PF438 NKP30‐BASED CHIMERIC ANTIGEN RECEPTOR REDIRECTED HUMAN T LYMPHOCYTES INDUCE EFFECTIVE ANTITUMORAL IMMUNE RESPONSES TO ACUTE MYELOID LEUKEMIA AND MELANOMA
Authors: Riedel S. , Givi S. , Khan S. , Ploch P. , Theobald M. , Hartwig U. 
Published: June 2019

Background:

Adoptive cellular therapy (ACT) using chimeric antigen receptor (CAR) redirected human T lymphocytes has achieved remarkable clinical outcome for acute lymphocytic leukemia and also shows great promise for e.g. multiple myeloma. However, suitable antigens for a specific CAR therapy in acute myeloid leukemia (AML) without significant off‐target effects are still warranted as e.g. CD33 and CD123 are also expressed on hematopoietic stem cells (HSC). In contrast, B7H6, a member of the B7 family, is frequently expressed on various cancers including patient‐derived (primary) AML blasts and e.g. melanoma but not found on normal tissues and recognized by the natural killer (NK) cell activating receptor NKp30.

Aims:

In this study, we therefore evaluated human T cells as well as CRISPR/Cas9 engineered TCR− T cells expressing two different 2nd. generation NKp30 CARs for effective antileukemic immunity to AML in vitro and in vivo using a patient‐derived AML xenograft (PDX) model. Additionally, antitumoral responses of NKp30 CAR T cells to melanoma were examined.

Methods:

Expression of B7H6 in various AML lines, primary AML samples, solid tumors and primary fibroblasts was examined by RNA‐based qPCR. T cells were polyclonally stimulated and redirected with a CAR containing the extracellular region of the NKp30, a CD8‐ or, alternatively, a IgG1‐spacer, a CD28 costimulatory domain and a CD3ζ signaling domain by retroviral gene transfer. NKp30 CAR+ T cells were antigen‐specifically stimulated with the B7H6 expressing, HLA class I/II negative melanoma cell line MaMel‐86b (courtesy of Dr. C. Wölfel, III. Dept. of Medicine, UMM) and NKp30 expression was determined by flow cytometry. IFN‐γ ELISPOT and cytotoxicity assays were performed to analyze antileukemic responses to different AML samples as well as to Ma‐Mel 86b in vitro. Moreover, NKp30 CAR+ CRISPR/Cas9 engineered TCR− T cells were studied. Biological activity of redirected T cells was tested in an established AML‐PDX NSG mouse model.

Results:

qPCR assays revealed B7H6 expression at various levels in >95% of primary AML samples tested in addition to e.g. K562 and HL60 myeloid cell lines while no gene bank RNA expression data are reported for healthy tissues. Following transduction and antigen‐specific expansion ≥90% of CD3+ and TCR−CD3− T cells (more CD8+ than CD4+ T cells) expressed both NKp30 CARs. Upon coculture with the B7H6 expressing K562 and HL‐60 cells as well as primary AML samples NKp30‐redirected T cells elicited strong IFN –γ release and exhibited specific cytolytic activity to both leukemia lines and primary AML blasts but not to B7H6 negative U266 cells as shown by 51Cr‐release and colony‐forming cytotoxicity assays. Interestingly, reactivity of NKp30‐CD8‐CD28‐CD3ζ CAR T cells was significantly higher as compared to the other CAR. In addition, NKp30 CAR T cells induced strong responses to MaMel‐86b cells. Moreover, 3rdparty, TCR− NKp30 CAR T cells excluding alloreactivity also elicited potent responses to B7H6. To test for antitumor immunity in vivo, we first examined reactivity of NKp30‐redirected T cells to K562 engrafted into NSG mice and observed reduction of tumor burden. Further studies to evaluate reactivity to primary AML blasts in a PDX model are in progress.

Summary/Conclusion:

These studies demonstrate that NKp30 CAR T cells and additionally non allogeneic 3rdparty TCR− NKp30 CAR T cells, particularly expressing a CAR with a short spacer, can successfully elicit immunity to AML and also to solid tumors such as melanoma. As the B7H6 is not expressed on CD34+ HSC, this antigen might be a promising target for ACT to AML.