IL-12 immunotherapy for cancer

1. A pharmaceutical composition comprising (i) a population of transduced human cells secreting interleukin-12 (IL-12), wherein said population secretes IL-12 at a concentration of at least 1500 pg/ml when said cells are cultured at a density of 10 6 cells/ml for 2 hours, and (ii) a pharmaceutically acceptable carrier, diluent, or excipient, wherein the pharmaceutical composition is formulated for administration to a human patient having cancer.

2. The pharmaceutical composition of claim 1 , wherein the cells are established cancer cells, primary cancer cells, cancer cells derived from a subject or leukemic cells.

3. The pharmaceutical composition of claim 2 , wherein the cancer cells are leukemic cells selected from the group consisting of acute lymphoblastic leukemia (ALL) cells, chronic lymphoblastic leukemia (CLL) cells, chronic myeloid leukemia (CML) cells, and acute myeloid leukemia (AML) cells.

4. The pharmaceutical composition of claim 1 , wherein said population secretes IL-12 at a concentration of from about 1500 pg/ml to about 2500 pg/ml, from about 2500 pg/ml to about 5000 pg/ml, from about 5000 pg/ml to about 7500 pg/ml, from about 7500 pg/ml to about 10000 pg/ml, from about 10000 pg/ml to about 12500 pg/ml, from about 12500 pg/ml to about 15000 pg/ml, from about 15000 pg/ml to about 17500 pg/ml, from about 17500 pg/ml to about 20000 pg/ml, or from about 20000 pg/ml to about 40000 pg/ml when said cells are cultured at a density of 10 6 cells/ml for 2 hours.

5. The pharmaceutical composition of claim 1 , wherein the cells are transduced with a vector construct comprising a lentiviral vector, wherein the lentiviral vector comprises:

(i) a cPPT; a WPRE; a 5′ LTR; HIV signal sequence; HIV Psi signal 5′ SD; delta-GAG element; RRE; 3′ SA; a promoter selected from the group consisting of a CMV promoter and an EF1-alpha promoter; and a 3′ SIN-LTR;

(ii) an IL-12 expression cassette, wherein the IL-12 expression cassette comprises a polynucleotide operably linked to the promoter, wherein the polynucleotide encodes (a) a p35 polypeptide and, separately, a p40 polypeptide; or (b) an IL-12 fusion polypeptide comprising fused p35 and p40 subunits, wherein the IL-12 fusion polypeptide activates an IL-12 receptor; and

(iii) an activator polynucleotide encoding an activator polypeptide that converts a prodrug to a drug, wherein the activator polynucleotide encodes a mutant tmpk polypeptide with increased kinase activity relative to wild-type tmpk and that has at least 90% sequence identity to SEQ ID NO: 16, 17 or 31.

6. The pharmaceutical composition of claim 5 , wherein said population secretes IL-12 at a concentration of from about 1500 pg/ml to about 2500 pg/ml, from about 2500 pg/ml to about 5000 pg/ml, from about 5000 pg/ml to about 7500 pg/ml, from about 7500 pg/ml to about 10000 pg/ml, from about 10000 pg/ml to about 12500 pg/ml, from about 12500 pg/ml to about 15000 pg/ml, from about 15000 pg/ml to about 17500 pg/ml, from about 17500 pg/ml to about 20000 pg/ml, or from about 20000 pg/ml to about 40000 pg/ml when said cells are cultured at a density of 10 6 cells/ml for 2 hours.

7. The pharmaceutical composition of claim 1 , wherein the cells are transduced with a vector construct comprising a lentiviral vector comprising an IL-12 expression cassette, wherein the IL-12 expression cassette comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide encodes an IL-12 fusion polypeptide comprising fused p35 and p40 subunits, wherein the p40 subunit has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 22; and the p35 subunit has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 24.

8. The pharmaceutical composition of claim 7 , wherein said population secretes IL-12 at a concentration of from about 1500 pg/ml to about 2500 pg/ml, from about 2500 pg/ml to about 5000 pg/ml, from about 5000 pg/ml to about 7500 pg/ml, from about 7500 pg/ml to about 10000 pg/ml, from about 10000 pg/ml to about 12500 pg/ml, from about 12500 pg/ml to about 15000 pg/ml, from about 15000 pg/ml to about 17500 pg/ml, from about 17500 pg/ml to about 20000 pg/ml, or from about 20000 pg/ml to about 40000 pg/ml when said cells are cultured at a density of 10 6 cells/ml for 2 hours.

9. The pharmaceutical composition claim 1 , wherein the cells are transduced with a vector construct comprising a lentiviral vector, wherein the lentiviral vector comprises:

(i) an IL-12 expression cassette, wherein the IL-12 expression cassette comprises a polynucleotide operably linked to a promoter, wherein the polynucleotide encodes an IL-12 fusion polypeptide comprising fused p35 and p40 subunits, wherein the IL-12 fusion polypeptide activates an IL-12 receptor, and

(b) an activator polynucleotide encoding an activator polypeptide that converts a prodrug to a drug, wherein the activator polynucleotide encodes a mutant tmpk polypeptide with increased kinase activity relative to wild-type tmpk and that has at least 90% sequence identity to SEQ ID NO: 16, 17 or 31.

10. The pharmaceutical composition of claim 9 , wherein said population secretes IL-12 at a concentration of from about 1500 pg/ml to about 2500 pg/ml, from about 2500 pg/ml to about 5000 pg/ml, from about 5000 pg/ml to about 7500 pg/ml, from about 7500 pg/ml to about 10000 pg/ml, from about 10000 pg/ml to about 12500 pg/ml, from about 12500 pg/ml to about 15000 pg/ml, from about 15000 pg/ml to about 17500 pg/ml, from about 17500 pg/ml to about 20000 pg/ml, or from about 20000 pg/ml to about 40000 pg/ml when said cells are cultured at a density of 10 6 cells/ml for 2 hours.

11. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is formulated for intravenous, intratumoral, intramuscular, intraperitoneal, or stereotactic administration to the human patient.