Methods for improving the efficacy and expansion of immune cells
The invention provides methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR), compositions and reaction mixtures comprising the same, and methods of treatment using the same.
1. A method of expanding and/or activating a population of immune cells, comprising:
providing a first CAR-expressing cell population, said first CAR-expressing cell population comprising a transiently expressed first CAR molecule, and said CAR molecule comprises an antigen binding domain of an antibody molecule; and
contacting said first CAR-expressing cell population with a ligand of the CAR molecule chosen from a cognate antigen molecule or an anti-antigen idiotypic antibody molecule that binds to said CAR molecule, under conditions such that immune cell expansion and/or activation occurs, thereby producing an expanded and/or activated immune cell population, and
contacting the expanded and/or activated immune cell population with a nucleic acid encoding a second CAR molecule, wherein the second CAR molecule is stably expressed, thereby producing a second CAR-expressing cell population;
wherein:
(i) the first CAR molecule comprises a CD19 CAR and the ligand comprises a CD19 cognate antigen molecule or an anti-idiotypic CD19 antibody molecule
(ii) the method does not comprise contacting the first CAR-expressing cell population with an agent that stimulates a CD3/TCR complex.
2. The method of claim 1 ,
wherein providing the first CAR-expressing cell population comprises introducing a nucleic acid encoding a first Chimeric Antigen Receptor (CAR) molecule into the immune cell population, under conditions suitable for transient expression of the CAR molecule, thereby producing a first CAR-expressing cell population, wherein the CAR molecule comprises an antigen binding domain of an antibody molecule.
3. The method of claim 1 , wherein the expansion and/or activation of the population of immune cells is carried out in vitro, ex vivo or in vivo.
4. The method of claim 1 , wherein:
the population of immune cells is acquired from a blood sample from a subject;
the population of immune cells comprises immune effector cells chosen from T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, myeloid-derived phagocytes, or a combination thereof;
the population of immune cells comprises primary T cells or a subset of lymphocytes chosen from anergized T cells, naïve T cells, T-regulatory cells, Th-17 cells, stem T cells, or a combination thereof; or
the population of immune cells comprises peripheral blood mononucleated cells (PBMCs), cord blood cells, or a combination thereof.
5. The method of claim 1 , wherein the ligand is present on a surface of a cell.
6. The method of claim 1 , wherein the nucleic acid encoding the first CAR molecule is an RNA molecule.
7. The method of claim 1 , wherein:
the first CAR molecule is transiently expressed in the immune cell population for a finite period of time or number of cell replications;
the first CAR molecule is internalized post a single ligand stimulation;
the immune cell does not receive repeated ligand stimulation;
the level of one or both of: the first CAR-surface density, or the affinity of the CAR antigen binding domain to the ligand is adjusted;
the first CAR-expressing immune cells are cultured in the presence of the ligand of the CAR molecule for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21, 22, 23 or 24 hours, or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 days;
the first CAR-expressing cells are cultured for a period of 8 days or less;
the first CAR-expressing cells shows at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or higher population doublings;
the first CAR-expressing immune cell population expands to a total of 400-600, or about 500 cells, wherein the cell expansion is measured between 10 and 25 days after stimulation with the ligand;
the expanded and/or activated immune cell population comprises immune effector cells having a less differentiated phenotype; or
wherein the first CAR-expressing cell population comprises a naïve T cell (T N ), a memory stem cell (T SCM ), a central memory T cell (T CM ), or a combination thereof.
8. The method of claim 1 , wherein the nucleic acid encoding the second CAR molecule is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.
9. The method of claim 1 , wherein the first and second CAR molecules are directed to the same antigen.
10. The method of claim 1 , wherein the first and second CAR molecules are the same or different CAR molecules.
11. The method of claim 1 , wherein the second CAR molecule is chosen from a CD19 CAR, a BCMA CAR, a CD33 CAR, a CLL-1 CAR, EGFRvIII CAR, a GFR alpha 4 CAR, an ROR1 CAR, a CD20 CAR, a CD22 CAR, a CD123 CAR, a CD10 CAR, a CD34 CAR, a FLT-3 CAR, a CD79b CAR, a CD179b CAR, a mesothelin CAR or a CD79a CAR, or any combination thereof.
12. The method of claim 1 , wherein the immune cell population transiently expressing the first CAR is expanded and/or activated in vitro or ex vivo by contacting said immune cell population with a CD19-antigen or anti-CD19 idiotypic antibody immobilized onto a non-cellular substrate.
13. The method of claim 1 , wherein the first and second CAR molecules an CD19 CAR and mesothelin CAR molecules, respectively.
14. The method of claim 1 , wherein the population of cells is expanded in the presence of a cytokine chosen from IL2 or IL-15 and IL-7.
15. The method of claim 1 , further comprising removing T regulatory cells from the immune cell population, to thereby provide a population of T regulatory-depleted cells.
16. The method of claim 1 , further comprising removing cells from the acquired immune effector cell population which express a check point inhibitor chosen from one or mom of PD+ cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of T regulatory-depleted cells, and check point inhibitor depleted cells.
17. An immune cell preparation or reaction mixture, comprising a population of immune effector cells made according to the method of claim 1 , wherein the population of immune cells comprises:
(i) cells that do not have a functional T cell receptor; or
(ii) cells that express a mutated or truncated form of one or more subunit of the TCR.
18. The method of claim 1 , wherein the first CAR-expressing population of immune cells comprises cells that do not have a functional T cell receptor.
19. The method of claim 1 , wherein the first CAR-expressing population of immune cells comprises cells that express a mutated or truncated form of one or more of a subunit of the TCR.
20. The method of claim 1 , wherein the method does not comprise stimulating TCRs on the first CAR-expressing cell population.
21. The method of claim 1 , wherein the ligand is attached to a non-naturally occurring substrate.
22. The method of claim 1 , wherein the method does not comprise contacting the first CAR-expressing cell population with an anti-CD3 antibody or an anti-CD28 antibody.
23. The method of claim 1 , wherein the anti-idiotypic CD19 antibody molecule comprises the same CDRs as the CD19-specific CAR mAb clone no. 136.20.1.
24. The method of claim 1 , wherein the anti-idiotypic CD19 antibody molecule comprises the CD19-specific CAR mAb clone no. 136.20.1.
25. The method of claim 1 , wherein the CD19 CAR comprises an antigen binding domain comprising a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 122, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 123, 124, 125, or 126, a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 127, a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 128, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO: 129, and a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO: 130.
26. The method of claim 1 , wherein the nucleic acid encoding the second CAR molecule enables stable expression of the second CAR molecule.
27. The method of claim 1 , wherein the ligand of the CAR molecule is a CD19 cognate antigen molecule.
28. The method of claim 1 , wherein ligand of the CAR molecule is an anti-idiotypic CD19 antibody molecule.