Screening of cell clones expressing polygenic transgenes through non-antibiotic dependent positive selection
Compositions and methods are provided for generating a clonal population of transfected eukaryotic cells derived from a single cell. The method includes transfecting a population of eukaryotic cells with a multi-cistronic nucleic acid vector followed by non-antibiotic selection and characterization of the selected cells. The multi-cistronic nucleic acid vector encodes a selection element which may be an autocrine protein, miRNA, and/or shRNA.
1 . A method for generating a clonal population of transfected NK-92 cells, comprising:
transfecting NK-92 cells with a multi-cistronic nucleic acid vector comprising a positive selection marker and at least one transgene, wherein the positive selection marker is ER-IL2 or ER-IL15;
culturing the transfected NK-92 cells in a cell culture medium in absence of IL-2;
diluting the cultured NK-92 cells by clonal dilution, in absence of IL-2, to form a plurality of individual transfected NK-92 clones; and
phenotypic and genomic screening the plurality of transfected NK-92 clones to select clones that (i) express the at least one transgene and (ii) displays single, non-exonic integration of the at least one transgene.
2 . The method of claim 1 , wherein the phenotypic screening is by flow cytometry and/or ELISA.
3 . The method of claim 1 , wherein the genomic screening is by whole genome sequencing and/or genome walking.
4 . The method of claim 1 , wherein the at least one transgene is selected from the group consisting of: an Fc Receptor, a homing receptor, G protein-coupled receptor (GPCR), a chemokine receptor, a cytokine receptor, secreted cytokine, a cell adhesion molecule, a selectin, an integrin, antigen binding protein, and a tumor associated antigen
5 . The method of claim 4 , wherein the Fc Receptor is CD16 or a high affinity CD16.
6 . The method of claim 4 , wherein the chemokine receptor is selected from CCR7, CXCR2, or the receptor for CXCL14, and the cell adhesion molecules is selected from L-selectin (CD62L), α4β7 integrin, LPAM-1, and LFA-1.
7 . The method of claim 4 , wherein the secreted cytokine or cytokine receptor is IL-12, TGF-beta trap, an extracellular domain of a TGFβRII molecule, and/or a single chain dimer of the TGF-beta Receptor II ectodomain.
8 . The method of claim of claim 4 , wherein the antigen binding protein binds an immune modulator protein in a tumor selected from CTLA-4, PD-1, IDO-1, CD39, or CD73.
9 . The method of claim 4 , wherein the antigen binding protein specifically binds a tumor associated antigen selected from CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.
10 . The method of claim 4 , wherein the antigen binding protein comprises a chimeric antigen receptor (CAR)
11 . The method of claim 10 , wherein the CAR is CD19-CAR, PD-L1-CAR, HER2CAR, BMCA-CAR, and/or CD33-CAR.
12 . The method of claim 1 , wherein the nucleic acid vector comprises a promoter.
13 . The method of claim 12 , wherein the promoter comprises at least one nuclear factor of activated T (NFAT) binding domain.
14 . The method of claim 1 further comprising characterizing the clones for the function of the expressed transgenic factors.
15 . The method of claim 1 , wherein the functional characterization comprises antibody dependent cellular cytotoxicity (ADCC), natural cytotoxicity, CAR-mediated cytotoxicity, doubling time, and/or secretion of a recombinant protein.
16 . The method of claim 1 , further comprising characterizing the clones for unchanged intrinsic, non-transgene related functions.
17 . The method of claim 1 , further comprising transfecting the population of eukaryotic cells with at least one proliferation enhancing factor.
18 . The method of claim 17 , wherein the at least one proliferation enhancing factor is selected from hTERT, Ras, SV40, Myc, CDK4, or combinations thereof.
19 . A clonal population of transfected NK-92 cells generated by the method of any one of claims 1 - 18 .
20 . A method of treating a cancer in a patient in need thereof, comprising:
administering to the patient a clonal population of transfected NK-92 cells, wherein the clonal population of transfected NK-92 cells are generated by the process of:
(a) transfecting NK-92 cells with a multi-cistronic nucleic acid vector comprising a positive selection marker and at least one transgene, wherein the positive selection marker is ER-IL2 or ER-IL15;
(b) culturing the transfected NK-92 cells in a cell culture medium in absence of IL-2;
(c) diluting the cultured NK-92 cells by clonal dilution, in absence of IL-2, to form a plurality of individual transfected NK-92 clones; and
(d) phenotypic and genomic screening the plurality of transfected NK-92 clones to select clones that (i) express the at least one transgene and (ii) displays single, non-exonic integration of the at least one transgene.
21 . The method of claim 20 , wherein the at least one transgene is selected from the group consisting of: an Fc Receptor, a homing receptor, G protein-coupled receptor (GPCR), a chemokine receptor, a cytokine receptor, secreted cytokine, a cell adhesion molecule, a selectin, an integrin, antigen binding protein, and a tumor associated antigen