Engineering and delivery of therapeutic compositions of freshley isolated cells

Abstract

The present invention relates to the transient modification of cells. In particular embodiments, the cells are immune systems, such as PBMC, PBL, T (CD3+ and/or CD8+) and Natural Killer (NK) cells. The modified cells provide a population of cells that express a genetically engineered chimeric receptor which can be administered to a patient therapeutically. The present invention further relates to methods that deliver mRNA coding for the chimeric receptor to unstimulated resting PBMC, PBL, T (CD3+ and/or CD8+) and NK cells and which delivers the mRNA efficiently to the transfected cells and promotes significant target cell killing.

1 . A method for preparing transiently modified unstimulated resting peripheral blood mononuclear cells (PBMCs), the method comprising:
 (a) obtaining freshly collected unstimulated resting PBMCs from a peripheral blood sample; and   (b) electroloading the PBMCs with an mRNA encoding for a chimeric receptor to generate electroloaded PBMCs expressing the chimeric receptor on their surface;   wherein the electroloading comprises flow electroporation.   
     
     
         2 . The method of  claim 1 , wherein the PBMCs are electroloaded less than 5 hours from the time the PBMCs were originally obtained from a subject. 
     
     
         3 . The method of  claim 1 , wherein the PBMCs do not undergo cell division prior to (b). 
     
     
         4 . The method of  claim 1 , wherein the chimeric receptor is an anti-CD19 chimeric receptor. 
     
     
         5 . The method of  claim 1 , wherein the chimeric receptor is an anti-CD20, anti-FBP, anti-TAG-72, anti-CEA, anti-carboxyanhydrase IX, anti-CD171, anti-IL-13 receptor, anti-G(D)2, anti-PSMA, anti-mesothelin, anti-Lewis-Y, or anti-CD30 chimeric receptor. 
     
     
         6 . The method of  claim 1 , wherein the chimeric receptor comprises a CD28 intracellular domain. 
     
     
         7 . The method of  claim 1 , wherein the chimeric receptor does not comprise a CD28 intracellular domain. 
     
     
         8 . A method for administering cells to a subject, the method comprising:
 (a) obtaining freshly collected unstimulated resting PBMCs from a peripheral blood sample;   (b) electroloading the PBMCs with an mRNA encoding for a chimeric receptor to generate electroloaded PBMCs expressing the chimeric receptor on their surface; and   (c) administering the electroloaded PBMCs to the subject,   wherein the electroloading comprises flow electroporation.   
     
     
         9 . The method of  claim 8 , wherein the PBMCs are electroloaded less than 5 hours from the time the PBMCs were originally obtained from a subject. 
     
     
         10 . The method of  claim 8 , wherein the PBMCs do not undergo cell division prior to (b). 
     
     
         11 . The method of  claim 8 , wherein the chimeric receptor is an anti-CD19 chimeric receptor. 
     
     
         12 . The method of  claim 8 , wherein the chimeric receptor is an anti-CD20, anti-FBP, anti-TAG-72, anti-CEA, anti-carboxyanhydrase IX, nati-CD171, anti-IL-13 receptor, anti-G(D)2, anti-PSMA, anti-mesothelin, anti-Lewis-Y, or anti-CD30 chimeric receptor. 
     
     
         13 . The method of  claim 8 , wherein the chimeric receptor comprises a CD28 intracellular domain. 
     
     
         14 . The method of  claim 8 , wherein the chimeric receptor does not comprise a CD28 intracellular domain. 
     
     
         15 . The method of  claim 8 , wherein the PBMCs are autologous to the subject. 
     
     
         16 . The method of  claim 8 , wherein the PBMCs are allogenic to the subject. 
     
     
         17 . The method of  claim 8 , wherein (a) comprises collecting the peripheral blood sample from a donor and separating the PBMCs in the sample from the non-PBMCs in the sample. 
     
     
         18 . The method of  claim 8 , wherein the method comprises treating or preventing a cancer in the subject. 
     
     
         19 . The method of  claim 18 , wherein the cancer is breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head & neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, or leukemia. 
     
     
         20 . The method of  claim 19 , wherein the leukemia is acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), or mantle cell lymphoma (MCL). 
     
     
         21 . The method of  claim 8 , wherein the electroloaded PBMCs are administered to the subject by intravenous injection. 
     
     
         22 . The method of  claim 8 , wherein the electroloaded PBMCs are administered to the subject by intratumoral injection.