Engineering and delivery of therapeutic compositions of freshly 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 - 24 . (canceled) 
     
     
         25 . A composition comprising post-electroloaded blood mononuclear cells, wherein said post-electroloaded blood mononuclear cells are produced by the method comprising:
 (a) electroloading a pre-electroloaded composition comprising unstimulated resting blood mononuclear cells with a mRNA encoding a protein; and   (b) transiently expressing the protein encoded by said mRNA,   wherein the viability of said post-electroloaded composition of said electroloaded blood mononuclear cells 1 day after electroloading is at least 80% if normalized to un-electroloaded blood mononuclear cells.   
     
     
         26 . The composition of  claim 25 , wherein the post-electroloaded composition comprises lymphocytes, T cells, CD3+ T cells, CD8+ T cells, Natural Killer (NK) cells, monocytes, or antigen presenting cells (APCs). 
     
     
         27 . The composition of  claim 25 , wherein the unstimulated resting blood mononuclear cells comprise lymphocytes, T cells, CD3+ T cells, CD8+ T cells, Natural Killer (NK) cells, monocytes, or antigen presenting cells (APCs). 
     
     
         28 . The composition of  claim 25 , wherein the unstimulated resting blood mononuclear cells are derived from peripheral blood (PB), bone aspirates, lipoaspirates, or tissue-specific perfusates/isolates. 
     
     
         29 . The composition of  claim 25 , wherein the unstimulated resting blood mononuclear cells comprise peripheral blood mononuclear cells (PBMCs). 
     
     
         30 . The composition of  claim 29 , wherein the PBMCs are frozen after collection. 
     
     
         31 . The composition of  claim 29 , wherein the PBMCs are cultured for less than 2 days. 
     
     
         32 . The composition of  claim 25 , wherein the unstimulated resting blood mononuclear cells comprise peripheral blood lymphocytes (PBLs). 
     
     
         33 . The composition of  claim 32 , wherein the PBLs are isolated by culturing PBMCs in a container and removing the cells that attach to the surface after about 1-2 hours. 
     
     
         34 . The composition of  claim 25 , wherein the unstimulated resting blood mononuclear cells comprise lymphocytes. 
     
     
         35 . The composition of  claim 34 , wherein the lymphocytes comprise a population of T cells. 
     
     
         36 . The composition of  claim 35 , wherein the T cells are separated from non-T cells by centrifugation. 
     
     
         37 . The composition of  claim 35 , wherein the T cells are separated from non-T cells by depleting non-T cells with an antibody that binds to CD56 and/or CD16. 
     
     
         38 . The composition of  claim 35 , wherein the population of T cells are enriched for CD3+ T cells. 
     
     
         39 . The composition of  claim 35 , wherein the population of T cells are enriched for CD8+ T cells. 
     
     
         40 . The composition of  claim 35 , wherein the T cells are cultured for less than 2 days. 
     
     
         41 . The composition of  claim 25 , where the protein is a chimeric receptor. 
     
     
         42 . The composition of  claim 41 , wherein the chimeric receptor is selected from an anti-CD19 chimeric receptor, an anti-CD19BBz encoding a single chain antibody conjugated with the 4-1 BB intercellular domain and the CD34 domain, an anti-CD20 chimeric receptor, an anti-FBP chimeric receptor, an anti-TAG-72 chimeric receptor, an anti-CEA chimeric receptor, an anti-carboxyanhydrase IX chimeric receptor, a nati-CD171 chimeric receptor, an anti-IL-13 chimeric receptor, an anti-G(D)2 chimeric receptor, an anti-PSMA chimeric receptor, an anti-mesothelin chimeric receptor, an anti-Lewis-Y chimeric receptor, or an anti-CD30 chimeric receptor. 
     
     
         43 . The composition of  claim 41 , wherein the chimeric receptor comprises an extracellular domain, a transmembrane domain, a costimulatory signaling region, and a CD3-zeta signaling domain. 
     
     
         44 . The composition of  claim 43 , wherein the extracellular domain comprises an antigen-binding moiety. 
     
     
         45 . The composition of  claim 44 , wherein the antigen-binding moiety binds to a tumor antigen. 
     
     
         46 . The composition of  claim 45 , wherein the tumor antigen is an antigen associated with a cancer selected from the group consisting of 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, leukemia, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and mantle cell lymphoma (MCL). 
     
     
         47 . The composition of  claim 41 , wherein the chimeric receptor is an anti-mesothelin chimeric receptor. 
     
     
         48 . The composition of  claim 47 , wherein the anti-mesothelin chimeric receptor comprises an anti-mesothelin scFv. 
     
     
         49 . The composition of  claim 48 , wherein the anti-mesothelin scFv is ss1. 
     
     
         50 . The composition of  claim 41 , wherein the chimeric receptor comprises an intracellular domain selected from either 41BB or CD28, and the cytoplasmic portion of the CD3-zeta chain. 
     
     
         51 . The composition of  claim 47 , wherein the anti-mesothelin chimeric receptor comprises an anti-mesothelin scFv, a 4-1 BB intracellular domain, and a CD3-zeta domain. 
     
     
         52 . A method of treating a subject, wherein said method comprises administering to a subject in need thereof the composition of  claim 25 . 
     
     
         53 . The method of  claim 52 , wherein the subject has a hyperproliferative disease. 
     
     
         54 . The method of  claim 53 , wherein the hyperproliferative disease is cancer. 
     
     
         55 . The method of  claim 54 , wherein the cancer is selected from the group consisting of 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, leukemia, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and mantle cell lymphoma (MCL). 
     
     
         56 . A method of treating a subject, wherein said method comprises administering to a subject in need thereof the composition of  claim 29 . 
     
     
         57 . The method of  claim 56 , wherein the subject has a hyperproliferative disease. 
     
     
         58 . The method of  claim 57 , wherein the hyperproliferative disease is cancer. 
     
     
         59 . The method of  claim 58 , wherein the cancer is selected from the group consisting of 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, leukemia, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and mantle cell lymphoma (MCL). 
     
     
         60 . A method of treating a subject, wherein said method comprises administering to a subject in need thereof the composition of  claim 35 . 
     
     
         61 . The method of  claim 60 , wherein the subject has a hyperproliferative disease. 
     
     
         62 . The method of  claim 61 , wherein the hyperproliferative disease is cancer. 
     
     
         63 . The method of  claim 62 , wherein the cancer is selected from the group consisting of 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, leukemia, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and mantle cell lymphoma (MCL). 
     
     
         64 . A method of treating a subject, wherein said method comprises administering to a subject in need thereof the composition of  claim 38 . 
     
     
         65 . The method of  claim 64 , wherein the subject has a hyperproliferative disease. 
     
     
         66 . The method of  claim 65 , wherein the hyperproliferative disease is cancer. 
     
     
         67 . The method of  claim 66 , wherein the cancer is selected from the group consisting of 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, leukemia, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and mantle cell lymphoma (MCL).