Process for producing donor-batched cells expressing a recombinant receptor
Provided herein are methods of producing engineered T cell compositions enriched for CD57 negative and/or CD27 positive T cells, such as from a plurality of donors. In some embodiments, the T cells are engineered with a recombinant receptor, such as a chimeric antigen receptor (CAR). Also provided herein are engineered T cell compositions containing T cells enriched for CD57 negative and/or CD27 positive T cells derived from a plurality of different donors, including compositions in which the T cells are engineered with or express a recombinant receptor (e.g. CAR). Also provided are methods of using the engineered T cell compositions in adoptive therapy, including in connection for cancer immunotherapy, such as for allogeneic therapies or for administration to one or more subjects in which the T cells are not derived from the subject(s) to whom the compositions are administered.
1 . A method of preparing a T cell composition from a donor pool, the method comprising:
(a) obtaining a plurality of engineered T cell compositions from a plurality of different donors, wherein each engineered T cell composition is enriched for T cells surface negative for CD57 (CD57−) from a donor sample from an individual donor of the plurality of different donors, and each engineered T cell composition comprises T cells genetically engineered with a recombinant receptor that is a chimeric antigen receptor (CAR); and
(b) combining the plurality of engineered T cell compositions to produce a donor pooled engineered T cell composition.
2 . The method of claim 1 , wherein each of the plurality of engineered T cell compositions is generated by a process comprising:
(i) selecting T cells surface negative for CD57 (CD57−) from the donor sample from the individual donor, thereby generating a CD57 depleted T cell population; and
(ii) introducing a heterologous polynucleotide encoding the recombinant receptor into the CD57 depleted cell population, thereby generating the engineered T cell composition.
3 . The method of claim 2 , wherein prior to step (ii), the method comprises stimulating the CD57 depleted T cell population under conditions to activate T cells in the population.
4 . The method of claim 2 , wherein the method further comprises (iii) incubating the cells of the engineered T cell composition for up to 96 hours subsequent to the introducing.
5 . The method of claim 4 , wherein the incubating is carried out under conditions in which the cells of the engineered T cell composition are not expanded or are not substantially expanded, compared to the number of cells of the engineered T cell composition at the initiation of the incubating.
6 . The method of claim 2 , wherein the method further comprises (iii) cultivating the cells of the engineered T cell composition under conditions for expansion of T cells in the composition.
7 . The method of claim 2 , wherein the selecting T cells surface negative for CD57 (CD57−) comprises:
(1) selecting one of (a) cells surface positive for a T cell marker(s) and (b) cells surface negative for CD57 (CD57−) from the donor sample from the individual donor, thereby generating an enriched population of cells; and
(2) selecting, from the enriched population of cells, for the other of (a) cells surface positive for the T cell marker(s) and (b) CD57− cells, thereby generating the CD57 depleted T cell population.
8 . The method of claim 2 , wherein the method further comprises knocking out expression of (i) an endogenous major histocompatibility complex (MHC) or a component thereof; and/or (ii) an endogenous T cell receptor (TCR) or a component thereof, in the T cells of the CD57 depleted T cell population and/or the engineered T cell composition prior to or during one or more of the steps of the method.
9 . The method of claim 1 , wherein the method is repeated for each of the individual donors of the plurality of different donors.
10 . A method of preparing a T cell composition from a donor pool, the method comprising:
(a) selecting T cells surface negative for CD57 (CD57−) from a donor sample from an individual donor, thereby generating a CD57 depleted T cell population;
(b) genetically engineering the CD57 depleted T cell population, thereby producing an engineered T cell composition, the genetic engineering comprising:
(1) knocking out expression of (i) an endogenous major histocompatibility complex (MHC) or a component thereof; and/or (ii) an endogenous T cell receptor (TCR) or a component thereof, in cells of the CD57 depleted T cell population; and
(2) introducing a heterologous polynucleotide encoding a recombinant receptor into the cells of the CD57 depleted T cell population, wherein the recombinant receptor is a chimeric antigen receptor (CAR);
wherein the knocking out in (1) and the introducing in (2) are carried out concurrently or successively in either order;
(c) repeating steps (a) and (b) for a plurality of different donors to produce a plurality of donor engineered T cell compositions, wherein each donor engineered T cell composition is generated from cells from the donor sample from the individual donor of the plurality of different donors; and
(d) combining the plurality of donor engineered T cell compositions from the plurality of different individual donors to produce a donor pooled engineered T cell composition.
11 . The method of claim 2 , wherein the CD57 depleted T cell population comprises greater than or greater than at or about 75% CD3+/CD57− cells.
12 . The method of claim 1 , wherein each of the plurality of engineered T cell compositions independently comprises greater than or greater than at or about 40% CD57−/CAR+ cells.
13 . The method of claim 1 , wherein each of the plurality of engineered T cell compositions comprise CD4+ and CD8+ T cells.
14 . A method of preparing a T cell composition from a donor pool, the method comprising:
(i) selecting for one of (a) cells surface positive for a T cell marker(s) and (b) cells surface negative for CD57 (CD57−) from a donor sample from a plurality of different donors, thereby generating an enriched population of cells; and
(ii) selecting, from the enriched population of cells, the other of (a) cells surface positive for the T cell marker(s) and (b) CD57− cells, thereby generating a CD57 depleted T cell population, wherein:
(1) the donor sample is a pooled sample comprising cells from the plurality of different donors, whereby the method produces a pooled CD57 depleted T cell population; or
(2) the donor sample is a sample from an individual donor, and steps (i) and (ii) are repeated separately for each donor sample from the plurality of different donors, whereby the method produces a CD57 depleted T cell population for each individual donor.
15 . The method of claim 14 , wherein the method of (2) further comprises combining the CD57 depleted T cell populations for each individual donor together to produce a pooled CD57 depleted T cell population.
16 . The method of claim 7 , wherein the T cell marker(s) is CD3, CD4, and/or CD8.
17 . The method of claim 14 , wherein:
(a) cells of the CD57 depleted T cell population or cells of the pooled CD57 depleted T cell population are knocked out (KO) for expression of (i) an endogenous major histocompatibility complex (MHC) or a component thereof; and/or
(b) the method further comprises knocking out expression of (i) an endogenous major histocompatibility complex (MHC) or a component thereof.
18 . The method of claim 14 , wherein:
(a) a heterologous polynucleotide encoding a recombinant receptor is introduced into cells of the CD57 depleted T cell population or cells of the pooled CD57 depleted T cell population, wherein the recombinant receptor is a chimeric antigen receptor (CAR); and/or
(b) the method further comprises introducing into cells of the CD57 depleted T cell population or cells of the pooled CD57 depleted T cell population a heterologous polynucleotide encoding a recombinant receptor that is a chimeric antigen receptor (CAR),
the method thereby generating an engineered T cell composition.
19 . The method of claim 14 , wherein the frequency of CD57+ T cells in the CD57 depleted T cell population and/or the pooled CD57 depleted T cell population is less than about or about 35% of the frequency of CD57+ T cells in the donor sample.
20 . The method of claim 1 , wherein the donor sample comprises an apheresis product or a leukapheresis product.
21 . The method of claim 1 , wherein:
the individual donor is healthy or is not suspected of having a disease or condition at the time the donor sample is obtained from the individual donor; and/or
each of the donors of the plurality of different donors is healthy or is not suspected of having a disease or condition at the time the donor sample is obtained from each of the different donors.
22 . The method of claim 14 , further comprising introducing a heterologous polynucleotide encoding a recombinant receptor into the CD57 depleted T cell population, thereby generating an engineered CD57 depleted T cell population, wherein the recombinant receptor is a chimeric antigen receptor (CAR).
23 . The method of claim 2 , wherein the introducing comprises targeted insertion of the heterologous polynucleotide with a viral vector comprising the heterologous polynucleotide.
24 . The method of claim 2 , wherein the heterologous polynucleotide is inserted into the β2M gene locus or the TRAC gene locus.