Prolonged, Low-Dose Anti-Thymocyte Globulin, Combined with CTLA4-Ig, Promotes Engraftment in a Stringent Transplant Model

Background: Despite significant nephrotoxicity, calcineurin inhibitors (CNIs) remain the cornerstone of immunosuppression in solid organ transplantation. We, along with others, have reported tolerogenic properties of anti-thymocyte globulin (ATG, Thymoglobulin®), evinced by its ability both to spare Tregs from depletion in vivo and, when administered at low, non-depleting doses, to expand Tregs ex vivo. Clinical trials investigating B7/CD28 blockade (LEA29Y, Belatacept) in kidney transplant recipients have proven that the replacement of toxic CNI use is feasible in selected populations. Methods: Rabbit polyclonal anti-murine thymocyte globulin (mATG) was administered as induction and/or prolonged, low-dose therapy, in combination with CTLA4-Ig, in a stringent, fully MHC-mismatched murine skin transplant model to assess graft survival and mechanisms of action. Results: Prolonged, low-dose mATG, combined with CTLA4-Ig, effectively promotes engraftment in a stringent transplant model. Our data demonstrate that mATG achieves graft acceptance primarily by promoting Tregs, while CTLA4-Ig enhances mATG function by limiting activation of the effector T cell pool in the early stages of treatment, and by inhibiting production of anti-rabbit antibodies in the maintenance phase, thereby promoting regulation of alloreactivity. Conclusion: These data provide the rationale for development of novel, CNI-free clinical protocols in human transplant recipients

A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy

T-bet plays a crucial role in Th1 development. We investigated the role of T-bet in the development of allograft rejection in an established MHC class II–mismatched (bm12 into B6) model of chronic allograft vasculopathy (CAV). Intriguingly, and in contrast to IFN-γ−/− mice that are protected from CAV, T-bet−/− recipients develop markedly accelerated allograft rejection accompanied by early severe vascular inflammation and vasculopathy, and infiltration by predominantly IL-17–producing CD4 T cells. Concurrently, T-bet−/− mice exhibit a T helper type 1 (Th1)–deficient environment characterized by profound IFN-γ deficiency, a Th2 switch characterized by increased production of interleukin (IL) 4, IL-5, IL-10, and IL-13 cytokines, as well as increased production of the proinflammatory cytokines IL-6, IL-12p40, and IL-17. Neutralization of IL-17 inhibits accelerated allograft rejection and vasculopathy in T-bet−/− mice. Interestingly, CD4 but not CD8 T cell deficiency in T-bet−/− mice affords dramatic protection from vasculopathy and facilitates long-term graft acceptance. This is the first study establishing that in the absence of Th1-mediated alloimmune responses, CD4 Th17 cells mediate an aggressive proinflammatory response culminating in severe accelerated allograft rejection and vasculopathy. These results have important implications for the development of novel therapies to target this intractable problem in clinical solid organ transplantation

IL-10 Receptor Signaling Is Essential for T R 1 Cell Function In Vivo

Interleukin-10 (IL-10) is essential to maintain intestinal homeostasis. CD4+ T regulatory type 1 (TR1) cells produce large amounts of this cytokine and being therefore currently examined in clinical trials as T-cell therapy in patients with inflammatory bowel disease (IBD). However, factors and molecular signals sustaining TR1 cell regulatory activity still need to be identified in order to optimize the efficiency and to ensure the safety of these trials. We investigated the role of IL-10 signaling in mature TR1 cells in vivo

IL-21 Is an Antitolerogenic Cytokine of the Late-Phase Alloimmune Response

Objective: Interleukin-21 (IL-21) is a proinflammatory cytokine that has been shown to affect Treg/Teff balance. However, the mechanism by which IL-21 orchestrates alloimmune response and interplays with Tregs is still unclear. Research design and methods: The interplay between IL-21/IL-21R signaling, FoxP3 expression, and Treg survival and function was evaluated in vitro in immunologically relevant assays and in vivo in allogenic and autoimmune models of islet transplantation. Results: IL-21R expression decreases on T cells and B cells in vitro and increases in the graft in vivo, while IL-21 levels increase in vitro and in vivo during anti-CD3/anti-CD28 stimulation/allostimulation in the late phase of the alloimmune response. In vitro, IL-21/IL-21R signaling (by using rmIL-21 or genetically modified $CD4^+$ T cells [IL-21 pOrf plasmid–treated or hIL-21-Tg mice]) enhances the T-cell response during anti-CD3/anti-CD28 stimulation/allostimulation, prevents Treg generation, inhibits Treg function, induces Treg apoptosis, and reduces FoxP3 and FoxP3-dependent gene transcripts without affecting FoxP3 methylation status. In vivo targeting of IL-21/IL-21R expands intragraft and peripheral Tregs, promotes Treg neogenesis, and regulates the antidonor immune response, whereas IL-21/IL-21R signaling in Doxa-inducible ROSA-rtTA-IL-21-Tg mice expands Teffs and $FoxP3^−$ cells. Treatment with a combination of mIL-21R.Fc and CTLA4-Ig (an inhibitor of the early alloimmune response) leads to robust graft tolerance in a purely alloimmune setting and prolonged islet graft survival in NOD mice. Conclusions: IL-21 interferes with different checkpoints of the FoxP3 Treg chain in the late phase of alloimmune response and, thus, acts as an antitolerogenic cytokine. Blockade of the IL-21/IL-21R pathway could be a precondition for tolerogenic protocols in transplantation

Tackling major hurdles in cell therapy translational research: Different products, common challenges

Cell-based immunotherapies address a number of unmet medical needs and some have shown success. All cell-based immunotherapy products undergo extensive preclinical and proof-of-concept investigations for only a few to reach the bedside. Thus, several hurdles need to be overcome in cell therapy translational research for wider access to such innovative treatments. Major hurdles include, but are not limited to: definitive characterization of cell therapy products, feasibility of upscaling a laboratory-grade cell manufacturing protocol, automation of critical manufacturing processes, standardized quality controls, choice of therapeutic window, comparability between clinical trials and commercial success. The aim of this thesis is to tackle these common challenges faced by different products during preclinical, translational and clinical phases. With several market authorizations and withdrawals by the EMA for advanced therapy medicinal products, it is timely to evaluate the translational activity around cell-based immunotherapy products. Adoptive transfer of ex vivo expanded T regulatory cells is currently under clinical investigation in solid organ transplantation, autoimmunity, and graft-versus-host disease, yet definitive characterization of their many modes of action remains to be identified. Such mechanisms are reviewed in Chapter 2. In Chapter 3, a preclinical investigation dissects one mechanism by which T regulatory type 1 (Tr1 cells) are therapeutically-induced in vivo in a mouse model of Tr1-induced tolerance to allogeneic islet graft transplantation, identifying macrophages as key players in this induction. In a translational context, feasibility of upscaling a laboratory-grade Tr1 cell manufacturing protocol using reagents and procedures compatible with good manufacturing practices (GMP) is reported along with data supporting the choice of therapeutic window for cell infusion in the upcoming clinical trial (Chapter 4). In an effort to reduce variations in manufacturing and cost-of-goods, feasibility of automating a critical step in hematopoietic stem cell transplantation (HSCT), washing and concentrating thawed cells is established (Chapter 5). To address the need for standardized and accredited quality controls, validation of a quantitative immune-phenotyping method is reported in Chapter 6, of relevance during collection of apheresis products, manufacturing and release of cell-based immunotherapy products. Heterogeneity in manufacturing NK cells for clinical use is reviewed in Chapter 7 while expanding on tools for harmonization of processes and quality controls to address the challenge of comparability between clinical trials. The hurdle of reaching larger patient populations and achieving commercial success is dissected for CAR-T cells in Chapter 8. Management of toxicities, investigating new indications and target tumor antigens, different manufacturing options and off-the-shelf products are addressed. This thesis documents a unique career path covering the spectrum from bench to bedside to provide arguments to many hurdles facing cell therapy translational research. Others surely remain, for instance role of patients in product development. Activating and suppressive cell-based immunotherapies alike face these challenges nevertheless possible solutions can be extrapolated from one product to another for the benefit of larger patient populations

Tackling major hurdles in cell therapy translational research: Different products, common challenges

Cell-based immunotherapies address a number of unmet medical needs and some have shown success. All cell-based immunotherapy products undergo extensive preclinical and proof-of-concept investigations for only a few to reach the bedside. Thus, several hurdles need to be overcome in cell therapy translational research for wider access to such innovative treatments. Major hurdles include, but are not limited to: definitive characterization of cell therapy products, feasibility of upscaling a laboratory-grade cell manufacturing protocol, automation of critical manufacturing processes, standardized quality controls, choice of therapeutic window, comparability between clinical trials and commercial success. The aim of this thesis is to tackle these common challenges faced by different products during preclinical, translational and clinical phases. With several market authorizations and withdrawals by the EMA for advanced therapy medicinal products, it is timely to evaluate the translational activity around cell-based immunotherapy products. Adoptive transfer of ex vivo expanded T regulatory cells is currently under clinical investigation in solid organ transplantation, autoimmunity, and graft-versus-host disease, yet definitive characterization of their many modes of action remains to be identified. Such mechanisms are reviewed in Chapter 2. In Chapter 3, a preclinical investigation dissects one mechanism by which T regulatory type 1 (Tr1 cells) are therapeutically-induced in vivo in a mouse model of Tr1-induced tolerance to allogeneic islet graft transplantation, identifying macrophages as key players in this induction. In a translational context, feasibility of upscaling a laboratory-grade Tr1 cell manufacturing protocol using reagents and procedures compatible with good manufacturing practices (GMP) is reported along with data supporting the choice of therapeutic window for cell infusion in the upcoming clinical trial (Chapter 4). In an effort to reduce variations in manufacturing and cost-of-goods, feasibility of automating a critical step in hematopoietic stem cell transplantation (HSCT), washing and concentrating thawed cells is established (Chapter 5). To address the need for standardized and accredited quality controls, validation of a quantitative immune-phenotyping method is reported in Chapter 6, of relevance during collection of apheresis products, manufacturing and release of cell-based immunotherapy products. Heterogeneity in manufacturing NK cells for clinical use is reviewed in Chapter 7 while expanding on tools for harmonization of processes and quality controls to address the challenge of comparability between clinical trials. The hurdle of reaching larger patient populations and achieving commercial success is dissected for CAR-T cells in Chapter 8. Management of toxicities, investigating new indications and target tumor antigens, different manufacturing options and off-the-shelf products are addressed. This thesis documents a unique career path covering the spectrum from bench to bedside to provide arguments to many hurdles facing cell therapy translational research. Others surely remain, for instance role of patients in product development. Activating and suppressive cell-based immunotherapies alike face these challenges nevertheless possible solutions can be extrapolated from one product to another for the benefit of larger patient populations

MANUFACTURING NATURAL KILLER CELLS AS MEDICINAL PRODUCTS

Natural Killer (NK) cells are Innate Lymphoid Cells (ILC) with cytotoxic and regulatory properties. Their functions are tightly regulated by an array of inhibitory and activating receptors, and their mechanisms of activation strongly differ from antigen recognition in the context of HLA presentation as needed for T-cell activation. NK cells thus offer unique opportunities for new and improved therapeutic manipulation, either in vivo or in vitro, in a variety of human diseases, including cancers. NK cell activity can possibly be modulated in vivo through direct or indirect actions exerted by small molecules or monoclonal antibodies. NK cells can also be adoptively transferred following more or less substantial modifications through cell and gene manufacturing, in order to empower them with new or improved functions and ensure their controlled persistence and activity in the recipient. In the present review, we will focus on the technological and regulatory challenges of NK cell manufacturing, and discuss conditions in which these innovative cellular therapies can be brought to the clinic

Manufacturing Natural Killer Cells as Medicinal Products

International audienceNatural Killer (NK) cells are innate lymphoid cells (ILC) with cytotoxic and regulatory properties. Their functions are tightly regulated by an array of inhibitory and activating receptors, and their mechanisms of activation strongly differ from antigen recognition in the context of human leukocyte antigen presentation as needed for T-cell activation. NK cells thus offer unique opportunities for new and improved therapeutic manipulation, either in vivo or in vitro, in a variety of human diseases, including cancers. NK cell activity can possibly be modulated in vivo through direct or indirect actions exerted by small molecules or monoclonal antibodies. NK cells can also be adoptively transferred following more or less substantial modifications through cell and gene manufacturing, in order to empower them with new or improved functions and ensure their controlled persistence and activity in the recipient. In the present review, we will focus on the technological and regulatory challenges of NK cell manufacturing and discuss conditions in which these innovative cellular therapies can be brought to the clinic