1. At least one DNA cassette with ‘suicidal genes cytosine deaminase and or herpes thymidine kinase’ and ‘chimeric receptor antigen’ to attack specific markers like CD19, wherein the cassette has the appropriate selection marker like Bleomycin/ Zeocin but not exclusively the mentioned selection markers. The gene(s) in the DNA cassette can be tagged with the expression of Green fluorescent protein or any small DNA tag (His/FLAG tag etc.) so as to confirm the functional expression of the construct visually by Fluorescent Microscopy or Mass Spectrometry, ELISA and or Western Blot for the gene or tagged gene. The invention and application for patent is about the said use of ‘genes’ in one DNA cassette and does not exclude use of any system or cells for the purpose of treating any cancer or treatment of an auto-immune or infectious disease.

2. A method of production of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette coding for ‘chimeric receptor antigen’ of camelid variety or from a source including llama and camel such that the said fraction is a nano-antibody capable of binding the respective receptor.

3. A method of production of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette coding for ‘chimeric receptor antigen’ of human variety capable of binding the respective receptor along with luciferase gene.

4. A method of production of a group of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette coding for ‘chimeric receptor antigen’ of human variety capable of binding the respective receptor without a reporter gene like luciferase.

5. A method of production of a group of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette coding for ‘costimulatory molecules’ along with the chimeric antigen receptor.

6. A method of production of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette consisting of ‘luciferase’ and ‘suicide genes cytosine deaminase and herpes simplex thymidine kinase’ where luciferase can offer a biological assay to monitor the expression of said genes and suicide genes offer a safety control over the survival of modified T cells.

7. A method of treating the patients of leukemia and other malignancies including cerebral tumors by infusing the transgenic T cells and keeping a safety control on the T cells by virtue of suicide genes.

8. A method of treating the patients post-surgery for rapid recovery from metastatic cancers.

9. A method of using the temporary, non-genome-integrative gene expression in the T cells by virtue of adenoviral vectors, or adeno-associated viral vectors or lipofection or electroporation whereas the transgenic cells express the factors only for a limited amount of time and thus provide a natural self-limiting mechanisms to exposure of transgenic factors when a permanent expression is not required.

10. A method of using Chimeric Antigen Receptor utilizing CD1a and CD207 binding chimeric antibody fragments for treatment of histiocytoma.

11. A method of using Chimeric Antigen Receptor utilizing CD13, CD33, CD117 binding chimeric antibody fragments for treatment of myeloid cell diseases including cancers.

12. A method of using Chimeric Antigen Receptor utilizing CD23 and CD5 binding chimeric antibody fragments for treatment of chronic lymphocytic leukemia.

13. A method of using Chimeric Antigen Receptor utilizing CD5 binding chimeric antibody fragments for treatment of mantle cell lymphoma.

14. A method of using Chimeric Antigen Receptor utilizing CD30 binding chimeric antibody fragments for treatment of anaplastic large cell lymphoma.

15. A method of using Chimeric Antigen Receptor utilizing CD99 binding chimeric antibody fragments for treatment of Ewing’s sarcoma.

16. A method of using Chimeric Antigen Receptor utilizing CD117 binding chimeric antibody fragments for treatment of gastrointestinal stromal tumor.

17. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of B cell leukemia

18. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of malignant melanoma

19. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of lung carcinoma

20. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of hepatic carcinoma

21. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of glioblastoma

22. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of renal cell carcinoma

23. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of rhabdomyosarcoma

24. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of osteosarcoma

25. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of prostate cancer

26. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of medulloblastoma

27. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of neuroblastoma

28. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of viral diseases

29. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of other malignant or abnormal rapidly growing or slow growing diseases

30. A method of using chimeric antigen receptor with co-expression of suicidal genes delivered by virtue of non-viral circular DNA molecules where the bacterial plasmids have eliminated the bacterial genes and bacterial DNA so as to generate the CAR generating minicircles of DNA so as to eliminate the ex-vivo gene transfer and simplify the therapy process by simple injections of DNA cassettes directly into the body for all carcinomas mentioned above. , Description:Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies or diseases described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. The present invention describes a DNA cassette for the constitutive or inducible expression of the therapeutic genes within the selected carrier cells. The said DNA cassette comprises of therapeutic chimeric antigen receptor sequence along with EGFR truncated (EGFRt) or herpes simplex viral thymidine kinase (HSV-tk) or cytosine deaminase (CD) as suicidal switch genes along with Guassia luciferase or any other kind of luciferase or fluorescent gene. The DNA cassette can be used for gene therapy involving direct delivery of genes to natural tissues. Our invention manages to get the therapeutic effect on cancer by virtue of genetically modified T-cells. T Cells of our invention will attack the cancer cells, their level can be monitored by taking blood and urine samples of patients for measuring the luciferase activity and their growth and death can be controlled by virtue of a suicidal switch. Chimeric antigen receptor binds a specific antigen expressed on the cancer cells or cells involved in other diseases. Chimeric receptor antigen targets the T-cells to the specific cells that need to be destroyed for the treatment of the disease. For example the cell therapy using CD19-targeted CAR-T cells can be used for treatment of acute lymphoblastic leukemia. To get the best control over this therapy, in this invention, the T cells which are carrier cells, are armed with suicidal genes and luciferase gene. Thus when the number of T cells grows over the required limit or when the patient experiences severe reactions due to particularly high levels of IL-6, the number of T cells can be controlled by virtue of suicidal genes. In addition to this benefit, when the CAR therapy is used against solid tumors, the T cells can be actually induced to undergo suicidal apoptosis so that the cancer cells can die along with the dying T cell. In one of the embodiment of the present invention, the said chimeric antigen receptor DNA consists of light chain and heavy chain linked with costimulatory molecules. The said DNA cassette consisting of therapeutic genes either alone or in combination is incorporated into the carrier cells of choice by use of an established plasmid. The DNA cassette can be inserted into a bacterial plasmid or viral particles during the process of propagation and transduction. Bacterial culture can be used for multiplication of Plasmid number. Once expressed, the therapeutic genes will be available for an extended but controlled period of time at the site of tumor/ malignancies providing optimal clinical benefit. Further, as the carrier cells are appropriately transfected with the said DNA cassette, the cells will be intravenously administered to patient. Two populations of said carrier cells expressing, individual genes like luciferase and suicidal genes can also be separately infused to the patient, if the size of overall construct is too big for expression in one instance. In another embodiment of the present invention the said DNA cassette can be tagged with the expression of Bleomycin/ Zeocin, Green fluorescent protein or small engineered DNA tag namely His/FLAG tag etc. to confirm the functional expression of the said DNA cassette visually by Green fluorescent protein or Mass Spectrometry or ELISA or Western Blot. The novelty of the present invention lies in the therapeutic mechanism offered by ingenuously allowing the T cells expressing luciferase gene, co-stimulatory molecules and suicidal genes along with the chimeric antigen receptor sequence. In one embodiment of this invention, humanized nano-antibodies generated in camels or Llamas will create effective antibody fragments for CAR for various tumor antigens. The suicidal genes offer an excellent safety switch such that the T-cell number can be kept in check in case of an unwanted reaction faced by the patient. In one embodiment, the DNA construct for treatment of acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin’s lymphoma utilizes the antibody for CD19 antigen. CD19 is absent on bone marrow progenitor cells. In fact our “DartBriteTM” or CD19 Chimeric Antigen Receptor Therapy may also be administered without the viral means of DNA delivery by use of supercoiled DNA molecules that can be administered straight into the patient’s body by intravenous or other routes of administration.

Claims:1. At least one DNA cassette with ‘suicidal genes cytosine deaminase and or herpes thymidine kinase’ and ‘chimeric receptor antigen’ to attack specific markers like CD19, wherein the cassette has the appropriate selection marker like Bleomycin/ Zeocin but not exclusively the mentioned selection markers. The gene(s) in the DNA cassette can be tagged with the expression of Green fluorescent protein or any small DNA tag (His/FLAG tag etc.) so as to confirm the functional expression of the construct visually by Fluorescent Microscopy or Mass Spectrometry, ELISA and or Western Blot for the gene or tagged gene. The invention and application for patent is about the said use of ‘genes’ in one DNA cassette and does not exclude use of any system or cells for the purpose of treating any cancer or treatment of an auto-immune or infectious disease.
2. A method of production of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette coding for ‘chimeric receptor antigen’ of camelid variety or from a source including llama and camel such that the said fraction is a nano-antibody capable of binding the respective receptor.
3. A method of production of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette coding for ‘chimeric receptor antigen’ of human variety capable of binding the respective receptor along with luciferase gene.
4. A method of production of a group of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette coding for ‘chimeric receptor antigen’ of human variety capable of binding the respective receptor without a reporter gene like luciferase.
5. A method of production of a group of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette coding for ‘costimulatory molecules’ along with the chimeric antigen receptor.
6. A method of production of transgenic T cells or other somatic cells where the transgenesis is conducted with the specifically engineered DNA cassette consisting of ‘luciferase’ and ‘suicide genes cytosine deaminase and herpes simplex thymidine kinase’ where luciferase can offer a biological assay to monitor the expression of said genes and suicide genes offer a safety control over the survival of modified T cells.
7. A method of treating the patients of leukemia and other malignancies including cerebral tumors by infusing the transgenic T cells and keeping a safety control on the T cells by virtue of suicide genes.
8. A method of treating the patients post-surgery for rapid recovery from metastatic cancers.
9. A method of using the temporary, non-genome-integrative gene expression in the T cells by virtue of adenoviral vectors, or adeno-associated viral vectors or lipofection or electroporation whereas the transgenic cells express the factors only for a limited amount of time and thus provide a natural self-limiting mechanisms to exposure of transgenic factors when a permanent expression is not required.
10. A method of using Chimeric Antigen Receptor utilizing CD1a and CD207 binding chimeric antibody fragments for treatment of histiocytoma.
11. A method of using Chimeric Antigen Receptor utilizing CD13, CD33, CD117 binding chimeric antibody fragments for treatment of myeloid cell diseases including cancers.
12. A method of using Chimeric Antigen Receptor utilizing CD23 and CD5 binding chimeric antibody fragments for treatment of chronic lymphocytic leukemia.
13. A method of using Chimeric Antigen Receptor utilizing CD5 binding chimeric antibody fragments for treatment of mantle cell lymphoma.
14. A method of using Chimeric Antigen Receptor utilizing CD30 binding chimeric antibody fragments for treatment of anaplastic large cell lymphoma.
15. A method of using Chimeric Antigen Receptor utilizing CD99 binding chimeric antibody fragments for treatment of Ewing’s sarcoma.
16. A method of using Chimeric Antigen Receptor utilizing CD117 binding chimeric antibody fragments for treatment of gastrointestinal stromal tumor.
17. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of B cell leukemia
18. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of malignant melanoma
19. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of lung carcinoma
20. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of hepatic carcinoma
21. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of glioblastoma
22. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of renal cell carcinoma
23. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of rhabdomyosarcoma
24. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of osteosarcoma
25. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of prostate cancer
26. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of medulloblastoma
27. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of neuroblastoma
28. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of viral diseases
29. A method of using Chimeric Antigen Receptor with co-expression of luciferase and suicidal genes for treatment of other malignant or abnormal rapidly growing or slow growing diseases
30. A method of using chimeric antigen receptor with co-expression of suicidal genes delivered by virtue of non-viral circular DNA molecules where the bacterial plasmids have eliminated the bacterial genes and bacterial DNA so as to generate the CAR generating minicircles of DNA so as to eliminate the ex-vivo gene transfer and simplify the therapy process by simple injections of DNA cassettes directly into the body for all carcinomas mentioned above.
, Description:Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies or diseases described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results.
The present invention describes a DNA cassette for the constitutive or inducible expression of the therapeutic genes within the selected carrier cells. The said DNA cassette comprises of therapeutic chimeric antigen receptor sequence along with EGFR truncated (EGFRt) or herpes simplex viral thymidine kinase (HSV-tk) or cytosine deaminase (CD) as suicidal switch genes along with Guassia luciferase or any other kind of luciferase or fluorescent gene.
The DNA cassette can be used for gene therapy involving direct delivery of genes to natural tissues. Our invention manages to get the therapeutic effect on cancer by virtue of genetically modified T-cells. T Cells of our invention will attack the cancer cells, their level can be monitored by taking blood and urine samples of patients for measuring the luciferase activity and their growth and death can be controlled by virtue of a suicidal switch.
Chimeric antigen receptor binds a specific antigen expressed on the cancer cells or cells involved in other diseases. Chimeric receptor antigen targets the T-cells to the specific cells that need to be destroyed for the treatment of the disease. For example the cell therapy using CD19-targeted CAR-T cells can be used for treatment of acute lymphoblastic leukemia. To get the best control over this therapy, in this invention, the T cells which are carrier cells, are armed with suicidal genes and luciferase gene. Thus when the number of T cells grows over the required limit or when the patient experiences severe reactions due to particularly high levels of IL-6, the number of T cells can be controlled by virtue of suicidal genes.
In addition to this benefit, when the CAR therapy is used against solid tumors, the T cells can be actually induced to undergo suicidal apoptosis so that the cancer cells can die along with the dying T cell.
In one of the embodiment of the present invention, the said chimeric antigen receptor DNA consists of light chain and heavy chain linked with costimulatory molecules.
The said DNA cassette consisting of therapeutic genes either alone or in combination is incorporated into the carrier cells of choice by use of an established plasmid. The DNA cassette can be inserted into a bacterial plasmid or viral particles during the process of propagation and transduction. Bacterial culture can be used for multiplication of Plasmid number.
Once expressed, the therapeutic genes will be available for an extended but controlled period of time at the site of tumor/ malignancies providing optimal clinical benefit. Further, as the carrier cells are appropriately transfected with the said DNA cassette, the cells will be intravenously administered to patient. Two populations of said carrier cells expressing, individual genes like luciferase and suicidal genes can also be separately infused to the patient, if the size of overall construct is too big for expression in one instance.
In another embodiment of the present invention the said DNA cassette can be tagged with the expression of Bleomycin/ Zeocin, Green fluorescent protein or small engineered DNA tag namely His/FLAG tag etc. to confirm the functional expression of the said DNA cassette visually by Green fluorescent protein or Mass Spectrometry or ELISA or Western Blot.
The novelty of the present invention lies in the therapeutic mechanism offered by ingenuously allowing the T cells expressing luciferase gene, co-stimulatory molecules and suicidal genes along with the chimeric antigen receptor sequence.
In one embodiment of this invention, humanized nano-antibodies generated in camels or Llamas will create effective antibody fragments for CAR for various tumor antigens. The suicidal genes offer an excellent safety switch such that the T-cell number can be kept in check in case of an unwanted reaction faced by the patient.
In one embodiment, the DNA construct for treatment of acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin’s lymphoma utilizes the antibody for CD19 antigen. CD19 is absent on bone marrow progenitor cells.
In fact our “DartBriteTM” or CD19 Chimeric Antigen Receptor Therapy may also be administered without the viral means of DNA delivery by use of supercoiled DNA molecules that can be administered straight into the patient’s body by intravenous or other routes of administration.