth rate (see Figure 7C), and statistically significantly prolonged survival (compared with the control Ad5 group and the negative control group, p<0.05, see Figure 8). There was no significant change in the body weight of the three groups of mice (see Figure 9). III(A)(ii) Orthotopic pancreatic cancer model

[0120] Tumors grow in the pancreas of mice. Because orthotopic pancreatic tumors cannot be measured with a vernier caliper like subcutaneous tumors, tumor growth was monitored by luminescence. Significant differences are marked with * (p<0.05) in the figure.

[0121] C57BL / 6 mice aged 6-8 weeks were selected and inoculated with 1×10 Panc02-car cells (suspended in 10% Matrigel in PBS, with each mouse inoculated with 1×10 6 cells, a total of 18 mice (see Figure 10) to establish an orthotopic pancreatic cancer model. When the tumor volume reaches about 100mm 3 The mice were randomly divided into three groups: negative control group: injected with PBS (0.1 mL); control Ad5 group: injected with Ad5 control virus (AdV-Ctrl, 1.5 × 10 9 ifu / dose); treatment group: injected with Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EFla, 1.5×10 9 Ifu / dose). All treatments were performed with a single intratumoral injection (see Figure 10). Tumor volume was measured by luminescence, and the survival time of mice after natural death was recorded.

[0122] Compared with the control Ad5 group (p=0.0032, AdV-Ctrl, see Figures 11A-11B) and the negative control group injected with PBS only (p=0.0002, PBS, see Figures 11A-11B), the AdV-GLP-1(7-36)-CMV / EF1a treatment group showed a statistically significantly slower tumor growth rate (see Figures 11A-11B) and a statistically significantly prolonged survival (p<0.05 compared with the control Ad5 group and the negative control group, see Figure 12). Ⅲ(B). In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla on colorectal cancer animal model

[0123] C57BL / 6 mice aged 6-8 weeks were selected and inoculated with 1×10 MC38-car cells (suspended in 10% Matrigel in PBS, with each mouse inoculated) on the right flank.6 cells, a total of 21 mice (see Figure 13) to establish a subcutaneous tumor model. When the tumor volume reaches about 100mm 3 They were randomly divided into three groups: Negative control group: injected with PBS (0.1 mL); control Ad5 group: injected with Ad5 control virus (AdV-Ctrl, 5×10 8 ifu / dose); treatment group: injected with Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EFla, 5×10 8 Ifu / dose). All treatments were intratumorally injected once every other day for a total of three doses (see Figure 13). The tumor volume, body weight changes and survival period of the mice after natural death were recorded.

[0124] Compared with the control Ad5 group (p<0.0001, see Figure 14B) and the negative control group injected with PBS only (p<0.0001, see Figure 14A), the AdV-GLP-1 (7-36) -CMV / EF1a treatment group showed a statistically significantly slower tumor growth rate (see Figure 14C), and statistically significantly prolonged survival (compared with the control Ad5 group, p<0.01; compared with the negative control group, p<0.05, see Figure 15). There was no significant change in the body weight of the three groups of mice (see Figure 16). Ⅲ(C). In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla on melanoma animal models

[0125] C57BL / 6 mice aged 6-8 weeks were selected and inoculated with 1×10 B16 cells (suspended in 10% Matrigel in PBS, with each mouse inoculated with 1×10 6 100 cells, a total of 21 mice (see Figure 17) to establish a subcutaneous tumor model. When the tumor volume reaches about 100 mm 3 The mice were randomly divided into three groups: negative control group: injected with PBS (0.1 mL); control Ad5 group: injected with Ad5 control virus (AdV-Ctrl, 5 × 10 8 ifu / dose); treatment group: injected with Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EF1a, 5×10 8 Ifu / dose). All treatments were intratumorally injected once every other day for a total of three doses (see Figure 17). The survival time of mice after natural death was recorded (see Figure 18). Ⅲ(D). In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla on glioma animal models

[0126] C57BL / 6 mice aged 6-8 weeks were selected and GL261 glioma cells (suspended in 10% Matrigel in PBS, 1×10 per mouse) were inoculated. 6 cells, a total of 11 mice (see Figure 19) to establish a subcutaneous tumor model. When the tumor volume reaches about 100mm 3 The mice were randomly divided into three groups: negative control group: injected with PBS only (0.1 mL, n = 2); control Ad5 group: injected with Ad5 control virus (AdV-Ctrl, 5 × 10 8 pfu / dose, n=4); treatment group: injected with Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EF1a, 5×10 8 pfu / dose, n=5). All treatments were intratumorally injected once every other day, for a total of three doses (see Figure 19). The tumor volume and survival time of mice after natural death were recorded.

[0127] Compared with the control Ad5 group (p=0.0001, AdV-Ctrl, see Figure 20) and the negative control group injected with PBS only (p=0.0001, PBS, see Figure 20), the AdV-GLP-1(7-36)-CMV / EFla treatment group showed a statistically significantly slower tumor growth rate (see Figure 20) and a statistically significantly prolonged survival (compared with the control Ad5 group, p=0.0027; compared with the negative control group, p=0.0082, see Figure 21). Ⅲ(E). In vivo antitumor effect of AdV-GLP-1(7-36)-CMV / EFla on renal cancer animal model

[0128] Balb / c mice aged 6-8 weeks were selected and inoculated with 1×10 Renca cells (suspended in 10% Matrigel in PBS) on the right flank of each mouse. 6 The subcutaneous tumor model was established by inoculating 24 mice on day 0 (see Figure 22). The tumor volume reached about 100 mm on day 10. 3 The mice were randomly divided into three groups: negative control group: injected with PBS (0.1 mL); control Ad5 group: injected with Ad5 control virus (AdV-Ctrl, 5 × 10 8 pfu / dose); treatment group: injected with Ad5 virus containing target DNA (AdV-GLP-1(7-36)-CMV / EF

[0129] The tumor growth rate of the recombinant adenovirus AdV-GLP-1 (7-36) -CMV / EF1a treatment group was significantly slower than that of the control group (AdV-Ctrl, Figures 23B and 23D) and the negative control group treated with PBS only (Figures 23A and 23D). Specifically, compared with the control group, the tumor growth rate of the AdV-GLP-1 (7-36) -CMV / EF1a treatment group was significantly reduced (*p < 0.001), and compared with the negative control group, it also showed a significant difference (*p < 0.05). Example IV: CD8 of recombinant adenovirus AdV-GLP-1(7-36)-CMV / EF1a + T cell-mediated anti-tumor effects

[0130] The recombinant adenovirus AdV-GLP-1(7-36)-CMV / EF1a was prepared according to the method of Example I, wherein the target protein sequence was SEQ ID NO: 1 and the target DNA sequence was SEQ ID NO: 5. The replication ability of the virus, the expression of the GLP-1 receptor agonist and the biological activity of the expressed GLP-1 receptor agonist were verified by the method of Example II. V(A): AdV-GLP-1(7-36)-CMV / EF1a therapy combined with anti-CD8, anti-CD4 or anti-NK therapy

[0131] Pancreatic cancer cells were subcutaneously inoculated into the right axilla of C57BL / 6 mice. When the tumor volume reached 100 mm 3 At the same time, the mice were randomly divided into several treatment groups and received the following injections: intratumoral injection of PBS (PBS group, 0.1 mL / time); intratumoral injection of control adenovirus (AdV-Ctrl group, 5×10 8 Infectious units / time); intraperitoneal injection of anti-CD8 antibody, anti-CD4 antibody or anti-NK antibody, followed by intratumoral injection of AdV-GLP-1(7-36)-CMV / EF1a (5×10 8 Infection units / time), where intratumoral injection was once every other day, twice in total. Tumor volume and survival were subsequently monitored. Each group of experiments contained 6-8 mice.

[0132] The tumor growth rates of the groups that received AdV-GLP-1(7-36)-CMV / EF1a treatment only (AdV-GLP1(7-36), Figure 24), and the groups that received anti-CD4 antibody (GLP1(7-36)+anti-CD4, Figure 24) or anti-NK antibody (GLP1(7-36)+anti-NK, Figure 24) treatment before AdV-GLP-1(7-36)-CMV / EF1a treatment were significantly slower than those of the groups that received anti-CD8 antibody (GLP1(7-36)+anti-CD8, Figure 24) treatment before AdV-GLP-1(7-36)-CMV / EF1a treatment, the control adenovirus group (AdV-Ctrl, Figure 24), and the negative control group that received PBS treatment only (PBS, Figure 24).

[0133] The group that received anti-CD8 antibody (GLP1(7-36)+anti-CD8, Figure 25) treatment before receiving AdV-GLP-1(7-36)-CMV / EF1a treatment showed no significant difference in survival rate compared with the control adenovirus group (AdV-Ctrl, Figure 25); however, its survival rate was significantly reduced compared with the group that received only AdV-GLP-1(7-36)-CMV / EF1a treatment (AdV-GLP1(7-36), Figure 25).

[0134] The group that received AdV-GLP-1(7-36)-CMV / EF1a treatment only (AdV-GLP1(7-36), Figure 25), and the group that received anti-CD4 antibody (GLP1(7-36)+anti-CD4, Figure 25) or anti-NK antibody (GLP1(7-36)+anti-NK, Figure 25) treatment before receiving AdV-GLP-1(7-36)-CMV / EF1a treatment, had significantly higher survival rate than the control adenovirus group (AdV-Ctrl, Figure 25; *: *p<0.5). However, there was no significant difference in survival rate between the groups that had received pre-treatment with anti-CD4 antibodies (GLP1(7-36)+anti-CD4, Figure 25) or anti-NK antibodies (GLP1(7-36)+anti-NK, Figure 25) and the group that had received only AdV-GLP-1(7-36)-CMV / EF1a treatment (AdV-GLP1(7-36), Figure 25). IV(B):AdV-GLP-1(7-36)-CMV / EF1a treatment significantly enhanced antitumor activity and reduced CD8 + T cell depletion

[0135] Pancreatic cancer cells were subcutaneously inoculated into the right axilla of C57BL / 6 mice. When the tumor volume reached about 200 mm3 At the same time, the mice were randomly divided into several treatment groups and received the following intratumoral injections: AdV-GLP-1(7-36)-CMV / EF1a (5×10 8 Infectious units / times); control adenovirus (AdV-Ctrl, 5×10 8 Infectious units / time); PBS (0.1mL / time). Each group was given the drug once every other day for a total of two times. 48 hours after the second dose, tumor tissue was isolated and single cell suspension was obtained for the detection of specific markers (such as IFN-γ + CD8 + T cells, Figure 26A; GzMB + CD8 + T cells, Figure 26B; PD1 + CD8 + T cells, Figure 26C). Each experiment contained 5 mice. The results showed that there were significant differences between the AdV-GLP-1(7-36)-CMV / EF1a treatment group (AdV-GLP1(7-36), Figures 26A-26C) and the control adenovirus group (AdV-Ctrl, Figures 26A-26C).

[0136] Pancreatic cancer cells were subcutaneously inoculated into the right axilla of C57BL / 6 mice. When the tumor volume reached 100 mm 3 At the same time, the mice were randomly divided into several treatment groups and received the following injections: intratumoral injection of PBS (PBS group, 0.1 mL / time); intratumoral injection of control adenovirus (AdV-Ctrl group, 5×10 8 Infectious units / time); intraperitoneal injection of anti-CD8 antibody (200 μg), anti-CD4 antibody (200 μg) or anti-NK antibody (200 μg), followed by intratumoral injection of AdV-GLP-1(7-36)-CMV / EF1a (5×10 8 Infectious units / time). Intratumoral injection was performed once every other day for a total of two times. Tumor volume and survival were subsequently monitored. Each group of experiments contained 6-8 mice.

[0137] T cells were isolated from mouse tumor tissues (samples taken 48 hours after treatment with AdV-GLP-1(7-36)-CMV / EF1a, control adenovirus, or PBS) and phenotyped by flow cytometry (FACScan).

[0138] Lipocalin 2 (LCN2), also known as Neutrophil Gelatinase-Associated Lipocalin (NGAL), is a secretory protein of the lipocalin superfamily. LCN2 is involved in the occurrence and development of a variety of inflammatory diseases. Studies have shown that LCN2 can promote neutrophil recruitment and inhibit CD8 + T cell activity. AdV-GLP-1(7-36)-CMV / EF1a treatment significantly reduced CD8 + The expression level of LCN2 in T cells (*p<0.05, AdV-GLP1(7-36), Figure 27A) significantly enhanced the CD8 + T cell anti-tumor response; control adenovirus treatment (AdV-Ctrl, Figure 27A) significantly increased CD8 + The expression level of LCN2 in T cells was significantly higher than that in the PBS group (***p<0.0001, compared with the PBS group). + In T cells, there was no significant difference in the expression level of LCN2 between the AdV-GLP-1(7-36)-CMV / EF1a treatment group (AdV-GLP1(7-36), Figure 27B) and the control adenovirus group (AdV-Ctrl, Figure 27B). IV(C): GLP-1(7-36) or GLP-1 analog (liraglutide) significantly reduced CD8 + LCN2 expression in T cells

[0139] Panc02 cells and CD8 + T cells were co-cultured for 24 hours, and GLP-1(7-36) was added to the experimental group. Then, CD8 + The expression level of LCN2 in T cells was significantly reduced in the presence of GLP-1 (7-36). + The expression level of LCN2 in T cells (**p<0.01, PANCO-2-GLP1, Figure 28A) was significantly higher than that in co-cultured Panc02 cells and CD8 + There was a significant difference compared with the control group of T cells (PANCO-2, Figure 28A).

[0140] After Panc02 cells were cultured in DMEM medium for 48 h, the supernatant was collected and CD8 +The T cells were further cultured for 24 hours. GLP-1 (7-36) or GLP-1 analog (liraglutide) was added to the experimental groups, and then CD8 + The expression level of LCN2 in T cells (Figure 28B). The results showed that the presence of GLP-1 (7-36) (***p < 0.001, CD8-GLP1, Figure 28B) or liraglutide (***p < 0.001, CD8-GLP1, Figure 28B) significantly reduced the expression of LCN2 in CD8 + The expression level of LCN2 in T cells was compared with that in co-cultured Panc02 cells and CD8 + There was a significant difference compared with the control group (CD8 T cells, Figure 28B). It is worth noting that GLP-1 (7-36) and liraglutide reduced CD8 + There was no significant difference in the effect on LCN2 expression levels in T cells (Figure 28B). IV(D):AdV-GLP-1(7-36)-CMV / EF1a significantly inhibits CD8 + T cell-mediated neutrophil recruitment

[0141] Panc02 cells were infected with AdV-GLP-1(7-36)-CMV / EF1a, control adenovirus (AdV-Ctrl), or PBS and cultured for 24 h. + T cells were co-cultured for 24 hours. + The T cell suspension was added to the lower chamber of the Transwell chamber, and the neutrophils isolated from mice were added to the upper chamber. After 4 hours, the cell suspension in the lower chamber was collected and the Ly6G neutrophil marker was detected ( FIG. 29 ).

[0142] Compared with the control adenovirus treatment group (***p<0.001, CD8-Panc02-AdV-Ctrl, Figure 29), the AdV-GLP-1(7-36)-CMV / EF1a treatment group (CD8-Panc02-AdV-GLP1(7-36), Figure 29) significantly inhibited the CD8 + T cell-mediated neutrophil recruitment.

[0143] Previous studies have shown that GLP-1 analogs (liraglutide) can induce natural killer (NK) cell-mediated anti-tumor effects through intratumoral injection, showing significant effects in hepatocellular carcinoma models. However, in this study, the recombinant adenovirus AdV-GLP-1(7-36)-CMV / EF1a expressing GLP-1(7-36) (prepared according to Example 1) induced CD8 +T cell-mediated anti-tumor effect, rather than NK cell-mediated anti-tumor effect. In addition, AdV-GLP-1(7-36)-CMV / EF1a also significantly reduced CD8 + The expression level of LCN2 in T cells was significantly reduced, and the infiltration of neutrophils in the tumor was significantly reduced, which was significantly different from the control adenovirus treatment group. It is worth noting that the infiltration of neutrophils in the tumor may play an immunosuppressive role in some solid tumors. Example V: Antitumor effects of recombinant Ad5 adenovirus AdV-GLP-1 (7-37 / A8G)-CMV in vivo and in vitro

[0144] AdV-GLP-1(7-37 / A8G)-CMV was prepared according to the method of Example I(A), and its target protein sequence was SEQ ID NO: 2 and its target DNA sequence was SEQ ID NO: 6. The viral replication ability, expression of GLP-1 receptor agonist and its biological activity were verified by the method of Example II. V(A): Antitumor effect of AdV-GLP-1(7-37 / A8G)-CMV on non-small cell lung cancer cells in vitro

[0145] A549 lung cancer cells were infected with AdV-GLP-1(7-37 / A8G)-CMV and control adenovirus (Ad5) at a multiplicity of infection (MOI) of 1 to 100. After 72 hours of infection, cell viability was determined by MTT assay to evaluate the oncolytic effect of Ad5 GLP1. VII(B): In vivo antitumor effect of AdV-GLP-1(7-37 / A8G)-CMV on pancreatic cancer animal model

[0146] C57BL / 6 mice aged 6-8 weeks were selected to establish a right axillary subcutaneous tumor model (Panc02 cells were suspended in PBS containing 10% Matrigel, and 1×10 6 cells, 18 mice in total, Figure 31). When the tumor volume reached about 100 mm 3 The mice were randomly divided into three groups: negative control group (only PBS was injected, 0.1 mL); control adenovirus group (AdV-Ctrl, 5×10 7 infection units / time); treatment group (AdV-GLP-1(7-37 / A8G)-CMV, 5×10 7 Infectious units / time). All treatment groups were injected intratumorally once every other day for a total of three times (Figure 31). The tumor volume and mouse body weight were then monitored, and the survival time of the mice after natural death was recorded.

[0147] Compared with the control adenovirus group (AdV-Ctrl, p<0.001, Figure 32), the tumor growth rate of the AdV-GLP-1 (7-37 / A8G)-CMV treatment group was significantly slowed down (AdV-GLP1 (7-37 / A8G), Figure 32); compared with the negative control group injected with PBS only (PBS, Figure 32), the tumor growth rate of the AdV-GLP-1 (7-37 / A8G)-CMV treatment group was also significantly slowed down. In addition, compared with the control adenovirus group (AdV-Ctrl, p<0.05, Figure 33), the survival rate of the AdV-GLP-1 (7-37 / A8G)-CMV treatment group was significantly improved (AdV-GLP1 (7-37 / A8G), Figure 33); compared with the negative control group injected with PBS only (PBS, Figure 33), the survival rate of the AdV-GLP-1 (7-37 / A8G)-CMV treatment group was also improved. There was no significant change in body weight among the three groups of mice ( FIG. 34 ). Example VI: In vivo antitumor effects of recombinant Ad5 adenoviruses AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a and AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a

[0148] AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a and AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a were prepared according to the method of Example I(B). The target protein sequence of AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a is SEQ ID NO:3, and the target DNA sequence is SEQ ID NO:7; the target protein sequence of AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a is SEQ ID NO:4, and the target DNA sequence is SEQ ID NO:8. The viral replication ability, the expression of the GLP-1 receptor agonist, and its biological activity were verified by the method of Example II.

[0149] C57BL / 6 mice aged 6-8 weeks were selected to establish a right axillary subcutaneous tumor model (panc02-car cells were suspended in PBS containing 10% Matrigel, and 1×10 6 cells, 15 mice in total, Figure 35). When the tumor volume reached about 100 mm 3At the same time, the mice were randomly divided into three groups: negative control group (PBS injection only, Figure 36A); control adenovirus group (AdV-Ctrl); treatment group (AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a, Figure 36B or AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a, Figure 36C). Each group of mice received intratumoral injections (5×10 8 infection units / time or 0.1 mL PBS), and the tumor volume was monitored ( FIG. 35 ).

[0150] Compared with the negative control group injected with PBS only (Figure 36A), the tumor growth rates of the AdV-GLP-1(7-36)-4GS-PLGF2-CMV / EF1a(36B) and AdV-PLGF2-4GS-GLP-1(7-36)-CMV / EF1a(Figure 36C) treatment groups were significantly slowed down.

Claims

1. A recombinant adenovirus or adenovirus vector comprising a first promoter, an E1A early activation replication element, an optional second promoter, and a target nucleotide sequence encoding a target protein, wherein the target protein comprises a glucagon-like peptide-1 (GLP-1) receptor agonist; wherein: The first promoter is a constitutive promoter; The optional second promoter is a constitutive promoter, which may be the same as or different from the first promoter. 2 . The recombinant adenovirus or adenoviral vector according to claim 1 , wherein the constitutive promoter is selected from the group consisting of CMV promoter and EF1a promoter.

3. The recombinant Ad5 adenovirus or adenoviral vector according to any one of the preceding claims, wherein the first promoter is a CMV promoter.

4. The recombinant adenovirus or adenoviral vector according to any preceding claim, wherein the second promoter is the EF1a promoter.

5. The recombinant adenovirus or adenoviral vector according to any preceding claim, further comprising a nucleotide sequence encoding an IL-2 signal peptide.

6. A recombinant adenovirus or adenoviral vector according to any of the preceding claims, wherein the GLP-1 receptor agonist comprises an amino acid sequence having 80% or higher homology with a sequence selected from the following group: an active fragment of GLP-1 (e.g., GLP-1(7-36), GLP-1(7-37)) and a GLP-1 analog.

7. The recombinant adenovirus or adenoviral vector according to any of the preceding claims, wherein the target protein comprises a GLP-1 receptor agonist and a tumor targeting protein.

8. The recombinant adenovirus or adenoviral vector according to any of the preceding claims, wherein the GLP-1 receptor agonist and the tumor targeting protein are connected via a linker sequence (GGGGS)n, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

9. The recombinant adenovirus or adenoviral vector according to any of the preceding claims, wherein the GLP-1 analog is a GLP-1 fragment modified by one or more amino acid substitutions, deletions and / or additions.

10. The recombinant adenovirus or adenoviral vector according to any one of the preceding claims, wherein the GLP-1 receptor agonist is secreted glucagon-like peptide-1 protein.

11. The recombinant adenovirus or adenoviral vector according to any of the preceding claims, wherein one or more tumors contacted with the recombinant adenovirus or adenoviral vector produce one or more effects selected from the group consisting of: tumor growth inhibition, tumor regression and prolonged survival.

12. The recombinant adenovirus or adenoviral vector according to any preceding claim, which, when administered to a subject, induces CD8+ T cell-mediated anti-tumor effects in the subject's tumor.

13. A pharmaceutical composition comprising the recombinant adenovirus or adenoviral vector according to any one of the preceding claims and a pharmaceutically acceptable carrier.

14. A method for inducing CD8+ T cell-mediated anti-tumor effects in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant adenovirus or adenoviral vector or pharmaceutical composition according to any preceding claim.

15. A method for treating or preventing one or more cancers in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant adenovirus or adenoviral vector or pharmaceutical composition according to any preceding claim.

16. The method of claim 15, wherein the one or more cancers are selected from the group consisting of liver cancer, pancreatic cancer, colon cancer, glioma, lung cancer, esophageal cancer, gastric cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, head and neck squamous cell carcinoma, melanoma, multiple myeloma, and lymphoma. The method according to claim 16 , wherein the lung cancer is non-small cell lung cancer.

18. The method according to any preceding claim, further comprising inhibiting tumor invasion and / or metastasis.

19. The method according to any of the preceding claims, further comprising one or more effects selected from the group consisting of reducing LCN2 expression in CD8+ T cells, reducing neutrophil infiltration within tumors, inhibiting tumor inflammatory pathways, inhibiting IDO-1, and restoring anti-tumor immune surveillance.

20. The method of any preceding claim, further comprising delaying the progression of cachexia in the subject.

21. The method of any preceding claim, further comprising extending the lifespan of the subject.

22. The method according to any of the preceding claims, wherein the recombinant adenovirus or adenoviral vector or pharmaceutical composition according to any of the preceding claims is administered as a monotherapy, adjuvant therapy or combination therapy.

23. The method according to any preceding claim, wherein the recombinant adenovirus or adenoviral vector or pharmaceutical composition according to any preceding claim is administered by intratumoral injection.

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