Search

Advances in cancer immunotherapies utilizing engineered hematopoietic cells have recently generated significant clinical successes. Of great promise are immunotherapies based on chimeric antigen receptor-engineered T (CAR-T) cells that are targeted toward malignant cells expressing defined tumor-associated antigens. CAR-T cells harness the effector function of the adaptive arm of the immune system and redirect it against cancer cells, overcoming the major challenges of immunotherapy, such as breaking tolerance to self-antigens and beating cancer immune system-evasion mechanisms. In early clinical trials, CAR-T cell-based therapies achieved complete and durable responses in a significant proportion of patients. Despite clinical successes and given the side effect profiles of immunotherapies based on engineered cells, potential concerns with the safety and toxicity of various therapeutic modalities remain. We discuss the concerns associated with the safety and stability of the gene delivery vehicles for cell engineering and with toxicities due to off-target and on-target, off-tumor effector functions of the engineered cells. We then overview the various strategies aimed at improving the safety of and resolving toxicities associated with cell-based immunotherapies. Integrating failsafe switches based on different suicide gene therapy systems into engineered cells engenders promising strategies toward ensuring the safety of cancer immunotherapies in the clinic.

Supplemental Digital Content is available in the text

Supplemental Digital Content is available in the text

Immune thrombocytopenia or ITP is a debilitating and life-threatening disorder affecting more than 4 in every 10, 000 adults annually. Following a basic understanding of the immunopathology underlying ITP, namely that production of anti-platelet antibodies results in accelerated platelet clearance and thrombocytopenia, animal models of ITP were quickly developed. Rodent models that develop ITP spontaneously or by passive transfer of anti-platelet sera or antibodies have become instrumental in investigating the mechanisms responsible for the breakdown of tolerance in human ITP, understanding the immunopathology that underlies the progression of platelet destruction, elucidating the mechanism(s) of therapeutic amelioration of the ITP, and driving the development of new therapeutic modalities. This review aims to capture the development history and methodology of currently available ITP disease models, and review their advantages and limitations in the study of various aspects of ITP. We also review how closely the various ITP models reflect the pathobiology of human ITP and their usefulness in advancing the development of new therapeutics, which are particularly needed to address the unmet need of patients who are refractory to the currently available repertoire of interventions.

Allogeneic transplantation of hematopoietic stem cells and lymphocytes is a curative treatment for malignant and non-malignant disease. However, the primary complication limiting the safety of transplantation is graft-versus-host disease (GvHD), which is mediated by donor T cells. Strategies for pre- and post-transplant manipulation of graft cells are not yet optimal for balancing GvHD severity with beneficial Graft-versus-Leukemia (GvL) effects. Emerging cell fate control gene therapy (CFCGT)-based strategies, such as ‘suicide’ gene therapy for donor T cell regulation, can supplement existing transplantation approaches by providing a safety element to reduce GvHD. Past uses of CFCGT in the clinic have provided proof-of-principle that GvHD can be controlled by such a strategy. However, there exists a need for improved transgene delivery and suicide control systems. Recently, lentiviral vectors (LVs) have emerged as effective gene delivery vehicles for the clinic. Combining lentiviral gene delivery with newer generations of ‘suicide’ systems that possess improved enzyme/prodrug specificities, activities, and reduced immunogenicity, could provide the necessary degree of control required to more successfully manage GvHD. Improving the safety of transplantation through successful CFCGT will serve to expand the potential donor pool and the spectrum of disorders that can be treated by this therapeutic schema.

Immune thrombocytopenia (ITP) is a debilitating, life‐threatening autoimmune disorder affecting more than 4 in every 100,000 adults annually, stemming from the production of antiplatelet antibody resulting in accelerated platelet destruction and thrombocytopenia. Numerous animal models of ITP have been developed that contributed to the basic understanding of the underlying mechanisms of ITP onset, progression, and maintenance. Rodent models that develop ITP spontaneously, or by passive transfer of an antiplatelet sera or antibody, play an instrumental role in the investigation of ITP mechanisms responsible for the breakdown of tolerance in human ITP, in studies of the immunopathology underlying the progression of platelet destruction, and in elucidation of the mechanisms of therapeutic amelioration of ITP by existing and new therapeutic modalities. This unit captures the protocols for the implementation and readout of passive antibody transfer mouse models of ITP, established by the infusion of a commercially‐available monoclonal rat anti‐mouse CD41 platelet antibody. © 2016 by John Wiley & Sons, Inc.

Donor Lymphocyte Infusion (DLI) is employed in the treatment of various malignancies, as donor-derived allogeneic T lymphocytes elicit strong anti-tumor immune responses (GvL/GvT). Unfortunately, these are often accompanied by GvHD, mediated by donor T cells that are allo-activated against host tissues. GvHD manifests with immunosupression, multi-organ dysfunction, severe morbidity and high mortality. It is suggested that CD4+Th1 and CD8+Tc1 cell subsets mediate both strong GvL/GvT and strong GvHD, while being cross-regulated by CD4+Th2 and CD8+Tc2 cells that mediate only moderate effects. Patients transplanted with T cell-depleted BM experience little GvHD, but have high rates of cancer relapse. It is possible to maintain beneficial GvL/GvT responses while controlling GvHD by transferring a drug-activating ‘suicide’ gene into donor T cells prior to transplant. Cells expressing the ‘suicide’ gene-product convert a non-toxic prodrug into a cytotoxic antimetabolite, and are eliminated by apoptosis. We have constructed 3rd generation lentiviral vectors (LVs) for the expression of human converting enzymes. We are using novel enzymes, endogenously present in human cells, to avoid unwanted immune responses against vector-transduced cells, recently proven a major drawback in clinical trials. Our approaches are based on the human thymidylate kinase (Tmpk), which acts on Zidovudine (AZT), and the human deoxycytidine kinase (dCK), which potently activates a number of prodrugs, including Cytarabine (AraC), Gemcitabine and Cladribine. We have developed active-site engineered mutants of Tmpk and dCK that are up to two orders of magnitude more catalytically active than wild-type (wt) enzymes. Our LVs efficiently transduce both immortalized and primary cells (up to 99%). The LV expression cassette also encodes a non-signaling form of human CD19 molecule that serves as a marker for ex vivo enrichment and in vivo tracking of transduced cells. We demonstrate efficient, selective and prompt killing of transduced cells in a dose-dependent manner by a number of prodrugs in cell lines in vitro and in a murine solid tumor model in vivo. For example, R16GLL Tmpk mutant-transduced Jurkat cells (human T cell leukemia line) are completely killed by apoptosis within 4 days of culture in media containing 100uM AZT (IC50 of 2µM), while wt cells are unaffected at the same conditions. In a NOD/SCID murine tumor model, K562 erythroid leukemia cells transduced with either empty vector, wt Tmpk, and R16GLL or F105Y Tmpk mutants were implanted subcutaneously. Mice bearing tumor cells expressing either R16GLL or F105Y Tmpk and receiving a low daily dose of 2.5mg/kg of AZT have developed significantly reduced tumors following 2 weeks of treatment (tumor size of 24–200mm2), compared to mice bearing wt Tmpk and empty vector transduced cells, or untreated mice (tumor size of over 1000mm2). We are currently evaluating the efficiency with which transduced cells can be cleared from circulation in a murine leukemia model and are evaluating the complete GvHD-treatment strategy in a murine model of GvHD/GvT based on the CD45 congenic strains. This much improved therapeutic strategy may offer a safe and complete solution for GvHD in patients undergoing BMT.

Donor Lymphocyte Infusion (DLI) is employed in the treatment of various malignancies, as donor-derived allogeneic T lymphocytes elicit strong anti-tumor immune responses (GvL/GvT). Unfortunately, these are often accompanied by GvHD, mediated by donor T cells that are allo-activated against host tissues. GvHD manifests with immunosupression, multi-organ dysfunction, severe morbidity and high mortality. It is suggested that CD4+Th1 and CD8+Tc1 cell subsets mediate both strong GvL/GvT and strong GvHD, while being cross-regulated by CD4+Th2 and CD8+Tc2 cells that mediate only moderate effects. Patients transplanted with T cell-depleted BM experience little GvHD, but have high rates of cancer relapse. It is possible to maintain beneficial GvL/GvT responses while controlling GvHD by transferring a drug-activating ‘suicide' gene into donor T cells prior to transplant. Cells expressing the ‘suicide' gene-product convert a non-toxic prodrug into a cytotoxic antimetabolite, and are eliminated by apoptosis. We have constructed 3rdgeneration lentiviral vectors (LVs) for the expression of human converting enzymes. We are using novel enzymes, endogenously present in human cells, to avoid unwanted immune responses against vector-transduced cells, recently proven a major drawback in clinical trials. Our approaches are based on the human thymidylate kinase (Tmpk), which acts on Zidovudine (AZT), and the human deoxycytidine kinase (dCK), which potently activates a number of prodrugs, including Cytarabine (AraC), Gemcitabine and Cladribine. We have developed active-site engineered mutants of Tmpk and dCK that are up to two orders of magnitude more catalytically active than wild-type (wt) enzymes. Our LVs efficiently transduce both immortalized and primary cells (up to 99%). The LV expression cassette also encodes a non-signaling form of human CD19 molecule that serves as a marker for ex vivoenrichment and in vivotracking of transduced cells. We demonstrate efficient, selective and prompt killing of transduced cells in a dose-dependent manner by a number of prodrugs in cell lines in vitroand in a murine solid tumor model in vivo. For example, R16GLL Tmpk mutant-transduced Jurkat cells (human T cell leukemia line) are completely killed by apoptosis within 4 days of culture in media containing 100uM AZT (IC50 of 2μM), while wt cells are unaffected at the same conditions. In a NOD/SCID murine tumor model, K562 erythroid leukemia cells transduced with either empty vector, wt Tmpk, and R16GLL or F105Y Tmpk mutants were implanted subcutaneously. Mice bearing tumor cells expressing either R16GLL or F105Y Tmpk and receiving a low daily dose of 2.5mg/kg of AZT have developed significantly reduced tumors following 2 weeks of treatment (tumor size of 24–200mm2), compared to mice bearing wt Tmpk and empty vector transduced cells, or untreated mice (tumor size of over 1000mm2). We are currently evaluating the efficiency with which transduced cells can be cleared from circulation in a murine leukemia model and are evaluating the complete GvHD-treatment strategy in a murine model of GvHD/GvT based on the CD45 congenic strains. This much improved therapeutic strategy may offer a safe and complete solution for GvHD in patients undergoing BMT.

Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the most widely used approach for suicide gene therapy. This ‘suicide’ strategy allows direct reduction of tumors and clearance of donor cells should graft-versus-host disease (GvHD) arise after bone marrow transplantation. Given recent clinical outcomes, this suicide approach may also provide a key safety component for therapeutic gene transfer vectors that integrate. Although suicide gene therapy using HSV1-TK-encoding oncoretroviral vectors has been evaluated in the clinic, the success of this approach has been relatively modest. Reasons for this include: low gene transfer efficacy, reduced expression of the suicide gene, and insufficient conversion of substrate. Our goal is to overcome these limitations by using a novel lentiviral vector (LV) encoding an alternative kinase/prodrug combination. The rational for our innovative suicide gene therapy strategy is two-fold: 1) Lentiviral vectors can efficiently transduce not only dividing cells but also non-dividing cells. 2) Applying a faster viral enzyme like equine herpes virus type 4 thymidine kinase (EHV4-TK) could be advantageous as it has been shown to be kinetically superior to HSV1-TK at GCV phosphorylation. The aim of this study is to evaluate whether LV-mediated gene modification of target cells with EHV4-TK can lead to efficient killing following GCV treatment. We first constructed a LV expression system carrying the wild-type EHV4-TK cDNA with an IRES element followed by a truncated form of human CD19 (hCD19Δ). Human CD19 was chosen as a cell surface marker to allow functional titering of virus and for immuno-enrichment of transduced cells prior to infusion since it is not expressed in the T cell lineage. The truncated form lacks the intracellular domain and therefore does not signal. Use of an IRES element can abrogate some variegated expression seen with vectors having dual promoters. The LV was pseudotyped with VSV-g and concentrated by ultracentrifugation. After one infection, Jurkat cells (human T cell leukemia) showed a more than 80% functional and stable transduction efficiency (MOI = 10). Using hCD19Δ as a selective marker, transduced Jurkat cells were enriched to over 95% positive by immuno-affinity sorting. EHV4-TK-transduced Jurkat cells exhibited increased cell killing in response to GCV treatment (the apoptotic cell indexes with or without GCV were 69.4 ± 1.5 % and 18.8 ± 1.7 %, respectively; n=3). Highly efficient transduction (more than 60%) of primary human T cells was accomplished by a three time exposure to virus over 36 hours at MOI of 20. Next, we found that GCV efficiently killed transduced primary human T cells in a dose dependent manner. We are now comparing the efficiency of GCV conversion by HSV1-TK and EHV4-TK using LV-transduced cells that express the similar protein levels. We are also evaluating intracellular levels of GCV metabolites by HPLC. These results demonstrate that our novel suicide gene therapy strategy has significant potential for many clinical applications.

Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the most widely used approach for suicide gene therapy. This ‘suicide' strategy allows direct reduction of tumors and clearance of donor cells should graft-versus-host disease (GvHD) arise after bone marrow transplantation. Given recent clinical outcomes, this suicide approach may also provide a key safety component for therapeutic gene transfer vectors that integrate. Although suicide gene therapy using HSV1-TK-encoding oncoretroviral vectors has been evaluated in the clinic, the success of this approach has been relatively modest. Reasons for this include: low gene transfer efficacy, reduced expression of the suicide gene, and insufficient conversion of substrate. Our goal is to overcome these limitations by using a novel lentiviral vector (LV) encoding an alternative kinase/prodrug combination. The rational for our innovative suicide gene therapy strategy is two-fold: 1) Lentiviral vectors can efficiently transduce not only dividing cells but also non-dividing cells. 2) Applying a faster viral enzyme like equine herpes virus type 4 thymidine kinase (EHV4-TK) could be advantageous as it has been shown to be kinetically superior to HSV1-TK at GCV phosphorylation. The aim of this study is to evaluate whether LV-mediated gene modification of target cells with EHV4-TK can lead to efficient killing following GCV treatment. We first constructed a LV expression system carrying the wild-type EHV4-TK cDNA with an IRES element followed by a truncated form of human CD19 (hCD19Δ). Human CD19 was chosen as a cell surface marker to allow functional titering of virus and for immuno-enrichment of transduced cells prior to infusion since it is not expressed in the T cell lineage. The truncated form lacks the intracellular domain and therefore does not signal. Use of an IRES element can abrogate some variegated expression seen with vectors having dual promoters. The LV was pseudotyped with VSV-g and concentrated by ultracentrifugation. After one infection, Jurkat cells (human T cell leukemia) showed a more than 80% functional and stable transduction efficiency (MOI = 10). Using hCD19Δ as a selective marker, transduced Jurkat cells were enriched to over 95% positive by immuno-affinity sorting. EHV4-TK-transduced Jurkat cells exhibited increased cell killing in response to GCV treatment (the apoptotic cell indexes with or without GCV were 69.4 ± 1.5 % and 18.8 ± 1.7 %, respectively; n=3). Highly efficient transduction (more than 60%) of primary human T cells was accomplished by a three time exposure to virus over 36 hours at MOI of 20. Next, we found that GCV efficiently killed transduced primary human T cells in a dose dependent manner. We are now comparing the efficiency of GCV conversion by HSV1-TK and EHV4-TK using LV-transduced cells that express the similar protein levels. We are also evaluating intracellular levels of GCV metabolites by HPLC. These results demonstrate that our novel suicide gene therapy strategy has significant potential for many clinical applications.

The infusion of donor lymphocytes in allogenic BMT recipients provides potent antitumor activity to treat recurrent malignancies. One complication, however, is severe GvHD, which is mediated by T cells in the graft. One approach to control GvHD is to employ ‘suicide' gene therapy. In the customary adaptation of this approach, the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene, combined with the antiviral prodrug ganciclovir (GCV), is used to control GvHD after introduction of this suicide gene into donor T lymphocytes. However, the efficiency of HSV1-tk is suboptimal and the issue of host immunogenicity against this heterologous effector gene product can hamper outcomes. In addition, prophylactic GCV is often used to control cytomegalovirus infection after BMT. This confounds the broad clinical implementation of this approach. Our novel suicide gene therapy strategy combines the use of human thymidylate monophosphate kinase (tmpk) and the prodrug Zidovudine (AZT) in a lentiviral vector (LV) format. Since tmpk is endogenously expressed in human cells, immunogenic responses will be limited. We constructed a LV expression system carrying wild-type or one of two modified forms of tmpk. These engineered tmpk mutants (F105Y and BL) show substantially increased catalytic conversion of AZT compared to wild-type tmpk. Our vector also includes a truncated form of human CD19 (hCD19Δ), not normally expressed on the T cell lineage, that can be used to enrich and track transduced cells. Highly efficient (95%) transduction of Jurkat cells (human T cell leukemia line) was attained by a single infection with our LVs (MOI of 10). Transduced cells were efficiently and selectively killed in a dose-dependent manner by AZT (IC50 of 2 μM), while wild-type tmpk transduced cells were unaffected by AZT up to 100 μM. In response to AZT treatment, the apoptotic cell indices of cells transduced with wild-type tmpk, F105Y, or BL were 6.2 ± 0.3 %, 40.7 ± 1.7 %, and 46.1 ± 4.6 %, respectively (n=3). We next established by HPLC that cells transduced with a LV encoding a mutant form of tmpk effectively convert AZT into its active anti-metabolite form, AZT-triphosphate (AZT-TP). Intracellular ratio of AZT-TP to AZT-monophosphate (MP) is 11.3 in cells transduced with a LV encoding the BL mutant of tmpk, compared to 0.02 in non-transduced cells and 0.10 in wild-type tmpk transduced cells. Our findings also revealed that following incubation with indirubin-3′-oxime, which inhibits cellular proliferation, and AZT treatment, transduced cells were successfully killed. Thus the cytotoxic mechanism differs from HSV1-tk mediated cell killing and is independent of cell proliferation. We also succeeded in the infection of primary mouse and human T cells to over 40% and 70% transduction efficiency, respectively. Lastly, we have shown that in vivo growth of tumor cells transduced with these mutant tmpk LVs was totally inhibited by treatment with AZT. These results demonstrate that our novel suicide gene therapy system has significant potential for many clinical applications.

The infusion of donor lymphocytes in allogenic BMT recipients provides potent antitumor activity to treat recurrent malignancies. One complication, however, is severe GvHD, which is mediated by T cells in the graft. One approach to control GvHD is to employ ‘suicide’ gene therapy. In the customary adaptation of this approach, the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene, combined with the antiviral prodrug ganciclovir (GCV), is used to control GvHD after introduction of this suicide gene into donor T lymphocytes. However, the efficiency of HSV1-tk is suboptimal and the issue of host immunogenicity against this heterologous effector gene product can hamper outcomes. In addition, prophylactic GCV is often used to control cytomegalovirus infection after BMT. This confounds the broad clinical implementation of this approach. Our novel suicide gene therapy strategy combines the use of human thymidylate monophosphate kinase (tmpk) and the prodrug Zidovudine (AZT) in a lentiviral vector (LV) format. Since tmpk is endogenously expressed in human cells, immunogenic responses will be limited. We constructed a LV expression system carrying wild-type or one of two modified forms of tmpk. These engineered tmpk mutants (F105Y and BL) show substantially increased catalytic conversion of AZT compared to wild-type tmpk. Our vector also includes a truncated form of human CD19 (hCD19Δ), not normally expressed on the T cell lineage, that can be used to enrich and track transduced cells. Highly efficient (95%) transduction of Jurkat cells (human T cell leukemia line) was attained by a single infection with our LVs (MOI of 10). Transduced cells were efficiently and selectively killed in a dose-dependent manner by AZT (IC50 of 2 μM), while wild-type tmpk transduced cells were unaffected by AZT up to 100 μM. In response to AZT treatment, the apoptotic cell indices of cells transduced with wild-type tmpk, F105Y, or BL were 6.2 ± 0.3 %, 40.7 ± 1.7 %, and 46.1 ± 4.6 %, respectively (n=3). We next established by HPLC that cells transduced with a LV encoding a mutant form of tmpk effectively convert AZT into its active anti-metabolite form, AZT-triphosphate (AZT-TP). Intracellular ratio of AZT-TP to AZT-monophosphate (MP) is 11.3 in cells transduced with a LV encoding the BL mutant of tmpk, compared to 0.02 in non-transduced cells and 0.10 in wild-type tmpk transduced cells. Our findings also revealed that following incubation with indirubin-3′-oxime, which inhibits cellular proliferation, and AZT treatment, transduced cells were successfully killed. Thus the cytotoxic mechanism differs from HSV1-tk mediated cell killing and is independent of cell proliferation. We also succeeded in the infection of primary mouse and human T cells to over 40% and 70% transduction efficiency, respectively. Lastly, we have shown that in vivo growth of tumor cells transduced with these mutant tmpk LVs was totally inhibited by treatment with AZT. These results demonstrate that our novel suicide gene therapy system has significant potential for many clinical applications.