LENTIVIRAL TRANSDUCTION METHODS
This invention relates to methods of transducing mammalian cells that comprise exposing a population of mammalian cells to a poloxamer in the absence of a lentiviral vector for 6 hours or more to produce a transduction-primed mammalian cell population, exposing the transduction-primed mammalian cell population to a lentiviral vector, such that the T cells are transduced with the lentiviral vector; and then separating the transduced mammalian cells from the poloxamer. Suitable lentiviral vectors may comprise heterologous nucleic acid that encodes an antigen receptor, such as a T Cell Receptor (TCR) or chimeric antigen receptor. This may be useful, for example, in the transduction of T cells or progenitor cells that differentiate into T cells.
1 . A method of transducing mammalian cells comprising:
(i) exposing a population of mammalian cells to a poloxamer in the absence of a lentiviral vector for 6 hours or more to produce a transduction-primed mammalian cell population and
(ii) exposing the transduction-primed mammalian cell population to a lentiviral vector, such that the T cells are transduced with the lentiviral vector; and
(iii) separating the transduced mammalian cells from the poloxamer.
2 . A method according to claim 1 wherein the population of mammalian cells is exposed to the poloxamer in the absence of the lentiviral vector for 12 hours or more to produce the transduction-primed mammalian cell population.
3 . A method according to claim 2 wherein the population of mammalian cells is exposed to the poloxamer in the absence of a lentiviral vector for about 24 hours to produce the transduction-primed mammalian cell population.
4 . A method according to any one of the preceding claims wherein the transduced mammalian cells are separated from the poloxamer after 48 to 96 hours of exposure to the lentiviral vector.
5 . A method according to claim 4 wherein the transduced mammalian cells are separated from the poloxamer after about 72 hours of exposure to the lentiviral vector.
6 . A method according to any one of the preceding claims wherein the poloxamer has an average molecular weight of 10.0 kDa to 15 kDa
7 . A method according to claim 6 wherein the poloxamer is poloxamer 407 or poloxamer 338.
8 . A method according to any one of the preceding claims wherein the mammalian cell population is exposed to the poloxamer by culturing the mammalian cells in a priming medium comprising the poloxamer.
9 . A method according to claim 8 wherein the priming medium comprises 10 μg/ml to 100 mg/ml poloxamer.
10 . A method according to any one of the preceding claims wherein the lentiviral vector comprises a nucleic acid encoding a heterologous antigen receptor.
11 . A method according to claim 10 wherein the transduced mammalian cells express the heterologous antigen receptor.
12 . A method according to claim 10 or claim 11 wherein the heterologous antigen receptor is a chimeric antigen receptor (CAR).
13 . A method according to claim 10 or claim 11 wherein the heterologous antigen receptor is a T cell receptor (TCR).
14 . A method according to claim 13 wherein the heterologous TCR is HLA-A*02-restricted
15 . A method according to claim 13 or claim 14 wherein the heterologous TCR is an affinity enhanced TCR.
16 . A method according to any one of claims 10 to 15 wherein the heterologous antigen receptor binds to a tumour antigen or tumour associated antigen.
17 . A method according to claim 16 wherein the tumour antigen is alpha-fetoprotein (AFP), NY-ESO1, MAGE-A10 or MAGE-A4.
18 . A method according to any one of the preceding claims wherein the mammalian cell population is exposed to the lentiviral vector by culturing the mammalian cells in a transduction medium comprising the lentiviral vector.
19 . A method according to any one of the preceding claims wherein the transduction medium further comprises the poloxamer.
20 . A method according to any one of the preceding claims wherein the mammalian cells are cultured in the transduction medium for 1 to 4 days.
21 . A method according to any one of the preceding claims comprising isolating or purifying the transduced mammalian cell population.
22 . A method according to claim 21 wherein transduced mammalian cell population isolated from the population by fluorescence-activated cell sorting.
23 . A method according to any one of the preceding claims comprising expanding the population of transduced mammalian cells.
24 . A method according to according to any one of the preceding claims comprising concentrating the population of transduced mammalian cells.
25 . A method according to according to any one of the preceding claims comprising storing the population of transduced mammalian cells.
26 . A method according to any one of the preceding claims comprising formulating the population of transduced mammalian cells with a pharmaceutically acceptable excipient.
27 . A method according to any one of the preceding claims wherein the mammalian cells are T cells.
28 . A method according to any one of claims 1 to 26 wherein the mammalian cells are progenitor cells capable of differentiation into T cells.
29 . A method according to claim 28 wherein the progenitor cells are iPSCs, mesoderm cells, haemogenic endothelial cells, haematopoietic progenitor cells or progenitor T cells.