Bala Sai Sundarasetty

Dendritic Cell–Mediated Immune Humanization of Mice: Implications for Allogeneic and Xenogeneic Stem Cell Transplantation

De novo regeneration of immunity is a major problem after allogeneic hematopoietic stem cell transplantation (HCT). HCT modeling in severely compromised immune-deficient animals transplanted with human stem cells is currently limited because of incomplete maturation of lymphocytes and scarce adaptive responses. Dendritic cells (DC) are pivotal for the organization of lymph nodes and activation of naive T and B cells. Human DC function after HCT could be augmented with adoptively transferred donor-derived DC. In this study, we demonstrate that adoptive transfer of long-lived human DC coexpressing high levels of human IFN-α, human GM-CSF, and a clinically relevant Ag (CMV pp65 protein) promoted human lymphatic remodeling in immune-deficient NOD.Rag1−/−.IL-2rγ−/− mice transplanted with human CD34+ cells. After immunization, draining lymph nodes became replenished with terminally differentiated human follicular Th cells, plasma B cells, and memory helper and cytotoxic T cells. Human Igs against pp65 were detectable in plasma, demonstrating IgG class-switch recombination. Human T cells recovered from mice showed functional reactivity against pp65. Adoptive immunotherapy with engineered DC provides a novel strategy for de novo immune reconstitution after human HCT and a practical and effective tool for studying human lymphatic regeneration in vivo in immune deficient xenograft hosts.

Lentiviral vectors for immunization: an inflammatory field.

Lentiviruses are retroviruses that are able to transduce both dividing and nondividing cells. Dendritic cells are key players in the innate and adaptive immune responses, and are natural targets for lentiviruses. Lentiviral vectors (LVs) have recently reached the clinical gene therapy arena, prompting their use as clinical vaccines. In recent years, LVs have emerged as a robust and practical experimental platform for gene delivery and rational genetic reprogramming of dendritic cells. Here, we present the status quo of the LV system for protective or therapeutic vaccine development. This vector system has been extensively evaluated for ex vivo and in vivo (immuno)gene delivery. Improvements of the LV design in order to further grant a higher biosafety profile for vaccine development are presented.

Lentivirus-induced ‘Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma

Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

Identity, potency, in vivo viability, and scaling up production of lentiviral vector-induced dendritic cells for melanoma immunotherapy

SmartDCs (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) consist of highly viable dendritic cells (DCs) induced to differentiate with lentiviral vectors (LVs) after an overnight ex vivo transduction. Tricistronic vectors co-expressing cytokines (granulocyte-macrophage-colony stimulating factor [GM-CSF], interleukin [IL]-4) and a melanoma antigen (tyrosine related protein 2 [TRP2]) were used to transduce mouse bone marrow cells or human monocytes. Sixteen hours after transduction, the cells were dispensed in aliquots and cryopreserved for identity, potency, and safety analyses. Thawed SmartDCs readily differentiated into highly viable cells with a DC immunophenotype. Prime/boost subcutaneous administration of 1×10(6) thawed murine SmartDCs into C57BL/6 mice resulted into TRP2-specific CD8(+) T-cell responses and protection against lethal melanoma challenge. Human SmartDC-TRP2 generated with monocytes obtained from melanoma patients secreted endogenous cytokines associated with DC activation and stimulated TRP2-specific autologous T-cell expansion in vitro. Thawed human SmartDCs injected subcutaneously in NOD.Rag1(-/-).IL2rγ(-/-) mice maintained DC characteristics and viability for 1 month in vivo and did not cause any signs of pathology. For development of good manufacturing practices, CD14(+) monocytes selected by magnetic-activated cell separation were transduced in a closed bag system (multiplicity of infection of 5), washed, and cryopreserved. Fifty percent of the monocytes used for transduction were recovered for cryopreservation. Thawed SmartDCs produced in two independent runs expressed the endogenous cytokines GM-CSF and IL-4, and the resulting homogeneous SmartDCs that self-differentiated in vitro contained approximately 1.5-3.0 copies of integrated LVs per cell. Thus, this method facilitates logistics, standardization, and high recovery for the generation of viable genetically reprogrammed DCs for clinical applications.

Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells

Development of lentiviral vectors (LVs) in the field of immunotherapy and immune regeneration will strongly rely on biosafety of the gene transfer. We demonstrated previously the feasibility of ex vivo genetic programming of mouse bone marrow precursors with LVs encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which induced autonomous differentiation of long-lived dendritic cells (DCs), referred to as self-differentiated myeloid-derived antigen-presenting-cells reactive against tumors (SMART-DCs). Here, LV biosafety was enhanced by using a DC-restricted and physiological promoter, the major histocompatibility complex (MHC) II promoter, and including co-expression of the herpes simplex virus-thymidine kinase (sr39HSV-TK) conditional suicide gene. Tricistronic vectors co-expressing sr39HSV-TK, GM-CSF and IL-4 transcriptionally regulated by the MHCII promoter or the ubiquitous cytomegalovirus (CMV) promoter were compared. Despite the different gene transfer effects, such as the kinetics, levels of transgene expression and persistency of integrated vector copies, both vectors induced highly viable SMART-DCs, which persisted for at least 70 days in vivo and could be ablated with the pro-drug Ganciclovir (GCV). SMART-DCs co-expressing the tyrosine-related protein 2 melanoma antigen administered subcutaneously generated antigen-specific, anti-melanoma protective and therapeutic responses in the mouse B16 melanoma model. GCV administration after immunotherapy did not abrogate DC vaccination efficacy. This demonstrates proof-of-principle of genetically programmed DCs that can be ablated pharmacologically.

Monocytes transduced with lentiviral vectors expressing hepatitis C virus non-structural proteins and differentiated into dendritic cells stimulate multi-antigenic CD8+ T cell responses

Halting the spread of hepatitis C virus (HCV) and also eradicating HCV in subjects with chronic infection are major goals for global health. To this end, several years of research on HCV vaccine development have led to the conclusion that multi-antigenic and multi-functional vaccine types are necessary for effectiveness against HCV infection. In this study, we evaluated lentiviral vectors (LV) expressing clusters of HCV structural (LV-HCV-S) and non-structural (LV-HCV-NS) genes for future vaccine development. Batches of high titer LV were used to transduce differentiated dendritic cells (DC) and monocytes. We report successful delivery of HCV gene clusters, particularly into monocytes, leading to >80% LV-HCV-NS and >70% LV-HCV-S and transduced cells, respectively. Intracellular expression of HCV proteins in monocyte-derived DC resulted in immunophenotypic changes, such as downregulation of CD83 and CD86. Monocytes expressing NS proteins and differentiated into DC stimulated allogeneic and autologous CD8(+) and CD4(+) T cells in vitro and resulted in antigen-specific CD8(+) T cell responses against NS3, NS4a and NS5b. Hence, lentiviral-mediated expression of the multi-antigenic HCV-NS cluster in monocytes subsequently differentiated into DC is a novel potential anti-HCV vaccine modality.

Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV

Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 (“SmyleDCpp65”). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4+ and CD8+ T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34+ cells obtained from PB and CB were transplanted into irradiated NOD.Rag1−/−.IL2γc−/− mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8+ T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4+/CD8+ T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation

BackgroundReactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCpp65”) that accelerated antigen-specific T cell development.MethodsHere we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed.ResultsUpscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated.ConclusionThese results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.ZusammenfassungHintergrundDie Reaktivierung latenter Viren wie das humane Cytomegalovirus (HCMV) führt zu einer hohen Morbidität und Mortalität nach allogener Stammzelltransplantation (allo-HSZT). Aufgrund verzögerter T-Zell-Entwicklung nach allo-HSZT ist eine wirksame Immunisierung der Patienten gegen HCMV von großer klinischer Bedeutung. Dabei spielt die Immunrekonstitution Dendritischer Zellen (DCs) eine wichtige Rolle. Frühere Verfahren zur ex vivo Generierung von DCs zur klinischen Anwendung sind komplex und wenig reproduzierbar, insbesondere im Hinblick auf die Vitalität und Potenz der Zellen nach der Kryopreservierung. In früheren Arbeiten konnten wir in humanisierten Stammzelltransplantations-Maus-Modellen eine neue Methode mittels Lentivirus-induzierten DCs (“SmyleDCpp65”) vorstellen, die zu einer beschleunigten Entwicklung antigen-spezifischer T-Zellen führt.VerfahrenIn der vorliegenden Arbeit zeigen wir die Möglichkeit, Monozyten mit einem Integrase-defekten lentiviralen Vektor (IDLV) unter guter Herstellungspraxis (GMP) zu transduzieren zur Ko-expression von GM-CSF, IFN-α und pp65 Zytomegalovirus Antigen. Nach Transduktion wurden die Zellen kryokonserviert.ErgebnisseDie standardisierte Produktion des IDLVs und die Herstellung von SmyleDCpp65 (n=3) unter GMP-konformen Bedingungen konnte demonstriert werden. Analytische Parameter zur Qualitätskontrolle der SmyleDCpp65 Identität nach dem Auftauen und Potenz nach der Kultivierung wurden definiert. Zellgewinnung, Uniformität der Zellen, Effizienz des Gentransfers, Reinheit und Vitalität waren hoch und konsistent. SmyleDCpp65 Zellen zeigten geringe IDLV Integrationen im Genom und ein polyklonales Integrationsmuster ohne Präferenz zu Protoonkogenen. Letztendlich wurde ein Verfahren zur Stimulation autologer T-Zellen durch GMP-SmyleDCpp65 validiert.FazitDie weitere Entwicklung dieser individuellen Zellvakzine für klinische Studien ist von hoher Relevanz, um die Immunrekonstitution gegen Zytomegalovirus nach allo-HSZT zu beschleunigen.

Multidimensional Analysis Integrating Human T-Cell Signatures in Lymphatic Tissues with Sex of Humanized Mice for Prediction of Responses after Dendritic Cell Immunization

Mice transplanted with human cord blood-derived hematopoietic stem cells (HSCs) became a powerful experimental tool for studying the heterogeneity of human immune reconstitution and immune responses in vivo. Yet, analyses of human T cell maturation in humanized models have been hampered by an overall low immune reactivity and lack of methods to define predictive markers of responsiveness. Long-lived human lentiviral induced dendritic cells expressing the cytomegalovirus pp65 protein (iDCpp65) promoted the development of pp65-specific human CD8+ T cell responses in NOD.Cg-Rag1tm1Mom-Il2rγtm1Wj humanized mice through the presentation of immune-dominant antigenic epitopes (signal 1), expression of co-stimulatory molecules (signal 2), and inflammatory cytokines (signal 3). We exploited this validated system to evaluate the effects of mouse sex in the dynamics of T cell homing and maturation status in thymus, blood, bone marrow, spleen, and lymph nodes. Statistical analyses of cell relative frequencies and absolute numbers demonstrated higher CD8+ memory T cell reactivity in spleen and lymph nodes of immunized female mice. In order to understand to which extent the multidimensional relation between organ-specific markers predicted the immunization status, the immunophenotypic profiles of individual mice were used to train an artificial neural network designed to discriminate immunized and non-immunized mice. The highest accuracy of immune reactivity prediction could be obtained from lymph node markers of female mice (77.3%). Principal component analyses further identified clusters of markers best suited to describe the heterogeneity of immunization responses in vivo. A correlation analysis of these markers reflected a tissue-specific impact of immunization. This allowed for an organ-resolved characterization of the immunization status of individual mice based on the identified set of markers. This new modality of multidimensional analyses can be used as a framework for defining minimal but predictive signatures of human immune responses in mice and suggests critical markers to characterize responses to immunization after HSC transplantation.

476. No GVHD, but Human Inflammatory “Cytokine Storm” and Mouse Macrophage Activation Upon Accelerated Development of Human CD4+ Effector Memory T Cells in Long-Term Humanized Mice

Non clinical assessment of immunotoxicity of human cellular products in vivo is limited by the lack of availability of pharm-tox models enabling a full range of human innate and adaptive responses. We previously demonstrated improved development of human adaptive T and B cell responses in mice receiving human hematopoietic stem cell transplantation (HSCT) in combination with prime/boost immunization with induced dendritic cells (iDCs), i.e. monocytes derived the stem cell donor reprogramed with a lentiviral vector for co-expression of GM-CSF, IFN-alpha and the CMV antigen pp65 that self differentiate into DCs in vivo (Salguero et al, 2014 J. Immunology; Daenthanasanmak et al, 2015 Mol. Ther. Meth.). For these previous studies, mice were kept for 6-10 weeks after iDC administration, and we did not observe signs of tumorigenicity or graft-versus-host disease (GVHD). After consultation with the German regulatory authorities, we were requested to perform pharm-tox analyses 26 weeks after iDC immunization. We report here a pilot-feasibility study with humanized mice showing long-term (more than 33-36 weeks after HSCT) robust immune reconstitution with human T cells (control mice, n=2 Vs. iDC-immunized mice, n=6). Longitudinal analyses of human immune reconstitution in blood samples confirmed accelerated development of CD4+ effector memory T cells 5-10 weeks after iDC immunization. No weight loss, GVHD or malignancies were observed after iDC immunization. 3/6 immunized mice developed skin erythema and inflammation around 23 weeks after iDC immunization and were sacrificed earlier for analyses. Analyses of all mice were performed at autopsy by macroscopic observations, H&E histopathology, immunohistochemistry and flow cytometry of target organs. Positivity for anti-human nuclei antibody, anti-human HLA-DR and anti-human CD3 leucocytes were detected in several tissues (spleen, bone marrow, brain, lungs, skin, eyelids, liver, and kidneys), confirming the long-term persistency of human T cells in the humanized mice. In inflamed skin, abundant human CD4+ T cells were localized in the basal layers of the epidermis and hair follicles in the dermis. Human macrophages (CD68+) could be detected in several tissues, but were rare in inflamed skin. However, in the inflamed dermis areas of ulceration, mouse macrophages (F4/80+) were observed frequently with strongly positive cytoplasm. Mice with ulcerations had significantly higher levels of several human inflammatory cytokines in plasma (IFN-gamma, IL-6, GM-CSF and IL-8) than immunized mice with no skin inflammation or control mice. In summary, a long-term pilot study exploring a hybrid human-mouse model system was feasible and iDCs accelerated mature human T cell reconstitution with no signs of tumorigenicity or GVHD. However, accumulation of human CD4+ memory T cells and activated mouse macrophages in inflamed skin was associated with elevated levels of human inflammatory cytokines resembling a “cytokine storm”.