Therapeutic Solutions International Inc (TSOI)

[centsability4me]

TSOI New Patent published 9/29/22

https://patents.justia.com/patent/20220306994

CHIMERIC CELLS COMPRISING DENDRITIC CELLS AND ENDOTHELIAL CELLS RESEMBLING TUMOR ENDOTHELIUM

Disclosed are means, methods and compositions of matter useful for induction of immunological responses towards tumor endothelial cells. In one embodiment the invention teaches fusion of dendritic cells and cells resembling tumor endothelial cells and administration of such chimeric cells as an immunotherapy for stimulation of tumor endothelial cell destruction. In other embodiments pluripotent stem cells are utilized to generate dendritic cells, wherein said dendritic cells are fused with pluripotent stem cell derived endothelial cells created in a manner to resemble tumor endothelial cells.

1. A hybrid cell comprising of: a) a dendritic or dendritic like cell and b) an endothelial cell generated in a manner to resemble tumor endothelium.

2. The hybrid cell of claim 1, wherein said hybrid cells is generated by fusion of a dendritic or dendritic like cell and an endothelial cell generated in a manner to resemble tumor endothelium.

3. The hybrid cell of claim 3, wherein said fusion is created by placement of both cell types in physical proximity while treating both cells with an agent capable of causing fusion of plasma membrane.

4. The hybrid cell of claim 3, wherein said fusion agent is one or more agents selected from a group comprising of: a) polyethylene glycol; b) ultrasound waves; c) radio waves; and d) phosphatidylcholine.

5. The hybrid cell of claim 1, wherein said dendritic cell is generated from a group of cells comprising of: a) a stem cell; b) a pluripotent stem cell; c) an inducible pluripotent stem cell; d) a parthenogenic stem cell; e) a somatic cell nuclear transfer derived stem cell; f) a pluripotent stem cell is generated by cytoplasmic transfer from an immature cell to a mature cell; and g) embryoid bodies from said pluripotent stem cells.

6. The hybrid cell of claim 5, wherein said embryoid bodies are dissociated and cells are cultured in cytokines capable of expanding dendritic cell progenitors.

7. The hybrid cell of claim 5, wherein said dendritic cell progenitors are cultured in GM-CSF.

8. The hybrid cell of claim 5, wherein said dendritic cell progenitors are cultured in flt-3 ligand.

9. The hybrid cell of claim 5, wherein said dendritic cell progenitors are cultured in IL-4.

10. The hybrid cell of claim 1, wherein said endothelial cells are derived from a pluripotent stem cell.

11. The hybrid cell of claim 10, wherein said pluripotent stem cell is an inducible pluripotent stem cell.

12. The hybrid cell of claim 10, wherein said pluripotent stem cell is a parthenogenic stem cell.

13. The hybrid cell of claim 10, wherein said pluripotent stem cell is a somatic cell nuclear transfer derived stem cell.

14. The hybrid cell of claim 10, wherein said pluripotent stem cell is generated by cytoplasmic transfer from an immature cell to a mature cell.

15. The hybrid cell of claim 1, wherein said hybrid cell is utilized to induce an immune response against tumor endothelial cells.

16. The hybrid cell of claim 10, wherein said endothelial cells are generated by culture of endothelial progenitor cells in a media replicating the tumor microenvironment.

17. The hybrid cell of claim 10, wherein said endothelial cells are generated by culture of endothelial progenitor cells in a media replicating the tumor microenvironment.

18. The hybrid cell of claim 17, wherein said media contains one or more agents selected from a group comprising of: a) prostaglandin E2; b) TGF-beta; c) IL-10; d) VEGF; e) PDGF-BB; f) EGF; g) FGF-1 and h) FGF-2.

19. The hybrid cells of claim 1, wherein antigen presenting activity of said cells is augmented as compared to baseline conditions by treatment with a toll like receptor agonist.

20. The hybrid cells of claim 19, wherein said toll like receptor agonist is HMGB-1 or a peptide derived thereof.

[0001] This application claims priority to U.S. Provisional Application Ser. No. 63/165,056, filed on Mar. 23, 2021, entitled "Chimeric Cells Comprising Dendritic Cells and Endothelial Cells Resembling Tumor Endothelium", which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The field relates to compositions of matter useful for induction of immunological responses towards tumor endothelial cells, including the fusion of dendritic cells and cells resembling tumor endothelial cells.

BACKGROUND OF THE INVENTION

[0003] The concept of treating cancer by blocking new blood vessel formation, angiogenesis, was pioneered by Judah Folkman who provided convincing arguments that it is not necessary to actively kill the tumor mass, but by suppressing its ability to grow through cutting off blood supply, malignant tumors may be converted into benign masses that eventually regress [1, 2]. Unfortunately, despite discovery of angiostatin, and endostatin, naturally derived inhibitors of angiogenesis, neither of these approaches translated into successful therapies. Nevertheless, the concept of targeting new blood vessel formation led to thousands of publications describing various antiangiogenic agents, of which several eventually proceeded through clinical trials and regulatory approval. Broadly anti-angiogenic agents approved by regulators can be classified into antibodies, such as Bevacizumab (Avastin) which binds VEGF [3], and Ramucirumab (Cyramza) [4], which binds VEGF-R2, as well as small molecules which bind multiple receptor kinases associated with angiogenesis such as Sunitinib [5-7], Cabozantinib [8-11], Pazopanib [12-14], and Regorafenib [15-17].

[0004] These approaches have augmented the standard of care for various tumor types and have achieved some level of progress. Unfortunately, the concept of blocking angiogenesis of cancer was not as simple as originally envisioned. One of the major hurdles in blocking angiogenesis was that even though de novo blood vessels are derived from nonmalignant cells, the malignant cells appear to possess ability to induce mutations in the new blood vessels. One example of the heterogeneity of tumor endothelial cells compared to endothelial cells from low and high metastatic tumors by Ohga et al [18]. The investigators extracted two types of tumor endothelial cells (TEM) from high-metastatic (HM) and low-metastatic (LM) tumors and compared their characteristics. HM tumor-derived TECs (HM-TECs) showed higher proliferative activity and invasive activity than LM tumor-derived TECs (LM-TECs). Moreover, the mRNA expression levels of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF) receptors 1 and 2, VEGF, and hypoxia-inducible factor-1a, were higher in HM-TECs than in LM-TECs. The tumor blood vessels themselves and the surrounding area in HM tumors were exposed to hypoxia. Furthermore, HM-TECs showed higher mRNA expression levels of the stemness-related gene stem cell antigen and the mesenchymal marker CD90 compared with LM-TECs. HM-TECs were spheroid, with a smoother surface and higher circularity in the stem cell spheroid assay. HM-TECs differentiated into osteogenic cells, expressing activated alkaline phosphatase in an osteogenic medium at a higher rate than either LM-TECs or normal ECs. Furthermore, HM-TECs contained more aneuploid cells than LM-TECs. The investigators concluded that the results indicate that TECs from HM tumors have a more pro-angiogenic phenotype than those from LM tumors. It appears that the aggressiveness of the tumor not only can alter endothelial cell function but also drug resistance ability. In another study, Akiyama et al. [19]compared murine TECs and normal ECs. It was found that TECs were more resistant to paclitaxel with the up-regulation of multidrug resistance (MDR) 1 mRNA, which encodes the P-glycoprotein, compared with normal ECs. Normal human microvascular ECs were cultured in tumor-conditioned medium (CM) and became more resistant to paclitaxel through MDR1 mRNA up-regulation and nuclear translocation of Y-box-binding protein 1, which is an MDR1 transcription factor. Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Akt were activated in human microvascular ECs by tumor CM. The investigators observed that tumor CM contained a significantly high level of VEGF. A VEGFR kinase inhibitor, Ki8751, and a phosphatidylinositol 3-kinase-Akt inhibitor, LY294002, blocked tumor CM-induced MDR1 up-regulation. MDR1 up-regulation, via the VEGF-VEGFR pathway in the tumor microenvironment, is one of the mechanisms of drug resistance acquired by TECs. It was observed that VEGF secreted from tumors up-regulated MDR1 through the activation of VEGFR2 and Akt. This process is a novel mechanism of the acquisition of drug resistance by TECs in the tumor microenvironment. Yet another study demonstrated that tumors can induce a "dedifferentiation" of tumor endothelium. Specifically, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. The stem cell marker aldehyde dehydrogenase (ALDH) mRNA expression and activity were higher in TECs than those in NECs. Next, ALDHhigh/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDHlow TECs, ALDHhigh TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDHhigh TECs than that in ALDHlow TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDHhigh TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis [20].

[0005] What is it that causes the tumor to evoke changes in the endothelium? As suggested above, there is some support for growth factor mediated alterations, additionally, horizontal gene transfer may also play a role [21-29]. Although the field of horizontal gene transfer has historically been controversial one of the strongest evidences supporting this concept is the phenomena of donor-derived relapse in leukemic patients. In these situations patients with leukemia who relapse after bone marrow transplant have the relapsing cells originate from donor cells that transformed into malignant cells [30, 31]. Another issue that affected efficacy of anti-angiogenesis therapies is that in some tumors, the tumor cells themselves transdifferentiate into endothelial-like cells, termed tumor vascular channels, which possess ability to mutate around either antibody or kinase inhibitor drugs [32-37].

[0006] The previously mentioned means by which tumor endothelial cells can protect themselves against anti-angiogenic agents has resulted in relatively low clinical efficacy of these drugs. To understand the general lack of efficacy in the initial registration trial.sup.ii, median progression free survival (PFS) of ovarian cancer patients who received bevacizumab plus chemotherapy was 6.8 months (95 percent CI: 5.6, 7.8) compared with 3.4 months (95 percent CI: 2.1, 3.8) for those who received chemotherapy alone. There was no statistically significant difference in overall survival (OS) for patients treated with bevacizumab plus chemotherapy compared with chemotherapy alone (median OS: 16.6 months versus 13.3 months; HR 0.89; 95 percent CI: 0.69, 1.14). Subset analysis led to identification that the group of patients that received paclitaxel with the antibody had the largest improvement, resulting in a 5.7-month improvement in median PFS (9.6 months versus 3.9 months; HR 0.47; 95 percent CI: 0.31, 0.72), an improvement in the objective response rate (53 percent versus 30 percent), and a 9.2-month improvement in median OS (22.4 months versus 13.2 months, HR 0.64; 95 percent CI: 0.41, 1.01).sup.iii. Multiple other trials where conducted for different indications using bevacizumab, unfortunately, progression free survival and overall survival was not increase more than a year in any of the studies [38-42], and neither in studies with small molecule kinase inhibitors [43-48].

[0007] This clinical translation, although highly beneficial in some patients, overall the effect was mediocre, highlights the disparity between animal studies, in which some studies complete regression was observed in established tumors [49, 50], whereas in clinical trials, relatively minimal effect compared to animal studies was observed [51]. One lesson from these studies is that the large heterogeneity of the patient and of the tumors, which calls for large patient populations in order to achieve an overall survival advantage.

[0008] Innovations in pharmacogenomics and personalized medicine will help identify specific patients and tumors that are likely to respond. Unfortunately, at present, patients with metastatic disease have limited options and a statistically significant extension of survival does equate to large market demand, as seen by the overall sale of angiogenesis inhibitors for cancer being over 20 billion annually.



From the PR announcing the filing of this patent on 3/23/21 :

Therapeutic Solutions International Creates Hybrid Cell Designed to Educate Immune System to Choke Cancer Blood Vessels

Novel Strategy Demonstrates Potent Results in Animal Model of Lung Cancer through Fusing StemVacs-V™ Immunotherapy with In-Vitro Generated Cancer Blood Vessels

https://www.prnewswire.com/news-releases/therapeutic-solutions-international-creates-hybrid-cell-designed-to-educate-immune-system-to-choke-cancer-blood-vessels-301253715.html

ELK CITY, Idaho, March 23, 2021 /PRNewswire/ — Therapeutic Solutions International, Inc., (OTC Markets: TSOI), announced today filing of a patent with new data on a unique cell created by the Company capable of training the immune system to kill blood vessels feeding cancer, but sparing healthy blood vessels. These data are an extension of previous findings from the Company showing that StemVacs is capable of suppressing new blood vessel production.

“Killing malignant blood vessels is the basis for Avastin, an antibody-based drug which created 7 billion in revenue last year.1 Another company, Batu Biologics, previously demonstrated vaccination against cancer blood vessels is safe and was cleared by the FDA for clinical trials,” stated Dr. James Veltmeyer, Chief Medical Officer of the Company. “The approach developed by our Company is completely unique because it merges the potent antigen presenting capabilities of StemVacs-V together with the reproducibility and consistency of iPSC generated tumor-like endothelial cells.”

StemVacs-V™ is comprised of “cancer resistant” dendritic cells which are developed from an engineered iPSC cell line. The novel chimeric cell product is generated by fusing StemVacs-V with iPSC derived endothelial cells which are cultured in a manner to make them replicate cancer stem cells. Proof of selective killing of cancer blood vessels was obtained by demonstration of immune response to proteins found only on cancer blood vessels.

“Numerous companies such as Fate Therapeutics, NantKwest, and Century Therapeutics have all achieved significant valuations through pioneering novel uses of iPSC derived immune cells,” said Famela Ramos, Vice President of Business Development. “To our knowledge we are the first company to use iPSC derived cells to stimulate naturally occurring immunity to kill what some call the ‘Achilles Heel’ of cancer.”

“Generation of a new cell that didn’t previously exist is a significant milestone for our Company. We believe the current animal studies in lung cancer can be extrapolated to other cancers, since no cancer can grow more than 2 millimeters without creating new blood vessels,” said Timothy Dixon, President and CEO of the Company and co-inventor of the patent. “By selectively killing cancer blood vessels the problem of mutations is solved since cancer blood vessels are not generated from tumor tissue and they do not mutate. Furthermore, since blood vessels are in direct contact with the blood and immune cells that travel through the blood, it is substantially easier for the immune system to kill malignant vessels as opposed to tumors.”