NKG2D的CAR-T细胞在小鼠体内的急性毒反应.ppt

October 10, 2016,*Center for Synthetic Immunity and the Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756; Celdara Medical, LLC, Lebanon, NH 03766; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215; xDepartment of Pediatric Oncology, Dana- Farber Cancer Institute, Boston, MA 02215; Division of Hematology-Oncology, Boston Childrens Hospital, Boston, MA 02115; Harvard Medical School, Boston, MA 02115; and #Exploratory Immuno-oncology, Novartis Institutes for BioMedical Research, Cambridge, MA 02139,神奇的大蛇丸 2018/04/09,Mouse and human,Background,Introduction of NKG2D-CAR,NKG2D ligands are expressed on various types of tumor cells and immunosuppressive cells (e.g. Tregs and myeloid-derived suppressor cells (MDSCs) within tumor microenvironments, these ligands provide attractive targets for cancer therapy.,Although attractive targets for immunotherapy, the potential of NKG2D ligands to be induced or expressed during immunotherapy resulting in off-tumor on-target toxicity remains unknown. Another challenge of immunotherapy is the potential to overactivate the immune system, such as occurs during CRS.,Results,Determination of the maximum tolerated dose,To evaluate tumor-dependent, receptor-specific, and nonspecific toxicity after injection of NKG2D CAR T cells, dose escalation studies in tumor-bearing and nontumor-bearing mice were conducted .,5 106, 107 , or 2 107 wtNKG2D T cells, NKG2D CAR T cells, or HBSS,MTD (maximum tolerated dose),Only mice receiving NKG2D CAR T cells at the highest dose appeared ill.,Health status The health status of mice was graded on a scale from 1 to 4 as follows: 1,normal and healthy; 1.5, some lethargy, walking a bit slowly; 2, moving slowly and (in tumor-bearing mice) a slight dragging of a limb; 2.5, hunched posture, moving slowly, and (in tumor-bearing mice) dragging limbs; 3,hunched posture with little movement; 3.5, little movement upon touch; 4,death.,RMA-RG tumor cells (105 cells),Assessment of toxicity following multidoses at the maximum tolerated dose,To determine whether multiple injections of CAR T cells would result in toxicity due to accumulation of T cells, tumor-bearing and nontumor-bearing mice were treated with up to three injections of 107 wtNKG2D T cells, 107 NKG2D CAR T cells, or HBSS.,No differential body weight changes after the second or third NKG2D CAR T cell injection compared with controls. Thus, multiple doses of NKG2D CAR T cells were well tolerated.,Mouse T lymphoma cell line,AST(aspartate amino transferase, 天冬氨酸转氨酶) and Creatinine ( 血清肌酐酸) as the markers of liver and kidney injury,Overall,treatment with NKG2D CAR T cells did not appear to affect kidney or liver function.,Analysis of serum analytes,An analysis of cytokines 20 h after injection of CAR T cells showed an increase in G-CSF, IL-6, IFN-g, MCP-1, IL-10, and MIG, with the highest cell dose having significant increases compared with controls,Only in mice injected with 2 107 NKG2D CAR T cells were clinical signs of illness observed (mean health score of 3.1）,There were no significant differences in serum cytokine amounts (IL-6, IFN-g, MCP-1, TNF-a) 3, 8, or 12 d after injection of NKG2D CAR T cells compared with controls,Table 3 Histological analysis of tissues harvested after a dose of 2 x 107 CAR T cells.,Analysis of histopathology,Despite their health status, mice treated with 2 107 NKG2D CAR T cells exhibited little pathology.,There was no evidence of lysis of tissues or infiltration of lymphocytes into specific tissues after injection of NKG2D CAR T cells. These findings of high amounts of serum inflammatory cytokines and no obvious tissue damage are consistent with CRS.,The role of CAR T cell effector mechanisms in acute toxicity,To determine which effector mechanisms of CAR T cells contributed to CRS, 2 107 NKG2D CAR T cells derived from mice that were deficient in perforin, IFN-g, or GM-CSF were injected into mice.,IFN- -/-CAR-T,Analysis of serum cytokines 20 h after T cell injection,GM-CSF-/-CAR-T,Perforin-/-CAR-T,The role of host immune cells,To investigate the role of host immune cells in this process, NSG mice, NOD/SCID mice, IL-2Rgdeficient mice, and Rag-1deficient mice were injected with a high dose (2 107 cells) of either NKG2D CAR T cells or wtNKG2D T cells.,The NSG and NOD/SCID mice remained healthy throughout the experiment, whereas the NOD, Rag- 1deficient, and IL-2Rgdeficient mice became acutely ill and lost body weight after receiving the high dose of NKG2D CAR T cells, similar to B6 mice,PK136 (anti-NK1.1 mAbs), 1A8 (anti-NK1.1 or anti-Ly6G mAb ) prior to injection of a high dose of NKG2D CAR T cells did not affect the development of CRS,mice defective of myeloid cells, T cells, and B cells did not develop CRS, but mice with more restricted deficiencies in immune cell populations did develop CRS.,The role of host cytokines,the hosts ability to respond to IL-1 or produce IL-6 was irrelevant to the outcome,2 107 NKG2D CAR T cells or wtNKG2D T cells were injected into mice deficient in IL-1R, IL-6, GM-CSF, IFN-g, or IFN-gR.,neither the production of IFN-g nor the response to the IFN-g present by the host was necessary for CRS like illness.,These signaling pathway was not essential in the acute illness observed,hosts deficient in NKG2D, DR5 (TRAIL receptor), orMyD88 also developed CRS after injection of a high dose of NKG2D CAR T