USE OF CHIMERIC ANTIGEN RECEPTOR MODIFIED CELLS TO TREAT CANCER
The present disclosure relates to compositions and methods for compositions, methods, and kits for treating cancer using chimeric antigen receptor (CAR) modified cells. Some embodiments of the present disclosure relate to an isolated nucleic acid sequence encoding CAR. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ.
1 . A method of measuring a cytokine in a T cell-mediated immune response, the method comprising:
contacting a population of cells comprising guanylate cyclase 2C (GUCY2C) comprising the amino acid sequence of SEQ ID NO: 33, with a population of modified T cells comprising a chimeric antigen receptor (CAR) in a medium, the CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising the amino acid sequence SEQ ID NO: 14;
collecting a supernatant from the medium; and
measuring a concentration of the cytokine in the supernatant, the cytokine including INF-γ.
2 . The method of claim 1 , wherein the population of modified T cells and the population of cells comprising GUCY2C are cocultured at a ratio of effector cells to target cells is 1:1, 1:3, 1:10, or 1:30.
3 . The method of claim 1 , wherein the population of cells comprising GUCY2C is a population of K562 cells transfected to express GUCY2C.
4 . The method of claim 1 , wherein the extracellular domain of the CAR comprises a scFv antigen binding domain.
5 . The method of claim 1 , wherein the intracellular domain of the CAR comprises a CD3-zeta (CD37) signaling domain.
6 . The method of claim 1 , wherein the supernatant is collected after coculturing the populations of cells for 24 hours.
7 . The method of claim 1 , further comprising measuring a concentration of IFN-γ released when the population of modified T cells is cultured with a control population of cells not expressing GUCY2C, and comparing the amounts of IFN-γ released in each case.
8 . The method of claim 7 , wherein IFN-γ release is observed when the population of modified T cells is cultured with the population of cells comprising GUCY2C and IFN-γ release is not observed when the population of modified T cells is cultured with the control population of cells.
9 . The method of claim 1 , wherein the concentration of the cytokine is measured using an ELISA assay.
10 . The method of claim 1 , wherein the intracellular domain further comprises a costimulatory domain.
11 . The method of claim 10 , wherein the costimulatory domain is a 4-1BB costimulatory domain.
12 . The method of claim 1 , wherein the CAR transduces a signal in response to binding of the extracellular domain to GUCY2C on the surface of the population of cells.
13 . The method of claim 1 , wherein transduced T cells expressing the chimeric antigen receptor release greater than 100 pg/ml of IFN-γ.
14 . The method of claim 1 , wherein the medium comprises interleukin-2 (IL-2) or interleukin-15 (IL-15).
15 . The method of claim 1 , wherein the population of cells is contacted with the population of transduced T cells for at least 18 hours.
16 . The method of claim 1 , wherein the concentration of IFN-γ measured is at least 2-fold greater than a baseline level of IFN-γ release by non-transduced T cells.
17 . The method of claim 1 , wherein IFN-γ release is lower when the CAR T cells are cultured with non-transduced K562 cells compared to K562 cells expressing GUCY2C.
18 . The method of claim 1 , wherein the CAR T cells exhibit IFN-γ release in response to tumor cells engineered to express GUCY2C but not in response to normal tissues intrinsically expressing GUCY2C.
19 . A method of treating patient with cancer, wherein the method comprises contacting a population of cells comprising guanylate cyclase 2C (GUCY2C) comprising the amino acid sequence of SEQ ID NO: 33 in a patient with a population of modified T cells comprising a chimeric antigen receptor (CAR), the CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising the amino acid sequence SEQ ID NO: 14; and
detecting release of one or more cytokines in the patient;
wherein release of one or more cytokines indicates activation of T cell-immune response for treating the patient.
20 . The method of claim 19 , wherein the one or more cytokines include INF-γ.
21 . The method of claim 19 , wherein the cancer is colorectal cancer.