Compositions and methods regarding engineered and non-engineered γ δ T-cells for treatment of hematological tumors
Aspects of the invention include compositions and methods for treatment of hematological 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-CD20 binding domain or anti-B cell maturation antigen (BCMA) binding domain, a CD8 hinge and transmembrane domain, a costimulatory domain, a CD3 ζ 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 CD20;
(ii) a CD8α hinge domain;
(iii) a CD8 α transmembrane domain;
(iv) a costimulatory signaling region; and
(v) a CD3α 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 binding domain comprises anti-CD20 antibody 3H7; or
the binding domain comprises the complementary determining regions of anti-CD20 antibody 3H7.
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 binding domain encodes:
a. a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR and LCVR sequences are SEQ ID NO: 99 and 107 respectively; or
b. a heavy chain complementarity determining region 1, 2, and 3 sequence of SEQ ID NOs: 101, 103, and 105 respectively, and a light chain complementarity determining region 1, 2, and 3 sequence of SEQ ID NOs: 109, 111, and 113 respectively.
4 . 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 CD33 signaling domain comprising:
(i)
SEQ ID NO: 4
(RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQ
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT
YDALHMQALPPR);
or
(ii)
SEQ ID NO: 5
(RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY
DALHMQALPPR).
5 . The δ1 γδ T cell of claim 4 , 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 isolated nucleic acid encodes the 4 - 1 BB costimulatory signaling region comprising SEQ ID NO:6 and the CD27 costimulatory signaling region comprising SEQ ID NO:7.
6 . 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, preferably IL 15 , and a multi-cistronic linker region amino terminal to the interleukin or interleukin secretion signal.
7 . The δ1 γδ T cell of claim 1 , wherein the nucleic acid encodes SEQ ID NO:10.
8 . The δ1 γδ T cell of claim 7 , wherein the nucleic acid comprises the sequence of SEQ ID NO:14.
9 . The δ1 γδ T cell of claim 1 , wherein the δ1 γδ T cell proliferates in response to contact with cells that exhibit cell surface expression, or overexpression, of CD20; wherein the cells that exhibit cell surface expression of CD20 are normal hematological cells.
10 . The δ1 γδ T cell of claim 9 , wherein the cells that exhibit cell surface expression of CD20 are normal B cells.
11 . The δ1 γδ T cell of claim 1 , wherein the δ1 γδ T cell proliferates in response to contact with cells that exhibit cell surface expression, or overexpression, of CD20; wherein the cells that exhibit cell surface expression of CD20 are hematological tumor cells.
12 . A cell population comprising a plurality of δ1 γδ T cells according to claim 1 .
13 . The cell population of claim 12 , wherein the plurality comprises a least about 10 8 δ1 γδ T cells, preferablyor from about 10 11 δ1 γδ T cells to about 10 11 δ1 γδ T cells.
14 . The cell population of claim 12 , wherein the plurality comprises composition that is at least 60%, 80%, or from about 60% or 80% to about 90% or 95% δ1 γδ T cells.
15 . A method of making the δ1 γδ T cell of claim 1 or the cell population of claims 12 , wherein the method comprises transfecting δ1 γδ T cell(s) with a construct comprising the nucleic acid as defined in claim 1 .
16 . The method of claim 15 , wherein the method comprises retroviral transduction.
17 . The method of claim 15 , 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.
18 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the δ1 γδ T cell of claim 1 or the cell population of claim 12 .
19 . A method of killing a hematological tumor cell that exhibits cell surface expression of CD20, the method comprising contacting the hematological tumor cell with a tumor cell killing effective amount of the δ1 γδ T cell of claim 1 ; the cell population of claim 12 ; or the pharmaceutical composition of claim 18 .
20 . The method of claim 19 , 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 hematological tumor cell.
21 . The method of claim 20 , wherein the method comprises introducing into a host organism comprising the hematological tumor cell a therapeutically effective amount of the δ1γδ T cell(s) or the pharmaceutical composition and simultaneously or sequentially administering one or more methods to elevate common gamma chain cytokine(s).
22 . The method of claim 21 , 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).
23 . The method of claim 22 , 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 δ1 γδ T cell(s).
24 . The method of claim 21 , wherein the one or more methods to elevate common gamma chain cytokine(s) comprises lymphodepletion before introducing the δ1 γδ T cell(s).
25 . The method of claim 21 , 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).
26 . The method of claim 20 , 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.
27 . The method of claim 19 , wherein the method is a method of treating cancer in a subject in need thereof.
28 . A method of treating cancer in a subject in need thereof, the method comprising:
a. administering a therapeutically effective amount of δ1γδ T cells according to claim 1 , wherein the cancer comprises hematological tumor cells that exhibit cell surface expression of CD20.
29 . The method of claim 28 , 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).
30 . The method of claim 28 or 29 , 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.
31 . The δ1 γδ T cell of claim 1 , wherein the costimulatory signaling region is a 4-1BB (CD137) costimulatory signaling region or a CD27 costimulatory signaling region.
32 . The δ1 γδ T cell of claim 6 , wherein the IL-15 in c comprises the sequence of SEQ ID NO:34.
33 . The δ1 γδ T cell of claim 32 , wherein the IL-15 comprises the sequence of SEQ ID NO: 34 operably linked to a secretion signal sequence of SEQ ID NO:33, or the IL-15 comprises the sequence of SEQ ID NO:34 operably linked to a secretion signal sequence of SEQ ID NO:49.
34 . The δ1 γδ T cell of claim 6 , wherein the secreted common gamma chain interleukin in d is IL-15.
35 . The δ1 γδ T cell of claim 6 , wherein the multicistronic linker region in d comprises any one of SEQ ID NOs:43-45, 47, or 52-55 or a combination thereof, or encodes an internal ribosome entry site, e.g., SEQ ID NO: 56 or 60.
36 . The method of claim 22 , wherein the method further comprises administering IL-2.
37 . The method of claim 22 , wherein the method further comprises administering IL-15.