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As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell’s HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.

Dipeptidyl peptidase 9 (DPP9) is a ubiquitously expressed intracellular prolyl peptidase implicated in immunoregulation. However, its physiological relevance in the immune system remains largely unknown. We investigated the role of DPP9 enzyme in immune system by characterizing DPP9 knock-in mice expressing a catalytically inactive S729A mutant of DPP9 enzyme (DPP9ki/ki mice). DPP9ki/ki mice show reduced number of lymphoid and myeloid cells in fetal liver and postnatal blood but their hematopoietic cells are fully functional and able to reconstitute lymphoid and myeloid lineages even in competitive mixed chimeras. These studies demonstrate that inactivation of DPP9 enzymatic activity does not lead to any perturbations in mouse hematopoiesis.

Dipeptidyl peptidase 9 (DPP9) is an intracellular N-terminal post-proline-cleaving enzyme whose physiological function remains largely unknown. We investigated the role of DPP9 enzyme in vivo by characterizing knock-in mice expressing a catalytically inactive mutant form of DPP9 (S729A; DPP9 ki/ki mice). We show that DPP9 ki/ki mice die within 12–18 h after birth. The neonatal lethality can be rescued by manual feeding, indicating that a suckling defect is the primary cause of neonatal lethality. The suckling defect results from microglossia, and is characterized by abnormal formation of intrinsic muscles at the distal tongue. In DPP9 ki/ki mice, the number of occipital somite-derived migratory muscle progenitors, forming distal tongue intrinsic muscles, is reduced due to increased apoptosis. In contrast, intrinsic muscles of the proximal tongue and extrinsic tongue muscles, which derive from head mesoderm, develop normally in DPP9 ki/ki mice. Thus, lack of DPP9 activity in mice leads to impaired tongue development, suckling defect and subsequent neonatal lethality due to impaired survival of a specific subset of migratory tongue muscle progenitors.

As the recent rise of immune checkpoint inhibitors has resulted in durable clinical outcomes in cancer patients, the need for a newer and more widely effective target for immunotherapy has been vigorously sought in the field; T cell immunoreceptor with Ig and ITIM domain (TIGIT) is an immune checkpoint molecule expressed on CD8+ T cells, CD4+ T cells, NK cells, and regulatory T cells (Treg). TIGIT induces exhaustion of effector T cells (Teff) and NK cells in the tumor microenvironment via engagement with its ligand PVR (CD155) which is dominantly expressed on malignant tumor cells. After initial screening processes from a synthetic library, MG1131 clone was chosen based on the strongest binding affinity to human TIGIT and the superior PVR-blocking activity. MG1131 was cross-reactive with the cynomolgus monkey TIGIT, but not with the mouse TIGIT. Our in vitro efficacy data demonstrated that MG1131 activated NF-κB signaling in T cells, and enhanced NK-mediated tumor killing activities in a PVR-dependent manner. In vitro Treg suppression assays showed that MG1131 inhibited immunosuppressive functionality of Treg, leading to restoration of proliferation and IFN-γ secretion capacities of Teff even in the presence of Treg. In addition, expression levels of TIGIT on CD8+ T cells from PBMCs of cancer patient samples were higher compared with other immune inhibitory molecules such as PD-1, Tim-3, CTLA-4, or LAG-3, and the blockade of TIGIT by MG1131 resulted in increased IFN-γ secretion. Thus, our data indicate that MG1131 is a promising candidate for cancer immunotherapy by modulating NK and T cell functions. Citation Format: Jeewon Lee, Munkyung Kim, Hye-mi Nam, Joong Hyuk Sheen, Eun Jung Song, Hye In Yum, So Jung Lim, Hye-Young Park. MG1131, a novel TIGIT-targeting monoclonal antibody, enhances anti-tumor immune responses by modulating NK and T cell activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2230.

Mesothelin (MSLN) is an antigen overexpressed in several malignancies, including mesothelioma and ovarian and pancreatic adenocarcinoma. It has been studied as a marker for diagnosis and a target for immunotherapy. Here, we adopted a bispecific antibody format for recruiting cytotoxic T cells to kill tumor cells. MG1122 is a novel, whole IgG-like bispecific antibody which recognizes CD3 on T cells and MSLN on tumor cells. MG1122 induced effective killing of MSLN-expressing tumor cells. This response was associated with robust activation of T cells as shown by nuclear factor of activated T cells (NFAT) activation, CD25 and CD69 upregulation and increased cytokine release. In addition, using a live cell analysis system, we found that levels of activated caspase 3/7 were amplified in the tumor cells by MG1122 treatment. Although MSLN is a surface antigen, it also exists as a secreted isoform, which can lead to tumor evasion against MSLN targeting antibodies. However, despite the high concentration of soluble MSLN, MG1122 still effectively bound and showed strong killing effects against MSLN-expressing tumor cells. Taken together, our MSLN/CD3 bispecific antibody, MG1122 offers a promising opportunity to redirect T cells to kill MSLN-expressing cancer cells. Citation Format: Yun-Jung Lee, Okjae Lim, Munkyung Kim, Jeewon Lee, Kisu Kim, Junhong Jung, SuA Lee, Sung Keun Kim, Haenaem Kwon, Yangmi Lim, Yong Yea Park, Jonghwa Won. MG1122, a whole IgG-like bispecific antibody targeting mesothelin and CD3, induces T cell-mediated killing of MSLN-expressing tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5768.

Alport syndrome is a genetic disease of collagen IV (α3, 4, 5) resulting in renal failure. This study was designed to investigate sex-phenotype correlations and evaluate the contribution of macrophage infiltration to disease progression using Col4a3 knock out (Col4a3KO) mice, an established genetic model of autosomal recessive Alport syndrome. No sex differences in the evolution of body mass loss, renal pathology, biomarkers of tubular damage KIM-1 and NGAL, or deterioration of kidney function were observed during the life span of Col4a3KO mice. These findings confirm that, similar to human autosomal recessive Alport syndrome, female and male Col4a3KO mice develop renal failure at the same age and with similar severity. The specific contribution of macrophage infiltration to Alport disease, one of the prominent features of the disease in human and Col4a3KO mice, remains unknown. This study shows that depletion of kidney macrophages in Col4a3KO male mice by administration of clodronate liposomes, prior to clinical onset of disease and throughout the study period, does not protect the mice from renal failure and interstitial fibrosis, nor delay disease progression. These results suggest that therapy targeting macrophage recruitment to kidney is unlikely to be effective as treatment of Alport syndrome.