METHODS FOR CONTROLLED ELIMINATION OF THERAPEUTIC CELLS
The technology relates in part to methods for controlling elimination of therapeutic cells, for example, cells that express a chimeric antigen receptor. The technology further relates to a two-step method of controlling destruction of therapeutic cells in a patient following an adverse event. The two-step system may include a rapamycin or rapamycin analog-based level of control and a second, rimiducid, level of control. The technology also relates in part to methods for cell therapy using cells that express the inducible caspase polypeptide and the rapamycin-sensitive polypeptide, where the proportion of therapeutic cells eliminated by apoptosis is related to the choice and amount of the administered ligand.
1 . A modified cell, comprising
a) a first polynucleotide encoding a first chimeric polypeptide, wherein the first chimeric polypeptide comprises a membrane-associated polypeptide region and a first multimerizing region; and
b) a second polynucleotide encoding a second chimeric polypeptide, wherein the second chimeric polypeptide comprises a pro-apoptotic polypeptide region and a second multimerizing region, wherein the second multimerizing region has a different amino acid sequence than the first multimerizing region;
wherein the first and second multimerizing regions bind to a first multimeric ligand.
2 . The modified cell of claim 1 , wherein the second multimerizing region binds to the first multimeric ligand and binds to a second multimeric ligand that does not significantly bind to the first multimerizing region.
3 . The modified cell of claim 2 , wherein:
the first ligand comprises a first portion,
the first multimerizing region binds to the first portion, and
the second multimerizing region does not significantly bind to the first portion.
4 . The modified cell of claim 2 , wherein the first multimerizing region is not capable of binding to the second multimeric ligand.
5 . The modified cell of claim 4 , wherein the first and second multimerizing regions bind to a rapamycin or to a rapalog.
6 . The modified cell of claim 4 , wherein the first multimerizing region comprises an FKBP12-Rapamycin Binding (FRB) region or FRB variant region.
7 . The modified cell of claim 6 , wherein the first multimerizing region comprises FRB L .
8 . The modified cell of claim 4 , wherein the first multimerizing region comprises at least two FRB or FRB variant regions.
9 . The modified cell of claim 6 , wherein the second multimerizing region comprises an FKBP12 or FKBP12 variant region.
10 . The modified cell of claim 9 , wherein the second multimerizing region comprises an FKBPv36 region.
11 . The modified cell of claim 6 , wherein the second ligand is selected from the group consisting of AP1903, AP20187, and AP1510.
12 . The modified cell of claim 1 , wherein the membrane-associated polypeptide comprises a T cell receptor.
13 . The modified cell of claim 1 , wherein the membrane-associated polypeptide comprises a chimeric antigen receptor.
14 . The modified cell of claim 1 , wherein the pro-apoptotic polypeptide is a Caspase-9 polypeptide.
15 . A nucleic acid, comprising a promoter, operatively linked to
a) a first polynucleotide encoding a first chimeric polypeptide, wherein the first chimeric polypeptide comprises a membrane-associated polypeptide region and a first multimerizing region; and
b) a second polynucleotide encoding a second chimeric polypeptide, wherein the second chimeric polypeptide comprises a pro-apoptotic polypeptide region and a second multimerizing region, wherein the second multimerizing region has a different amino acid sequence than the first multimerizing region;
wherein the first and second multimerizing regions bind to a first multimeric ligand.
16 . A nucleic acid, comprising
a) a first polynucleotide encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises (i) a transmembrane region, (ii) a T cell activation molecule, (iii) an antigen recognition moiety, and (iv) a FRB or FRB variant region; and
b) a second polynucleotide encoding a chimeric caspase polypeptide, wherein the chimeric caspase polypeptide comprises (i) an FKBP12 or FKBP12 variant region, and (ii) a caspase polypeptide.
17 . A method of controlling survival of transplanted modified cells in a subject, comprising:
a) transplanting a modified cell of claim 9 into the subject; and
b) after (a), administering to the subject rapamycin or a rapalog, in an amount effective to kill at least 30% of the modified cells that express the second chimeric polypeptide comprising the pro-apoptotic polypeptide region-optionally wherein
i) the second multimerizing region is a FKBP12 or FKBP12 variant region, further comprising administering a ligand that binds to the FKBP12 or FKBP12 variant region on the second chimeric polypeptide comprising the pro-apoptotic polypeptide region in an amount effective to kill at least 90% of the modified cells that express the second chimeric polypeptide; or
ii) alloreactive modified cells are present in the subject and the number of alloreactive modified cells is reduced by at least 90% after administration of rapamycin, the rapalog.
18 . (canceled)
19 . A method of controlling survival of transplanted modified cells in a subject, comprising:
a) transplanting modified cells of claim 9 into the subject; and
b) after (a), administering to the subject a ligand that binds to the FKBP12 or FKBP12 variant region on the second chimeric polypeptide comprising the pro-apoptotic polypeptide region in an amount effective to kill at least 90% of the modified cells that express the second chimeric polypeptide.
20 . (canceled)
21 . A method for treating a subject having a disease or condition associated with an elevated expression of a target antigen expressed by a target cell, comprising
(a) administering to the subject an effective amount of a modified cell of claim 9 , wherein the modified cell comprises a polynucleotide coding for a chimeric antigen receptor or a T cell receptor that bind to the target antigen; and
(b) after a), administering an effective amount of rapamycin or a rapalog.
22 . A method of controlling survival of transplanted modified cells in a subject, wherein modified cells of claim 9 have been transplanted into the subject comprising identifying a presence or absence of a condition in the subject that requires the removal of the modified cells from the subject, and
administering a rapamycin or a rapalog, or a ligand that binds to the FKBP12 or FKBP12 variant region, maintaining a subsequent dosage, or adjusting a subsequent dosage to the subject based on the presence or absence of the condition identified in the subject.