阅读说明：本技术 用于癌症治疗的工程化干细胞 (Engineered stem cells for cancer therapy ) 是由 徐伟成 林振寰 陈建霖 郑隆宾 蔡长海 李玮 于 2019-03-18 设计创作，主要内容包括：本揭示内容提供工程化干细胞,其包含载体,所述载体包含含有自杀基因的核酸序列、免疫检查点基因的核酸序列的聚核苷酸及天然细胞毒性触发受体或TNF相关的细胞凋亡诱导配体,其中所述干细胞系靶向肿瘤的细胞。本揭示内容亦提供治疗个体的癌症或增强肿瘤内免疫性或增强肿瘤微环境中的免疫性的方法,其包含向所述个体投与有效量的本揭示内容的工程化干细胞。(The present disclosure provides an engineered stem cell comprising a vector comprising a polynucleotide comprising a nucleic acid sequence of a suicide gene, a nucleic acid sequence of an immune checkpoint gene, and a natural cytotoxicity triggering receptor or TNF-related apoptosis-inducing ligand, wherein the stem cell line targets cells of a tumor. The present disclosure also provides methods of treating cancer or enhancing immunity within a tumor or enhancing immunity in a tumor microenvironment in a subject comprising administering to the subject an effective amount of the engineered stem cells of the present disclosure.)

1. An engineered stem cell comprising a vector comprising a polynucleotide comprising a nucleic acid sequence of a suicide gene, a nucleic acid sequence of an immune checkpoint gene and a natural cytotoxicity triggering receptor sequence or a TNF-related apoptosis-inducing ligand (TRAIL) sequence; wherein the stem cells are tumor-targeted cells.

2. The engineered stem cell of claim 1, wherein the suicide gene is a cytosine deaminase gene, a varicella zoster virus thymidine kinase gene, a nitroreductase gene, an Escherichia coli (Escherichia coli) gpt gene, an Escherichia coli Deo gene, a thymidine kinase gene (TK), caspase 1, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, Fas, or Cytosine Deaminase (CD).

3. The engineered stem cell of claim 1, wherein the suicide gene is TK.

4. The engineered stem cell of claim 3, wherein the TK gene comprises a sequence as set forth in SEQ ID NO1 or 2.

5. The engineered stem cell of claim 1, wherein the immune checkpoint gene is E3 ubiquitin ligase Cbl-b, CTLA-4, PD-1, TIM-3, killer cytostatic receptor (KIR), LAG-3, CD73, Fas, aryl hydrocarbon receptor, Smad2, Smad4, TGF- β receptor, ILT-3, IDO, KIR, or LAG 3.

6. The engineered stem cell of claim 1, wherein the immune checkpoint gene is PD-1.

7. The engineered stem cell of claim 6, wherein the PD-1 gene has a sequence as set forth in SEQ ID NO 3.

8. The engineered stem cell of claim 1, wherein the natural cytotoxicity triggering receptor is NCR1, NCR2, or NCR 3.

9. The engineered stem cell of claim 1, wherein the natural cytotoxicity triggering receptor is NCR 3.

10. The engineered stem cell of claim 9, wherein the NCR3 gene comprises a sequence as set forth in SEQ ID No. 4.

11. The engineered stem cell of claim 1, wherein the TRAIL is TIC 10.

12. The engineered stem cell of claim 1, wherein the TRAIL comprises the sequence set forth in SEQ ID NO. 5.

13. The engineered cell of claim 1, wherein the stem cell is selected from the group consisting of: embryonic stem cells, bone marrow stromal cells, hematopoietic stem cells, and neural stem cells.

14. The engineered cell of claim 13, wherein the stem cell is an MSC.

15. A combination comprising an engineered cell according to any one of claims 1 to 14 and optionally another active agent.

16. Use of an engineered cell according to any one of claims 1 to 14 or a combination according to claim 15 for the manufacture of a medicament for treating cancer or enhancing immunity in a tumor in an individual.

17. The use of claim 16, wherein the cancer is breast cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, prostate cancer, skin cancer, brain cancer, bladder cancer, endometrial cancer, kidney cancer, pancreatic cancer, thyroid cancer, melanoma, leukemia, fibrosarcoma, sarcoma, adenocarcinoma, or glioma.

18. The use of claim 17, wherein the cancer is a metastatic cancer.

19. The use of claim 16, wherein the effective amount is at 100,000(1 x 10)⁵) To 2,000,000 (2X 10)⁶) Within a single cell.

20. The use of claim 16, wherein the method is via increasing tumor-specific CD8 with central memory potential⁺IFN-γ⁺CD44⁺T cells enhance immunity in the tumor microenvironment.

21. The use of claim 16, wherein the method induces a significant reduction in tregs and by this reverses the intratumoral CD8⁺And CD4⁺T cell to Treg ratio, the method also reduces the number of TAMs, which increases CD8 in the TME⁺And CD4⁺Ratio of T cells to TAMs.

22. The use according to claim 16, wherein the engineered stem cells according to any one of claims 1 to 14 or the 15 combination according to claim can be administered to the individual intravenously or intraarterially.

23. The use of claim 16, wherein the engineered stem cells can be administered in combination with another active agent.

24. The use of claim 16, wherein the other active agent is GCV.

25. The use of claim 16, wherein the engineered stem cell and another active agent are administered separately, simultaneously or concurrently.

Technical Field

The present invention relates to engineered stem cells for the treatment of cancer. Specifically, the engineered stem cells comprise at least a suicide gene and an immune checkpoint gene.

Background

Checkpoint immunotherapy by interacting with the PD-1/PD-L1 pathway is at the forefront of cancer treatment (cancer) and offers promise for cure of cancer. The protein encoded by this gene is the natural cytotoxic receptor (NCR3), which can help NK cells lyse tumor cells. However, up to 70% of patients do not respond to treatment and even cause severe complications in some Clinical cases (The Journal of Clinical endoscopy & Metabolism2013,98(4): 1361-. There is a need for improvements that allow inhibitors to selectively accumulate within tumors without eliciting autoimmune responses in peripheral normal tissues.

US20180214544 provides a combination of immune checkpoint blockade and hematopoietic stem cell transplantation and/or hematopoietic stem cell mobilization that produces synergistic effects in disease therapy. However, there is still a need to improve the effect of immune checkpoint inhibitors.

Disclosure of Invention

The present disclosure provides an engineered stem cell comprising a vector comprising a polynucleotide comprising a nucleic acid sequence of a suicide gene, a nucleic acid sequence of an immune checkpoint gene and a natural cytotoxicity triggering receptor sequence or a TNF-related apoptosis-inducing ligand sequence, wherein the stem cell is a tumor-targeted cell.

Some embodiments of the engineered stem cells include embryonic stem cells, bone marrow stromal cells, hematopoietic stem cells, and neural stem cells. A particular example of such an engineered stem cell is an MSC. Another specific example of the engineered stem cell is a umbilical cord mesenchymal stem cell (UMSC).

Some examples of such suicide genes include cytosine deaminase gene, varicella zoster virus thymidine kinase gene, nitroreductase gene, Escherichia coli (Escherichia coli) gpt gene, Escherichia coli Deo gene, thymidine kinase gene (TK), caspase 1, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9 and Fas or Cytosine Deaminase (CD).

Certain examples of such immune checkpoint genes include E3 ubiquitin ligase Cbl-b, CTLA-4, PD-1, TIM-3, killer cytostatic receptor (KIR), LAG-3, CD73, Fas, arene receptor, Smad2, Smad4, TGF- β receptor, ILT-3, IDO, KIR and LAG 3.

Certain examples of the natural cytotoxicity triggering receptors include NCR1, NCR2, and NCR 3.

One embodiment of a TRAIL gene comprises TIC 10.

The present disclosure provides kits or combinations comprising a vector or engineered cell of the present disclosure and optionally another active agent.

The present disclosure also provides methods for treating cancer or enhancing immunity within a tumor in a subject comprising administering to the subject an effective amount of the engineered stem cells of the present disclosure. In one embodiment, the effective amount is at 100,000(1 × 10)⁵) To 2,000,000 (2X 10)⁶) Within a single cell. In one embodiment, the cancer is a metastatic cancer.

Drawings

FIGS. 1A to 1G show in vitro characterization of UMSC and UMSC-TRAIL-TK-PD-1. A. Cellular morphology and biological properties of umbilical cord mesenchymal stem cells (UMSC) from Wharton's jelly, WJ. B. The flow cytometry plots showed that the cells were negative for CD1q, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, and HLA-DR, but positive for CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, and HLA-ABC. Results of RFP and PD-1 flow cytometry and transduction with transgenes (UMSC-PD-1 and UMSC-TRAIL-TK-PD-1). UMSC-TRAIL-TK-PD-1-Luc retained luciferase expression for more than 100 days, and cell proliferation analysis of E by BrdU incorporation and migration by transwell analysis revealed that genetic modification did not affect UMSC-TRAIL-TK-PD-1 cell viability (E- (a)), cell proliferation (E- (b)), or migration (E- (c)) in vitro compared to unlabeled UMSC after 14h of incubation. UMSC-TRAIL-TK-PD-1 exhibits similar behavior to ordinary UMSC without plasmid labeling. G. Identifying glial cell differentiation of UMSC-TRAIL-TK-PD-1 by immunofluorescence using MAP-2, Tuj-1 and GFAP; the results show a refractive cell morphology as common UMSC and extended neurite-like structures arranged in a network.

FIGS. 2A to 2D show in vitro immunological evaluation of UMSC-TRAIL-TK-PD-1. Binding affinity of hrp-conjugated PD-1 protein increased significantly from a dose-dependent manner. B. The gating strategy was based on the rationality of the first gate, exclusion of doublets by FSC-A and FSC-H, selection of 7-AAD⁺(R&D Systems)/CD45⁺Or FSC-A excludes dead cells (B- (a) and B- (B)). Umsc (at a ratio of 1: 1) significantly inhibits CD4⁺And CD8⁺Proliferation of both T cells (C- (a) and C- (b)). However, UMSC-TRAIL-TK-PD-1 significantly increased CD4 at 1:1 or 1:10 ratios⁺And CD8⁺Proliferation of both T cells (C- (a) and C- (b)). D. Compared to UMSC, CD3-CD28 stimulated UMSC-TRAIL-TK-PD-1 exhibits CD4⁺INF-γ⁺Is significantly increased (D- (a)) and CD8⁺CD122⁺Decreases (D- (b)).

FIGS. 3A to 3E show the in vitro suicide and bystander effect of UMSC-TRAIL-TK-PD-1-GFP. A. An increased TK content was found in UMSC-TRAIL-TK-PD-1 by Western blot (Western blot) compared to UMSC-Akt and UMSC. Gcv itself does not affect cell proliferation of UMSC. Phosphorylated GCV induced UMSC-TRAI at 24h and 48h after GCV treatment according to immunohistochemistryApoptosis-like cell damage in L-TK-PD-1-GFP (white arrow) (B- (B)). Cell proliferation of UMSC-TRAIL-TK-PD-1-GFP was inhibited from a dose-dependent manner (B- (a)). C. UMSC-TRAIL-TK-PD-1-GFP significantly attenuated the growth of 4T1-Luc cells (C- (a), C- (b)), Hep55.1C (C- (C) and C- (d)), Pan18-Luc (C- (e) and C- (f)), CT26-Luc (C- (g) and C- (h)) and GL261-Luc (C- (i) and C- (j)) in the presence of 0. mu.g/mL, 1. mu.g/mL, 10. mu.g/mL, 100. mu.g/mL GCV after co-cultivation for 24h,48h and 72 h. In this co-culture system, the cell death rate due to suicide effects slowly reached about one third of the total system during the first two days, and then subsequently accelerated from day 3 to day 6. The same findings showed that most 4T1-Luc cells were killed from day 3 to day 5. Furthermore, quantitative evaluation of apoptotic cells under this bystander effect by PI/annexin-V staining using flow cytometry showed significant cytotoxicity (E- (a) to E- (d)) in a GCV dose-dependent and time-dependent manner. FIGS. 4A to 4C show that UMSC-TRAIL-TK-PD-1 expressing TRAIL exhibits in vitro anti-tumor activity in 4T1-Luc and Hep55.1C-Luc cells. A. Genetically modified UMSC-TRAIL-TK-PD-1 allows for expression of the associated TRAIL protein on the surface of UMSC cells (90%) as measured by FACS analysis. B. UMSC-TRAIL-TK-PD-1 expressing TRAIL induces apoptosis especially at 72 hours after co-culture (4T1-Luc, Hep55.1C-Luc), reduction of apoptosis from cell shrinkage, adherent 4T1-Luc cells (B- (a)) and Hep55.1C-Luc with the appearance of cell debris (B- (B)) as represented by propidium iodide staining (PI staining) (B- (c)). C. Quantitatively, cell death (C- (a) and C- (b)) occurred at 24 hours, 48 hours and 72 hours as measured by FACS analysis, and a large amount of annexin-V was detected in the co-culture⁺PI⁺Dead cells (. gtoreq.70%), in which UMSC-TRAIL-TK-PD-1 is present in a dose-dependent manner.

Fig. 5A to 5G show tumor targeting of UMSC-TRAIL-TK-PD-1-Luc in some tumor models. A. Bioluminescence intensity increased from UMSC-TRAIL-TK-PD-1-Luc cells in a dose-dependent manner as measured by in vitro IVIS. umsc-TRAIL-TK-PD-1-Luc survived and relocated to subcutaneous 4T1 tumors. IVIS ghosts were initially observed 5 days after intravenous UMSC-TRAIL-TK-PD-1-Luc injectionThe bioluminescent signal of the subcutaneous tumor area in the image, after which the intensity gradually increased and peaked at day 14. Intraarterial UMSC-TRAIL-TK-PD-1-Luc transplantation recruited directly to the orthotopic 4T1 tumor region (C) without lung compression (lung entrampent) 2 hours after intrafemoral injection (also applicable to hep55.1c (D) and pan18 tumor region (E)). Subsequently, UMSC-TRAIL-TK-PD-1-Luc survived and relocated to the tumor site. F. Metastatic tumors from the original 4T 1-tumor model significantly recruited UMSC-TRAIL-TK-PD-1-Luc, increasing bioluminescence intensity, as measured by IVIS in multiple metastatic sites. G. Multiple GFP s were found in 4T1 tumors 1 day after treatment, according to immunohistochemical analysis⁺Luciferase enzymes⁺Cells, indicating recruitment of UMSC-TRAIL-TK-PD-1-GFP into the tumor microenvironment.

FIGS. 6A to 6G show the therapeutic effect of UMSC-TRAIL-TK-PD-1 in the 4T1-Luc model. A. The tumoricidal effect of luciferase-expressing 4T1-Luc and Hep55.1C-Luc tumor-expressing mice treated with various strategies of genetically modified UMSC was evaluated by IVIS, tumor volume and survival time after a q4dx3 treatment regimen. B. Prior to treatment, each group of test cells was exposed to 3% O₂Overexpression of CXCR4 (by western blot) was induced from a time-dependent manner in hypoxic pretreatment cultures, thereby enhancing stem cell homing. UMSC-PD-1(UP) and UMSC-trail (ut) groups exhibited therapeutic effects that reduced tumor volume compared to those of IgG control groups as measured by IVIS. In addition, the UMSC-TK-PD-1+ gcv (utpg) group, UMSC-TRAIL-TK + gcv (uttg) group, and UMSC-TRAIL-PD-1(UTP) group showed stronger antitumor effects and exhibited inhibition of tumor growth, respectively. D. Intravenous UTP significantly prolonged survival of 4T1 and the hempa55.1c model compared to the other groups (D- (a) to D- (D)). E. Fewer than 5 lung metastatic nodules were found in UTP-treated mice compared to more than 20 metastases in the lungs of control mice. However, UMSC-tk (ut), UP and UTP did not show a significant reduction in metastasis compared to the control. F-G. Next, to verify whether intraarterial injection of UMSC-TRAIL-TK-PD-1 showed significantly robust therapeutic effects in the 4T1-Luc and Hep55.1C-Luc models following the q7dx2 course of treatment, four groups (UMSC-TK-PD-1+ GCV (UTPG) group, UMSC-TRAIL-TK-PD-1(UTTP) group and UMSC-TRAIL-TK-PD-1+ GCV (UTTPG) group) from examination of tumor growth and median survival time (F- (a)). Prior to the analysis of intra-arterial injections, intravenous administration of the UMSC-TRAIL-TK-PD-1+ gcv (uttpg) group showed stronger antitumor effects (F- (b)) than the IgG control, UTPG and other groups of UTTP, respectively. Importantly, intra-arterial implants revealed a potent therapeutic effect over intravenous implants. Furthermore, in the 4T1-Luc (F- (c-d)) and Hep55.1C-Luc (G- (a-b)) models, the UTTPG group significantly inhibited tumor growth and prolonged median survival of mice compared to the IgG control, UTPG and other groups of UTTP, respectively. Unfortunately, administration of anti-PD-L1 did not show any significant therapeutic effect (F- (c)) in the 4T1-Luc model.

Figures 7A to 7D show that UTTPG treatment enhances immunity in the Tumor Microenvironment (TME). A. The gating strategy was based on the rationality of the first gate, exclusion of doublets by FSC-A and FSC-H, selection of 7-AAD⁺(R&D Systems)/CD45⁺Or FSC-A excludes dead cells (A- (a)). Tumor-infiltrating CD45 across UTTPG treatment groups and other treatment groups⁺There was an overall increase in the percentage of leukocytes (A- (b)). In UTTPG treatment, CD3 compared to the other groups⁺CD8⁺And CD3⁺CD4⁺The frequency of both T cells is significantly increased (A- (c) and A- (d)). Uttpg induced a significant reduction in Treg and TAM (B- (a) and B- (B)), and by this reversed intratumoral CD8⁺(C- (b)) and CD4⁺(C- (a)) ratio of T cells to Tregs. In addition, the number of TAMs decreased dramatically in response to UTTPG treatment, which increased CD8 in TME⁺(C- (d)) and CD4⁺(C- (C)) ratio of T cells to TAM. D. Intracellular granzyme B (Grb)⁺) (D- (b)) and Ki67⁺Significant upregulation of (D- (a)) cells indicates that UTTPG treatment not only increases the anti-tumor immune population, but also effectively achieves activation and proliferation of TIL.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory methods described below are those well known and commonly employed in the art.

As used herein, the terms "a" and "an" and "the" and similar references are to be construed as covering both the singular and the plural.

As used herein, the terms "genetically modified cell," "redirected cell," "genetically engineered cell," or "modified cell" refer to a cell that expresses a recombinant polynucleotide of the invention.

The terms "polynucleotide", "nucleic acid", and "oligonucleotide" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof.

As used herein, the term "gene" refers to a polynucleotide containing at least one Open Reading Frame (ORF) that is capable of encoding a particular polypeptide or protein after transcription and translation.

As used herein, the term "encode" as it applies to a polynucleotide refers to a polynucleotide that is considered to "encode" a polypeptide, which if manipulated from its native state or by methods well known to those skilled in the art, can be transcribed and/or translated to produce mRNA for the polypeptide and/or fragments thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

As used herein, the term "operably linked" refers to a functional linker between a regulatory sequence and a heterologous nucleic acid sequence, such that the latter is expressed. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first and second nucleic acid sequences are in functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence.

As used herein, the term "expression" refers to the process of transcription of a polynucleotide into mRNA and/or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein.

As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system.

As used herein, the term "thymidine kinase" or "TK" means the thymidine kinase suicide gene "TK", which is known in the art to provide biological safety to recombinant vectors. Unless otherwise specified, the term "TK" refers to wild-type (WT) and/or mutated forms of genes known in the art.

As used herein, the term "bone marrow stromal cells," also known as "mesenchymal stem cells" or MSCs, are pluripotent stem cells that can differentiate into multiple cell types.

As used herein, the terms "subject," individual "or" patient "are used interchangeably and refer to a vertebrate, preferably a mammal, more preferably a human.

As used herein, the term "treating" should be understood to include any sign of success in treating, mitigating, or ameliorating an injury, pathology, or condition. This may include parameters such as: (iii) mitigation; (iii) alleviating; reduction of symptoms; slowing the rate of degeneration or decline; less attenuation of the degradation endpoint; improving physical or mental health of a patient; or to prevent the onset of disease.

As used herein, the term "therapeutically effective amount," when used in reference to the symptoms of a disease/condition, refers to the amount and/or concentration of a compound that ameliorates, attenuates, or eliminates the symptoms of one or more diseases/conditions or prevents or delays the onset of one or more symptoms.

Mesenchymal Stem Cells (MSCs) are considered to be cell mediators expressing therapeutic proteins by gene transfer and show a unique tumor homing tropism for targeted delivery of anticancer substances to animal models of various tumors, including melanoma, glioblastoma and breast cancer. There are several advantages, such as ease of isolation and expansion, immune tolerance properties, and systemic or local delivery. While current methods of genetic engineering by viral transduction of DNA to MSCs are applicable as diagnostic and therapeutic strategies for cancer treatment, they can induce deleterious transformations that increase the risk of secondary malignant disease.

It is essential to test whether MSCs can represent an effective vehicle for delivering genetic material for anti-cancer function. Tumor Necrosis Factor (TNF) -related apoptosis-inducing ligand (TRAIL), a promising anti-Cancer death ligand with sequence homology to TNF and FasL, mediates apoptotic effects by binding to its Death Receptor (DR), in particular because upon activation of TRAIL-R1/DR4 and TRAIL-R2/DR5, homotrimers (protein complexes) cause caspase-8 activation, triggering apoptosis (Nat Rev Cancer 2008; 8: 782-98; Science 1998; 281: 1305-8; Eur Jcancer 2006; 42: 2233-40). In addition, suicide gene therapy is based on the transfer of a gene encoding the suicide protein of herpes simplex virus thymidine kinase (HSV-TK), which is selectively sensitized to GCV by the preferential monophosphorylation of the non-toxic prodrug Ganciclovir (GCV) to a toxic compound by the viral TK enzyme (Mol Biol Cell 2002; 13: 4279-95). Chimeric antigen receptor-T cell (CAR-T) immunotherapy combined with suicide gene modification has been shown to not only inhibit tumor overgrowth, but also to improve the safety profile to facilitate clinical development (Journal of Cancer 2011; 2: 378-382).

It has not been demonstrated whether PD-1 or NCR3 overexpressing MSCs will enhance migration and immunosensitization into tumors, induce tumor death, and reduce inflammation. The present disclosure develops natural nanoparticles with inherent anti-tumor capabilities, thereby playing an important role in therapeutic genetic engineering.

In one embodiment, the present disclosure provides an engineered stem cell comprising a vector comprising a polynucleotide comprising a nucleic acid sequence of a suicide gene, an immune checkpoint gene nucleic acid sequence and a natural cytotoxicity triggering receptor sequence or a TNF-related apoptosis-inducing ligand sequence; wherein the stem cells are tumor-targeted cells.

In one embodiment, the tumor-targeted cell is a stem cell selected from the group consisting of: embryonic stem cells, bone marrow stromal cells, hematopoietic stem cells, and neural stem cells.

In one embodiment, the stem cells are MSCs. In one embodiment, the MSCs have the phenotype CD34^-/CD45^-/CD105⁺/CD90⁺/CD73⁺. MSCs have been shown to differentiate in vitro or in vivo, including osteoblasts, chondrocytes, myocytes, and adipocytes. Mesenchyme is embryonic connective tissue, which is derived from the mesoderm and differentiates into hematopoietic and connective tissue, whereas MSCs do not differentiate into hematopoietic cells. Stromal cells are connective tissue cells that form a supporting structure in which the functional cells of the tissue reside.

In genetics, suicide genes will cause cells to kill themselves via apoptosis. In some embodiments, the suicide gene is a cytosine deaminase gene, a varicella zoster virus thymidine kinase gene, a nitroreductase gene, an escherichia coli gpt gene, an escherichia coli Deo gene, a thymidine kinase gene (TK), caspase 1, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, Fas, or Cytosine Deaminase (CD). In one embodiment, the suicide gene is a thymidine kinase gene. In one embodiment, the TK gene is a wild-type TK gene. In another embodiment, the TK gene is a mutated form of the gene. In some embodiments, thymidine kinase sequences include, but are not limited to, the following.

HSV1-TK sequence

CpG-free HSV1-TK sequence

Immune checkpoints are regulators of the immune system. Immune checkpoint molecules are considered as targets for cancer immunotherapy due to their potential for use in various types of cancer. Examples of immune checkpoint genes include, but are not limited to, E3 ubiquitin ligase Cbl-b, CTLA-4, PD-1, TIM-3, killer cytostatic receptor (KIR), LAG-3, CD73, Fas, arene receptor, Smad2, Smad4, TGF-beta receptor, ILT-3, IDO, KIR, and LAG 3. In a certain embodiment, the immune checkpoint gene is PD-1. In some embodiments, the PD-1 sequence includes (but is not limited to) the following sequences.

PD-1 sequence

Natural cytotoxic trigger receptors may also be used in the vectors of the present disclosure. Examples of natural cytotoxicity triggering receptors include, but are not limited to, NCR1, NCR2, and NCR 3. In a certain embodiment, the natural cytotoxicity triggering receptor is NCR 3. In some embodiments, the NCR3 sequence includes (but is not limited to) the following sequences.

NCR3 sequence

TNF-related apoptosis-inducing ligand (TRAIL) is a protein that functions as a ligand to induce cell death processes. Examples of TRAIL genes include, but are not limited to, TIC 10. In some embodiments, the TIC10 sequence includes (but is not limited to) the following sequence.

TRAIL sequences

The vectors of the present disclosure comprise one or more control sequences to regulate expression of the polynucleotides of the present disclosure. The isolated polynucleotide may need or be manipulated prior to insertion into the vector, depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, the control sequences include, inter alia, promoters, leaders, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, suitable promoters are selected based on the host cell selected.

A recombinant expression vector is disclosed comprising a polynucleotide of the present disclosure and one or more expression control regions, such as promoters and terminators, origins of replication, and the like, depending on the type of host into which it is designed to be introduced. Non-limiting examples of constitutive promoters include SFFV, CMV, PKG, MDNU3, SV40, Ef1a, UBC, and CAGG.

In some embodiments, the various nucleic acids and control sequences described herein are joined together to produce a recombinant expression vector that includes one or more convenient restriction sites to allow for insertion or substitution of the polynucleotides of the disclosure at such sites. Alternatively, in some embodiments, the polynucleotides of the present disclosure are expressed by inserting the polynucleotides or nucleic acid constructs comprising the sequences into an appropriate expression vector. In some embodiments involving the production of expression vectors, the coding sequence is positioned in the vector such that the coding sequence is operably linked with the appropriate control sequences for expression. A recombinant expression vector is any suitable vector (e.g., a plasmid or virus) that can conveniently be subjected to recombinant DNA procedures and can bring about the expression of the polynucleotides of the present disclosure. The choice of the vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or a closed loop plasmid. In one embodiment, the vector is a viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alpha virus (alphavirus) vectors, and the like. In a certain embodiment, the viral vector is a lentiviral vector. Lentiviral vectors are based on or derived from oncogenic retroviruses (a subset of MLV-containing retroviruses) and lentiviruses (a subset of HIV-containing retroviruses). Examples of such viruses include, but are not limited to, Human Immunodeficiency Virus (HIV), Equine Infectious Anemia Virus (EIAV), Simian Immunodeficiency Virus (SIV), and Feline Immunodeficiency Virus (FIV). Alternatively, it is contemplated that other retroviruses may be used as the basis for the vector backbone, such as Murine Leukemia Virus (MLV).

In some embodiments, the vectors used in the present disclosure are pLAS3w, pLAS3w. ppuro, pLAS3w. pneo, pLAS3w.phyg, and pLAS3w.pbsd, pCMV- Δ R8.91, or pmd.g.

In another embodiment, the invention provides a kit or combination comprising a vector or engineered cell of the disclosure and optionally another active agent. In one embodiment, the additional active agent is GCV.

The vectors or engineered cells of the present disclosure are typically combined with another carrier, such as a compound or composition, an inert (e.g., a detectable agent or label), or an active, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, or the like, and including a pharmaceutically acceptable carrier. Carriers also include pharmaceutical excipients and additives, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, derivatized sugars, such as alditols, aldonic acids, esterified sugars, and the like, and polysaccharides or sugar polymers). Exemplary protein excipients include serum albumin, such as Human Serum Albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. The carrier further comprises a buffering agent or a pH regulator; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as citrate, ascorbate, gluconate, carbonate, tartrate, succinate, acetate, or phthalate; tris, tromethamine hydrochloride or phosphate buffer.

Any of the compositions described herein may be included in a kit. In a non-limiting example, cells for cell therapy or one or more reagents to produce cells may be included in the kit. The kit may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent. Where more than one component is present in the kit, the kit will typically also contain a second, third or other additional container in which the other component(s) may be separately placed. However, various combinations of components may be included in the vial. The kit may have a single container means, and/or it may have different container means for each compound. The kit of the invention will also generally include means for receiving any container in a closed, restricted form for commercial sale. These containers may include injection or blow molded plastic containers in which the vial is desired to be stored.

In another embodiment, the present invention provides a method for treating cancer or enhancing immunity within a tumor in a subject, comprising administering to the subject an effective amount of an engineered stem cell of the present disclosure. In one embodiment, the effective amount is at 100,000(1 × 10)⁵) To 2,000,000 (2X 10)⁶) Within a single cell. In some embodiments, the effective amount is at 1 × 10⁵To 1X 10⁶Within a single cell.

In one embodiment, the cancer is a metastatic cancer.

In one embodiment, the method is via increasing tumor specific CD8 with central memory potential⁺IFN-γ⁺CD44⁺T cells enhance immunity in the tumor microenvironment. In one embodiment, the method induces a significant reduction in tregs and by this reverses intratumoral CD8⁺And CD4⁺Ratio of T cells to tregs. The method also reduces the number of TAMs, which increases CD8 in the TME⁺And CD4⁺Ratio of T cells to TAMs. In one embodiment, the effective amount is at 100,000(1 × 10)⁵) To 2,000,000 (2X 10)⁶) Within a single cell. In some embodiments, the effective amount is at 1 × 10⁵To 1X 10⁶Within a single cell.

Exemplary cancers to be treated using the methods and compositions as set forth herein are breast cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, prostate cancer, skin cancer, brain cancer, bladder cancer, endometrial cancer, kidney cancer, pancreatic cancer, thyroid cancer, or melanoma or metastatic cancers thereof. Exemplary cancer cells include, but are not limited to, carcinomas, melanomas, leukemias, fibrosarcomas, sarcomas, adenocarcinomas, and gliomas.

Delivery methods include, but are not limited to, intra-arterial, intramuscular, and intravenous. In particular embodiments, it may be desirable to administer the pharmaceutical compositions and/or cells of the present disclosure locally to the area in need of treatment; this can be achieved, for example but not limited to, by local infusion during surgery, by injection or by means of a catheter. In some embodiments, the compositions or cells are administered by intravenous injection. In another embodiment, the compositions or cells are administered by intramuscular injection. These compositions can be administered from one injection or from multiple injections. Solutions containing these cells can be prepared in suitable diluents such as water, ethanol, glycerol, liquid polyethylene glycols, various oils and/or mixtures thereof and other diluents known to those skilled in the art. In some embodiments, the engineered stem cells of the present disclosure can be administered to a subject intravenously or intraarterially. The present disclosure surprisingly finds that the above administration of the engineered stem cells of the present disclosure has beneficial efficacy in treating cancer, enhancing immunity within a tumor, or enhancing immunity in a tumor microenvironment. In particular, intraarterial administration exhibits superior efficacy over intravenous administration.

In one embodiment, the engineered stem cells of the present disclosure can be administered with another active agent. In some embodiments, the engineered stem cell and the another active agent can be administered concurrently, separately, or simultaneously. In one embodiment, the engineered stem cells and the another active agent can be administered periodically. In another embodiment, the additional active agent is GCV.

It will be understood that, if any prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the art.

Although the disclosure has been provided in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing description and examples should not be construed as limiting.

Examples of the invention

The method and the material are as follows:

preparation, isolation and characterization of UMSC and other Stem cells

Collected human umbilical cord tissue approved by the Institutional Review Board (IRB) of the Chinese Medical University Hospital (Taichung) was used without Ca²⁺And Mg²⁺Washed three times with PBS (DPBS, Life Technology). It was mechanically cut in the midline direction by scissors and the umbilical artery, the blood vessels of the vein and the outer membrane (outlining membrane) were separated from the gordonia gel (WJ). The jelly contents were then roughly cut to less than 0.5cm³Treated with collagenase type 1 (Sigma, St Louis, USA) and treated at 37 ℃ in 95% air/5% CO₂Incubate for 3h under a humid atmosphere. The explants were then placed in DMEM containing 10% Fetal Calf Serum (FCS) and antibiotics at 37 ℃ in 95% air/5% CO₂Culturing under humid atmosphere. It was allowed to stand for 5-7 days to allow migration of cells from the explant. The cell morphology of umbilical cord-derived mesenchymal stem cells (UMSCs) became uniformly spindle-shaped in culture after 4-8 passages and specific surface molecules from WJ were characterized by flow cytometric analysis. Cells were detached with 2mM EDTA in PBS, washed with PBS containing 2% BSA and 0.1% sodium azide (Sigma, USA) and incubated with individual antibodies conjugated with Fluorescein Isothiocyanate (FITC) or Phycoerythrin (PE), including CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1q, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC and HLA-DR (BD, PharMingen). Thereafter, cells were analyzed using a Becton Dickinson flow cytometer (Becton Dickinson, San Jose, Calif.).

Other types of stem cells can be obtained and cultured according to procedures known in the art.

Plasmid construction:

the plasmid TK, NCR3, TRAIL, PD-1 and GFP cDNA from TK (0.1. mu.g) (pUNO1-HSV1TK, InvivoGen), NCR3 (0.1. mu.g) (pLenti-C-mGFP-NCR3, Origene), TRAIL (0.1. mu.g) (pCMV6-Myc-DDK-TRAIL, Origene) or PDCD-1 (0.1. mu.g) (pLenti-C-Myc-DDK-PDCD1, Origene) was transfected into pIRES (Clontech) or pSF-CMV-UMSbfI (pIRES-TK-1, RES-TK-1, pIRES-pIRES, etc. by specific restriction enzyme linkers (EcoR 1 and Nhe1 in TK, and BamH1 and Not1 in PD-1) to construct pIRES-TK-1, pIRES-TK-1, pIRES-1, and so as described in the plasmid, UMSC-TK-GFP and UMSC-PD-1-GFP. The above constructs can be transfected into other types of stem cells.

Lentiviral plasmids:

lentiviral vectors (pLAS3w) and packaging (psPAX 2)/encapsulating plasmids (pmd2.g) were obtained from academy Sinica, Taiwan. cDNAs encoding full-length human TK, NCR3, TRAIL, PD-1 and control GFP, which were recombined from cDNAs (pUNO1-HSV1TK, InvivoGen; pLenti-C-mGFP-NCR3, Origene; pCMV6-Myc-DDK-TRAIL, Origene pLenti-C-Myc-DDK-PDCD1, Origene) were transferred to pUltra-CMV-Sbf 1(Oxford Genetics) by specific restriction enzyme linkers (EcoR 1 and Nhe1 in TK, BamH1 and Not1 in PD-1) to construct pUltra-TK-PD-1, pUltra-TK-PD, pUltra-CMV-1-and TRAIL-GFP. Subsequently, these templates were amplified by PCR using specific primers and subcloned into lentiviral vector backbone plasmids pLAS2w and pLAS3w (Academia Sinica, Taiwan) by restriction enzyme digestion (Lenti-TK-GFP, Lenti-PD-1-GFP, Lenti-TRAIL-GFP, Lenti-TK-PD-1-GFP, Lenti-TRAIL-PD-1-GFP and Lenti-TRAIL-TK-PD-1-GFP). To generate recombinant lentiviruses carrying TK, PD-1, TRAIL and control GFP, the recombinant plasmids and the vector and packaging and encapsulating plasmids were co-transfected into 293T cells at a ratio of 3:3:1 by XtreemeGene HP DNA (Roche). The culture supernatant containing the virus particles was collected after 36 hours and half the volume of the culture supernatant was again collected after another 24 hours, and then centrifuged at 15,000rpm/min for 10min to remove debris, and then transferred to a 36-mL ultracentrifuge tube to ultracentrifuge at 25,000rpm/min for 3 h. The pellet containing lentivirus was resuspended. The virus was thawed immediately prior to titration and cell transduction. UMSC is infected with a suitable lentivirus, wherein the gene transfer efficiency reaches at least 80%.

Lentiviral transduction

Lentiviral plasmid transduction was performed in 6-well plates. Unless otherwise specified, UMSC is 1 × 10⁵Individual cells/well were seeded in triplicate, with a final volume of 1 ml/well and a multiplicity of infection (MOI) of 5. Protamine sulfate (Sigma-Aldrich) was added from 8mg/ml stock (in DMEM-LG, sterile filtered) to obtain the desired final concentration. The cells were transduced for 24 hours, and then replaced with 1.5 ml/well to construct UMSC-TRAIL-TK-PD-1, UMSC-TRAIL, UMSC-TK, and UMSC-PD-1. Overgrown cells were seeded onto 6-well plates for drug screening using 1.0mg/ml G418 or puromycin (puromycin) solution (Sigma). The medium was changed every 2 days. Expression of Green Fluorescent Protein (GFP) was observed using an inverted fluorescence microscope based on the color of the medium and the state of the cells. After 7 days of selection, the complete medium without G418 was replaced and the culture was continued.

Construction of piggyBac translocation subsystem for stabilizing cell lines

The piggyBac vector pPB-CMV-MCS-EF1 α -RedPuro, which contains a Multiple Cloning Site (MCS), piggyBac terminal repeat (PB-TR), Core Insulator (CI) and puromycin selectable marker (BSD) and is fused with RFP driven by human EF1 α, was used as a base vector (System Bioscience). DNA fragments containing TRAIL-TK-PD-1, TRAIL, TK and PD-1 (from pUltra-TRAIL-TK-PD-1, pUltra-TRAIL, pUltra-TK and pUltra-PD-1) were PCR amplified and sub-cloned into pPB-CMV-MCS-EF1 alpha-RedPuro vector, which precedes the coding region of EF1 alpha. Detailed information about the construction of the vector may be obtained on demand. To generate UMSC stable cells, the above plasmids were co-transfected with piggyBac transposase expression vectors (System Biosciences) into UMSC cells by electroporation method using Amaxa Nucleofector II (Lonza). Stable cells (UMSC-TRAIL-TK-PD-1, UMSC-TRAIL, UMSC-TK and UMSC-PD-1) were selected in the presence of puromycin.

In vitro proliferation, migration and differentiation assay

To examine cell proliferation and migration, bromodeoxyuridine (BrdU) incorporation and transwell migration assays were performed to compare UMSC-TRAIL-TK-PD-1 or UMSC. Proliferation of UMSC-TRAIL-TK-PD-1 or UMSC was tested by measuring BrdU incorporation (10 μ M) using the BrdU chemiluminescence immunoassay kit (Roche) and further confirmed by counting Trypan blue (Trypan blue) cells. After 4-6h starvation (incubation in serum-deficient medium), UMSC was incubated in medium for 2 days and pulse-loaded with 10. mu.M BrdU for 12h as previously described (J Clin Invest 2009; 119: 1997). The UMSC was then incubated with anti-BrdU-peroxidase for 90min and the staining developed by incubation with the substrate solution for 3 min. Plates were read using an Lmax microplate luminometer (Molecular Devices). The results as shown in fig. S5 were analyzed and presented as percent increase (%) relative to the control.

As previously described and modified to evaluate cell migration assays (EMBO Mol Med 2013; 5: 1227-. Briefly, UMSC-TK-PD-1 or UMSC was placed in the upper chamber (transwell: 6.5-mm diameter, 5.0-mm pore size) according to the manufacturer's instructions (Costar, code 3421). SDF-1 α (100ng/mL, R) was used in the lower chamber&D System, positive control). At 5% CO₂The analysis was performed in an incubator at 37 ℃ over a 4-h incubation period. Since almost all cells stay on the bottom side of the membrane after migration, quantification can be performed by simply counting these cells. Adherent cells at the bottom side of the membrane were counted under a microscope as previously described.

Adipogenic differentiation was induced according to the methods previously described (J Orthop Res 2002; 20: 1060). Briefly, a confluent monolayer culture of UMSC-TK-PD-1 or UMSC is grown in adipogenic differentiation medium consisting of: DMEM-high glucose (DMEM-HG, Sigma), 100U/mL penicillin, 100mg/mL streptomycin, 100mM insulin (Sigma), 500mM 3-isobutyl-1-methylxanthine (Sigma), 1mM dexamethasone (Sigma), 100mM indomethacin (Sigma), and 10% FCS. Cells maintained in normal UMSC medium were used as negative controls. Adipogenic differentiation was performed three times a week. To assess adipogenic differentiation, cells were stained with 0.3% oil red o (sigma) for 10min at room temperature (to label intracellular lipid accumulation) and counterstained with hematoxylin.

To induce osteogenic differentiation, confluent monolayers of UMSC-TK-PD-1 or UMSC cultures were grown in DMEM-high glucose (DMEM-HG, Sigma) containing 100U/mL penicillin (Sigma), 100mg/mL streptomycin (Sigma), 50mg/mL L-ascorbic acid 2-phosphate (Sigma), 10mM b-glycerophosphate (Sigma), 100nM dexamethasone (Sigma) and 10% FCS. Cells maintained in normal UMSC medium were used as negative controls. Osteogenic differentiation medium was changed three times per week. Calcium mineralization was detected using alizarin red S staining (1%, Sigma) to determine the extent of osteogenesis (J Biomed Mater Res 1998,42, 433).

A high density pellet cell culture system (J Biomed Mater Res 1998,42,433) was used to induce chondrogenic differentiation of UMSC-TK-PD-1 or UMSC. Washing cells in serum-free chondrogenic differentiation medium, said medium consisting of: DMEM-HG, 100U/mL penicillin, 100mg/mL streptomycin, 50mg/mL L-ascorbic acid 2-phosphate, 40mg/mL proline (Sigma), 100mg/mL sodium pyruvate (Sigma), 100nM dexamethasone, and ITS-plus (10mg/mL bovine insulin, 5.5mg/mL transferrin, 5mg/mL sodium selenite, 4.7mg/mL linoleic acid, and 0.5mg/mL bovine serum albumin, Sigma). Aliquots of 250,000 cells were resuspended in chondrogenic differentiation medium and centrifuged at 250 Xg, and then 10ng/mL TGF-. beta.1 (R & D Systems) was added. Pellets maintained in chondrogenic differentiation medium without TGF-. beta.1 served as a negative control. Media was changed twice weekly. Chondrogenic differentiation of pellet cultures was confirmed histologically using an Alcian blue (Alcian blue) stain (Sigma) of sulfated proteoglycans. In addition, as previously described, endothelial cells were induced to differentiate into vascular tubes by culturing UMSC-TK-PD-1 or UMSC for 2-3 days in EBM (Cambrex) in 24-well plates pre-coated with matrigel (300. mu.L/well; Becton Dickinson) and vascular endothelial growth factor (VEGF,10ng/ml, Sigma).

To induce neural cell differentiation, UMSC-TK-PD-1 or UMSC was cultured with DMEM using a modified three-step method (Stem Cells Transl Med.2015; 4: 775-88). Briefly, in the neural induction step, cells were plated from low density onto 6-well plates containing fibronectin (Sigma) and then sequentially exposed to (1) DMEM-HG (Sigma) containing 10% FCS and 10ng/mL bFGF (R & D Systems) (Sigma) for 24h, (2) DMEM-HG containing 1mM β -mercaptoethanol (β ME, Sigma) and 10ng/mL NT-3(R & D Systems) for 2 days in the neural restriction step, and (3) DMEM-HG containing NT-3(10ng/mL, R & D Systems), NGF (10ng/mL, R & D Systems) and BDNF (50/mL, R & D Systems) for 3 to 7 days in the neural differentiation step. After cell differentiation, the cells were left for immunohistochemical analysis.

Flow cytometry

For analysis of cell surface marker expression, cells were detached with 2mM EDTA in PBS, washed with PBS containing BSA (2%) and sodium azide (0.1%), and then incubated with respective antibodies conjugated with Fluorescein Isothiocyanate (FITC) or Phycoerythrin (PE) until analysis. According to the previous document (Mucosal Immunol 2013,6(3):498-510), duplex exclusion by FSC-A and FSC-H, selection of 7-AAD based on adjustment of the first gate⁺(R&D Systems)/CD45⁺Or FSC-A excludes dead cells for gating. As a control, cells were stained with mouse IgG1 isotype control antibody. Antibodies to PD-1, PD-L1, CD3, CD8, CD4, CD25, Foxp3, CD44, CD45, CD11b, F4/80, IFN-. gamma., CD206, TRAIL and GFP for flow cytometry were purchased from BD Biosciences. Cells were analyzed using facscan (BD) with CellQuest Analysis (BD Biosciences) and FlowJo software v.8.8(TreeStar Inc.). Results are expressed as a percentage of positively stained cells relative to total cells. For quantitative comparison of surface protein expression, the fluorescence intensity of each sample was presented as Median Fluorescence Intensity (MFI). For the intracellular staining of Ki-67 and granzyme B, TIL was incubated in the presence of 1. mu.g/ml anti-CD 3 for 48 h. The cells were then incubated with anti-CD 8, followed by permeabilization using Triton x100, and then stained with antibodies to Ki-67(Millipore) and granzyme B. Data were analyzed using a facscan (BD) with CellQuest Analysis (BD Biosciences) and FlowJo v.8.8 (TreeStar).

In vitro analysis of antigen-specific T cell responses

Splenocytes from BALB/c mice (2X 10)⁶) Cultures were performed in 24-well plates in RPMI-1640 medium (Gibco) supplemented with 10% fbs (sigma), 1% penicillin/streptomycin (Gibco). Then, splenocytes (2X 10) co-cultured with UMSC-TRAIL-TK-PD-1 were allowed to co-culture⁵) Kept unstimulated or incubated with CD3-CD28 beads (Dynabeads, Thermo). For proliferation assays, splenocytes were stained with carboxyfluorescing yellow succinimidyl ester (CFSE) (Invitrogen) as previously described (Nat Protoc.2007; 2: 2049-56). Proliferation Index (PI) is used to estimate proliferation/division of cells, which can be calculated by the following formula: PI is the total number after proliferation/the total number before proliferation. After 6 days of culture, cells were harvested and stained to analyze the proliferation of subsets of tregs, CD 4-and CD8-T cells. Alternatively, to analyze proliferation after 6 days of culture in longitudinal samples with limited cell numbers, non-CFSE stained splenocytes were cultured as previously described and stained with Ki67 or isotype control antibody. Fold change in proliferation (FC proliferation) was calculated as the ratio of proliferation under UMSC-TRAIL-TK-PD-1 conditions divided by proliferation under control conditions.

In addition, by mixing 1X 10 in flat-bottomed 96-well microtiter plates⁵Cell responder CD4 from mouse spleen and enriched by Nylon fleece column (Polysciences)⁺T cells were co-cultured with allogeneic Dendritic Cells (DCs) at a ratio of 10:1 (T: DCs) to perform Mixed Lymphocyte Reaction (MLR) analysis. Make CD4⁺T cells and allogeneic DCs in UMSC-TK-PD-1 (10)²、10³And 10⁴) Incubate for 6 days in the absence or presence. Effector T cells were stimulated a total of three times in succession. Culture supernatants were harvested on day 5 for ELISA analysis of IFN-. gamma.and IL-12 secretion (R)&D)。

Suicide effect of UMSC-TK-PD-1 and Ganciclovir (GCV) in vitro

To study the biological effects in vitro, the suicidal ability of the combination of UMSC-TK-PD-1 and GCV was analyzed. At 37 ℃ in 5% CO₂After 24h incubation, various doses of GCV (0.1. mu.g/mL, 1. mu.g/mL, 10. mu.g/mL and 100. mu.g/mL) were added daily to eachIn wells, for 7 consecutive days. Cell viability was assessed by MTT assay (Invitrogen) and GFP fluorescence intensity was assessed by luminometer (Promega).

In vitro bystander effect analysis

Will be at 37 ℃ in 5% CO₂4T1-Luc (BCRC, Taiwan), CT26-Luc (BCRC, Taiwan) or Hep-55.1C-Luc (BCRC, Taiwan) cells (1X 10) co-cultured in DMEM containing 10% FBS⁴Individual cells) and various numbers of UMSC-TRAIL-TK-PD-1 cells (UMSC-TRAIL-TK-PD-1: tumor cell ratios ═ 1:1, 1:4, 1:16, 1:32, and 1:64) were seeded on 24-well plates. The medium was replaced daily with fresh medium containing 100. mu.g/mL GCV for 7 consecutive days. UMSC-TRAIL-TK-PD-1 and 4T1-Luc cells were also inoculated in DMEM medium containing 10% FBS without GCV as respective controls. After 8 days, luciferase fluorescence intensity according to photometer (Promega) was taken from 5 random fields to determine cell density. Further studies of the time course of bystander effect of the above co-culture system were performed daily in the same number of UMSC-TRAIL-TK-PD-1 and 4T1-Luc cells seeded in 12-well plates containing GCV (100. mu.g/mL). The cell death rate was measured as percent fluorescence intensity of GFP and luciferase by a luminometer (Promega).

In vitro apoptosis assay

To investigate the pro-apoptotic potential against various tumor cells, co-cultures were performed at a 1:2 ratio and cytotoxicity was assessed at 24h by annexin-V-FITC/Propidium Iodide (PI) staining (eBioscience) using FACScanto II. Tumor cell populations were gated based on Forward Scatter (FSC) and Side Scatter (SSC) parameters.

Mouse model and tumor inoculation

All Animal experiments were conducted according to the institutional guidelines for Animal Research of Chinese Medical University (institutional guidelines on Animal Research of Chinese Medical University). A mouse cancer model was established using 4T1, CT26, Hep-55.1C, CT26-Luc, 4T1-Luc or Hep-55.1C-Luc, using female BALB/C mice 6 to 8 weeks old. Briefly, 4T1 cells (1X 10)⁶) Implantation of female BALB/c mice in the 4 th mouse mammary fat pad on the right flank and on the fourth post-tumor implantationTreatment was started on day 8.

In vivo UMSC migration assay

To examine the biodistribution of stem cells injected intravenously or intraarterially, the luciferase gene (pHAGE PGK-GFP-IRES-LUC-W, Addgene) was sub-cloned into pUltra-TRAIL-TK-PD-1, and then sub-cloned into pLAS3W to construct Lenti-TRAIL-TK-PD-1-Luc. UMSC (UMSC-TRAIL-TK-PD-1-Luc) (2X 10) engineered by Lenti-TRAIL-TK-PD-1-Luc 7 days after tumor inoculation⁶Individual cells) were injected into the femoral vein or artery of 4T1 tumor-bearing mice for several days. Ex vivo imaging was performed by placing whole animals in the IVIS luminea imaging system (Xenogen) at the indicated time points (6h, 1d, 3d, 6d, 9d and 14d) after UMSC-TRAIL-TK-PD-1-Luc injection and analyzing fluorescence based on manufacturer's recommendations. Fluorescence intensity was measured as photons/sec/cm by Living Image software (Xenogen)². Mice were sacrificed 24 hours after UMSC injection and then various organs (lung, liver, spleen, heart, kidney and brain) were isolated. Each organ was minced, treated with collagenase, and prepared for flow cytometry analysis.

Bioluminescence imaging (BLI)

Animals were imaged using IVIS imaging System 200series (Xenogen) to record bioluminescent signals (luciferase expression) emitted from 4T1-Luc, CT26-Luc, Hep-55.1C-Luc. Animals were anesthetized with isoflurane and thus received an intraperitoneal injection of D-luciferin (Caliper) at a dose of 270mg/g body weight. Imaging acquisition was performed 15min after intraperitoneal injection of luciferin. For BLI analysis, the IVIS System (Xenogen) was used to define regions of interest encompassing the intracranial signal region, and the total photon flux was recorded. To facilitate comparison of cell engraftment rates, the luminescence score for each animal was normalized to its own luminescence reading on day 14, by which each mouse was allowed to serve as its own control.

In vivo therapeutic Effect of UMSC-TRAIL-TK-PD-1 on tumor-bearing mice

In the 4T1-Luc and Hep55.1C-Luc mouse models, it was first examined whether intravenous injection of UMSC-TRAIL-TK-PD-1 could significantly induce tumoricidal effects compared to anti-PD-1 (Roche) or IgG controls. The treatment groups were then subdivided into six groups(FIG. 6A): IgG-control group; UMSC-PD-1(UP) group; UMSC-TRAIL (UT) group; UMSC-TRAIL-PD-1(UTP) group; UMSC-TRAIL-TK + GCV (UTTG) group; and UMSC-TK-PD-1+ GCV (UTPG) group. Prior to each treatment, cells were subjected to a hypoxic pretreatment regimen in which 3% O was used₂Incubation for 24 hours in content induced upregulation of CXCR4(Millipore), thereby enhancing tumor homing effects (Cancer Research 2012; 73: 2333-2344). By repeating the second and third injections in each group at 10-day intervals 5X 10⁵Individual cells were evaluated for the anti-tumor effect of sequential therapy. GCV (50mg/kg) was administered intraperitoneally starting on day 2 after each treatment administration for 7 consecutive days.

Next, to further confirm whether intra-arterial injection of UMSC-TRAIL-TK-PD-1 could significantly induce tumoricidal effects compared to UMSC-TK-PD-1, the treatment group was subdivided into six groups (fig. 6F): IgG-control group; UMSC-TK-PD-1+ GCV (UTPG) group; UMSC-TRAIL-TK-PD-1(UTTP) group; and UMSC-TRAIL-TK-PD-1+ GCV (UTTPG) group.

Survival study

To determine the therapeutic effect of UMSC-TRAIL-TK-PD-1 on the survival of 4T1-Luc and hep55.1c-Luc tumor mice in vivo, mice were treated with six different therapeutic targets via the right femoral vein (n ═ 8) three consecutive times every 4 days (q4d × 3) within 10 days after tumor inoculation. Tumor volume was monitored using a digital caliper (Mitutoyo) every 2-3d using the following formula: tumor(s)(equation 1), where W is the width of the tumor and L is the length of the tumor (W)<L). For ethical reasons, when the volume exceeds 3,000mm³When needed, animals were euthanized. Mice were sacrificed when tumor size reached a maximum diameter of 2cm or when their body weight was reduced to less than 80%. Survival was reported from median and mean survival time with 95% confidence intervals using the Kaplan-Meier survival assay. The statistical difference between these different conditions was determined by log rank analysis (n-8).

Isolation of infiltrating leukocytes (TIL), splenocytes, and Peripheral Blood Mononuclear Cells (PBMC)

Leukocytes in tumor, spleen or peripheral blood were collected from freshly euthanized mice 4 weeks after the last treatment. Single cell suspensions of Tumor Infiltrating Lymphocytes (TILs) were prepared using the methods previously described (Blood 2005; 06: 2339). Briefly, collagenase type IV (2.5mg ml) was used^-1Gibco) digested tumor tissue for 20min, TIL was isolated, and concentrated by discontinuous percoll gradient (GE Healthcare) centrifugation. CD8 in TIL suspension was isolated by mixing with α CD8 microbeads (Miltenyi Biotec) on a MACS column or staining with anti-CD 8 antibody on a FACSAria (BD biosciences) sorter⁺T cells (purity)>95%). From CD8⁺The percentage of T cells multiplied by the total number of lymphocytes from the percoll gradient, which was divided by 100 and the weight of the tumor, to obtain the infiltrative CD8⁺Total T cells/gram tumor. Tumor-associated macrophages (TAM, CD11 b) in TIL suspensions were examined on FACSAria (BD biosciences) after staining with anti-CD 11b, anti-CD 206 or anti-F4/80 antibodies⁺CD206⁺F4/80⁺Cell) (purity)>95%). Purification of regulatory T lymphocytes (Treg, CD4+ CD25+) using a separation kit (Miltenyi Biotec) (purity)>90%). Regulatory T lymphocytes (CD 4) were analyzed on FACSAria (BD biosciences) after staining with anti-CD 4, anti-CD 25, and anti-Foxp 3 antibodies⁺CD25⁺Foxp3⁺Cell) (purity)>95%). The spleens were teased open and filtered with a nylon mesh screen to obtain a single cell suspension. To further generate single cell spleen cell suspensions, red blood cells were removed by using RBC lysis buffer. Spleen CD8 was isolated by mixing cells with α CD8 microbeads (Miltenyi Biotec) on a MACS column or staining with anti-CD 8 antibodies on a FACSAria (BD biosciences) sorter⁺T cells (purity)>95％)。

Peripheral Blood Mononuclear Cells (PBMC) were isolated from each mouse (blood.2001; 98: 3520-6). The cells were harvested using Ficoll-Histopaque (Sigma Aldrich) centrifugation (science.1997; 275:964-7) and washed twice with 1mM EDTA in PBS for further experiments.

Flow cytometry

The TIL suspension was washed with PBS containing BSA (2%) and sodium buntanide (0.1%). Cells were stained with individual fluorescent dye-conjugated monoclonal antibodies to cell surface markers as follows: anti-PD-L1 (MIH5), anti-CD 3(145-2C11), anti-CD 8(53-6.7), anti-CD 11b (M1/70), anti-CD 45(30-F11), anti-IFN-gamma (XMG1.2), anti-CD 44(IM7.8.1R), anti-CD 4(GK1.5), anti-CD 25(PC61.5), anti-Foxp 3(MF23), anti-F4/80 (BM8), and anti-CD 206(MR5D 3). As controls, cells were stained with mouse IgG1 isotype control or IgG2 isotype control antibody. Cells were analyzed using facscan (BD) with CellQuest Analysis (BD Biosciences) and FlowJo software v.8.8(TreeStar Inc.).

According to the previous literature (Mucosal Immunol.2013; 6:498-510), duplex was excluded by FSC-A and FSC-H, dead cells were excluded and further selected as 7AAD based on the correct rationality of the first gate⁺/CD45⁺(or FSC-A) to perform gating. CD8 from TIL or splenocyte suspension was then analyzed using FACScan (BD) with CellQuest Analysis (BD Biosciences) and FlowJo software v.8.8(Treestar Inc.)⁺T cell, CD4⁺T cells, tregs and TAMs. Results are expressed as a percentage of positively stained cells relative to total cells. Differences between groups were assessed by two-way ANOVA and Newman-Keuls post hoc tests. P value<0.05 was considered significant.

Isolation of CD8 from TIL⁺CD44⁺IFN-γ⁺T cells

Separation of CD8 in TIL suspensions by mixing with α CD8 microbeads (Miltenyi Biotec) on a MACS column⁺T cells. To examine the expression of IFN-. gamma.and CD44, isolated CD8 was transfected with anti-mouse CD28 mAb (0.5. mu.g), Monensin (Monensin) and brefeldin A (brefeldin A)⁺T cells were treated for 3 hours. At the same time, make it and 1 × 10⁶Irradiated 4T1-Luc cells (at 84cGy min^-10.5-mm Cu filter, Philipsx ray unit) were co-incubated at 37 ℃ for 24 h. Flow cytometric analysis of IFN- γ and CD44 expression was then performed using the BD Cytofix/Cytoperm Plus set, following the manufacturer's instructions.

CD8⁺Evaluation of Ki-67 and granzyme B expression in T cells

Intracellular binding of Ki-67 and granzyme BStaining, TIL in anti-CD 3 (1. mu.g ml)^-1) Culturing in the presence for 48 h. The cells were then incubated with anti-CD 8, followed by permeabilization with triton 100 and staining with antibodies against Ki-67 and granzyme B (Millipore).

CFSE test

Tregs isolated from tumors were incubated with splenic CD8 treated with Fluorosuccinimidyl Carbodiacetate (CFSE)⁺T cells were co-cultured in the presence of CD3 antibody, and the fluorescence intensity of CFSE was used to monitor CD8⁺Proliferation of T cells. CFSE^{Is low in}Cells are defined as cells with lower fluorescence intensity than the original population, which represents expanded CD8⁺T cells.

Interleukin measurement

TIL from mice treated with different regimens at 2X 10⁵cells/mL were cultured directly for 48h in 6-well plates in PRMI-1640(Invitrogen) medium containing 2mM L-glutamic acid (Sigma-Aldrich). ELISA kit Using Quantikine (R)&D Systems) to measure the levels of TNF- α, VEGF, IL-10, and TGF- β. Semi-quantitative analysis of TNF-. alpha.VEGF, IL-10 and TGF-. beta.contents in the culture supernatant and serum was performed. Optical density was measured using a spectrophotometer (Molecular Devices) and standard curves were generated using the program softmax (Molecular Devices).

Evaluation of transfer resistance

Mice challenged with 4T1-Luc, CT26-Luc, or Hep-55.1C-Luc tumors were examined for lung metastasis by direct visual counting of metastatic nodules. The lungs were then excised and washed once in water and further fixed by dipping into 4% PFA and dehydrated in 30% sucrose at room temperature. Surface metastases were then expressed as white nodules and counted under the microscope.

Immunohistochemical evaluation

Animals were anesthetized with chloral hydrate (0.4g/kg, ip) and their abdominal skin tissue was fixed by transcardial perfusion with saline, followed by immersion in 4% paraformaldehyde. Tissue samples were dehydrated in 30% sucrose, frozen on dry ice, and then cut into a series of adjacent 6- μm thick coronal sections using a cryostat. Sections were stained with H & E and Prussian blue (for iron discrimination) for observation by optical microscopy (Nikon, E600). Each section was immunostained using a secondary antibody (1: 500; Jackson Immunoresearch) coupled with FITC or Cy-3, with antibodies against CD4(1: 100; BD), CD8(1: 400; BD), and then analyzed in three-dimensional images using a Carl Zeiss LSM510 laser scanning confocal microscope. The total number of cells co-stained with cell type specific markers was measured as previously described (J.Cereb. blood Flow Metab.2008; 28,1804-.

TUNEL assay

Apoptosis was analyzed by immunohistochemistry using the commercially available TUNEL staining kit (DeadEnd Fluorimetric TUNEL system; Promega), as previously described (Proceedings of the National Academy of sciences 2009; 106, 9391-9396). Percent TUNEL labeling was expressed as the number of TUNEL positive nuclei divided by the total number of DAPI stained nuclei (nat. protocols 2016; 11: 688-713; PLoS Genet. 2009; 5: e 1000379). The apoptosis index is expressed as the percentage of TUNEL positive apoptotic nuclei divided by the total number of nuclei visualized by DAPI counterstaining obtained from counts of randomly selected microscopic fields.

Evaluation of immune-related adverse events (irAE)

Groups were evaluated for irAE after treatment, including: (1) weight monitoring, (2) histology, (3) immune cell infiltration and (4) liver and kidney function. Body weight of mice was monitored during the treatment period. In addition, groups of treated mice (n-6) were evaluated for H4 weeks after tumor inoculation&E stained liver, lung, spleen, kidney and colon sections for histological analysis. Examination of liver, colon, kidney and lung by IHC for CD8⁺And CD4⁺T cell infiltration (Cancer Res 2016; 76:5288-²The number of positive cells in 10 high power fields was scored. In addition, biochemical profiles of ALT, AST, creatinine and glucose were measured by a Beckman Unicell DxC800 analyzer using each group (n ═ 6) of mouse sera from consecutive time points (0d, 5d, 10d, 15d, 20d, 25d and 30 d).

Statistical analysis

All measurements in this study were performed in a blinded design. Results are expressed as mean ± SEM. The significance of the mean difference between the control and treated groups was assessed using the Two-tailed Student's t test. Differences between groups were assessed by two-way ANOVA and Newman-Keuls post hoc tests. P values <0.05 were considered significant.

Example 1 in vitro characterization of UMSC and UMSC-TK-PD-1

Primary cultures of umbilical cord mesenchymal stem cells (UMSC) were prepared from Wharton's Jelly (WJ) and analyzed for cell morphology and biological properties (fig. 1A). Flow cytometry revealed that cells were negative for CD1q, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, and HLA-DR, but positive for CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49B, and HLA-ABC (fig. 1B). These observations indicate that UMSC has the same surface markers as Mesenchymal Stem Cells (MSCs), which is consistent with the observations of bone marrow MSCs (J Cell Sci 2004,117,2971).

To evaluate the transfection efficiency of UMSC, UMSC-TRAIL-TK-PD-1 was analyzed for RFP fluorescence and PD-1 expression by flow cytometry studies. The uptake potency was confirmed to be 55% -65% on average by RFP and PD-1 flow cytometry results 36h to 48h post-transfection (fig. 1C). Subsequently, 3-5 days after puromycin or G418 selection, more than 90% of the cells were completely transduced with the transgene (fig. 1C).

UMSC-TRAIL-TK-PD-1-Luc retained luciferase expression for more than 100 days (fig. 1D), and cell viability by MTT assay, cell proliferation assay by BrdU incorporation and migration by transwell assay (fig. 1E) revealed that pLAS3w-TRAIL-TK-PD-1 labeling did not affect UMSC-TRAIL-TK-PD-1 cell viability, cell proliferation or migration in vitro after 14h of incubation compared to unlabeled UMSC.

To confirm whether UMSC-TRAIL-TK-PD-1 still has pluripotent differentiation potential, adipogenic, chondrogenic, osteogenic and angiogenic assays demonstrated that UMSC-TRAIL-TK-PD-1 exhibited similar behavior to normal UMSC without plasmid labeling (fig. 1F). Glial cell differentiation of UMSC-TRAIL-TK-PD-1 was identified by immunofluorescence of MAP-2, Tuj-1 and GFAP and exhibited a refractive cell body morphology as common UMSC and extended neurite-like structures arranged in a network (fig. 1G). Therefore, UMSC-TRAIL-TK-PD-1 does not lose cell differentiation potential in vitro.

Example 2 binding of PD-L1 to specific proteins of UMSC-TRAIL-TK-PD-1 in vitro

Since tumor cells express PD-L1 for the purpose of immune escape (Trends immunol. 2006; 27:195-201), genetically modified UMSCs of UMSC-TRAIL-TK-PD-1 were created, in which the presented PD-1 can capture tumor cells via PD-1/PD-L1 interactions. To demonstrate the protein-ligand binding affinity of UMSC-TRAIL-TK-PD-1, RLU of HRP-conjugated PD-1 protein was analyzed by ELISA at various concentrations. UMSC-TRAIL-TK-PD-1 was incubated with HRP-conjugated PD-L1 protein at 37 ℃ for 2 hours. The binding affinity of HRP-conjugated PD-1 protein increased significantly from a dose-dependent manner (fig. 2A). The results indicate that UMSC-TRAIL-TK-PD-1 has high binding efficiency with PD-L1.

EXAMPLE 3 in vitro Activity of UMSC-TRAIL-TK-PD-1 in human T cells

To determine whether the stimulatory effect was a direct interaction between T cells and MSCs, splenocytes T cells were stimulated with CD3-CD28 beads. The gating strategy was based on the rationality of the first gate depicted in FIG. 2B, exclusion of doublets by FSC-A and FSC-H, selection of 7-AAD⁺(R&D Systems)/CD45⁺Or FSC-A excludes dead cells. T cells were labeled with CFSE and then co-cultured with UMSC or UMSC-TRAIL-TK-PD-1 stimulated with CD3-CD28 beads for 6 days. UMSC (at a ratio of 1: 1) significantly inhibits CD4⁺And CD8⁺Proliferation of both T cells (fig. 2C), but not at the 1:10 ratio. However, UMSC-TRAIL-TK-PD-1 significantly increased CD4 at either 1:1 or 1:10 ratio⁺And CD8⁺Proliferation of both T cells (fig. 2C). Furthermore, UMSC-TRAIL-TK-PD-1 stimulated by CD3-CD28 beads significantly increased CD4 compared to UMSC⁺INF-γ⁺And reducing the content of CD8⁺CD122⁺(FIG. 2D). These results indicate that any ratio of UMSC-TRAIL-TK-PD-1 can support T cell proliferation, while higher ratios are inhibitory.

Example 4 suicide Effect of UMSC-TRAIL-TK-PD-1 in vitro

To investigate the Thymidine Kinase (TK) -induced cell killing effect in UMSC-TRAIL-TK-PD-1, the suicide effect was tested by assessing the cell viability of UMSC-TRAIL-TK-PD-1 in the presence of various concentrations of GCV. First, a significant increase in TK content from a time and dose dependent manner was found in UMSC-TRAIL-TK-PD-1 (fig. 3A). GCV itself did not affect cell proliferation of UMSC (fig. 3B). Following GCV treatment, phosphorylated GCV induced apoptosis-like cell damage in UMSC-TRAIL-TK-PD-1 (fig. 3B). Cell proliferation of UMSC-TRAIL-TK-PD-1 was inhibited from a dose-dependent manner (fig. 3B). This indicates that UMSC-TRAIL-TK-PD-1 can express TK after transfection of a plasmid of TRAIL-TK-PD-1, and that GCV can be activated to its toxic form by inducing cytotoxicity to UMSC itself.

In vitro sensitivity of tumor cells to bystander effects of UMSC-TRAIL-TK-PD-1

To examine bystander effects via UMSC-TRAIL-TK-PD-1, cell viability was assessed for both 4T1(hep55.1c, Pan18, CT26) and UMSC-TRAIL-TK-PD-1 by co-culturing each cell at different ratios directly at various concentrations of GCV (fig. 3C). UMSC-TRAIL-TK-PD-1 significantly attenuated the growth of 4T1-Luc cells (hep55.1c, Pan18-Luc, CT26-Luc and GL261-Luc) (n ═ 3) after co-culture in the presence of 100 μ g/mL GCV for 7 days when the ratio was at most 1:32 and at least 1: 1. Furthermore, it was confirmed that the optimal inhibition efficiency was at a ratio of 1:1 (FIGS. 3D-3E).

To further confirm the bystander effect of UMSC-TRAIL-TK-PD-1, the time course of both suicide and bystander effect of UMSC-TRAIL-TK-PD-1 was studied (before 7 days). In this co-culture system, the cell death rate due to suicide effects slowly reached about one third of the entire system during the first two days, and then subsequently accelerated from day 3 to day 6. In bystander effect experiments, the same findings showed that most 4T1-Luc cells were killed from day 3 to day 5 (FIGS. 3C-3E). Furthermore, flow cytometry studies also demonstrated that UMSC-TRAIL-TK-PD-1 co-cultured with 4T1 cells significantly increased apoptotic cells (PI) from a GCV dose-dependent manner⁺annexin-V⁺Cells) (fig. 3C). Thus, inIn the co-culture system, the suicide effect of UMSC-TRAIL-TK-PD-1 and the bystander effect on 4T1-Luc cells (Hep55.1C, Pan18-Luc, CT26-Luc and GL261-Luc) occurred from day 3 to day 5.

UMSC-TRAIL-TK-PD-1 expressing TRAIL exhibits in vitro anti-tumor activity in 4T1-Luc cells.

UMSC-TRAIL-TK-PD-1 may be genetically modified to express high levels of TRAIL. UMSC-TRAIL-TK-PD-1 was transduced by a vector encoding full-length human TRAIL. FACS analysis showed associated TRAIL protein expression (90%) on the surface of UMSC cells (fig. 4A).

To confirm whether UMSC-TRAIL-TK-PD-1 can exert a tumoricidal effect on cancer cells, a co-culture experiment between tumor cells and UMSC-TRAIL-TK-PD-1 was then performed. UMSC-TRAIL-TK-PD-1 expressing TRAIL induces apoptosis especially at 48 hours after co-culture (4T1-Luc, Hep55.1C-Luc), represented by apoptosis from cell shrinkage, reduction of adherent 4T1-Luc cells, and Hep55.1C-Luc with the appearance of cell debris, which was confirmed by propidium iodide staining (PI staining) (FIG. 4B). To quantify cell death at 24 hours, 48 hours and 72 hours, large amounts of annexin-V were detected in the co-cultures as measured by FACS analysis⁺PI⁺Dead cells (. gtoreq.70%) in which UMSC-TRAIL-TK-PD-1 existed in a dose-dependent manner (FIG. 4C).

Example 5 tumor targeting of UMSC-TRAIL-TK-PD-1-Luc in the 4T1 tumor model

To demonstrate the UMSC-TRAIL-TK-PD-1 homing effect, biological distribution of UMSC-TRAIL-TK-PD-1-Luc after intravenous or intra-arterial implantation was performed using IVIS. First, the bioluminescence intensity increased from a cell dose-dependent manner as measured in vitro by IVIS (fig. 5A). In healthy mice, intravenous UMSC-TRAIL-TK-PD-1-Luc transplantation was initially trapped in the pulmonary capillaries from one day after injection, which showed an enhancement of the bioluminescent image of IVIS in the lungs (fig. 5B). Homing of UMSC-TRAIL-TK-PD-1-Luc allowed UMSC-TRAIL-TK-PD-1-Luc to survive and relocate to subcutaneous 4T1 tumors. Bioluminescent signals from the subcutaneous tumor area in IVIS images were initially observed 5 days after UMSC-TRAIL-TK-PD-1-Luc injection, followed by a gradual increase in intensity and a peak at day 14 (fig. 5B).

At 2 hours post-femoral intra-arterial injection, UMSC-TRAIL-TK-PD-1-Luc transplantation recruited directly to the orthotopic 4T1 tumor region without lung compression (also applicable to hep55.1c and pan18 tumor regions), which showed enhanced bioluminescent imaging of IVIS (fig. 5C-5E).

Subsequently, homing of UMSC-TRAIL-TK-PD-1-Luc allowed UMSC-TRAIL-TK-PD-1-Luc to survive and relocate to the tumor site.

To further confirm whether UMSC-TRAIL-TK-PD-1-Luc could track metastatic loci derived from the 4T 1-tumor model, intraarterial implantation of UMSC-TRAIL-TK-PD-1-Luc was performed 21 days after induction of the 4T 1-tumor model. Consistently, metastatic lung tumors from the original 4T1 tumor model significantly recruited UMSC-TRAIL-TK-PD-1-Luc, increasing bioluminescence intensity in multiple metastatic sites as measured by IVIS (fig. 5F). Multiple GFP s were found in 4T1 tumors 1 day post-treatment via immunohistochemical analysis⁺Luciferase enzymes⁺Cells, indicating recruitment of UMSC-TRAIL-TK-PD-1-GFP into the tumor microenvironment (fig. 5G). .

Example 6 therapeutic Effect of UMSC-TRAIL-TK-PD-1 on the 4T1-Luc model

The tumoricidal effect of luciferase-expressing 4T1-Luc and Hep55.1C-Luc tumor-bearing mice treated with various strategies of genetically modified UMSC was evaluated by IVIS, tumor volume and survival time after a q4dx3 treatment regimen (FIG. 6A). Prior to treatment, each group of test cells was exposed to 3% O₂This induced overexpression of CXCR4 in order to enhance stem cell homing in a time-dependent manner upon exposure to hypoxic pretreatment culture (fig. 6B). Notably, the UMSC-PD-1(UP) and UMSC-trail (ut) groups exhibited therapeutic effects that reduced tumor volume compared to those of the IgG control group as measured by IVIS (fig. 6C). Furthermore, the UMSC-TK-PD-1+ gcv (utpg) group, UMSC-TRAIL-TK + gcv (uttg) group, and UMSC-TRAIL-PD-1(UTP) group each showed stronger antitumor effects, and each exhibited inhibition of tumor growth (fig. 6C). Median survival time for IgG, UP, UT, UTPG, UTTG and UTP treated mice was 24 days, 32 days, 34 days, 43 days and 44 days, respectively (fig. 6D). UTP significantly prolongs survival compared to other groupsThe survival time reached 63 days (FIG. 6D). Furthermore, UTP significantly prevented tumor metastasis in the lung compared to other treatments (fig. 6E). On average, less than 5 lung metastatic nodules were found in UTP-treated mice compared to more than 20 metastases in the lungs of control mice. However, UMSC-tk (ut), UP and UTP did not show a significant reduction in metastasis compared to the control. Thus, it is hypothesized that metastasis is not only inhibited by the UTP-induced bystander effect, but is largely influenced by the immunopotentiating effects from UTP in the TME, which allows for systematic analysis of intratumoral immunity.

Next, to verify whether intraarterial injection of UMSC-TRAIL-TK-PD-1 showed significant therapeutic effects in the 4T1-Luc and hep55.1c-Luc models after q7dx2 treatment course protocol (fig. 6F), tumor growth and median survival time were examined for four groups (UMSC-TK-PD-1+ gcv (utpg) group, UMSC-TRAIL-TK-PD-1(UTTP) group, and UMSC-TRAIL-TK-PD-1+ gcv (uttpg) group). Prior to the analysis of intra-arterial injections, intravenous administration of UMSC-TK-PD-1+ gcv (utpg) group, UMSC-TRAIL-TK-PD-1(UTTP) group, and UMSC-TRAIL-TK-PD-1+ gcv (uttpg) group showed stronger anti-tumor effects and exhibited inhibition of tumor growth, respectively (fig. 6F-6G). Importantly, intra-arterial implants revealed a potent therapeutic effect over intravenous implants. Furthermore, the UTTPG group significantly inhibited tumor growth and prolonged median survival in 4T1-Luc and hep55.1c-Luc models compared to the IgG control, UTPG, and other groups of UTTP, respectively (fig. 6F-6G). Unfortunately, administration of anti-PD-L1 did not show any significant therapeutic effect in the 4T1-Luc model and the hepa55.1c model (fig. 6F-6G).

Example 7UTTPG treatment enhances immunity in the Tumor Microenvironment (TME)

Treatment outcomes inspired, the immunological properties of TME in the 4T1 tumor model were evaluated. Importantly, UTTPG can reverse immune decline in TME. Tumor-infiltrating CD45 across UTTPG treatment groups and other treatment groups⁺There was an overall increase in the percentage of white blood cells (fig. 7A). The results revealed that CD3 was present in UTTPG treatment compared to the other groups⁺CD8⁺And CD3⁺CD4⁺The frequency of both T cells was significantly increased (fig. 7A). UTTPG also induced a significant reduction in Treg (fig. 7B), and by this reversed intratumoral CD8⁺And CD4⁺Ratio of T cells to tregs (fig. 7C). In addition, the number of TAMs decreased dramatically in response to UTTPG treatment (FIG. 7B), which increased CD8 in the TME⁺And CD4⁺Ratio of T cells to TAM (fig. 7C). Note that the intracellular granzyme B (Grb)⁺) And Ki67⁺Significant up-regulation of cells indicated that UTTPG treatment not only increased the anti-tumor immune population, but also effectively achieved activation and proliferation of TIL (fig. 7D).