The present invention relates to systems and methods for treating cancer with cold atmospheric plasma.
Recent progress in atmospheric plasmas led to the creation of cold plasmas with ion temperatures close to room temperature. Cold non-thermal atmospheric plasmas can have tremendous applications in biomedical technology. K. H. Becker, K. H. Shoenbach and J. G. Eden “Microplasma and applications” J. Phys. D.: Appl. Phys. 39, R55-R70 (2006). In particular, plasma treatment can potentially offer a minimum-invasive surgery that allows specific cell removal without influencing the whole tissue. Conventional laser surgery is based on thermal interaction and leads to accidental cell death i.e., necrosis and may cause permanent tissue damage. In contrast, non-thermal plasma interaction with tissue may allow specific cell removal without necrosis. In particular, these interactions include cell detachment without affecting cell viability, controllable cell death etc. It can be used also for cosmetic methods of regenerating the reticular architecture of the dermis. The aim of plasma interaction with tissue is not to denaturate the tissue but rather to operate under the threshold of thermal damage and to induce chemically specific response or modification. In particular, presence of plasma can promote chemical reactions that would have the desired effect. Chemical reaction can be promoted by tuning the pressure, gas composition and energy. Thus, the important issues are to find conditions that produce effect on tissue without thermal treatment. Overall plasma treatment offers the advantage that—can never be thought of in most advanced laser surgery. E. Stoffels, I. E Kieft, R. E. J Sladek, L. J. M van den Bedem, E. P van der Laan, M. Steinbuch “Plasma needle for in vivo medical treatment: recent developments and perspectives” Plasma Sources Sci. Technol. 15, S169-S180 (2006).
Several different systems and methods for performing Cold Atmospheric Plasma (CAP) treatment have been disclosed. For example, U.S. Published Patent Application No. 2014/0378892 discloses a two-electrode system for CAP treatment. U.S. Pat. No. 9,999,462 discloses a converter unit for using a traditional electrosurgical system with a single electrode CAP accessory to perform CAP treatment.
As a near-room temperature ionized gas, cold atmospheric plasma (CAP) has demonstrated its promising capability in cancer treatment by causing the selective death of cancer cells in vitro. See, Yan D, Sherman J H and Keidar M, “Cold atmospheric plasma, a novel promising anti-cancer treatment modality,” Oncotarget. 8 15977-15995 (2017); Keidar M, “Plasma for cancer treatment,” Plasma Sources Sci. Technol. 24 33001 (2015); Hirst A M, Frame F M, Arya M, Maitland N J and O'Connell D, “Low temperature plasmas as emerging cancer therapeutics: the state of play and thoughts for the future,” Tumor Biol. 37 7021-7031 (2016). The CAP treatment on several subcutaneous xenograft tumors and melanoma in mice has also demonstrated its potential clinical application. See, Keidar M, Walk R, Shashurin A, Srinivasan P, Sandler A, Dasgupta S, Ravi R, Guerrero-Preston R and Trink B, “Cold plasma selectivity and the possibility of a paradigm shift in cancer therapy,” Br. J. Cancer. 105 1295-301 (2011); Vandamme M, Robert E, Dozias S, Sobilo J, Lerondel S, Le Pape A and Pouvesle J-M, “Response of human glioma U87 xenografted on mice to non thermal plasma treatment,” Plasma Med. 1 27-43 (2011); Brulle L, Vandamme M, Ries D, Martel E, Robert E, Lerondel S, Trichet V, Richard S, Pouvesle J M and Le Pape A, “Effects of a Non thermal plasma treatment alone or in combination with gemcitabine in a MIA PaCa2-luc orthotopic pancreatic carcinoma model,” PLoS One. 7 e52653 (2012); and Chernets N, Kurpad D S, Alexeev V, Rodrigues D B and Freeman T A, “Reaction chemistry generated by nanosecond pulsed dielectric barrier discharge treatment is responsible for the tumor eradication in the B16 melanoma mouse model,” Plasma Process. Polym. 12 1400-1409 (2015).
The rise of intracellular reactive oxygen species (ROS), DNA damage, mitochondrial damage, as well as apoptosis have been extensively observed in the CAP-treated cancer cell lines. See, Ahn H J, Kim K II, Kim G, Moon E, Yang S S and Lee J S, “Atmospheric-pressure plasma jet induces apoptosis involving mitochondria via generation of free radicals,”. PLoS One. 6 e28154 (2011); Ja Kim S, Min Joh H and Chung T H, “Production of intracellular reactive oxygen species and change of cell viability induced by atmospheric pressure plasma in normal and cancer cells,” Appl. Phys. Lett. 103 153705 (2013); and Yan D, Talbot A, Nourmohammadi N, Sherman J H, Cheng X and Keidar M, “Toward understanding the selective anticancer capacity of cold atmospheric plasma—a model based on aquaporins (Review),” Biointerphases. 10 040801 (2015). The increase of intracellular ROS may be due to the complicated intracellular pathways or the diffusion of extracellular ROS through the cellular membrane. See, Yan D, Xiao H, Zhu W, Nourmohammadi N, Zhang L G, Bian K and Keidar M, “The role of aquaporins in the anti-glioblastoma capacity of the cold plasma-stimulated medium,” J. Phys. D. Appl. Phys. 50 055401 (2017). However, the exact underlying mechanism is still far from clear.
Cancer cells have shown specific vulnerabilities to CAP. See, Yan D, Talbot A, Nourmohammadi N, Cheng X, Canady J, Sherman J and Keidar M, “Principles of using cold atmospheric plasma stimulated media for cancer treatment,” Sci. Rep. 5 18339 (2015)
Understanding the vulnerability of cancer cells to CAP will provide key guidelines for its application in cancer treatment. Only two general trends about the cancer cells' vulnerability to CAP treatment have been observed in vitro based on just a few cell lines. First, one study just compared the cytotoxicity of CAP treatment on the cancer cell lines expressing p53 with the same treatment on the cancer cell lines without expressing p53. The cancer cells expressing the p53 gene were shown to be more resistant to CAP treatment than p53 minus cancer cells. Ma Y, Ha C S, Hwang S W, Lee H J, Kim G C, Lee K W and Song K, “Non-thermal atmospheric pressure plasma preferentially induces apoptosis in p53-mutated cancer cells by activating ROS stress-response pathways,” PLoS One. 9 e91947 (2014). p53, a key tumor suppressor gene, not only restricts abnormal cells via the induction of growth arrest or apoptosis, but also protects the genome from the oxidative damage of ROS such as H2O2 through regulating the intracellular redox state. Sablina A A, Budanov A V, Ilyinskaya G V, Larissa S, Kravchenko J E and Chumakov P M, “The antioxidant function of the p53 tumor suppressor,” Nat. Med. 11 1306 (2005). p53 is an upstream regulator of the expression of many anti-oxidant enzymes such as glutathione peroxidase (GPX), glutaredoxin 3 (Grx3), and manganese superoxide dismutase (MnSOD). Maillet A and Pervaiz S, “Redox regulation of p53, redox effectors regulated by p53: a subtle balance,” Antioxid. Redox Signal. 16 1285-1294 (2012). In addition, cancer cells with a lower proliferation rate are more resistant to CAP than cancer cells with a higher proliferation rate. Naciri M, Dowling D and Al-Rubeai M, “Differential sensitivity of mammalian cell lines to non-thermal atmospheric plasma,” Plasma Process. Polym. 11 391-400 (2014). This trend may be due to the general observation that the loss of p53 is a key step during tumorigenesis. Tumors at a high tumorigenic stage are more likely to have lost p53. See, Fearon E F and Vogelstein B, “A genetic model for colorectal tumorigenesis,” Cell. 61 759-767 (1990).
Despite the complicated interaction between CAP and cancer cells, the initial several hours after treatment has been found to be an important stage for the cytotoxicity of CAP. The anti-cancer ROS molecules in the extracellular medium are completely consumed by cells during this time period. After the initial several hours, replacing the medium surrounding the cancer cells does not change the cytotoxicity of CAP. See, Yan D, Cui H, Zhu W, Nourmohammadi N, Milberg J, Zhang L G, Sherman J H and Keidar M, “The specific vulnerabilities of cancer cells to the cold atmospheric plasma-stimulated solutions,” Sci. Rep. 7 4479 (2017).
Allogeneic or autologous chimeric antigen receptor (CAR) therapy has been the subject of many recent studies. In this therapy, T cells are collected from the patient or donor by apheresis, and the T cells are then expanded and genetically modified using one of several approaches. The CAR-T cells then are infused into the patient. In one prior study, a large panel of single-chain variable fragments of an antibody that bind to CD70 were generated and formatted into CARs. Anti-CD70 allogeneic CAR-T cells were identified, selected and studied in short- and long-term cytotoxicity assays, which confirmed their ability to kill renal cell carcinoma (RCC) cells in vitro and in multiple in vivo models. Anti-CD70 AlloCAR T therapy candidates were ranked based on tonic signaling, transduction efficiency, phenotype, activation status and expansion. The pre-clinical study also found that anti-CD70 AlloCAR T cells could be successfully manufactured in a large-scale process. See, “Allogene Therapeutics Presents Preclinical Data Demonstrating the Potential of AlloCAR T™ Therapy in Renal Cell Carcinoma (RCC) at the 2019 AACR Annual Meeting,” Apr. 1, 2019. See also, U.S. Published Patent Application No. 20120288512 entitled “Anti-CD70 Antibody-Drug Conjugates and Their Use for the Treatment of Cancer and Immune Disorders,” U.S. Published Patent Application No. 20180230224 entitled “CD70 BINDING MOLECULES AND METHODS OF USE THEREOF” and U.S. Published Patent Application No. 20180208671 entitled “ANTI-CD70 CHIMERIC ANTIGEN RECEPTORS,” all of which are hereby incorporated by referenced in their entirety.
In a preferred embodiment, the present invention is a method for producing microvesicles ex vivo for use in systemic treatment of cancer. The method comprises isolating patient cancerous tumor primary cells, culturing isolated patient cancerous tumor primary cells in appropriate culture media, treating cultured patient cancerous tumor primary cells non cold atmospheric plasma, after apoptosis of cultured patient cancerous tumor primary cells occurs, collecting apoptotic cell-derived extracellular microvesicles from the culture media by differential centrifugation, directly applying apoptotic cell-derived extracellular microvesicles to one of a naïve T cell culture or a dendritic cell culture, isolating antigen specific T cells from said one of a T cell culture and a dendritic cell culture, and storing said isolated antigen specific T cells.
Still other aspects, features, and advantages of the present invention are readily apparent from the following detailed description, simply by illustrating a preferable embodiments and implementations. The present invention is also capable of other and different embodiments and its several details can be modified in various obvious respects, all without departing from the spirit and scope of the present invention. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature, and not as restrictive. Additional objects and advantages of the invention will be set forth in part in the description which follows and in part will be obvious from the description or may be learned by practice of the invention.
For a more complete understanding of the present invention and the advantages thereof, reference is now made to the following description and the accompanying drawings, in which:
A preferred embodiment of the present invention is described with reference to
Thus, as shown in
An exemplary CAR-T therapy that may be used in the present invention is described in S. Panowski, et al., “AlloCART™ TARGETING CD70 FOR THE TREATMENT OF RENAL CELL CARCINOMA.” Renal Cell Carcinoma (RCC) is a highly T-cell infiltrated tumor type with responsiveness to immuno-oncology agents. T cells can be genetically modified to express chimeric antigen receptors (CARs). To translate this approach for RCC treatment, expression data were mined and CD70 was identified as an antigen expressed in a high proportion of patients with RCC, with limited normal tissue expression on a fraction of activated lymphocytes and dendritic cells. Since CD70 expression is present on activated T cells, targeting it with a CAR could lead to fratricide and T cell exhaustion. Screens were specifically designed to identify CARs that were less impacted by these issues. A large panel of scFvs that bind to CD70 were generated and formatted into CARs. CD70 CAR-T cells were ranked based on tonic signaling, transduction efficiency, phenotype, activation status and expansion. A subset of CD70 CAR-T cells were moved into in vitro short and long-term cytotoxicity assays. Target cells expressing high, medium, and low levels of CD70 were utilized. CART cells were evaluated in vivo and robust anti-tumor activity was observed. Some candidates performed better with CD70 knockout, and some worked irrespective of knockout. A cynomolgus monkey toxicity study was conducted with one clone formatted as a CD70-CD3 bispecific antibody and no unexpected findings were observed. Multiple off-switch CAR formats were evaluated. CD70 CAR-T cells were also successfully manufactured in a large-scale process. In summary, multiple CD70 CAR-T cells have been profiled and a subset selected for further investigation as potential clinical candidates.
The present invention may be used with other CAR-T therapies. Additionally, use of the CAP treatment may allow for reduced dosage for the CAR-T therapy as well as reduced dosages in chemotherapy and other treatments.
Recently immunotherapy such as chimeric antigen receptors (CARs) has brought new paradigm in cancer immunotherapy, wherein a patient's own T cells are bioengineered to express CARs that identify, attach to, and subsequently kill tumor cells. Moreover, checkpoint blockade, adoptive cell transfer, human recombinant cytokines and cancer vaccines have shown very encouraging signs for cancer treatment, however only a subset of patients show complete response to these treatments. The principle of cancer immunotherapy is based on the identification of tumor-associated antigens (TAAs) which are dysregulated mutated gene products that are presented as antigens and neutralization of these cells by engineered T cells. However, the sparse expression of these antigens and loss of neoantigen during malignancy are insufficient to prompt a full-blown immune response to neutralize the tumor. Moreover, these therapies have other limitations that directly affect patients, some of these are cytokine release syndrome (CRS) and CAR T-related encephalopathy syndrome (CRES), long vein-to-vein time, treatment is restricted to heavily pretreated patients, multistep process of generating autologous CAR T cells increases the risk of production failure and commercial scalability challenges. Recently we have found that the non-thermal or cold atmospheric plasma (CAP) treatment of cancer cells can oxidized proteins and induces cell death. These oxidized proteins are carried by apoptotic bodies and could act as antigen presenting cells (APC). Oxidized proteins derived from CAP treated apoptotic cells are better immunogenic epitopes that could induce a strong immune response against the tumor cells. See, Takada, K. et al., “Combined chronic toxicity/carcinogenicity test of tris(2-chloroethyl)phosphate (TCEP) applied to female mouse skin,” Eisei Shikenjo Hokoku, 18-24 (1991).
For several cancer cell lines apoptosis has been shown to be superior to necrosis in facilitating the cross-presentation of tumor-associated antigens to CD8+ T cells by dendritic cells (DCs) and DCs are principal initiators of CD4+ and CD8+ T cell-mediated immune responses. See, Banchereau, J. & Steinman, R. M., “Dendritic cells and the control of immunity,” Nature 392, 245-252 (1998). Previous studies shown that plasma treatment could induces cancer-specific long-term immune memory in mice by enhanced cytotoxic T cell infiltration into reinoculated tumors. See, Mizuno, K., et al., “Plasma-Induced Suppression of Recurrent and Reinoculated Melanoma Tumors in Mice,” IEEE 2, 353-359 (2018). Moreover, apoptotic cell-derived extracellular microvesicles (ApoEVs) act directly as antigen presentation units by carrying surface MHC molecules in complex with antigenic peptide to directly interact with naïve T cells. See, Braciale, T. J. et al., “Antigen presentation pathways to class I and class II MHC-restricted T lymphocytes,” Immunol Rev 98, 95-114 and Muhsin-Sharafaldine, M. R. et al., “Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles.” Oncotarget 7, 56279-56294.
In the present invention, cancer cells are isolated from the patient and are treated ex vivo with CAP to isolate the microvesicles, which are then administered to the patient to provide systemic treatment of the cancer within the patient.
The method includes the following steps:
A preferred embodiment of a CAP enabled generator is described with reference to the drawings. A gas-enhanced electrosurgical generator 200 in accordance with a preferred embodiment of the present invention is shown in
A generator housing front panel 210 is connected to housing 202. On the face front panel 210 there is a touch-screen display 212 and there may be one or a plurality of connectors 214 for connecting various accessories to the generator 200. For a cold atmospheric plasma generator such as is shown in
As shown in
As shown in
Another embodiment, shown in
The various valves and sensors in either embodiment of the module are electrically connected to a main PCB Board through a connector. The PCB connector is connected to a PCB Board that has a microcontroller (such as CPU). As previously noted, a plurality of gas modules can be in a single gas control unit or single electrosurgical generator to provide control of multiple differing gases. The plurality of gas control modules further may be connected to the same PCB Board, thus providing common control of the modules.
In the above-disclosed embodiment, a cold atmospheric plasma below 35° C. is produced. When applied to the tissue surrounding the surgical area, the cold atmospheric plasma induces metabolic suppression in only the tumor cells and enhances the response to the drugs that are injected into the patient.
The cold plasma applicator 500 may be in a form such as is disclosed in U.S. Pat. No. 10,405,913 and shown in
While the present application discloses a specific type of cold plasma, other types of plasma jets may be used in the present invention.
The foregoing description of the preferred embodiment of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiment was chosen and described in order to explain the principles of the invention and its practical application to enable one skilled in the art to utilize the invention in various embodiments as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto, and their equivalents. The entirety of each of the aforementioned documents is incorporated by reference herein.
The present application is a continuation-in-part of U.S. Non-provisional patent application Ser. No. 16/892,651, filed on Jun. 4, 2020, which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/857,022 filed on Jun. 4, 2019. The present application further claims the benefit of the filing date of U.S. Provisional Patent Application Ser. No. 63/339,704 filed by the present inventors on May 9, 2022. The aforementioned provisional patent applications are hereby incorporated by reference in their entirety.
Number | Date | Country | |
---|---|---|---|
63339704 | May 2022 | US | |
62857022 | Jun 2019 | US |
Number | Date | Country | |
---|---|---|---|
Parent | 16892651 | Jun 2020 | US |
Child | 18195223 | US |