T cell manufacturing compositions and methods
The generation of antigen specific T cells by controlled ex vivo induction or expansion can provide highly specific and beneficial T cell therapies. The present disclosure provides T cell manufacturing methods and therapeutic T cell compositions which can be used for treating subjects with cancer and other conditions, diseases and disorders personal antigen specific T cell therapy.
1. A method of preparing tumor antigen-specific T cells ex vivo suitable for use as an autologous therapy, the method comprising:
(a) depleting CD14+ cells and CD25+ cells from a population of immune cells comprising antigen presenting cells (APCs) and T cells, thereby forming a CD14/CD25 depleted population of immune cells comprising a first population of APCs and T cells, wherein the population of immune cells is from a biological sample from a human subject with cancer; and
(b) incubating the CD14/CD25 depleted population of immune cells for a first time period in the presence of:
(i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and
(ii) (A) a polypeptide comprising at least two tumor antigen epitope sequences expressed by cancer cells of a human subject, wherein each of the at least two tumor antigen epitope sequences contains a mutation and binds to an MHC protein expressed by the subject with a stronger affinity than a corresponding wild-type epitope sequence, or (B) a polynucleotide encoding the polypeptide; thereby forming a population of stimulated T cells; and
(c) expanding the population of stimulated T cells, thereby forming an expanded population of T cells, wherein expanding the population of stimulated T cells comprises expanding T cells derived from naïve CD8+ T cells or naïve CD4+ T cells, wherein the expanded population of T cells comprises (i) at least 1×10{circumflex over ( )}6 total CD8+ T cells, (ii) at least 1×10{circumflex over ( )}6 total CD4+ T cells and (iii) T cells that are specific to
(A) a first complex comprising (i) a first tumor antigen epitope sequence of the at least two tumor antigen epitope sequences and (ii) an MHC protein expressed by the cancer cells or APCs of the human subject, and
(B) a second complex comprising (i) a second tumor antigen epitope sequence of the at least two tumor antigen epitope sequences (ii) an MHC protein expressed by the cancer cells or APCs of the human subject; and
wherein at least 0.1% of the CD8+ T cells that are specific to the first complex or the second complex in the expanded population of T cells are derived from the naïve CD8+ T cells, and at least 0.1% of the CD4+ T cells that are specific to the first complex or the second complex in the expanded population of T cells are derived from the naïve CD4+ T cells.
2. The method of claim 1 , wherein steps (b) and (c) are performed in less than 28 days.
3. The method of claim 1 , wherein the fraction of CD8+ T cells that are specific to the first complex or the second complex of the total number of CD8+ T cells in the expanded population of T cells is at least two-fold higher than the fraction of CD8+ T cells that are specific to the first complex or the second complex of the total number of CD8+ T cells in the biological sample.
4. The method of claim 1 , wherein the fraction of CD4+ T cells that are specific to the first complex or the second complex of the total number of CD4+ T cells in the expanded population of T cells is at least two-fold higher than the fraction of CD4+ T cells that are specific to the first complex or the second complex of the total number of CD4+ T cells in the biological sample.
5. The method of claim 1 , wherein expanding comprises (A) contacting the population of stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs (i) has been incubated with FLT3L and (ii) presents the at least two tumor antigen epitope sequences; and (B) expanding the population of stimulated T cells for a second time period, thereby forming an expanded population of T cells.
6. The method of claim 5 , wherein the second population of mature APCs has been incubated with FLT3L for at least 1 day prior to contacting the population of stimulated T cells with the second population of mature APCs.
7. The method of claim 5 , wherein expanding further comprises (C) contacting the expanded population of T cells with a third population of mature APCs, wherein the third population of mature APCs (i) has been incubated with FLT3L and (ii) presents the at least two tumor antigen epitope sequences; and (D) expanding the expanded population of T cells for a third time period, thereby forming the expanded population of T cells of (c).
8. The method of claim 7 , wherein the third population of mature APCs has been incubated with FLT3L for at least 1 day prior to contacting the expanded population of T cells with the third population of mature APCs.
9. The method of claim 1 , wherein the biological sample is a peripheral blood sample, a leukapheresis sample or an apheresis sample.
10. The method of claim 1 , further comprising harvesting the expanded population of T cells, cryopreserving the expanded population of T cells or preparing a pharmaceutical composition containing the expanded population of T cells.
11. The method of claim 1 , wherein incubating comprises incubating the CD14/CD25 depleted population of immune cells in the presence of FLT3L and an RNA encoding the polypeptide.
12. The method of claim 1 , wherein the polypeptide is from 8 to 50 amino acids in length.
13. The method of claim 1 , wherein the polypeptide comprises at least three tumor antigen epitope sequences, each expressed by cancer cells of the human subject with cancer.
14. The method of claim 1 , wherein depleting CD14+ cells and CD25+ cells from the population of immune cells comprises contacting the population of immune cells with a CD14 binding agent and a CD25 binding agent.
15. The method of claim 1 , wherein depleting further comprises depleting CD19+ cells from the population of immune cells.
16. A pharmaceutical composition comprising the expanded population of T cells of claim 1 , and a pharmaceutically acceptable excipient.
17. The method of claim 1 , wherein the method further comprises administering a pharmaceutical composition comprising the expanded population of cells comprising tumor antigen specific T cells to the human subject with cancer.