METHODS FOR ENHANCING AND MAINTAINING CAR-T CELL EFFICACY
The technology relates generally to the field of immunology and relates in part to compositions and methods for activating T cells and other cells resulting in an immune response against a target antigen. The technology also relates to compositions and methods for enhancing and maintaining chimeric antigen receptor-expressing T cells, while reducing cytotoxic effects of CAR-T cell therapies
1 . A modified cell population, comprising modified T cells, wherein:
the modified T cells comprise a polynucleotide that encodes a chimeric antigen receptor, wherein the chimeric antigen receptor comprises:
(i) a transmembrane region;
(ii) a T cell activation molecule; and
(iii) an antigen recognition moiety
wherein the ratio of CD8 + to CD4 + T cells in the modified cell population is 3:2 or greater.
2 . The modified cell population of claim 1 , wherein the chimeric antigen receptor comprises
(i) a transmembrane region;
(ii) a costimulatory polypeptide cytoplasmic signaling region, a truncated MyD88 polypeptide region lacking the TIR domain, a truncated MyD88 polypeptide region lacking the TIR domain and a costimulatory polypeptide cytoplasmic signaling region, or a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain;
(iii) a T cell activation molecule; and
(iv) an antigen recognition moiety.
3 . The modified cell population of any one of claims 1 to 2 , wherein the modified T cells comprise a second polynucleotide that encodes an inducible chimeric pro-apoptotic polypeptide.
4 . The modified cell population of claim 1 , wherein the modified T cells comprise a second polynucleotide that encodes a chimeric signaling polypeptide, wherein the chimeric signaling polypeptide comprises:
(i) a costimulatory polypeptide cytoplasmic signaling region;
(ii) a truncated MyD88 polypeptide region lacking the TIR domain;
(iii) a truncated MyD88 polypeptide region lacking the TIR domain and a costimulatory polypeptide cytoplasmic signaling region; or
(iv) a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain.
5 . The modified cell population of claim 4 , wherein the chimeric signaling polypeptide comprises a membrane targeting region.
6 . The modified cell population of claim 4 , wherein costimulatory polypeptide cytoplasmic signaling region is a signaling region that activates the signaling pathways activated by MyD88, CD40 and/or MyD88-CD40 fusion chimeric polypeptide.
7 . The modified cell population of claim 1 , wherein the modified T cells comprise a nucleic acid comprising a promoter operably linked to
(i) a first polynucleotide encoding the chimeric antigen receptor; and
(ii) a second polynucleotide encoding a chimeric signaling polypeptide, wherein the chimeric signaling polypeptide comprises
a. a costimulatory polypeptide cytoplasmic signaling region;
b. a truncated MyD88 polypeptide region lacking the TIR domain;
c. a truncated MyD88 polypeptide region lacking the TIR domain and a costimulatory polypeptide cytoplasmic signaling region; or
d. a truncated MyD88 polypeptide region lacking the TIR domain and a CD40 cytoplasmic polypeptide region lacking the CD40 extracellular domain.
8 . The modified cell population of claim 7 , wherein the nucleic acid comprises, in 5′ to 3′ order, the first polynucleotide and the second polynucleotide.
9 . The modified cell population of any one of claim 7 or 8 , wherein the first polynucleotide encodes, in 5′ to 3′ order, an antigen recognition moiety, a transmembrane region, and a T cell activation molecule, and the second polynucleotide is 3′ of the polynucleotide sequence encoding the T cell activation molecule.
10 . The modified cell population of any one of claims 7 to 9 , wherein the nucleic acid comprises a third polynucleotide that encodes a linker polypeptide between the first and the second polynucleotides.
11 . The modified cell population of claim 10 , wherein the linker polypeptide comprises a 2A polypeptide.
12 . The modified cell population of any one of claims 10 to 11 , wherein the nucleic acid comprises a fourth polynucleotide encoding an inducible chimeric pro-apoptotic polypeptide.
13 . The modified cell population of any one of claims 2 to 12 , wherein the costimulatory polypeptide cytoplasmic signaling region is selected from the group consisting of CD27, CD28, 4-1BB, OX40, ICOS, RANK, TRANCE, and DAP10, or a signaling region that activates the signaling pathways activated by MyD88, CD40, CD27, CD28, 4-1BB, OX40, ICOS, RANK, TRANCE, and DAP10.
14 . The modified cell population of any one of claims 2 to 3 , wherein the chimeric antigen receptor comprises two costimulatory polypeptide cytoplasmic signaling regions selected from the group consisting of CD27, CD28, 4-1BB, OX40, ICOS, RANK, TRANCE, and DAP10, or a signaling region that activates the signaling pathways activated by CD27, CD28, 4-1BB, OX40, ICOS, RANK, TRANCE, and DAP10, or a signaling region that activates the signaling pathways activated by MyD88, CD40, CD27, CD28, 4-1BB, OX40, ICOS, RANK, TRANCE, and DAP10.
15 . The modified cell population of any one of claims 4 to 12 , wherein the chimeric signaling polypeptide comprises two costimulatory polypeptide cytoplasmic signaling regions selected from the group consisting of CD27, CD28, 4-1BB, OX40, ICOS, RANK, TRANCE, and DAP10, or a signaling region that activates the signaling pathways activated by MyD88, CD40, CD27, CD28, 4-1BB, OX40, ICOS, RANK, TRANCE, and DAP10.
16 . The modified cell population of any one of claims 1 to 15 , wherein 80% or more of the modified cells are CD8 + T cells.
17 . A method for stimulating a cell mediated immune response to a target cell or tissue in a subject, comprising administering a modified cell population of any one of claims 1 to 16 .
18 . A method for treating a subject having a disease or condition associated with an elevated expression of a target antigen, comprising administering to the subject an effective amount of a modified cell population of any one of claims 1 to 16 .
19 . A method for reducing the size of a tumor in a subject, comprising administering a modified cell population of any one of claims 1 to 16 to the subject, wherein the antigen recognition moiety binds to an antigen on the tumor.
20 . A method for preparing a modified cell population of any one of claims 1 to 16 , comprising contacting T cells with a nucleic acid that comprises a polynucleotide that encodes the chimeric antigen receptor with a cell population under conditions in which the nucleic acid is incorporated into the cells, and enriching the T cells to obtain a modified cell population wherein the ratio of CD8 + to CD4 + T cells in the cell population is 3:2 or greater.
21 . The method of claim 20 , comprising the step of administering the modified cell population to a subject.
22 . The method of claims 17 to 19 , further comprising administering a cytokine neutralizing agent.
23 . The method of claim 23 wherein the neutrailizing agent is an antibody.
24 . The method of claim 23 , wherein the neutrailizing agent is an anti-TNFα antibody.