A composition based on mesenchymal stem cells for treating liver cancer

Abstract

The present invention discloses a composition for treating liver cancer based on mesenchymal stem cells, which composition comprises human mesenchymal stem cells and a conditionally replicating adenovirus therapy system loaded in the human mesenchymal stem cells; the conditionally replicating adenovirus therapy system is composed of a conditionally replicating adenovirus vector and a target gene inserted therein, the conditionally replicating adenovirus molecule comprises a first expression regulatory element and a second expression regulatory element located downstream of the first expression regulatory element, and the first expression regulatory element and the second expression regulatory element regulate the expression of the target gene. The targeting delivery system of the present invention exhibits better anti-cancer activity and safety in a mouse orthotopic transplantation tumor model of liver cancer, increases intratumoral T cell infiltration, raises the proportion of activated T cells, has a scavenging effect on AFP-negative liver cancer cells in the tumor, and has good potential and prospects for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to a composition for treating liver cancer based on mesenchymal stem cells. Background Art

[0002] Liver cancer is a common malignant tumor in China. In 2020, the number of newly diagnosed cases in China was approximately 410,000. The mortality rate of liver cancer in China is 20.4 per 100,000, accounting for 18.8% of all malignant tumor deaths. At present, surgical resection remains the main treatment method for liver cancer. However, the postoperative recurrence rate of patients caused by microscopic tumor residues and small blood vessel invasions is extremely high, and the remaining tumor tissues after surgery also tend to grow faster. In addition, liver cancer is not sensitive to chemotherapy, and some chemotherapy drugs also have the effect of promoting tumor metastasis.

[0003] Conditionally replicating adenovirus (CRAd) refers to a therapeutic adenovirus vector that can selectively replicate in tumor cells, ultimately causing the tumor cells to disintegrate and release progeny virus particles. Inserting an adenovirus E1A gene expression cassette controlled by a tumor-specific promoter into a human adenovirus type 5 vector with deletions in the E1 and E3 genes is a common method for constructing CRAd. As an effective treatment method for clearing drug-resistant tumor cells, CRAd has been used in the treatment and research of various solid tumors. Alpha-fetoprotein (AFP) is an important marker for liver cancer and is highly specifically expressed in liver cancer cells. Therefore, the AFP promoter can be used as a specific promoter to construct CRAd targeting liver cancer cells. However, tumors have the characteristic of heterogeneity, and there are significant differences in the expression levels of AFP in the tumor tissues of liver cancer patients. The above system is difficult to play a role in tumor cells with negative AFP in the tumor.

[0004] At the same time, CRAd has the disadvantage of being unsuitable for systemic administration because it can be rapidly cleared by the immune system and is difficult to effectively reach the local tumor tissue. Loading CRAd into mesenchymal stem cells for in vivo transportation helps to solve the above problems. Mesenchymal stem cells derived from human umbilical cord Wharton's jelly (HUMSC) are an ideal in vivo cell carrier. It has the advantages of rapid amplification, easy genetic engineering modification, and no ethical problems. More importantly, it can migrate and home to the tumor tissue, and can transport the CRAd loaded on it to the local tumor tissue to facilitate the clearance of residual and metastatic micro-tumor foci. However, not all HUMSC homing to the local liver cancer can become packaging cells for any CRAd, which is related to the replication regulatory elements in CRAd.

[0005] To solve the above problems, it is necessary to find a new composition for treating liver cancer based on mesenchymal stem cells. Summary of the Invention

[0006] One aspect of the present invention is directed to the problems that hepatoma cells have heterogeneity in the prior art and how to successfully package CRAd in mesenchymal stem cells, and provides a composition for treating hepatoma based on mesenchymal stem cells.

[0007] The technical solution provided by the present invention is as follows:

[0008] A composition for treating hepatoma based on mesenchymal stem cells, the composition comprising human mesenchymal stem cells and a conditionally replicating adenovirus therapy system loaded in the human mesenchymal stem cells;

[0009] Wherein, the conditionally replicating adenovirus therapy system is composed of a conditionally replicating adenovirus vector and a target gene inserted therein, the conditionally replicating adenovirus vector includes a first expression regulatory element and a second expression regulatory element located downstream of the first expression regulatory element, the first expression regulatory element and the second expression regulatory element respectively regulate the expression of the target gene, and the polypeptide encoded by the target gene has the activity of treating hepatoma;

[0010] Wherein, the first expression regulatory element includes an AFP promoter, and the second expression regulatory element includes an hTERT promoter.

[0011] Preferably, in some embodiments of the present invention, the target gene directly or indirectly connected to the first expression regulatory element is one or more selected from the adenovirus E1A gene, the adenovirus E1B19K gene or the adenovirus E1B55K gene.

[0012] More preferably, in some embodiments of the present invention, the target gene directly or indirectly connected to the first expression regulatory element is the adenovirus E1A gene.

[0013] Preferably, in some embodiments of the present invention, the first expression regulatory element further includes a sequence as shown in SEQ ID No.1 directly or indirectly connected downstream of the E1A gene.

[0014] More preferably, in some embodiments of the present invention, the sequence is composed of 1 or 2 to 8 repeated sequences as shown in SEQ ID No.1.

[0015] Preferably, in some embodiments of the present invention, the target gene connected to the second expression regulatory element is a gene encoding a bispecific antibody, a single-chain antibody, a Fab, a Fab', an F(ab')2, an Fd, a dAb, a complementarity-determining region fragment or a chimeric antibody.

[0016] More preferably, in some embodiments of the present invention, the target gene connected to the second expression regulatory element is a gene encoding a bispecific antibody against human CD3 and PD-L1.

[0017] Further preferably, in certain embodiments of the present invention, the amino acid sequence of the bispecific antibody against human CD3 and PD-1 is as shown in SEQ ID No.1, or a sequence having more than 85% identity therewith and having substantially similar biological functions.

[0018] To enhance the effect of the composition of the present invention, preferably, in one embodiment of the present invention, the above composition further comprises 5-FU.

[0019] Another aspect of the present invention is to provide the use of the above composition in the preparation of a medicament for treating liver cancer.

[0020] The beneficial effects of the present invention are as follows:

[0021] The CRAd targeting and transporting system loaded with the bifunctional antibody provided by the present invention shows better anti-cancer activity and safety in the orthotopic transplantation tumor model of liver cancer mice compared with the system not loaded therewith, increases the infiltration of T cells in the tumor, raises the proportion of activated T cells, has a scavenging effect on AFP-negative liver cancer cells in the tumor, and has good potential and prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the shuttle plasmid structure constructed in the examples of the present invention;

[0023] Figure 2 Result diagram of the selective cytotoxic effect of CRAd in the examples of the present invention;

[0024] Figure 3 Result diagram of the content of the functional antibody in the culture supernatant after the tumor cells are infected with AdE1A-BsAb in the examples of the present invention;

[0025] Figure 4 Result diagram of the direct binding and competitive effect of the bifunctional antibody in the examples of the present invention;

[0026] Figure 5 Result diagram of the T cell killing effect mediated by the bifunctional antibody in the examples of the present invention;

[0027] Figure 6 Result diagram of the T cell activation mediated by the bifunctional antibody in the examples of the present invention;

[0028] Figure 7 Result diagram of the bifunctional antibody inhibiting apoptosis induced by PD-L1 in the examples of the present invention;

[0029] Figure 8 Result diagram of the packaging of CRAd in hepatocyte-like differentiated HUMSC in the examples of the present invention;

[0030] Figure 9 This is the replication result graph of CRAd in the supernatant in the embodiments of the present invention in HepG2;

[0031] Figure 10 This is the in vivo anti-hepatocellular carcinoma effect result graph of HUMSC loaded with CRAd combined with PBMC in the embodiments of the present invention;

[0032] Figure 11 This is the distribution result graph of adenovirus particles and bifunctional antibodies in tumor tissues in the embodiments of the present invention;

[0033] Figure 12 This is the infiltration and activation result graph of intratumoral T cells in the embodiments of the present invention;

[0034] Figure 13 This is the result graph of the levels of ALT and AST in peripheral blood in the embodiments of the present invention, wherein the levels of ALT and AST in tumor-free mice are shown by the dashed line;

[0035] Figure 14 This is the pathological graph of normal extrahepatic tissues in the embodiments of the present invention;

[0036] Figure 15 This is the result graph of the treatment of hepatocellular carcinoma heterogeneity model by HUMSC loaded with CRAd combined with PBMC in the embodiments of the present invention.

[0037] Sequence description

[0038] SEQ ID No.1 is the nucleotide sequence included in the first expression regulation element in the embodiments of the present invention;

[0039] SEQ ID No.2 is the amino acid sequence of the bispecific antibody against human CD3 and PD-L1 in the embodiments of the present invention. Detailed implementation manners

[0040] The present invention discloses a composition for treating hepatocellular carcinoma based on mesenchymal stem cells. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. And those skilled in the art can obviously make changes or appropriate changes and combinations to the content described herein without departing from the content, spirit and scope of the present invention to implement and apply the technical solution of the present invention.

[0041] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise clearly indicated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "having" etc. will be understood to include the stated elements or components, without excluding other elements or other components. Terms such as "such as", "for example", etc. are intended to indicate exemplary embodiments and are not intended to limit the scope of the present disclosure. The term "a" can mean "one", but can also mean "one or more", "at least one", and "one or more than one". The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of the alternatives.

[0042] The following explains some of the terms that appear in the present invention.

[0043] The term "conditional replication adenovirus vector" is an adenovirus vector that has been engineered to replicate under a predetermined condition. For example, gene functions essential for replication, such as those encoded by adenovirus early regions, can be operably linked to inducible, repressible, or tissue-specific transcriptional control sequences, such as promoters.

[0044] The term "expression regulatory element" refers to a nucleic acid sequence that affects the expression of an operably linked polynucleotide, including polynucleotide sequences that promote the transcription and translation of a heterologous polynucleotide. Expression regulatory elements that can be used in the present invention include, but are not limited to, promoters, enhancers, intron splicing signals, polyA (polyA), inverted terminal repeats (ITRs), etc.

[0045] The terms "upstream" or "downstream" refer to upstream and downstream along the protein translation direction of the coding region.

[0046] The term "alpha-fetoprotein" (AFP) is a glycoprotein that belongs to the albumin family and is mainly synthesized by fetal hepatocytes and yolk sacs. Alpha-fetoprotein has a relatively high concentration in the fetal blood circulation, and then decreases after birth. By 2-3 months after birth, alpha-fetoprotein is basically replaced by albumin and is difficult to detect in the blood, so its content in adult serum is extremely low. Alpha-fetoprotein has many important physiological functions, including transport function, bidirectional regulatory function as a growth regulator, immunosuppression, induction of apoptosis in T lymphocytes, etc. Alpha-fetoprotein is closely related to the occurrence and development of liver cancer and various tumors, and can show a relatively high concentration in various tumors, and can be used as a positive detection index for various tumors.

[0047] The term "hTERT" refers to human telomerase reverse transcriptase. The above-mentioned human telomerase reverse transcriptase synthesizes complementary DNA using the RNA template of telomerase. After forming an RNA-DNA hybrid form, it becomes double-stranded DNA and inserts into the chromosome of the host cell. In this way, in the above-mentioned host cell, telomerase attaches to the chromosome telomere, rather than the telomere attaching to the chromosome telomere, and continues to generate telomeres, thereby forming immortalized cells. The hTERT promoter has high transcriptional activity in more than 85% of tumor cells and is inactivated in normal cells, and is a commonly used tumor cell-specific promoter.

[0048] Embodiments:

[0049] In order to solve the problems of difficult-to-overcome heterogeneity of liver cancer tissue and insufficient activation of intratumoral T cells in the prior art, the present inventors constructed a novel conditionally replicating adenovirus. The conditionally replicating adenovirus is composed of a conditionally replicating adenovirus vector and a target gene inserted therein. In certain embodiments of the present invention, the inventors inserted the expression cassette of the target gene into the adenovirus shuttle plasmid pAdTrack, and packaged the CRAd carrying the target gene in 293A cells. In the present invention, the polypeptide encoded by the target gene has the activity of treating liver cancer. In certain embodiments of the present invention, the above-mentioned polypeptide can be, for example, cytokines, antibodies, antigen-binding portions, etc. In order to achieve certain specific effects of treating liver cancer, in certain embodiments of the present invention, the above-mentioned target gene can be one or more than two target genes. These genes are respectively regulated by the first expression regulatory element and the second expression regulatory element located downstream of the first expression regulatory element for the expression of the target gene. The first expression regulatory element includes the AFP promoter, and the second expression regulatory element includes the hTERT promoter. In certain embodiments of the present invention, a purification tag or a detection protein can also be operably linked upstream or downstream of the above-mentioned target gene, for example, including but not limited to, His tag, green fluorescent protein expression cassette, etc.

[0050] Preferably, in certain embodiments of the present invention, the target gene linked to the first expression regulatory element can be one or several selected from the adenovirus E1A gene, the adenovirus E1B19K gene, or the adenovirus E1B55K gene. More preferably, in certain embodiments of the present invention, the target gene linked to the first expression regulatory element is the adenovirus E1A gene.

[0051] Preferably, in certain embodiments of the present invention, the first expression regulatory element further includes the sequence shown in SEQ ID No.1 directly or indirectly linked downstream of the E1A gene.

[0052] More preferably, in some embodiments of the present invention, the sequence consists of 1 or 2 to 8 repeated sequences as shown in SEQ ID No.1. For example, it is composed of 2, 3 or 4 sequences in tandem.

[0053] Hepatocellular carcinoma also has the characteristic of high expression of programmed death ligand-1 (PD-L1). Moreover, adenovirus infection can increase interferon-γ in the tumor, and the latter can also increase the expression of PD-L1 in tumor cells, which will ultimately promote the generation of Treg cells and the apoptosis of activated T cells. Although PD-L1 antibodies can significantly increase the proportion of CD4 + and CD8 + T cells in the tumor tissues of liver cancer patients. However, simply blocking the PD-L1 / PD-1 axis is not sufficient to transform "cold tumors" into "hot tumors" because T cell activation is insufficient. Therefore, in some embodiments of the present invention, the inventors loaded a PD-L1 / CD3 bifunctional antibody expression cassette controlled by the human telomerase reverse transcriptase (hTERT) promoter onto the original CRAd, so that it can express the bifunctional antibody while replicating in tumor cells. In some embodiments of the present invention, the above bifunctional antibody is fused from a single-chain antibody against human CD3 and a modified protein of the extracellular region of human PD-1 (HAC). This bifunctional antibody can be strictly restricted to be expressed locally in the tumor, and can activate intratumoral T cells while blocking the PD-L1 / PD-1 axis, which is beneficial to enhancing the anti-cancer effect of the new CRAd and solving the AFP heterogeneity problem.

[0054] Preferably, in some embodiments of the present invention, the amino acid sequence of the bispecific antibody against human CD3 and PD-1 is as shown in SEQ ID No.1, or a sequence having more than 85% identity with it and having substantially similar biological functions. The above sequence having more than 85% identity with the amino acid sequence shown in SEQ ID No.1 means having an amino acid sequence identical to it by 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Those skilled in the art can randomly or engineer point mutations in the amino acid sequence of the bispecific antibody against human CD3 and PD-1 described in this specification in a suitable manner. The purposes can be, for example, to obtain better affinity and / or dissociation properties, improvement of expression performance, humanization, etc. These sequences can have the same or substantially the same biological functions as the bispecific antibody against human CD3 and PD-1, and these mutated amino acid sequences are all included in the protection scope of the present invention.

[0055] The pharmaceutical use of the conditionally replicating adenovirus in the present invention can be for the preparation of a drug for treating liver cancer, or for a treatment means other than administering a drug to a patient for treating liver cancer.

[0056] As a drug, human umbilical cord Wharton's jelly-derived mesenchymal stem cells (HUMSC) can be used as a drug delivery system. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells (HUMSC) are an ideal in vivo cell carrier, which has advantages such as fast amplification, easy genetic engineering modification, and no ethical issues. More importantly, it can migrate and home to tumor tissues, and can transport the loaded CRAd to the local tumor tissues to facilitate the clearance of residual and metastatic microtumor foci. The inventors of the present invention found in the research that when HUMSC homed to the local liver cancer, it could undergo hepatocyte-like differentiation, and during the hepatocyte-like differentiation process of HUMSC, the expression of AFP showed a trend of first increasing and then decreasing (representing the activity of the AFP promoter therein); the expression of miR-122 was continuously increasing, but lagged behind the increase of AFP. This can provide a sufficient time window for the replication of the CRAd loaded therein, enabling the HUMSC homed to the local liver cancer to successfully become the packaging cell of CRAd.

[0057] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0058] Example 1: Preparation of CRAd

[0059] An E1A expression cassette regulated by both the AFP promoter and miR-122 was inserted between the XhoI and KpnI restriction enzyme sites of the adenovirus shuttle plasmid pAdTrack to construct the CRAd shuttle plasmid pAdE1A. Then, a bifunctional antibody expression cassette controlled by the hTERT promoter was inserted between the KpnI and BglII restriction enzyme sites of pAdE1A to construct the CRAd shuttle plasmid pAdE1A-BsAb. The structure of the shuttle plasmid is as Figure 1 shown.

[0060] The shuttle plasmids pAdTrack, pAdE1A, and pAdE1A-BsAb were respectively recombined with the adenovirus backbone plasmid pAdEasy-1 in Escherichia coli BJ5183. After the recombined adenovirus plasmid was linearized by PacI digestion, it was transfected into logarithmic growth phase 293A cells in a 24-well plate. About 10 - 14 days later, cytopathic effect appeared in the cells. It was repeatedly frozen and thawed at -20 °C and 37 °C and shaken 3 times, and the culture medium was collected. This was the first-generation virus; after the virus was passaged 3 times in 293A cells, it was collected, purified, and concentrated. The titer was determined by the 50% tissue culture infective dose method (TCID50).

[0061] Example 2: Selective cytotoxic effect of CRAd

[0062] HepG2 and SMMC-7721 cells were seeded at 10 4 cells / well in 96-well plates. 50 MOI of each of the three adenoviruses was added to each well, and the survival rates of tumor cells were detected using the CCK-8 reagent on the 2nd, 3rd, and 4th days after infection. Tumor cells not infected with the virus served as negative controls (survival rate 100%). The results are as Figure 2 shown. In AFP-positive liver cancer cell lines, the survival rates of cells in the AdE1A and AdE1A-BsAb groups were significantly reduced: approximately 80% on the 3rd day; approximately 50% on the 4th day. In the AFP-negative liver cancer cell line SMMC-7721, the survival rates of cells in the AdE1A and AdE1A-BsAb groups did not change significantly and remained above 90% on the 4th day.

[0063] Example 3: Expression of bifunctional antibody in liver cancer cells

[0064] HepG2 and SMMC-7721 cells were infected with 50 MOI of AdE1A-BsAb, and then the culture supernatants were collected daily. The content of the bifunctional antibody in the supernatants was detected using a His-tag kit. The results are as Figure 3 shown. The content of the upper functional antibody in the supernatants of the two cell types was similar in the first three days. On the 4th day, the content of BsAb in the supernatant of SMMC-7721 reached approximately 800 ng / mL, while the supernatant of HepG2 contained approximately 400 ng / mL of BsAb, which was related to excessive cell death in HepG2 on the 4th day after infection.

[0065] Example 4: Binding activity of bifunctional antibody

[0066] SMMC-7721 cells were infected with 50 MOI of different adenoviruses, and the cell culture supernatants were collected 4 days later. Then, direct binding experiments of the bifunctional antibody and competitive experiments with commercial antibodies were performed using 7721-PD-L1 cells (SMMC-7721 cells stably expressing PD-L1) and Jurkat cells (CD3-positive cells). The results are as Figure 4 shown. Flow cytometry results showed that the bifunctional antibody in the culture supernatant could directly bind to the two target cells; the bifunctional antibody could also compete with the corresponding commercial antibody for cell surface target antigens.

[0067] Example 5: T cell killing effect mediated by bifunctional antibody

[0068] 7721-PD-L1 cells were seeded at 10 4 cells / well in 96-well plates, and then 5×10 4After co-culturing human peripheral blood mononuclear cells (PBMCs) with 100 μL of different culture supernatants (collected from the previous experiment) for 16 hours, the death rate of target cells was measured using a lactate dehydrogenase cell viability assay kit (Blank was the culture supernatant of uninfected virus cells). The results are as Figure 5 shown. The results showed that in the group of AdE1A-BsAb-infected cell supernatants containing bifunctional antibodies, the death rate of target cells exceeded 60%.

[0069] Example 6: T cell activation mediated by bifunctional antibody

[0070] The cell components in the co-culture system supernatant of Example 5 were collected, labeled with PE-CD69 antibody and APC-CD3 antibody, and the proportion of activated T cells was detected by flow cytometry. CD69 is a surface marker of T cell activation. The results are as Figure 6 shown. The proportion of CD69 + cells in CD3 + cells was significantly higher in the AdE1A-BsAb group than in other groups.

[0071] Example 7: PD-L1 blockade mediated by bifunctional antibody

[0072] 7721-PD-L1 cells and Jurkat cells were seeded in a 24-well plate at a ratio of 10:1, and co-cultured with the cell culture supernatant collected in Experiment 5 for 24 hours. The suspended cells in the co-culture system were collected, labeled with APC-CD3 antibody and FITC-AnnexinV, and the proportion of apoptotic cells in CD3 + cells was analyzed by flow cytometry. The results are as Figure 7 shown. The results showed that the apoptotic proportion of CD3+ cells in the co-culture system containing BsAb was significantly reduced, indicating that BsAb inhibited T cell apoptosis induced by the PD-L1 / PD-1 axis.

[0073] Example 8: Isolation, culture and hepatocyte-like differentiation of human umbilical cord blood mesenchymal stem cells

[0074] The umbilical cord was rinsed thoroughly with PBS, the arterial and venous vessel walls of the umbilical cord were dissected, the gelatinous part was taken, cut into pieces and placed in a pre-wetted T-75 cm plastic culture flask containing DF12 medium (containing 10% serum and 2 mM L-glutamine) for inverted culture. After 4-8 hours, the culture flask was turned over. After culturing for 10-14 days, the tissue blocks were removed and the culture was continued. Cells from passages 3-5 were used for the experiment.

[0075] Hepatocyte-like differentiation: HUMSCs were seeded at 1×10 4 cells / cm 2Inoculated at a density into a 10-cm cell culture dish and cultured in DMEM medium containing 5% FBS, 20 ng / mL HGF, 10 ng / mL bFGF, 10 ng / mL OSM, 0.61 mg / mL nicotinamide, and 10 -7 μg / mL dexamethasone for 1 week, and then an additional 10% of 100×ITS+1 liquid media supplement was added to the culture system and continued to be cultured, with the medium changed twice a week.

[0076] Example 9: Packaging of CRAd in hepatocyte-like differentiated HUMSC

[0077] Ten days after in vitro induction of hepatocyte-like differentiation of HUMSC, they were infected with AdE1A and AdE1A-BsAb respectively. Two days later, the cells were collected and the adenovirus particles in the cells were observed by transmission electron microscopy. The results were as Figure 8 shown. Adenovirus particles with a diameter of approximately 70-90 nm (indicated by arrows) were visible in the electron micrographs of both groups of cells, indicating that CRAd can be packaged during the hepatocyte-like differentiation process of HUMSC.

[0078] Example 10: Release of CRAd in hepatocyte-like HUMSC

[0079] Ten days after in vitro induction of hepatocyte-like differentiation of HUMSC, they were infected with AdTrack, AdE1A, and AdE1A-BsAb respectively, and the culture supernatant was collected. Then the supernatant was added to the cultured HepG2 cells respectively, and the green fluorescent protein was observed by fluorescence microscopy two days later. The results were as Figure 9 shown. Since AdE1A and AdE1A-BsAb can be packaged and secreted by differentiated HUMSC, the secreted adenovirus can replicate in HepG2 cells, resulting in the clustered distribution of green fluorescent protein.

[0080] Example 11: Establishment of a mouse orthotopic liver cancer transplantation tumor model

[0081] Take HepG2-Luci cells (HepG2 cells labeled with Luciferase) in the logarithmic growth phase and adjust the cell density to 5×10 7 cells / mL. Take 100 μL of the cell suspension and inject it subcutaneously into the dorsal side of the root of the right hind limb of BALB / c nude mice. After about 2 weeks of inoculation, a solid tumor with a size of 1 cm 3 grew. After sacrificing the mice, the fish-like tumor tissue was taken under sterile conditions, placed in physiological saline containing gentamicin, the tumor mass was rinsed, and trimmed into 1-2 mm 3Size for backup. Anesthetize BALB / c nude mice intraperitoneally with sodium pentobarbital (2%) at a dose of 100 mg / kg. Disinfect routinely, cut a 1 - 2 cm incision along the midline of the abdomen under the xiphoid process, extrude the liver, make a small incision with a scalpel, put the tumor mass into the incision about 3 mm deep with ophthalmic forceps, and stop bleeding with a gauze soaked in normal saline for 1 minute to prevent the tumor mass from falling off. Suture the incision and apply erythromycin eye ointment to the incision to prevent infection.

[0082] Example 12: In vivo anti - cancer effect of the in vivo targeting system mediated by HUMSC combined with PBMC

[0083] Ten days after establishing the orthotopic liver cancer xenograft mouse model, administer HUMSC loaded with CRAd twice via the micro - vein, with a 3 - day interval between the two administrations, and administer 1×10 6 cells each time. The loading method of CRAd is to infect HUMSC at 500 MOI for 48 hours. Three days after the second administration of HUMSC, administer PBMC twice via the micro - vein again, with a 3 - day interval between the two administrations, and inject 6×10 6 cells each time. One day before treatment, on the 8th day and the 16th day after treatment, observe the changes in the size of the orthotopic tumor with a small animal in - vivo imaging system, and record the fluorescence signal intensity. The results are as Figure 10 shown. On the 8th day, the tumor growth in both the MSC.AdE1A + PBMC and MSC.AdE1A - BsAb + PBMC treatment groups was significantly inhibited; on the 16th day, the anti - cancer effect of the MSC.AdE1A - BsAb + PBMC treatment group was significantly better than that of the MSC.AdE1A + PBMC treatment group.

[0084] Example 13: Distribution of adenovirus particles and bifunctional antibodies in cancer tissues

[0085] Collect the tumor tissue specimens of each treatment group in Example 12, prepare paraffin - embedded sections, label the adenovirus Hexon protein in the tissue and the His - tag of the bifunctional antibody with the corresponding antibodies, and observe their distribution in the tissue under a confocal microscope. The results are as Figure 11 shown. No bifunctional antibody was observed in the tissue of the MSC.AdE1A + PBMC treatment group. In the tissue of the MSC.AdE1A - BsAb + PBMC treatment group, the distribution of the bifunctional antibody was found to be consistent with that of the adenovirus particles, indicating that the bifunctional antibody can be secreted to play an anti - tumor role.

[0086] Example 14: Infiltration and activation of T cells in tumor tissues

[0087] Three tumor tissue samples were taken from each of the MSC.AdE1A+PBMC treatment group and the MSC.AdE1A-BsAb+PBMC treatment group, and intratumoral lymphocytes were isolated using Percoll. After labeling with PE-CD69 antibody and APC-CD3 antibody, flow cytometry was used to analyze the activation of T cells in the tumor. The results are as Figure 12 shown. The proportion of CD3+ T cells in the tumor of the MSC.AdE1A-BsAb+PBMC treatment group increased significantly, and the proportion of CD69+ activated T cells was also much higher than that of the MSC.AdE1A+PBMC treatment group.

[0088] Example 15: Determination of Transaminases in Mouse Peripheral Blood after Treatment

[0089] The peripheral sera of mice in each treatment group after treatment were collected, and the contents of ALT and AST were measured using a transaminase detection kit. The results are as Figure 13 shown. The levels of ALT and AST in both the MSC.AdE1A+PBMC treatment group and the MSC.AdE1A-BsAb+PBMC treatment group decreased significantly. Among them, the ALT level in the MSC.AdE1A-BsAb+PBMC treatment group was lower than that in the MSC.AdE1A+PBMC treatment group, indicating that MSC.AdE1A-BsAb caused less damage to normal hepatocytes.

[0090] Example 16: Pathological Observation of Normal Tissues outside the Liver

[0091] The lung, kidney, and spleen tissues of mice in each treatment group were collected, paraffin-embedded sections were prepared, and HE staining was performed to observe whether there were pathological changes. The results are as Figure 14 shown. The results showed that there were no severe pathological changes in each group, indicating that the treatment system of HUMSC loaded with CRAd was non-toxic to normal tissues outside the liver.

[0092] Example 17: Treatment of Hepatocellular Carcinoma Heterogeneity Model with HUMSC Loaded with CRAd Combined with PBMC

[0093] HepG2 and 7721-PD-L1-Luci (7721-PD-L cells labeled with Luciferase) were mixed at a ratio of 3:1, and the cell density was adjusted to 5×10 7 cells / mL. 100 μL of the cell suspension was subcutaneously injected into the dorsal side of the right hind limb root of BALB / c nude mice. After the tumor mass grew, an orthotopic liver cancer transplantation tumor model was further established. Then, using the same sequential cell treatment protocol as before, on the day before treatment and on the 8th and 16th days after treatment, a small animal in vivo imaging system was used for observation. The results are as Figure 15 shown. Since CRAd cannot directly lyse AFP-negative 7721-PD-L1 cells, but the BsAb released locally in the tumor can mediate PD-L1+ Cell death, so the MSC.AdE1A-BsAb + PBMC treatment group has a significant inhibitory effect on the growth of AFP-negative cells.

[0094] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composition for treating liver cancer based on mesenchymal stem cells, characterized in that, The composition comprises human mesenchymal stem cells and a conditionally replicating adenovirus therapy system loaded in the human mesenchymal stem cells; Wherein, the conditionally replicating adenovirus therapy system is composed of a conditionally replicating adenovirus vector and a target gene inserted therein. The conditionally replicating adenovirus vector comprises a first expression regulatory element and a second expression regulatory element located downstream of the first expression regulatory element. The first expression regulatory element and the second expression regulatory element respectively regulate the expression of the target gene, and the polypeptide encoded by the target gene has the activity of treating liver cancer; Wherein, the first expression regulatory element comprises an AFP promoter, and the second expression regulatory element comprises an hTERT promoter; The target gene directly or indirectly connected to the first expression regulatory element is the adenovirus E1A gene, and the target gene connected to the second expression regulatory element is a gene encoding a bispecific antibody against human CD3 and PD-L1. The amino acid sequence of the bispecific antibody against human CD3 and PD-L1 is as shown in SEQ ID No.

2.

2. The composition according to claim 1, wherein The first expression regulatory element further comprises a sequence as shown in SEQ ID No. 1 directly or indirectly connected downstream of the E1A gene.

3. The composition according to claim 2, wherein The first expression regulatory element further comprises a plurality of repeated sequences as shown in SEQ ID No. 1 directly or indirectly connected downstream of the E1A gene, and the plurality is 2 to 8.

4. The composition according to claim 1, wherein The composition further comprises 5-FU.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a medicament for treating liver cancer.

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