Method for transduction of t cells in the presence of malignant cells
The present invention provides an in-vitro method of reducing the efficiency of transducing malignant cells of the blood system of a subject that are not derived from T cells with lentiviral vector particles without reducing the efficiency of transducing T cells in a sample comprising T cells and said malignant cells. A combination of compositions comprising a first composition and a second composition is also disclosed, wherein said first composition comprises i) transduced T cells of a subject, wherein said transduced T cells express a CAR comprising an antigen binding domain, wherein the antigen binding domain of said CAR binds specifically to a tag of a tagged polypeptide, and ii) non-transduced malignant cells of the blood system of said subject, and wherein said second composition comprises said tagged polypeptide, wherein said tagged polypeptide binds specifically to an antigen expressed on the surface of said malignant cells. Alternatively, the transduced T cells of said first composition may comprise a nucleic acid encoding a CAR and an inducible gene expression system, and said second composition may comprise an induction agent inducing said gene system.
1 . An improved method for preparing a therapeutic population of T cells to treat a subject who has circulating malignant cells,
wherein the method comprises contacting T cells from the subject with lentiviral vector particles that encode a chimeric antigen receptor (CAR) specific for a tumor antigen on the malignant cells, thereby transducing the T cells to express the CAR;
wherein the improvement comprises:
(a) using lentiviral vector particles that are pseudotyped with an envelope protein that binds T cells, and that encode a CAR that binds the tag of a tagged polypeptide instead of said tumor antigen;
(b) contacting the T cells with said pseudotyped lentiviral vector particles in a mixed cell population from the subject that also contains circulating malignant cells,
whereupon the pseudotyped lentiviral vector particles selectively transduce the T cells in preference to the circulating malignant cells in the mixed cell population;
wherein the T cells obtained thereby specifically bind said tagged polypeptide, and are effective in treating the subject when administered to the subject in combination with said tagged polypeptide.
2 . The method according to claim 1 , wherein said malignant cells are circulating tumor cells (CTCs), malignant B cells, malignant B cell subsets, malignant B cell progenitors, malignant NK cells, malignant stem cells, malignant myeloid cells, malignant myeloid subsets or malignant myeloid progenitors.
3 . The method according to claim 1 , wherein said CAR binds to a tag selected from the group consisting of dextran, biotin, fluorescein isothiocyanate (FITC), phycoerythrin (PE), and a peptide.
4 . The method according to claim 1 , wherein said T cells of said mixed cell population from the subject is a population comprising activated T cells.
5 . The method according to claim 1 , wherein said circulating malignant cells are malignant B cells, malignant B cell subsets or malignant B cell progenitors,
and wherein said tumor antigen is selected from the group consisting of CD10, CD19, CD20, CD22, CD24, CD33, CD34, and CD79a.
6 . The method of claim 1 , wherein said envelope protein is selected from the group consisting of BaEV env, VSV-G, RD114-TR, GALV-TR, MV-H/F, NiV-G/F, and Ampho-MLV env.
7 . The method of claim 1 , wherein said pseudotyped lentiviral vector particles are pseudotyped with vesicular stomatitis virus G protein (VSV-G), which selectively binds to LDL receptors (LDLR) on the T cells in the mixed cell population from the subject.
8 . The method of claim 1 , wherein the mixed cell population is a sample of whole blood, a leukapheresis, a buffy coat, or peripheral blood mononuclear cells (PBMC).