Manufacturing process for making t cells expressing chimeric antigen receptors
Aspects of the present disclosure relate to methods for manufacturing genetically engineered T cells expressing a chimeric antigen receptor (CAR) that provide several improvements over conventional manufacturing methods, thereby enabling production of a robust supply of clinically useful CAR T-cell therapies.
1 . A method for manufacturing genetically engineered T cells, the method comprising:
(i) providing a first population of T cells obtained from human blood cells;
(ii) incubating the first population of T cells in the presence of a T cell activating agent in a cell culture vessel to produce a second population of T cells, wherein the second population of T cells comprises activated T cells;
(iii) introducing into the second population of T cells a first ribonucleoprotein (RNP) complex comprising a first Cas9 enzyme and a first guide RNA (gRNA) targeting a T cell receptor alpha chain constant region (TRAC) gene, and a second RNP complex comprising a second Cas9 enzyme and a second gRNA targeting a beta-2 microglobulin (β2M) gene to produce a third population of T cells, wherein the third population of T cells comprises T cells having the TRAC gene disrupted and the β2M gene disrupted, wherein the first RNP complex and the second RNP complex are introduced into the second population of T cells in one electroporation event;
(iv) incubating the third population of T cells with an adeno-associated viral (AAV) vector to produce a fourth population of T cells, wherein the AAV vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and wherein the nucleic acid sequence is flanked by homologous sequences to the TRAC gene locus, and wherein the fourth population of T cells comprises activated T cells expressing the CAR and having the TRAC gene disrupted and the β2M gene disrupted;
(v) expanding the fourth population of T cells thereby producing an expanded T cell population;
(vi) removing TCRαβ + T cells from the expanded T cell population to produce a population of genetically engineered T cells, wherein the population of genetically engineered T cells comprises T cells expressing the CAR and having the TRAC gene and the β2M gene disrupted; and
(vii) harvesting the population of genetically engineered T cells.
2 . The method of claim 1 , wherein the first population of T cells is derived from cryopreserved T cells enriched from human blood cells.
3 . The method of claim 1 , wherein the first population of T cells is prepared by a process comprising: (a) obtaining blood cells from a human donor; and (b) enriching CD4 + T cells and/or CD8 + T cells from the blood cells.
4 . The method of claim 3 , wherein step (b) is performed using magnetic beads conjugated with anti-CD4 and/or anti-CD8 antibodies.
5 . The method of claim 3 , further comprising (c) cryopreserving the enriched CD4 + T cells and CD8 + T cells produced in step (b).
6 . The method of claim 1 , wherein the first population of T cells has a cell viability of at least 80% and/or a purity of at least 80% of CD4 + and CD8 + T cells.
7 . The method of claim 1 , wherein the T cell activating agent comprises a CD3 agonist and a CD28 agonist, and wherein the CD3 agonist and CD28 agonist are attached to a nanomatrix particle.
8 . The method of claim 1 , wherein step (ii) is performed by incubating the first population of T cells with the T cell activating agent in the cell culture vessel at a cell seeding density of 2×10 6 /cm 2 and a cell concentration of 2×10 6 cells/mL for 48 hours.
9 . The method of claim 1 , wherein the ratio of the T cell activating agent to medium in the mixture is 1:12.5 (v/v).
10 . The method of claim 1 , further comprising diluting the T cell activating agent in the second population of T cells after step (ii) to reduce activation and to allow cells to recover before step (iii).
11 . The method of claim 1 , wherein the amount of the first Cas9 enzyme in the first RNP complex is the same as the amount of the second Cas9 enzyme in the second RNP complex.
12 . The method of claim 1 , wherein the concentration of the first Cas9 enzyme is 0.15 mg/mL, the concentration of the second Cas9 enzyme is 0.15 mg/mL, the concentration of the first gRNA targeting the TRAC gene is 0.08 mg/mL, and the concentration of the second gRNA targeting the β2M gene is 0.2 mg/mL.
13 . The method of claim 1 , wherein the cell concentration in step (iii) is 100×10 6 cells/mL to 300×10 6 cells/mL.
14 . The method of claim 1 , wherein the cell number in each vessel in step (iii) is 3×10 8 cells.
15 . The method of claim 1 , wherein the AAV vector has a multiplicity of infection (MOI) value of 10,000 to 80,000.
16 . The method of claim 15 , wherein the MOI of the AAV vector is 20,000.
17 . The method of claim 15 , wherein the AAV vector is AAV serotype 6 (AAV6) vector.
18 . The method of claim 1 , wherein step (v) is performed by culturing the fourth population of T cells in a cell culture vessel at a seeding density of 2×10 5 cells/cm 2 to 7×10 5 cells/cm 2 for 6 days to 12 days.
19 . The method of claim 18 , wherein the cell culture vessel is a static cell culture vessel allowing for cell expansion for 7 days to 9 days without medium change.
20 . The method of claim 1 , wherein step (v) is performed by culturing the fourth population of T cells in a cell culture vessel at a seeding density of 2×10 5 cells/cm 2 to 5×10 5 cells/cm 2 for 7 days to 9 days.
21 . The method of claim 20 , wherein the fourth population of T cells is cultured at a seeding density of 3×10 5 cells/cm 2 to 5×10 5 cells/cm 2 .
22 . The method of claim 20 , wherein the cell culture vessel is a static cell culture vessel allowing for cell expansion for 10 days to 12 days without medium change.
23 . The method of claim 1 , wherein step (vi) is performed by contacting the expanded cells to beads on which anti-TCRαβ antibodies are immobilized, and collecting unbound cells.
24 . The method of claim 1 , wherein the expanding step comprises seeding the T cells at a density between 150,000 cells/cm 2 and 500,000 cells/cm 2 , in a cell vessel.
25 . The method of claim 24 , wherein the expanding step comprises seeding the T cells at a density between 300,000 cells/cm 2 and 500,000 cells/cm 2 in a cell vessel.
26 . The method of claim 1 , wherein the first Cas9 enzyme, the second Cas9 enzyme, or both are Streptococcus pyogenes Cas9 nuclease (spCas9).
27 . The method of claim 1 , wherein the first Cas9 enzyme and the second Cas9 enzyme are the same.
28 . The method of claim 1 , wherein the first Cas9 enzyme comprises the amino acid sequence of SEQ ID NO: 1, and/or wherein the second Cas9 enzyme comprises the amino acid sequence of SEQ ID NO: 1.
29 . The method of claim 1 , wherein the first gRNA targeting the TRAC gene comprises a spacer sequence of SEQ ID NO: 4.
30 . The method of claim 29 , wherein the first gRNA targeting the TRAC gene comprises the nucleotide sequence of SEQ ID NO: 2.
31 . The method of claim 30 , wherein the first gRNA, the second gRNA, or both comprise one or more 2′-O-methyl phosphorothioate modification.
32 . The method of claim 1 , wherein the second gRNA targeting the β2M gene comprises a spacer sequence of SEQ ID NO: 8.
33 . The method of claim 32 , wherein the second gRNA targeting the β2M gene comprises the nucleotide sequence of SEQ ID NO: 6.
34 . The method of claim 1 , wherein the CAR comprises an extracellular domain targeting a cancer antigen, a transmembrane domain, a co-stimulatory domain, and a CD3z cytoplasmic signaling domain.
35 . The method of claim 1 , wherein the CAR binds CD19.
36 . The method of claim 35 , wherein the extracellular domain comprises a single-chain variable fragment (scFv), the transmembrane domain is derived from CD8a, and/or the co-stimulatory domain is derived from CD28.
37 . The method of claim 36 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 37.
38 . The method of claim 1 , wherein the CAR binds BCMA.
39 . The method of claim 38 , wherein the extracellular domain comprises a single-chain variable fragment (scFv), the transmembrane domain is derived from CD8a, and/or the co-stimulatory domain is derived from 4-1BB.
40 . The method of claim 39 wherein the CAR comprises the amino acid sequence of SEQ ID NO: 61.