Inoculation by Applying Nanosecond Pulsed Electric Fields to a Biopsy and Reintroducing the Treated Biopsy to the Subject

Abstract

A subject is inoculated from a disease by exposing a biopsy of a tumor or other abnormal growth to a nanosecond pulsed electric field (nsPEF). A sufficient treatment can be confirmed by detecting calreticulin on the tumor cell membranes, which indicates apoptosis occurring in the tumor cells. Treated tumor cells from the biopsy are then reintroduced into the subject. The calreticulin-exhibiting tumor cells activate the subject's immune system against the tumor, and any other like tumors in the body, and effectively vaccinates the subject against the disease. The treatment can be combined with CD47-blocking antibodies, doxorubicin, CTLA-4-blocking antibodies, and/or PD-1-blocking antibodies. The immune response may be measured at a later time. Specific electrical characteristics of the nsPEF treatments can be based on the type and/or strength of the tumor.

What is claimed is: 
     
         1 . A method of using a biopsy to treat a disease in a subject, the method comprising:
 extracting a sample of cells from an abnormal growth from a subject;   passing sub-microsecond pulsed electric fields through the extracted sample of cells sufficient to stimulate apoptosis in the cells;   confirming that the cells of the sample have initiated apoptosis; and then   reintroducing the cells into the subject, the cells that have initiated apoptosis inoculating the subject from the disease.   
     
     
         2 . The method of  claim 1  wherein the confirming includes detecting calreticulin on surface membranes of the cells. 
     
     
         3 . The method of  claim 1  further comprising:
 introducing CD47-blocking antibodies into the subject, the CD47-blocking antibodies neutralizing CD47 on surface membranes of the cells. 
 
     
     
         4 . The method of  claim 1  further comprising:
 introducing CD47-blocking antibodies to the extracted sample of cells, the CD47-blocking antibodies neutralizing CD47 on surface membranes of the cells. 
 
     
     
         5 . The method of  claim 4  wherein the introducing of CD47-blocking antibodies occurs before the passing of sub-microsecond pulsed electric fields. 
     
     
         6 . The method of  claim 4  wherein the introducing of CD47-blocking antibodies occurs after the passing of sub-microsecond pulsed electric fields. 
     
     
         7 . The method of  claim 1  further comprising:
 introducing doxorubicin into the subject. 
 
     
     
         8 . The method of  claim 1  further comprising:
 introducing doxorubicin to the extracted sample of cells. 
 
     
     
         9 . The method of  claim 8  wherein the introducing of doxorubicin occurs before the passing of sub-microsecond pulsed electric fields. 
     
     
         10 . The method of  claim 8  wherein the introducing of doxorubicin occurs after the passing of sub-microsecond pulsed electric fields. 
     
     
         11 . The method of  claim 1  further comprising:
 introducing CTLA-4-blocking antibodies into the subject. 
 
     
     
         12 . The method of  claim 1  further comprising:
 introducing CTLA-4-blocking antibodies to the extracted sample of cells. 
 
     
     
         13 . The method of  claim 12  wherein the introducing of CTLA-4-blocking antibodies occurs before the passing of sub-microsecond pulsed electric fields. 
     
     
         14 . The method of  claim 12  wherein the introducing of CTLA-4-blocking antibodies occurs after the passing of sub-microsecond pulsed electric fields. 
     
     
         15 . The method of  claim 1  further comprising
 introducing PD-1-blocking antibodies into the subject. 
 
     
     
         16 . The method of  claim 1  further comprising
 introducing PD-1-blocking antibodies to the extracted sample of cells. 
 
     
     
         17 . The method of  claim 16  wherein the introducing of PD-1-blocking antibodies occurs before the passing of sub-microsecond pulsed electric fields. 
     
     
         18 . The method of  claim 16  wherein the introducing of PD-1-blocking antibodies occurs after the passing of sub-microsecond pulsed electric fields. 
     
     
         19 . The method of  claim 1  wherein the abnormal growth includes a tumor. 
     
     
         20 . The method of  claim 1  wherein the subject is a human, a cat, or a dog. 
     
     
         21 . The method of  claim 1  further comprising:
 measuring an immune response biomarker in a second sample of the subject in order to confirm a stimulation of an immune response of the subject against the disease 
 measuring a concentration or level of white blood cells in the second sample. 
 
     
     
         22 . The method of  claim 21  wherein measuring the immune response biomarker comprises:
 gauging a concentration or level in the second sample of a member selected from a group consisting of white blood cells, inflammatory cytokines, C-reactive proteins, and antibodies of cancer cell markers. 
 
     
     
         23 . The method of  claim 1  wherein the sub-microsecond pulsed electric fields includes a number of pulses greater than 50, an amplitude of at least 10 kilovolts per centimeter, and a pulse length of between 0.1 and 1000 nanoseconds.