Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T-cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly-efficient multiplex gene editing in primary human T-cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T-cells deficient in expression of both their T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T-cells manufactured with this process do not mediate graft-versus-host reactions, and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T-cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19+ tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an “off-the-shelf” manner akin to other biological immunopharmaceuticals.

In the absence of a local inflammatory response, expression of MHC Class II molecules is mainly restricted to hematopoietic cells and thymus epithelium. However, certain tumors such as melanoma may acquire aberrant constitutive expression of MHC class II. In a set of primary melanoma cell populations and correspondingly expanded autologous tumor-infiltrating lymphocytes (TIL), we show how MHC class II expression on melanoma cells associates with strong MHC class II-restricted CD4+ T cell responses that are specific for tumors. Notably, we found that tumor-specific CD4+ T cell responses were dominated by TNF production. TNF reduced CD8+ T cell activation in IFN-γ rich environments resembling a tumor site. Conversely, direct CD4+ T cell responses had no influence on either the proliferation or viability of melanoma cells. Taken together, our results illustrate a novel immune escape mechanism that can be activated by aberrant expression of MHC class II molecules, which by attracting tumor-specific CD4+ T cells elicit a local inflammatory response dominated by TNF that in turn inhibits cytotoxic CD8+ T cell responses.

Chordomas are tumors which arise at vertebral bodies and the base of the skull. Although rare in incidence they are deadly owing to slow growth and a lack of effective therapeutic options. In this study, we addressed the need for chordoma cell systems that can be used to identify therapeutic targets and empower testing of candidate pharmacological drugs. Eight human chordoma cell lines that we established exhibited cytology, genomics, mRNA and protein profiles that were characteristic of primary chordomas. Candidate responder profiles were identified through an immunohistochemical analysis of a chordoma tissue bank of 43 patients. Genomic, mRNA and protein expression analyses confirmed that the new cell systems were highly representative of chordoma tissues. Notably, all cells exhibited a loss of CDKN2A and p16 resulting in universal activation of the CDK4/6 and Rb pathways. Therefore, we investigated the CDK4/6 pathway and responses to the CDK4/6-specific inhibitor palbociclib.
In the newly validated system, palbociclib treatment efficiently inhibited tumor cell growth in vitro and a drug responder vs non-responder molecular signature was defined based on immunohistochemical expression of CDK4/6/pRb (S780). Overall, our work offers a valuable new tool for chordoma studies including the development of novel biomarkers and molecular targeting strategies.

Myeloid-derived suppressive cells (MDSC) have been reported to promote metastasis, but the loss of cancer-induced B cells/B regulatory cells (tBreg) can block metastasis despite MDSC expansion in cancer. Here, using multiple murine tumor models and human MDSC, we show that MDSC populations that expand in cancer have only partially primed regulatory function and limited prometastatic activity unless they are fully educated by tBregs. Cancer-induced tBregs directly activate the regulatory function of both the monocyte and granulocyte subpopulations of MDSC, relying, in part, on TgfβR1/TgfβR2 signaling. MDSC fully educated in this manner exhibit an increased production of reactive oxygen species and NO and more efficiently suppress CD4+ and CD8+ T cells, thereby promoting tumor growth and metastasis. Thus, loss of tBregs or TgfβR deficiency in MDSC is sufficient to disable their suppressive function and to block metastasis. Overall, our data indicate that cancer-induced B cells/B regulatory cells are important regulators of the immunosuppressive and prometastatic functions of MDSC. Cancer Res; 75(17); 1–10. ©2015 AACR.

Myeloid-derived suppressive cells (MDSC) have been reported to promote metastasis, but the loss of cancer-induced B cells/B regulatory cells (tBregs) can block metastasis despite MDSC expansion in cancer. Here, using multiple murine tumor models and human MDSC, we show that MDSC populations which expand in cancer have only partially primed regulatory function and limited pro-metastatic activity unless they are fully educated by tBregs. Cancer-induced tBregs directly activate the regulatory function of both the monocyte and granulocyte subpopulations of MDSC, relying in part on TgfβR1/TgfβR2 signaling. MDSC fully educated in this manner exhibit an increased production of ROS and NO and more efficiently suppress CD4+ and CD8+ T cells, thereby promoting tumor growth and metastasis. Thus, loss of tBregs or TgfβR deficiency in MDSC is sufficient to disable their suppressive function and to block metastasis. Overall, our data indicate that cancer-induced B cells/B regulatory cells are important regulators of the immune suppressive and pro-metastatic functions of MDSC.