Compositions and methods for enhanced lymphocyte-mediated immunotherapy
Lymphocytes having a suppressed autophagy gene useful in immunotherapy are disclosed. The lymphocytes can express an antigen targeting receptor such as a chimeric antigen receptor (CAR) or endogenous or engineered T-cell receptor to target cells expressing a tumor-specific antigen. Methods of making and using such lymphocytes are disclosed. Some such lymphocytes are useful in conducting CAR-T or TCR-T therapy.
1 . A lymphocyte comprising an antigen targeting receptor and a suppressed autophagy gene, wherein the antigen targeting receptor comprises a chimeric antigen receptor (CAR) or an engineered T-cell receptor.
2 . The lymphocyte as defined in claim 1 , comprising a nucleic acid encoding the antigen targeting receptor inserted within a locus of the autophagy gene to disrupt expression of the autophagy gene.
3 . The lymphocyte as defined in claim 2 , wherein the nucleic acid encoding the antigen targeting receptor is inserted within the locus of the autophagy gene so that expression of the antigen targeting receptor is regulated by an endogenous promoter of the autophagy gene.
4 . The lymphocyte as defined in claim 1 , wherein the nucleic acid encoding the antigen targeting receptor is inserted within an exon of the autophagy gene.
5 . The lymphocyte as defined in claim 1 , wherein the nucleic acid encoding the antigen targeting receptor is inserted within an intron of the autophagy gene, optionally wherein the autophagy gene is ATG5 and the nucleic acid encoding the antigen targeting receptor is inserted within intron 2 of the ATG5 gene, and further optionally where the nucleic acid used to insert the antigen targeting receptor has the sequence of one of SEQ ID NO:8 or SEQ ID NO:9.
6 . The lymphocyte as defined in claim 1 , wherein the autophagy gene is at a first locus of a genome of the lymphocyte and the lymphocyte has been genetically modified to contain a nucleic acid encoding the antigen targeting receptor at a second locus of the genome, the first locus being different from the second locus.
7 . The lymphocyte as defined in claim 6 , wherein expression of the antigen targeting receptor is regulated by a heterologous promoter.
8 . The lymphocyte as defined in claim 1 , wherein the autophagy gene is ablated, or wherein the autophagy gene is suppressed by RNAi.
9 . The lymphocyte as defined in claim 1 , wherein:
the lymphocyte comprises a lymphocyte with activity in killing tumor cells;
the lymphocyte comprises a cytotoxic lymphocyte; and/or
the lymphocyte comprises a T-cell, a natural killer (NK) cell, or a B-cell, optionally wherein the T-cell comprises a T-cell derived from an induced pluripotent stem cell, a CD34+T-cell, a CD4+T-cell, a CD8+T-cell, a Treg cell, a tissue-resident memory T-cell (TRM), or a natural killer T-cell (NKT).
10 . The lymphocyte as defined in claim 1 , wherein the autophagy gene is ULK1, ULK2, ULK3, FIP200, Vps34, Beclin-1, p150, UVRAG, ATG1, ATG4, ATG5, ATG7, ATG8, ATG9, ATG10, ATG12, ATG13, ATG14L, ATG16L, ATG16L1, ATG18, VMP1, or GABARAP.
11 . The lymphocyte as defined in claim 1 , wherein the antigen targeting receptor is specific for a tumor-specific antigen, optionally wherein the tumor-specific antigen comprises the folate receptor (FR), the α-folate receptor, the B-folate receptor, the y-folate receptor, CD19, CD20, CD133, CD138, CEA, Claudin 18.2, EGFR, EGFRvlll, EphA2, EpCAM, GD2, GPC3, HER2, MSLN, MG7, MUC1, NY-ESO-1, LMP1, prostate specific membrane antigen (PSMA), Fra, NKG2DI, BCMA, IL13Ralpha2, LeY, CD70, B7-H3, ROR1, or PSCA.
12 . The lymphocyte as defined in claim 1 , wherein the antigen targeting receptor comprises a chimeric antigen receptor (CAR), and wherein the CAR is an α-folate receptor CAR, and wherein optionally the nucleotide construct used to insert the CAR in the lymphocyte has the nucleic acid sequence of either one of SEQ ID NO:8 or SEQ ID NO:9.
13 . The lymphocyte as defined in claim 1 , wherein the autophagy gene is ATG5, ATG14, or ATG16L1.
14 . The lymphocyte as defined in claim 13 , wherein the lymphocyte is a CD4+T-cell or a CD8+T-cell.
15 . A method of making the lymphocyte of claim 1 for use in immunotherapy, the method comprising a step of modifying the lymphocyte to suppress the autophagy gene, and a step of modifying the lymphocyte to express the antigen targeting receptor.
16 . The method as defined in claim 15 , wherein the step of modifying the lymphocyte to suppress the autophagy gene comprises using a gene editing method to insert a nucleic acid encoding the antigen targeting receptor at a locus of the autophagy gene, optionally wherein the gene editing method is used to insert the nucleic acid encoding the antigen targeting receptor so that expression of the antigen targeting receptor is regulated by an endogenous promoter of the autophagy gene.
17 . The method as defined in claim 15 , wherein the step of modifying the lymphocyte to express the antigen targeting receptor comprises inserting a nucleic acid encoding the antigen targeting receptor within a genome of the lymphocyte at a first locus, and wherein the step of modifying the lymphocyte to suppress an autophagy gene comprises knocking out the autophagy gene at a second locus of the genome, the second locus being different from the first locus.
18 . A method of conducting cancer immunotherapy comprising administering the lymphocyte as defined in claim 11 to a mammalian subject.
19 . A method of conducting cancer immunotherapy as defined in claim 18 , wherein the cancer is breast cancer, ovarian cancer, prostate cancer, or lung cancer.