Compositions and methods regarding engineered and non-engineered γδ T-cells for treatment of solid tumors
Aspects of the invention include compositions and methods for treatment of solid tumors with engineered or non-engineered γδ-T cells. In some embodiments, the γδ-T cells comprise a chimeric antigen receptor (CAR) construct. The CAR construct can contain an anti-TryD binding domain, a CD8α hinge and transmembrane domain, a costimulatory domain, a 003ζ signalling domain, a combination thereof, or all thereof. The CAR construct can contain an anti-GPC3 binding domain, a CD8α hinge and transmembrane domain, a costimulatmy domain, a CD3ζ signalling domain, a combination thereof, or all thereof. The CAR construct can contain a domain encoding for a secreted common gamma chain cytokine such as a sIL 15 domain.
1 . A δ1 γδ T cell comprising:
a. a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises:
(i) a binding domain that specifically binds to GPC3;
(ii) a CD8α hinge domain;
( 111 ) a CD8α transmembrane domain;
(iv) a costimulatory signaling region selected from a 4-1BB costimulatory signaling region and a CD27 costimulatory signaling region; and
(v) a CD3ζ C. signaling domain: or
b. a polypeptide comprising a CAR comprising an amino acid sequence encoded by the nucleic acid of (a);
wherein the δ1 γδ T cell functionally expresses the binding domain of the polypeptide or the nucleic acid encoded CAR on the surface of the δ1 γδ T cell, and wherein the nucleic acid encodes SEQ ID NO: 20.
2 . The δ1 γδ T cell of claim 1 , wherein the (i)-(v) are in 5′ to 3′ order.
3 . The δ1 γδ T cell of claim 1 , wherein the CAR comprises:
a. a CD8α hinge domain comprising SEQ ID NO:1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO:2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY);
b. a CD8α transmembrane domain comprising SEQ ID NO:3 (IWAPLAGTCGVLLLSLVITLYC); and/or
c. a CD35ζ signaling domain comprising:
(i)
SEQ ID NO: 4
(RVKFSRSADAPAYQQGQNQLYNELNLGRREEYD
VLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK
MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT
YDALHMQALPPR);
or
(ii)
SEQ ID NO: 5
(RVKFSRSADAPAYQQGQNQLYNELNLGRREEYD
VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM
AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY
DALHMQALPPR).
4 . The δ1 γδ T cell of claim 3 , wherein the CAR comprises:
a. a 4-1BB costimulatory signaling region comprising SEQ ID NO:6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL); or
b. a CD27 costimulatory signaling region comprising SEQ ID NO:7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP), or
wherein the nucleic acid encodes the 4-1BB costimulatory signaling region comprising SEQ ID NO:6 and the CD27 costimulatory signaling region comprising SEQ ID NO:7.
5 . The δ1 γδ T cell of claim 1 , wherein the nucleic acid further encodes:
a. a secreted cytokine;
b. a secreted common gamma chain interleukin;
c. a secreted IL-15;or
d. a secreted common gamma chain interleukin, and a multi-cistronic linker region amino terminal to the interleukin or interleukin secretion signal.
6 . The δ1 γδ T cell of claim 5 , wherein:
a. the secretion signal comprises a sequence of SEQ ID NO: 12 or SEQ ID NO: 26;
b. the sIL15 domain comprises a sequence of SEQ ID NO: 14;
c. the P2A cleavage sequence comprises a sequence of SEQ ID NO: 15 or SEQ ID NO: 25;
d. the furin cleavage sequence comprises a sequence of SEQ ID NO: 16; and/or
e. the CAR comprises, in amino to carboxy order, a sequence of SEQ ID NO: 17, SEQ ID NO: 12, and SEQ ID NO: 14.
7 . The δ1 γδ T cell of claim 1 , wherein the nucleic acid comprises the sequence of SEQ ID NO: 21.
8 . A cell population comprising a plurality of δ1 γδ T cells according to claim 1 .
9 . The cell population of claim 8 , wherein the plurality comprises at least about 10 8 δ1 γδ T cells, or from about 10 8 δ1 γδ T cells to about 10 11 δ1 γδ T cells.
10 . The cell population of claim 8 , wherein the plurality comprises a composition that is at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ1 γδ T cells.
11 . A method of making the δ1 γδ T cell of claim 1 , or the cell population of claim 8 , wherein the method comprises transfecting δ1 γδ T cell(s) with the nucleic acid according to claim 1 .
12 . The method of claim 11 , wherein the method comprises retroviral transduction.
13 . The method of claim 11 , wherein the method comprises ex vivo expansion of the δ1 γδ T cell(s), wherein the ex vivo expansion is performed before transfection and/or after transfection of the nucleic acid.
14 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a δ1 γδ T cell of claim 1 or the cell population of claim 8 .
15 . A method of killing a solid tumor cell that exhibits cell surface expression of GPC3, the method comprising contacting the solid tumor cell with a tumor cell killing effective amount of the δ1 γδ T cell of claim 1 ; the cell population of claim 8 ; or the pharmaceutical composition of claim 14 .
16 . The method of claim 15 , wherein the method comprises introducing a therapeutically effective amount of the δ1 γδ T cell(s) or the pharmaceutical composition into a host organism comprising the solid tumor cell.
17 . The method of claim 16 , wherein the method comprises introducing into a host organism comprising the solid tumor cell a therapeutically effective amount of the δ1 γδ T cell(s) or the pharmaceutical composition and simultaneously or sequentially administering one or more methods to elevate common gamma chain cytokine(s).
18 . The method of claim 17 , wherein the administering one or more methods to elevate common gamma chain cytokine(s) comprises administering simultaneously with introducing the δ1 γδ T cell(s) or sequentially an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced δ1 γδ T cell(s).
19 . The method of claim 18 , wherein the one or more methods to elevate common gamma chain cytokine(s) comprise administering an amount of common gamma chain cytokine(s) effective to increase proliferation, cytotoxic activity, persistence, or the combination thereof of the introduced δ1 γδ T cell(s) before and/or after introducing the γδ T cell(s).
20 . The method of claim 17 , wherein the one or more methods to elevate common gamma chain cytokine(s) comprises lymphodepletion before introducing the δ1 γδ T cell(s).
21 . The method of claim 17 , wherein the one or more methods to elevate common gamma chain cytokine(s) comprises secretion of one or more common gamma chain cytokine(s) from the introduced δ1 γδ T cell(s).
22 . The method of claim 16 , wherein the method reduces the in vivo tumor burden in the host organism, and/or increases the mean survival time of the host organism as compared to a control organism, wherein the control organism is not treated with the δ1 γδ T cell(s) or the pharmaceutical composition.
23 . The method of claim 15 , wherein the method is a method of treating cancer in a subject in need thereof.
24 . A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of δ1 γδ T cells according to claim 1 , wherein the cancer comprises solid tumor cells that exhibit cell surface expression of GPC3.
25 . The method of claim 24 , wherein the method comprises simultaneously with the administering of δ1 γδ T cells or sequentially, administering one or more methods to elevate common gamma chain cytokine(s).
26 . The method of claim 24 , wherein the method comprises performing a plurality of administrations of the δ1 γδ T cells, wherein the interval between the plurality of administrations is at least about a week, or at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and/or no more than once every 6 or 12 months.
27 . The δ1 γδ T cell of claim 1 , wherein the nucleic acid encodes SEQ ID NO: 20.
28 . The δ1 γδ T cell of claim 27 , wherein the nucleic acid comprises the sequence of SEQ ID NO: 23 or 24.
29 . The δ1 γδ T cell of claim 3 , wherein the IL-15 in c comprises the sequence of SEQ ID NO: 14.
30 . The δ1 γδ T cell of claim 3 , wherein the IL-15 in c comprises the sequence of SEQ ID NO: 14 operably linked to a secretion signal sequence of SEQ ID NO:12, or wherein the IL-15 comprises the sequence of SEQ ID NO: 14 operably linked to a secretion signal sequence of SEQ ID NO: 26.
31 . The δ1 γδ T cell of claim 3 , wherein the secreted common gamma chain interleukin in d is IL-15.
32 . The δ1 γδ T cell of claim 3 , wherein the multicistronic linker region comprises a sequence of any one of SEQ ID NOs: 15-17, 25, or 27-30, or a combination thereof, or encodes an internal ribosome entry site, e.g., SEQ ID NO: 31 or 32.
33 . The δ1 γδ T cell of claim 6 , wherein the secretion signal in a comprise SEQ ID NO: 12.
34 . The method of claim 12 , wherein the retroviral transduction is gammaretroviral transduction.
35 . The method of claim 18 , further comprising administering IL-2.
36 . The method of claim 18 , further comprising administering IL-15.