Early Cancer Detection And Enhanced Immunotherapy

Abstract

A method of therapy for a tumor or other pathology by administering a combination of thermotherapy, immunotherapy, and vaccination optionally combined with gene delivery. The combination therapy beneficially treats the tumor and prevents tumor recurrence, either locally or at a different site, by boosting the patient's immune response both at the time or original therapy and/or for later therapy. With respect to gene delivery, the inventive method may be used in cancer therapy, but is not limited to such use; it will be appreciated that the inventive method may be used for gene delivery in general. The controlled and precise application of thermal energy enhances gene transfer to any cell, whether the cell is a neoplastic cell, a pre-neoplastic cell, or a normal cell.

Claims

1. A cancer therapeutic method comprising administering to a patient having an early stage tumor a combination of thermotherapy and immunotherapy, where thermotherapy comprises systemically administering a plurality of tumor-antibody-coated nanoparticles coated with a thermosensitive polymer, the thermotherapy further comprises heating the tumor-antibody-coated nanoparticles using an energy source at the site of the tumor so as to damage one or more tumor cell membranes and release antigenic material in vivo that activates and stimulates an immunogenic response of the patient at the site of the tumor; and immunotherapy comprises systemically administering the patient's natural killer (NK) cells/dendritic cells pre-sensitized in vitro to the tumor.

2. The method of claim 1 where the step of heating the tumor-antibody-coated nanoparticles using the energy source comprises controllably increasing the temperature to which the tumor-antibody-coated nanoparticles are exposed from 37? C. to between 41? C. and 43? C. for a predetermined time period resulting in melting the thermosensitive polymer coating the tumor-antibody-coated nanoparticles, and releasing additional tumor antigens in the circulation of the patient from the thermally damaged tumor cells; the method further comprising the steps of: obtaining from the blood of the patient, the tumor antigens to build a new potent vaccine against many additional tumor specific antigens, the vaccine combined with tumor-antibody-coated nanoparticles conjugated with checkpoint inhibitors, and Rock inhibitors or Wnt inhibitors; and administering the vaccine with the tumor-antibody-coated nanoparticles conjugated with viral-like particles (VLP) while simultaneously releasing the conjugated checkpoint inhibitors, and Rock inhibitors or Wnt inhibitors, from the tumor-antibody-coated antibody coated nanoparticles to prevent new or old tumor cells or metastatic cells from being disguised from the T-lymphocytes or the patient's natural killer (NK) cells, thereby providing a vaccine for treatment of potential recurrences of the same tumor to the patient and enhancing the immune response at the specific location of one or more metastatic lesions, circulating tumor cells, or sessile tumor cells.

3. The method of claim 2 where the checkpoint inhibitors conjugated with the tumor-antibody-coated nanoparticles are selected from the group consisting of PD-1, PD-L1, CTLA-4, jagged 1 inhibitor 15D11, and combinations thereof.

4. The method of claim 2 where the tumor-antibody-coated nanoparticles are conjugated with Rock inhibitors, the Rock inhibitors selected from the group consisting of Fasudil, exoenzyme, Y27632, Botox, and combinations thereof.

5. The method of claim 2 where the tumor-antibody-coated nanoparticles are conjugated with Wnt inhibitors, the Wnt inhibitors selected from the group consisting of niclosamide, ivermectin, and combinations thereof.

6. The method of claim 2 further comprising the step of: repeating administration of the vaccine together with checkpoint inhibitors and Rock inhibitors once or twice a year, thereby reducing an auto-immune reaction.

7. The method of claim 1 where the tumor-antibody-coated nanoparticles are conjugated with a medication, and where thermotherapy includes exposing the tumor-antibody-coated nanoparticles and medication to a light pulse at a frequency in the range of 20 Hz to 60 Hz to decrease proliferation of the tumor cells.

8. The method of claim 1 where immunotherapy is administered at intervals to the patient after the initial therapy acting as a booster to the original immunotherapy and reduce or prevent tumor recurrences at a same or different site.

9. The method of claim 1 where immunotherapy further comprises obtaining NK cells/dendritic cells grown in culture under light pulses with a tumor biomarker from blood or a tumor biopsy specimen containing tumor lysate, killed circulating tumor cells (ct cell), and tumor extracellular vesicles (ECV).

10. The method of claim 1 where thermotherapy includes exposing the tumor-antibody-coated nanoparticles to a light pulse at a frequency in the range of 20 Hz-60 Hz to decrease proliferation of the tumor cell.

11. The method of claim 1 where the step of heating the tumor-antibody-coated nanoparticles using the energy source comprises using a thermoacoustic unit to control a thermal energy delivery unit using a processor to maintain the tumor-antibody-coated nanoparticles at a predetermined temperature as a closed circuit once the tumor-antibody-coated nanoparticles have attached to the tumor cells, then controllably increasing the temperature to which the tumor-antibody-coated nanoparticles are exposed from 37? C. to 41? C.-43? C. for a predetermined desired time period resulting in melting the thermosensitive polymer coating the tumor-antibody-coated nanoparticles, and releasing under control a medication or gene, which are attached to the thermosensitive tumor-antibody-coated nanoparticles locally at the desired site.

12. The method of claim 1 where the anti-tumor antibody is specific for at least one tumor biomarker in the patient's blood.

13. The method of claim 1 where the polymer contains an inhibitory gene(s) and a CRISPR/cas9 complex to stimulate or modify tumor genes at the desired site upon release from the polymer at a desired temperature that is obtained by incremental increase from 37? C. to 43? C. permitting precision nanoparticle assisted thermotherapy and imaging (NATTI) to release the gene(s) and optional medicament(s) and/or checkpoint inhibitor(s) from the nanoparticles.

14. The method of claim 1 further comprising conjugating the nanoparticles with a chimeric receptor on B cells and with T-cells cultured in vitro and expressing an antigen for the chimeric receptor to target abnormal B cells seen in leukemia.

15. The method of claim 1 further comprising performing precision nanoparticle assisted thermotherapy and generating a photoacoustic image of the cells to which the nanoparticles bind at an early cellular stage having a size between one and two millimeters in diameter not easily detectable by radiographic imaging.

16. A method of simultaneous localized thermotherapy and vaccination, the method comprising: administering a plurality of nanoparticles to a patient in need thereof, the administered nanoparticles having a size less than 10 nm in diameter and coated with an antitumor antibody, the patient provided nanoparticle assisted thermotherapy, the unbound nanoparticles undergoing renal elimination from the body within a plurality of hours of administration, and the tumor-bound nanoparticles remaining in the patient, wherein the nanoparticle assisted thermotherapy comprises heating the tumor-bound nanoparticles using an energy source at the site of the tumor so as to damage one or more tumor w cell membranes and release antigenic material in vivo that activates and stimulates an immunogenic response by the natural killer (NK) cells/dendritic cells of the patient.

17. A therapeutic method to treat a pathology implicating a cellular protein(s), the method comprising administering to a patient having a cellular pathology a combination of thermotherapy, and immunotherapy, with gene delivery, where: thermotherapy comprises systemically administering antibody-coated nanoparticles coated with a thermosensitive polymer and conjugated with one or more genes for gene delivery to a diseased cell, the thermotherapy further comprises heating the antibody-coated nanoparticles using an energy source to release the one or more genes at the site of the diseased cell to effect gene therapy; immunotherapy comprises systemically administering the patient's natural killer (NK) it) cells/dendritic cells pre-sensitized in vitro to the cellular protein(s); and wherein the gene therapy comprises use of a CRISPR/cas9 and/or CRISPRi to replace or modify at least one genetic component in the diseased cell.

18. The method of claim 17, further comprising performing plasmaphoresis on the patient post-therapy to purify a patient's blood from toxins and cellular components generated by the therapy.

19. The method of claim 17, further comprising: applying light energy to a tube containing the patient's blood cells post-therapy to achieve a temperature up to 60? C. to kill immune cells containing nanoparticles; passing the pulsed blood cells through a dielectrophoresis system to characterize and remove dead or live T-cells, sensitized killer cells, and tumor cells; and re-infusing the dielectrophoresis treated blood in the patient while simultaneously administering immunosuppressive agents, thus reducing the likelihood of a severe post-therapy autoimmune response in the patient.

Description

EXAMPLE 1

[0117] T cells and dendritic cells are obtained from a patient's blood, and grown in culture along with a tumor or other antigen, plus nanoparticles coated with thermosensitive polymers conjugated with antigen and VLP using culture methods known in the art.

[0118] The nanoparticle complex is injecting them along with checkpoint inhibitors and IL-2. The inventive method is applied, killing tumor cells, and increasing the response of T-cells and dendritic cell.

[0119] The patient's blood is assessed for new biomarkers from the dead cells.

[0120] The cultured T-cells and dendritic cells are harvested, along with the nanoparticle-coated antigen plus VLP or RNA or DNA phages. These are stored under appropriate conditions, and reinjected into the patient with low dose coated nanoparticles or systemic medicaments to be administered with checkpoint inhibitors such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rock inhibitor, Fasudil or Wnt inhibitor such as niclosamide as needed, e.g., semi-annually, annually, biannually, etc. as vaccination or in tumor recurrences in metastatic disease with repetition as needed. This is followed up with counting or quantifying circulating DNA, exosomes or circulating cells to recognize potential tumor recurrences.

EXAMPLE 2

[0121] A checkpoint inhibitors and rock inhibitors, e.g., Botox up to 50-100 picograms (pg), or 100 picograms (pg) to 1 nanogram (ng) or more or fasudil from 100 picograms (pg) to 10 nanograms (ng) or 50 nanograms (ng) to 1 milligrams (mg) or Wnt inhibitors, e.g., niclosamide is added to a thermosensitive polymer conjugated with and antibody coated pluralities of nanoparticle for controlled release with the checkpoint inhibitor using the inventive controlled thermotherapy, NATTI, to release the medication locally at temperature of 41-43 C degree along immune stimulators such as VLP to treat a patient with breast, colorectal, glioblastoms, prostate, eye or skin melanomas, pancreatic, lung cancer, and/or ovarian cancer etc. A checkpoint inhibitor, such as nivolumab or PD-1L, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, such as Fasudil or botox, or Wnt inhibitors, such as niclosamide or ivermectin, is combined with pluralities of antibody coated nanoparticle assisted targeted immunotherapy for adaptive T-cell transfer to overcome the limitations of standard immunotherapy and prevent a cytokine storm. In one embodiment the Rock inhibitor Fasudil can be taken orally at the dose of 40-80 mg as needed and niclosamide 1-2 gram once or repeated in a week ivermeting also can be given orally at a dose of 1 gram orally for a period of time during and shortly after thermotherapy for a few days as needed.

EXAMPLE 3

[0122] Nanoparticles are conjugated with a chimeric receptor, a CD19 protein that is found only on B cells, along with the T-cells cultured in vitro that expresses a chimeric antigen receptor (chimeric antigen receptor T (CAR T)-cells) to target abnormal B cells seen in leukemia along with PD-1L, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, such as Fasudil or botox, or Wnt inhibitors, such as niclosamide. The reappearance of new biomarkers as neoantigens in these patients can be also treated in the postoperative period using the inventive method repeated therapy as vaccination along with Rock inhibitor encourage the abnormal mature cells to undergo apoptotic degeneration rather cell proliferation with abnormal genetic changes which is characterized by the tumors occurring as a result of aging process and not a pre-existing genetic mutation.

[0123] Plasmaphoresis is simultaneously performed or performed after treatment.

[0124] This example treats acute and chronic hematologic malignancies such as acute lymphoblastic leukemia, non-Hodgkin lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, etc.

EXAMPLE 4

[0125] Pluralities of antibody coated nanoparticles are conjugated with all-trans retinoic acid (ATRA) and arsenic trioxide to target leukaemia cells in acute promyelocytic leukemia and used in the inventive method. The all-trans retinoic acid is released at the site of the tumor without exposing the entire body to the toxic medication, simultaneously, plasmophoresis is performed to clear all toxin released in the blood, along with leukemic cells. It is appreciated that other blood cell cancers are removed in the same session.

EXAMPLE 5

[0126] In a patient with a hematologic malignancy that is resistant to chemotherapeutic agents or immune therapy, NATTI is performed with gene delivery, along with chemotherapeutic agents, to target all immune cells initially without subjecting the patient to systemic heavy chemotherapy, followed by bone marrow transplantation, without exposing the entire body to systemic chemotherapy.

EXAMPLE 6

[0127] Pluralities of antibody coated nanoparticles are conjugated with RNA that contains an aptamer, ribosomes, and siRNA in a thermosensitive polymer and administered to using NATTI to target specific tumor cells.

EXAMPLE 7

[0128] The microenvironment of the cancer cell is modified by delivering medicaments that block the uptake of exosomal signals and prevent the uptake of ECV. Such medications include choloroquine, heparin, cytochalasin D, and ethyl-isopropyl amiloride are conjugated with polymeric coating and conjugated with antibody coated nanoparticles administered to the patient and released with NATTI. These medications are approved for patient use. The medicaments are provided using NATTI in conjunction with chemotherapeutic agents and rock inhibitors.

EXAMPLE 8

[0129] The inventive method provides nanoparticle assisted localized immunothermotherapy and thermotherapy for delivery of customized vaccines with or without VLP to target core mutations in a patient. The immune cells or T-cells that can attack those core mutations are identified via a cancer biomarker. The immune cells or T-cells are then cultured with the nanoparticles coated with thermosensitive particles and VLP and IL-2. The antibody coated nanoparticles with checkpoint inhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc., and Rock inhibitor, such as Fasudil or Botox etc., or Wnt inhibitor, such as niclosamide, etc., are injected into the patient, controllably heated using a thermal energy source, and imaged, for specific patient, or those with metastatic disease or recurrences as immunotherapy, such as in breast cancer, prostate cancer, glioblastoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, intestinal or colon cancer, etc.

EXAMPLE 9

[0130] Antibody coated nanoparticles are conjugated with RNA phage VLP, which is generally stable. VLPs of the related RNA phage PP7 are crosslinked with inter-subunit disulfide bonds, rendering them significantly more stable. They exhibit high immunogenicity. Such nanoparticles complement the inventive NATTI technology and can be employed in anti-cancer and antibacterial treatment. Lytic phages attach to receptors on the bacterial surface, inject their genetic material through the bacterial membrane, and overtake the bacterium's transcription and translation machinery to synthesize new phages. The application of thermotherapy damages all VLP, phages, virocides or foreign protein and eliminate their future growth and potential adverse reactions.

EXAMPLE 10

[0131] To prevent a severe autoimmune response after tumor immunotherapy, before extracorporeal plasmapheresis, one uses the nanoparticle assisted thermotherapy and imaging system to apply heavy thermal energy to a tube containing blood cells and to achieve a temperature as high as 60? C. to kill the sensitized immune cells containing nanoparticles. Blood is then passed through a dielectrophoresis system to characterize and remove dead or live T-cells, sensitized killer cells, and dead tumor cells prior to re-infusing blood in the patient while simultaneously administering immunosuppressive agents and Rock inhibitors, including biologics. This reduces the severe autoimmune response often seen after tumor immunotherapy.

[0132] The embodiments shown and described in the specification are only specific embodiments of the inventor who is skilled in the art and are not limiting in any way. Therefore, various changes, modifications, or alterations to those embodiments may be made without departing from the spirit of the invention in the scope of the following claims. The references cited are expressly incorporated by reference herein in their entirety.