Classical approaches to immunotherapy that show promise in some malignancies have generally been disappointing when applied to high-grade brain tumors such as glioblastoma multiforme (GBM). We recently showed that ex vivo expanded/activated γδ T cells recognize NKG2D ligands expressed on malignant glioma and are cytotoxic to glioma cell lines and primary GBM explants. In addition, γδ T cells extend survival and slow tumor progression when administered to immunodeficient mice with intracranial human glioma xenografts. We now show that temozolomide (TMZ), a principal chemotherapeutic agent used to treat GBM, increases the expression of stress-associated NKG2D ligands on TMZ-resistant glioma cells, potentially rendering them vulnerable to γδ T cell recognition and lysis. TMZ is also highly toxic to γδ T cells, however, and to overcome this cytotoxic effect γδ T cells were genetically modified using a lentiviral vector encoding the DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) from the O(6)-methylguanine methyltransferase (MGMT) cDNA, which confers resistance to TMZ. Genetic modification of γδ T cells did not alter their phenotype or their cytotoxicity against GBM target cells. Importantly, gene modified γδ T cells showed greater cytotoxicity to two TMZ resistant GBM cell lines, U373^TMZ-R and SNB-19^TMZ-R cells, in the presence of TMZ than unmodified cells, suggesting that TMZ exposed more receptors for γδ T cell-targeted lysis. Therefore, TMZ resistant γδ T cells can be generated without impairing their anti-tumor functions in the presence of high concentrations of TMZ. These results provide a mechanistic basis for combining chemotherapy and γδ T cell-based drug resistant cellular immunotherapy to treat GBM.

Relapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting. To circumvent this problem two approaches were investigated. First, a natural killer (NK) cell line, which does not express CD5, was used for CAR expression. Second, CRISPR-Cas9 genome editing technology was used to knockout CD5 expression in CD5-positive Jurkat T cells and in primary T cells, allowing for the use of CD5-negative T cells for CAR expression. Two structurally distinct anti-CD5 sequences were also tested, i) a traditional immunoglobulin-based single chain variable fragment (scFv) and ii) a lamprey-derived variable lymphocyte receptor (VLR), which we previously showed can be used for CAR-based recognition. Our results show i) both CARs yield comparable T-cell activation and NK cell-based cytotoxicity when targeting CD5-positive cells, ii) CD5-edited CAR-modified Jurkat T cells have reduced self-activation compared to that of CD5-positive CAR-modified T cells, iii) CD5-edited CAR-modified Jurkat T cells have increased activation in the presence of CD5-positive target cells compared to that of CD5-positive CAR-modified T cells, and iv) although modest effects were seen, a mouse model using the CAR-expressing NK cell line showed the scFv-CAR was superior to the VLR-CAR in delaying disease progression.

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus–based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

Genetically modified, autologous hematopoietic stem and progenitor cells (HSPCs) represent a new class of genetic medicine. Following this therapeutic paradigm, we are developing a product candidate, designated CD68-ET3-LV CD34 ⁺ , for the treatment of the severe bleeding disorder, hemophilia A. The product consists of autologous CD34 ⁺ cells transduced with a human immunodeficiency virus 1–based, monocyte lineage-restricted, self-inactivating lentiviral vector (LV), termed CD68-ET3-LV, encoding a bioengineered coagulation factor VIII (fVIII) transgene, termed ET3, designed for enhanced expression. This vector was shown capable of high-titer manufacture under clinical scale and Good Manufacturing Practice. Biochemical and immunogenicity testing of recombinant ET3, as well as safety and efficacy testing of CD68-ET3-LV HSPCs, were utilized to demonstrate overall safety and efficacy in murine models. In the first model, administration of CD68-ET3-LV-transduced stem-cell antigen-1 ⁺ cells to hemophilia A mice resulted in sustained plasma fVIII production and hemostatic correction without signs of toxicity. Patient-derived, autologous mobilized peripheral blood (mPB) CD34 ⁺ cells are the clinical target cells for ex vivo transduction using CD68-ET3-LV, and the resulting genetically modified cells represent the investigational drug candidate. In the second model, CD68-ET3-LV gene transfer into mPB CD34 ⁺ cells isolated from normal human donors was utilized to obtain in vitro and in vivo pharmacology, pharmacokinetic, and toxicology assessment. CD68-ET3-LV demonstrated reproducible and efficient gene transfer into mPB CD34 ⁺ cells, with vector copy numbers in the range of 1 copy per diploid genome equivalent without affecting clonogenic potential. Differentiation of human CD34 ⁺ cells into monocytes was associated with increased fVIII production, supporting the designed function of the CD68 promoter. To assess in vivo pharmacodynamics, CD68-ET3-LV CD34 ⁺ cell product was administered to immunodeficient mice. Treated mice displayed sustained plasma fVIII levels and no signs of product related toxicity. Collectively, the findings of the current study support the preclinical safety and efficacy of CD68-ET3-LV CD34 ⁺ .

Gamma delta (γδ) T cells recently emerged as an attractive candidate for cancer immunotherapy treatments due to their inherent cytotoxicity against both hematological and solid tumors. Moreover, γδ T cells provide a platform for the development of allogeneic cell therapies, as they can recognize antigens independent of MHC recognition and without the requirement for a chimeric antigen receptor. However, γδ T cell adoptive cell therapy depends on ex vivo expansion to manufacture sufficient cell product numbers, which remains challenging and limited by inter-donor variability. In the current study, we characterize the differences in expansion of γδ T cells from various donors that expand (EX) and donors that fail to expand, i.e. non-expanders (NE). Further, we demonstrate that IL-21 can be used to increase the expansion potential of NE. In order to reduce the risk of graft versus host disease (GVHD) induced by an allogeneic T cell product, αβ T cell depletions must be considered due to the potential for HLA mismatch. Typically, αβ T cell depletions are performed at the end of expansion, prior to infusion. We show that γδ T cell cultures can be successfully αβ depleted on day 6 of expansion, providing a better environment for the γδ T cells to expand, and that the αβ T cell population remains below clinically acceptable standards for T cell-depleted allogeneic stem cell products. Finally, we assess the potential for a mixed donor γδ T cell therapy and characterize the effects of cryopreservation on γδ T cells. Collectively, these studies support the development of an improved allogeneic γδ T cell product and suggest the possibility of using mixed donor γδ T cell immunotherapies.