Humanized NOG Mice Research Articles

Abstract B026: Development of tumor-targeted agonistic Immunomodulators using the DARPin® technology platform

Abstract Background: Antibodies against several agonistic immunomodulatory receptors proved to be highly efficacious in preclinical tumor models especially in combination with other immunomodulatory compounds and treatment modalities. However, their clinical application met with limited success and was associated with systemic toxicities which are believed to be driven by Fc-receptor-mediated cross-linking of antibody molecules. We developed a new class of biologics based on the DARPin® technology platform, which allow tumor-localized engagement of immunomodulatory agonists in the absence of Fc-receptors. Preclinical in vitro and in vivo results indicate that DARPin®-based compounds are at least comparable to conventional antibodies in agonistic potency. The localization of their activity to the tumor site relative to the systemic activity of agonistic MAbs should improve the safety and therefore the therapeutic index for drug candidates which exploit this mode of action. Methods: We generated multi-specific DARPin® therapeutics, which bind to human fibroblast activation protein (FAP) and either to 4-1BB, OX40 or CD40 receptors. Each of these receptors can effectively enhance immune responses through a set of distinct and non-redundant mechanisms. Functional properties of these molecules were characterized using appropriate sets of biochemical methods and cell-based assays utilizing reporter cells lines or various subsets of primary immune cells. The FAP-targeted 4-1BB agonist referred to as MP0310, which represents our most advanced drug candidate, was also characterized in vivo in terms of its pharmacokinetics and pharmacodynamics in mice as well as cynomolgus monkeys. The in vivo efficacy of MP0310 was analyzed both in monotherapy and in combination with tumor-specific T-cell engagers using a xenotransplantation human tumor model of immunodeficient mice engrafted with human PBMCs. Results and Conclusions: All analyzed FAP-targeted DARPin®-based agonistic immunomodulators efficiently induced activation of the NFkB pathway in reporter cell lines in a dose dependent manner but only upon crosslinking through binding to FAP. The same pattern of functional activity was exhibited by the molecules in assays with appropriately selected human primary immune cell subsets. We show that MP0310 promotes tumor regression in a dose dependent manner when co-administered with a tumor-specific T-cell engaging bi-specific antibody into tumor-bearing immunodeficient NGS mice engrafted with human PBMCs. The observed effect correlated well with 4-1BB receptor occupancy in blood and an increase of CD8 T lymphocytes in the tumors. Urelumab, a clinically tested 4-1BB agonistic antibody associated with liver toxicity, led to signs of severe graft-versus-host disease (GVHD) and weight loss in the humanized NOG mice. In contrast, mice treated with several doses of MP0310 showed only minor signs of GVHD and marginal weight loss. Thus, tumor targeted DARPin® agonists of co-stimulatory molecules can enhance T-cell activity in a manner independent of Fc-crosslinking and localized primarily to the tumor site, thereby avoiding the systemic toxicity seen with antibodies against the same immunologic targets. This approach with DARPin® therapeutics can be broadly applied to a range of tumor targets and immune receptors. Citation Format: Victor Levitsky, Alexand Link, Michael T. Stump, Elmar vom Baur. Development of tumor-targeted agonistic Immunomodulators using the DARPin® technology platform [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B026.

Abstract 3859: Hormone receptor and HER2/HER3 expression in preclinical breast cancer models of primary tumor and bone metastasis

Abstract Breast cancer expressing estrogen receptor (ER) and progesterone receptor (PR) is classified as hormone receptor positive, and triple-positive breast cancer expresses also human epidermal growth factor receptors (HER2). Despite the hormonal and HER2 targeted treatments, breast cancer metastasizes to bone in high frequency and may develop resistance against the used treatment. Prevention and treatment of bone metastases is challenging and moreover, hormones are strong regulators of both bone and immune system. Aim of the present study was to verify and compare ER, PR, HER2 and also HER3 status in preclinical primary and bone metastasis breast cancer models utilizing immunodeficient and human immune system engrafted mice. BT-474 human breast cancer cells were inoculated orthotopically into mammary fat pad of placebo or 17β-estradiol (E2) supplemented female immunodeficient NOG mice. In a bone tumor study, BT-474 cells were inoculated into the tibia of female NOG or humanized NOG mice (HSCFTL-NOG-F mice, Taconic Biosciences). Tumor growth was followed for 8 weeks and histopathological tumor evaluation and immunohistochemical stainings for ER, PR, HER2 and HER3 were performed. Orthotopic tumor growth of BT-474 was hormone dependent and only minor growth was observed in the absence of E2. In the presence of E2 supplement, the orthotopic tumor expressed ER, PR and HER2/HER3. However, in the absence of E2 supplement there was reduced PR expression but no major changes in the ER and HER2/HER3 expression. In contrast, when breast cancer cells were inoculated into the tibia, tumor growth was observed also without E2 supplement. In this case, tumor in the bone was positive for ER and HER2/HER3 but negative for PR. No significant changes were observed between immunodeficient and humanized mice regarding intratibial tumor growth or ER, PR and HER2/HER3 expression. As a summary, estrogen supplementation is needed to support breast cancer BT-474 tumor growth when cancer cells are inoculated orthotopically into mammary fat pad. In contrast, BT-474 tumor growth was observed in bone also in the absence of supplied E2 in immunodeficient and humanized mice. ER and HER2/HER3 expression was observed in primary and bone tumors, but PR expression was significantly reduced if no estrogen supplement was used. Taking together, when developing new therapies against breast cancer, treatment targets in preclinical models should be carefully verified. Focus should be addressed not only on primary tumor but also on bone metastasis where cancer cells are under influence of different tumor microenvironment and may express differently hormone receptors and HER2/HER3. While hormones influence breast cancer progression, they also regulate bone turnover and immune system, and therefore humanized mouse models provide an essential platform for novel therapy development. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni H. Mäki-Jouppila, Azusa Tanaka, Michael Seiler, Teppo Haapaniemi, Jenni Bernoulli. Hormone receptor and HER2/HER3 expression in preclinical breast cancer models of primary tumor and bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3859.

Abstract LB-278: In vivo evaluation model of human T cell activation by immune checkpoint inhibitors using human PBMC-transferred NOG mouse

Abstract Within the past decade, immune checkpoint inhibitors, such as anti-programmed death (PD)-1 antibodies (e.g. pembrolizumab; Keytruda and Nivolumab: OPDIVO), treatment has become one of the most advancing therapeutic options in oncology. The PD-1/PD-L1 pathway plays a crucial role in mediating self tolerance and controlling self damage to suppress effector/self-reactive T cells and protects against autoimmune reactions. Therapeutic treatment of anti-PD-1 antibodies clearly suppresses tumor growth and induces T cell activation in cancer patients by blockade of the PD-1/PD-L1 pathway, but over activation of T cells exhibits immune-related adverse effects with exacerbation of autoimmune reactions. A human peripheral blood mononuclear cells (hPBMC) transferred humanized NOG mouse has been used to evaluate the efficacy and safety of therapeutic antibodies due to the efficient engraftment of human T cells in the mice. The human T cells were spontaneously activated in the mice because of xenogenic-reaction and the mice frequently died of graft-versus-host disease (GVHD) (Ito R et al., Transplantation, 2009). In the present study, we evaluate the potential adverse effects of 2 therapeutic antibodies, Keytruda and OPDIVO by using conventional NOG mice transferred with hPBMC. These mice showed a weight loss, wasting, rough fur, and skin thickening suggesting of GVHD symptoms. These changes exacerbated in either antibody treated hPBMC-NOG mice with increase of activation marker for T cells, and they died earlier than saline-treated group. By immune-cytoloical analysis, human T cells was significantly decreased in number in hPBMC-NOG mice even though the antibodies were treated. These results demonstrated the hPBMC-NOG mice might become powerful preclinical tools to analyze human T cells activated by therapeutic immune checkpoint inhibitors. Citation Format: Chiyoko Nishime, Ikumi Katano, Eiko Nishinaka, Kenji Kawai, Ryoji Ito, Jun-ichi Hata, Taichi Yamamoto. In vivo evaluation model of human T cell activation by immune checkpoint inhibitors using human PBMC-transferred NOG mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-278.

Abstract B099: Importance of hormone receptor and HER2/HER3 status verification in preclinical breast cancer models using immunodeficient and humanized mice

Abstract Introduction: Breast cancer expressing estrogen receptor (ER) and progesterone receptor (PR) is classified as hormone receptor positive, and triple-positive breast cancer expresses also human epidermal growth factor receptor (HER2). Despite the hormonal and HER2 targeted treatments, breast cancer metastasizes to bone in high frequency and may develop resistance against the used treatment. Prevention and treatment of bone metastases is challenging; moreover, hormones are strong regulators of both bone and immune system. The aim of the present study was to verify ER, PR, HER2, and also HER3 status in preclinical primary and bone metastasis breast cancer models utilizing immunodeficient and human immune system engrafted mice in order to verify predictive models for drug development. Materials and Methods: BT-474 human breast cancer cells were inoculated orthotopically into mammary fat pad of placebo or 17β-estradiol (E2) supplemented female immunodeficient NOG mice. In a bone tumor study, BT-474 cells were inoculated into the tibia of female NOG or humanized NOG mice (HSCFTL-NOG-F mice, Taconic Biosciences). Tumor growth was followed for 8 weeks, and histopathologic tumor evaluation and immunohistochemical stainings for ER, PR, HER2, and HER3 were performed. Results: Orthotopic tumor growth of BT-474 was hormone dependent and only minor growth was observed in the absence of estrogen. In the presence of E2 supplement, the orthotopic tumor expressed ER, PR, and HER2/HER3. However, in the absence of E2 supplement there was reduced PR expression but no major changes in the ER and HER2/HER3 expression. In contrast, when breast cancer cells were inoculated into the tibia, tumor growth was observed also without E2 supplement. In this case, tumor in the bone was positive for ER and HER2/HER3 but negative for PR. No significant changes were observed between immunodeficient and humanized mice regarding intratibial tumor growth and ER, PR, and HER2/HER3 expression. Conclusions: Estrogen supplementation is needed to support breast cancer BT-474 tumor growth when cancer cells are inoculated orthotopically into mammary fat pad. In contrast, BT-474 tumor growth was observed in bone even in the absence of supplied estrogen. ER and HER2/HER3 expression was observed in primary and bone tumors, but PR expression was significantly reduced if no estrogen supplement was used. Differences in tumor growth depending on the site and estrogen level highlight the importance of tumor microenvironment in breast cancer, and also refer why tumor may shift resistant to used hormonal or HER2 targeted therapy. Taking together, when developing new therapies against breast cancer, treatment targets in preclinical models should be carefully verified. Focus should be placed not only on primary tumor but also on bone metastasis where cancer cells are under influence of different tumor microenvironment and may express differently hormone receptors and HER2/HER3. While hormones influence breast cancer progression, they also regulate bone turnover and immune system, and therefore humanized mouse models provide an essential platform for novel therapy development. Citation Format: Tiina Kähkönen, Mari I. Suominen, Jenni Mäki-Jouppila, Jussi M. Halleen, Azusa Tanaka, Michael Seiler, Teppo Haapaniemi, Jenni Bernoulli. Importance of hormone receptor and HER2/HER3 status verification in preclinical breast cancer models using immunodeficient and humanized mice [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B099.

Abstract A016: NOG-EXL mice have superior immune cell engraftment and lineage development in the human immune system (HIS) model, the ImmunoGraft

Abstract Background: The clinical success of immune checkpoint modulators and development of next-generation immune-oncology (IO) agents underscores the need for robust patient-derived models to validate novel IO therapeutics and better predict clinical outcomes and biomarkers of response. The Champions ImmunoGraft® model utilizing humanized NOG mice (PrkdcscidIl2rgtm1Sug) is an innovative preclinical model for assessing the efficacy of IO agents against solid tumors and has been described previously. Improved immunodeficient mouse strains, such as triple transgenic NOG-EXL mice (Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3,CSF2) expressing huGM-CSF and huIL-3, allow for superior huCD34+ engraftment and lineage development. In this study we compared human immune lineage development and solid tumor engraftment in NOG and NOG-EXL mice and tumor response to checkpoint inhibitors in a humanized mouse platform. Materials and Methods: NOG and NOG-EXL were humanized by intravenous transplantation of 12x104 CD34+ hematopoietic stem cells (HSC). Establishment of human immune components in peripheral blood was evaluated by flow cytometry. At 16 weeks post-transplantation, patient-derived xenograft (PDX) tissue from a NSCLC patient with known response to nivolumab was implanted in humanized NOG and NOG-EXL mice. Treatment with the checkpoint inhibitors nivolumab (α-PD-1; 10mg/kg) and ipilimumab (α-CTLA4; 5mg/kg) and a combination of both agents is planned once tumor implants reach approximately 150-200mm3. Immune cell populations in spleen, blood and the implanted tumor (T cell, macrophages and myeloid and dendritic cells) will be evaluated at the mid- and end-point of the study. Tumor responses will be assessed by changes in tumor volume and with immune-related RECIST criteria applied. Human immune cell proliferation and activation following treatment with the checkpoint inhibitors will be determined by flow cytometry and immunohistochemistry of peripheral blood, spleen and PDX tissue. Results: NOG-EXL mice engrafted human immune components more readily than NOG (take rate of 80% versus 100%, with proportions of huCD45+ cells greater than 25% consistently found in the peripheral blood of NOG-EXL mice. Donor-to-donor variability was observed in both mouse strains, consistent with literature reports. T cell lineage development was equivalent in NOG and NOG-EXL mice 12 weeks post-HSC transplantation. Improved myeloid lineage development was identified in NOG-EXL animals with higher circulating huCD33+ levels observed in comparison to NOG mice. Comparable growth rates have been observed for NSCLC tumor implants in both animal strains. Conclusions: While the ImmunoGraft platform is reflective of the human tumor microenvironment (both immune and tumor cell-based), mouse strains with improved human immune system development promise to further enhance its value for screening IO therapies. We demonstrated that NOG-EXL mice allow better engraftment of human immune components compared to NOG. Importantly, the presence of additional immune lineages in NOG-EXL mice did not adversely affect growth of NSCLC PDX models. A study to evaluate the efficacy of checkpoint inhibitors in NSCLC PDX models in this enhanced ImmunoGraft is ongoing. The NOG-EXL ImmunoGraft has the potential to advance translational IO drug discovery and development. Citation Format: Bhavna Verma, Maria Mancini, Angela Davies, David Sidransky, Neal Goodwin. NOG-EXL mice have superior immune cell engraftment and lineage development in the human immune system (HIS) model, the ImmunoGraft [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A016.

Abstract 1594: Enhancement of the anti-tumor activity of CEA TCB via combination with checkpoint blockade by PD-L1 and interleukin-2 variant immunocytokine

Abstract Cancer immunotherapy represents a promising therapeutic approach to extend the overall survival of cancer patients. However, several mechanisms within the tumor microenvironment orchestrate the suppression of host immune response requiring combination strategies to prolong the durability of effect. Among suppressive pathways, up-regulation of PD-1 ligand (PD-L1) and its interaction with PD-1 receptor plays a key role in suppression of T-cell activity, a mechanism also called adaptive immune resistance. Therefore, approaches able to inhibit the tumor immune suppressive mechanisms along with those expanding the frequency of intra-tumor T-cells and enhancing/prolonging their functionality are required. CEA TCB (RG7802, RO6958688) is a novel T cell bispecific antibody targeting carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells, currently being investigated as single agent and in combination with atezolizumab in Phase 1/1b studies in patients with advanced and/or metastatic CEA-expressing tumors (NCT02324257; NCT02650713). CEA TCB treatment leads to increased intra-tumoral T cell infiltration and T-cell activation along with up-regulation of PD-1/PD-L1 suppressive pathway. Here we show that combination of CEA TCB with PD-L1 blocking antibody in vitro enhances T cell activation as detected by increased CD3 signaling and secretion of pro-inflammatory cytokines. Combination in vivo performed in both stem cell humanized NOG mice (HSC mice engrafted with MKN45) and fully immunocompetent human CEA transgenic C56BL/6 mice (hCEA Tg mice engrafted with MC38-hCEA) demonstrated significantly improved anti-tumor activity of combination as compared to activity of single agents, yielding to increased number of tumor-free animals. Randomization of animals that progressed to CEA TCB monotherapy revealed that combination of CEA TCB with PD-L1 blocking antibody is required to control tumor outgrowth, as tumors treated with corresponding monotherapy arms progressed to treatment. Efficacy of CEA TCB was also potentiated when administered in combination with a half-life-extended IL-2 variant (untargeted (IgG-IL2v) or fibroblast-activating protein-targeted IL-2 variant (FAP-IL2v)), resulting in stronger tumor growth inhibition in MKN45-bearing HSC mice or prolonged survival in PanCO2-hCEA-bearing hCEA Tg C56BL/6 mice. Synergy likely reflects ability of IL2v to enhance anti-tumor efficacy by increasing number of effector T cells in tumors. In conclusion, CEA TCB treatment leads to intra-tumoral T cell infiltration and T-cell activation. This is accompanied by up-regulation of PD-1/PD-L1 suppressive pathway, which can be overcome by combination therapy with a PD-L1 inhibitor. In vivo efficacy of CEA TCB is further potentiated when administered in combination with immunotherapies that increase the pool of available tumor-infiltrating effector cells. Citation Format: Marina Bacac, Sara Colombetti, Linda Fahrni, Tanja Fauti, Valeria Nicolini, Johannes Sam, Petros Papastogiannidis, Marine Le Clech, Xavier Miot, Inja Waldhauer, Karolin Rommel, Christian Gerdes, Christian Klein, Pablo Umaña. Enhancement of the anti-tumor activity of CEA TCB via combination with checkpoint blockade by PD-L1 and interleukin-2 variant immunocytokine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1594. doi:10.1158/1538-7445.AM2017-1594

Abstract IA03: Patient-derived xenografts as translational tools to predict patient responses to oncology therapy

Abstract Successful drug development in oncology is grossly suboptimal, manifested by the very low percentage of new agents being developed that ultimately succeed in clinical approval. This poor success is in part due to the inability of standard cell-line xenograft models to accurately predict clinical success and to tailor chemotherapy specifically to a group of patients more likely to benefit from the therapy. Patient-derived xenografts (PDXs) maintain the histopathological architecture and molecular features of human tumors, and offer potential solution to maximize drug development success and ultimately generate better outcomes for patients. Over the past decade PDXs have been successfully used for pre-clinical evaluation of cancer therapeutics and to derive predictive biomarkers in various solid tumors. To assess the utility of tumor grafts in guiding therapy for individual patients, we analyzed a large set of correlations between PDX model drug responses and patient clinical outcomes. We obtained 113 clinical correlations across 78 patients with various solid tumors for whom a PDX model was successfully developed and drug testing completed. Some patients were included in more than one response group, having undergone multiple treatments as cycles of disease progression and therapy occurred. Treatment outcomes and PDX responses were evaluated using Response Evaluation Criteria In Solid Tumors (RECIST) criteria and diagnostic parameters calculated. A significant association was observed between drug responses in the patient and the corresponding PDX model in 89% (101/113) of the therapeutic analyses. We further stratified our population by whether or not the patient's treatment was the first or further line received after resection of the tumor to develop the PDX model. We found positive responses in the tumorgrafts reflected positive outcomes in the patient in virtually every instance, suggesting that PDX model drug responses correlate with patient clinical outcomes, even as patients undergo numerous treatments overtime. Although our data suggest that PDX models are a predictable platform for preclinical evaluation of a variety of treatments, the immunocompromised mice hosts reduce the similarity of PDX models to the original tumor environment and preclude the evaluation of cancer immunotherapeutic approaches. Hence, development of robust preclinical tools to test such drugs against human tumors in the context of an allogeneic immune system remains a challenge. Since it was demonstrated that transplantation of human hematopoetic stem cells (HSCs) into NOG or NSG mice result in reconstitution of the hematopoesis and functional human immunity, we have combined the PDX platform with the humanized NOG mice to create immunografts—tumorgrafts implanted into immunodeficient mice reconstituted with a homologous human immune system. Immunografts were treated with immune checkpoint inhibitors targeting CTLA4 and PD1 and human immune activation as well as tumor growth inhibition was evaluated. Our results indicate that immunografts produce a human tumor immune cell interaction and exhibit significant tumor growth inhibition in response to immune-oncology agents in several types of patient-derived tumors. Although this approach is technically challenging and may not be suitable for patients with aggressive, rapidly progressing disease, further development and optimization of this system may contribute to better selection of immune oncology drugs alone and in combination for pharmaceutical development. Taken together, our studies demonstrate that application of PDX models to guide treatment decisions in oncology may improve the identification of drugs most likely to benefit an individual patient and lead to better patient tumor responses. Moreover, given the clinical relevance of these models, they could also be deployed as real-time patient surrogates in the drug development process. Citation Format: Evgeny Izumchenko, David Sidransky. Patient-derived xenografts as translational tools to predict patient responses to oncology therapy. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA03.

Abstract 1494: Combination of CEA TCB, a novel T-cell bispecific antibody for the treatment of solid tumors, with PD-L1 checkpoint blockade

Abstract Recent results from clinical trials have shown that immune therapies, particularly immune checkpoint inhibitors, can extend the overall survival of cancer patients and lead to durable responses. Despite these promising results, current immune-based therapies are only effective in a proportion of patients and combination strategies are needed to improve therapeutic benefit. Programmed death-ligand 1(PD-L1) is found on the surface of immune and tumor cells and its expression is induced by interferon gamma (IFNg). It prevents the immune system from destroying cancer cells by interacting with the inhibitory programmed death-1 (PD-1) and B7.1 receptors on activated T cells, which results in a T-cell inhibitory signal. CEA TCB (RG7802, RO6958688) is a novel T cell bispecific antibody targeting the carcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells, currently being investigated as single agent in a Phase I study in patients with advanced and/or metastatic CEA-expressing tumors. CEA TCB-mediated killing of tumor cells led to T cell activation, IFNg secretion and subsequent up-regulation of the PD-1/PD-L1 immune suppressive pathway in vitro and in vivo, similarly to what happens during tumor adaptive immune resistance mechanisms where upon recognition of tumor antigens, TILs produce IFNg, which drives PD-L1 expression in the tumor microenvironment and delivers a suppressive signal to T cells upon binding to PD-1. Incubation of the high-CEA expressing gastric carcinoma cell line (MKN45) with human PBMCs (E:T 10:1) and increasing concentrations of CEA TCB, led to a dose-dependent up-regulation of PD-1 receptor on both CD4+ or CD8+ T cell subsets as well as of PD-L1 on surviving tumor cells as early as 24 h following treatment. PD-1 expression was specific as it did not occur in the absence of CEA-expressing tumor cells or upon treatment with un-targeted control TCB. In vivo studies performed using MKN45 tumor xenografts (MKN45) in fully humanized NOG mice demonstrated that CEA TCB treatment led to increased frequency of intra-tumoral T cells expressing PD-1 and to a strong induction of PD-L1 expression in tumors. Combination of CEA TCB with a PD-L1 blocking antibody showed significant increase of anti-tumor activity as compared to the respective single agents. These preclinical data indicate that CEA TCB treatment leads to up-regulation of the PD-1/PD-L1 immune suppressive pathway and that the combination of CEA TCB with PD-L1 blocking agents results in enhanced anti-tumor activity. Phase Ib clinical trials investigating the combination of CEA TCB and atezolizumab are currently ongoing. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Linda Fahrni, Valeria Nicolini, Christian Gerdes, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. Combination of CEA TCB, a novel T-cell bispecific antibody for the treatment of solid tumors, with PD-L1 checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1494.

Antibody-secreting plasma cells with unique CD5+IgG+CD21lo phenotype developed in humanized NOG mice

Antibody-secreting plasma cells with unique CD5+IgG+CD21lo phenotype developed in humanized NOG mice

Induction of APOBEC3B cytidine deaminase gene in HTLV-1 infected T-cells of ATL model mouse

A variety of mutations are accumulated in the genome of HTLV-1 infected T-cells during ATL development. To elucidate the mechanism of ATL development a mouse model of ATL was established by infecting HTLV-1 to humanized NOG mice and the infected mice recapitulate the ATL-like symptoms and die of leukemia within several months of infection. Analysis of gene expressions in the humanized mouse model of ATL demonstrated the induction of APOBEC3B (A3B) gene in the HTLV-1 infected human T-cells. A3B is a member of the APOBEC family of cellular cytidine deaminase and was recently identified as the mutational source in multiple human cancers. We have previously shown that HTLV-1 infected CD25 (-) CD4 T-cells but not CD25 (+) CD4 T-cells in ATL model mouse express a small amount of Tax mRNA even though both cell populations consist of identical infected-cell clones. As the A3B expression in HTLV-1 infected CD25 (+) T-cells was similar to, or rather higher than that in CD25 (-)T-cells, Tax appears not to be involved in the induction A3B. In contrast, ex vivo culture of infected T-cells, in which Tax expression was highly enhanced in both CD25 (+) and CD25 (-)T-cells, resulted in the suppression of A3B expression. However, ectopic expression of HTLV-1 in Jurkat cell by the transfection of HTLV-1 molecular clone plasmid enhanced the expression of A3B gene. It is thus suggested that HTLV-1 infection but not Tax induces the A3B expression. Interplay between HTLV-1 infection and A3B and the possible involvement of A3B in the mutagenesis of host cell genome are currently investigated.

Abstract 2481: CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors

Abstract T cell bispecific antibodies (TCBs) are potent molecules that upon simultaneous binding to tumor cells and T cells trigger strong T cell activation resulting in the killing of tumor cells. CEA TCB (RG7813) is a novel bispecific antibody targeting carcinoembryonic antigen (CEA), often overexpressed on solid tumors (e.g. colorectal, gastric, pancreatic, lung carcinoma etc.), and the CD3 epsilon chain present on T cells. CEA TCB bears several innovative technological features that distinguish it from other bispecific antibodies currently in (pre-)clinical development: (a) bivalency for tumor antigen translating into higher avidity, superior potency and better differentiation between high and low antigen-expressing cells; (b) head-to-tail fusion geometry for anti-tumor and CD3-binding domains, resulting in higher potency compared to conventional IgG-based TCBs; (c) extended half-life compared to non-Fc-based TCBs; (d) fully silent Fc ensuring lower risk of FcgR-mediated infusion reactions; and (e) robust production using standard manufacturing processes (enabled by “CrossMAb” and knob-into-hole bispecific antibody technologies). In vitro, CEA TCB mediates potent target-dependent T cell cytotoxicity, T cell activation, proliferation, and cytokine release in killing assays, exclusively in the presence of CEA-expressing target-cells. CEA TCB activity correlates with CEA expression level, showing higher potency against tumor cells with high expression of CEA. In vivo, CEA TCB induces dose- and time-dependent regression of CEA-expressing tumors with variable amounts of immune cell infiltrate. In fully humanized NOG mice, CEA TCB is efficacious in poorly-infiltrated tumors and converts non-inflamed into highly-inflamed tumors. Histological and FACS analyses revealed that CEA TCB recruits new T cells into tumors and/or expands pre-existing ones and is able to induce T cell re-localization from the tumor periphery into the tumor bed. Surprisingly, CEA TCB treatment also qualitatively alters the composition of intratumoral T cells resulting in an increased frequency of activated (CD69, CD25), proliferating (Ki67) and differentiated T cells (having effector memory phenotype) that are ready to kill (express high levels of Granzyme B). Taken together, these preclinical data show that CEA TCB is a novel tumor-targeted T cell bispecific antibody with promising anti-tumor activity and the novel ability to modify the tumor microenvironment. Phase 1 clinical trials with CEA TCB are currently ongoing. Future studies will focus on identification of combination partners that inhibit T cell suppression and unleash the full potential of T cell activity. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Johannes Sam, Valeria Nicolini, Nathalie Steinhoff, Oliver Ast, Peter Bruenker, Ralf Hosse, Thomas Hofer, Ekkehard Moessner, Christiane Jaeger, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2481. doi:10.1158/1538-7445.AM2015-2481

49. In Vivo Gene Therapy of Mobilized Hematopoietic Stem Cells With AD5/35 Vectors Encoding a Sleeping Beauty Transposase Integration Machinery

Current protocols for hematopoietic stem cell (HSC) gene therapy involve the collection of HSCs from donors/patients, in vitro culture, transduction with retrovirus vectors, and retransplantation into myelo-conditioned patients. Besides its technical complexity, disadvantages of this approach include the necessity for culture in the presence of multiple cytokines which can affect the pluripotency of HSCs and their engraftment potential. Furthermore, the requirement for myeloablative regimens in patients with non-malignant disorders creates additional risks. We therefore explored the potential for in vivo transduction and genome editing of HSCs. Previously, we and others reported that human HSCs express high levels of CD46, a complement regulatory protein, and can therefore be efficiently transduced by CD46-targeting adenovirus vectors such as Ad5/35 vectors.In a CD46-transgenic mouse model we showed that HSCs could be flushed from the bone marrow using a mobilizing regimen of GCSF and the CXCR4 antagonist AMD3100. These mobilized HSCs could then be transduced with intravenously injected, GFP-encoding Ad5/35 vectors. Upon transduction, mobilized HSCs relocalized back to the bone marrow, expressed the transgene and were clonogenically active in colony forming unit assays. When we followed transgene expressing HSCs in vivo over a course of 2 weeks we saw a loss of transgene expression over time. This is most likely due to leaky viral gene expression of the first generation Ad vector used in these studies. To avoid these cytotoxic effects, we employed a Helper-dependent Ad (HDAd) vector that is devoid of all viral genes. We compared the transduction performance of the HDAd to the first generation vector both in the CD46-transgenic mouse model as well as in a humanized NOG mouse model.Adenoviral vectors do not integrate into the genome of the host cell and we have therefore only been able to follow transduced cells for limited periods of time. In order to be able to track transduced HSCs and their progeny for extended periods of time and to assess their long-term repopulating potential we are developing a set of HDAd vectors that allow for genomic integration of the transgene using a sleeping beauty transposon approach. In in vitro transduction studies we were able to show that the HDAd-encoded integration machinery can be used to insert a GFP cassette into the genome of HSCs, that the transduced HSCs remain clonogenic and that the transgene is passed on to progeny cells. Preliminary in vivo transduction studies in mobilized CD46-tg mice show GFP expression in up to 2.5% of bone marrow-derived lineage-depleted cells at 4 weeks after vector injection, indicating stable in vivo transduction of HSCs.

The Critical Interaction Between Epstein-Barr Virus (EBV) Positive B-Cells and Tumor Associated Macrophages (TAMs)

Introduction: EBV positive diffuse large B-cell lymphoma of the elderly (EBV positive DLBCL, elderly) has been newly categorized in 2008 WHO classification. The incidence is higher in Asian countries than Western countries. The prognosis of DLBCL has been improved by the introduction of rituximab, while EBV positive DLBCL remains unknown. Recently we reported that EBV positive DLBCL of the elderly did have quit inferior prognosis than EBV negative DLBCL. The mechanism lying under the inferiority has yet been elucidated. We hypothesize that tumor microenvironment plays a role in the mechanism, as EBV related lymphoma are usually accompanied with massive infiltration of non-tumor cells. We have previously found that tumor secreted small RNAs were selectively taken by macrophages and dramatically change the character into tumor supporting phenotype in the EBV positive lymphoma. Accordingly, we focus the interaction between tumor cells and macrophages in EBV positive lymphoma microenvironment.Methods: We investigated the number of CD163-positive macrophage (CD163+M2) in the DLBCL specimen with and without EBV positivity. The effect of EBV positivity in the tumor cells on the macrophages infiltrated in the tumor were studied by use of the coculture system using human monocytic cell line (THP-1) and human Burkitt lymphoma cells lines (Akata) which has two subclones such as EBV positive and negative. PMA-induced macrophages from THP1 cells were cocultured with EBV positive or negative Akata, then the expression of the several cytokines such as TNF-a, IL-10, CXCL10, and VEGF were measured by real-time PCR. Finally, we tried to clarify the impact of the macrophages on tumor formation in vivo by using xeno-transplantation model. Hematopoietic humanized NOG mice were infected with EBV to induce EBV related lymphoproliferative disease (LPD). After the appearance of symptoms of the LPD such as body weight loss, mice were treated with Clodronate to deplete macrophages.Result: The multivariate analysis for DLBCL patients demonstrated the statistically significant association between both high scores of CD163+M2 macrophages (CD163-positive cell > 20%) and EBV positivity, and poor prognosis (p = 0.0084, 0.0020, respectively.), which implies that EBV affected the quantity of CD163+M2 macrophages in the tumor microenvironment. Co-culture of THP1 with EBV positive lymphoma cells significantly upregulated CXCL10 and VEGF, when compared with EBV negative cells. (p = 0.0073, 0.0161, respectively.). (Figure 1) Most surprisingly, EBER positive B cells almost completely disappeared by macrophage depletion by Clodronate treatment (Figure 2). These results suggest that the macrophages in the EBV positive tumor microenvironment are crucial for survival of EBV+ tumor cells.Conclusion: EBV status of the lymphoma cells affected on TAMs in the way such as up-regulation of CXCR10 and VGEF, angiogenetic factors which are presumed to support tumor survival. The in vivo depletion of macrophages by Clodronate also demonstrated that they were indispensable for EBV positive tumor cell survival.Taken together, the interaction of EBV positive tumor cells and tumor associated macrophages is of crucial importance in the biology and formation of EBV positive lymphoma. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 1207: Pharmacodynamic evaluation of ADCC mediated effects in humanized IL2 or IL15 NOG mice models

Abstract Humanized mice (i.e. immunodeficient NOG mice engrafted with human hematopoietic stem cells (HSC)) have demonstrated their usefulness for studying the human immune system. These models should help recapitulating some pathology such as viral infection and then are suitable system to evaluate new human specific therapies. Nevertheless, one issue of these models is the incomplete maturation of natural killer (NK) cell subpopulation. This lake of NK cell functionality was solved by the Central Institute for Experimental Animals (CIEA) with the second generation of transgenic NOG mice expressing human IL2 or IL15 (NK-NOG). The serum levels of hIL-2 and hIL-15 in mice were 1-2 and 0.1-0.2 ng/mL, respectively. In these mice, human NK cells differentiated from HSC are well distributed in the mouse body (peripheral blood, bone marrow, spleen…). They expressed various NK receptors such as NKp30, NKp44, NKp46, etc., granzyme A and perforin, indicating their maturity in the mice. They are also fully functional as evidenced using an ex vivo 51Cr release assay demonstrating lysis properties of human NK cells from collected spleen of HSC-humanized mice.In vivo proof of concept studies were successfully performed with humanized NK-NOG mice xenografted with either human Burkitt's disseminated lymphoma cells or with human solid tumors treated with humanized therapeutic antibodies. Prediction of ADCC-mediated antitumor activity of humanized antibodies is now routinely feasible for preclinical evaluation using these new in vivo NK-NOG models. Citation Format: Caroline Mignard, Jean-Francois Mirjolet, Olivier Duchamp, Yasuyuki Ohnishi, Ikumi Katano, Mamoru Ito. Pharmacodynamic evaluation of ADCC mediated effects in humanized IL2 or IL15 NOG mice models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2014-1207

Carrier model of HTLV-1 infection in humanized NOG mice

HTLV-1 infection leads to adult T-cell leukemia (ATL) after long latent infection period. We have established an HTLV-1-infected humanized NOG mouse model to analyze the mechanism of ATL development. Humanized mice were inoculated with Jurkat-based HTLV-1 producing cells, JEX, into peritoneal cavity to infect human T-cells with HTLV-1. Infected T-cells proliferated vigorously in infected mice and the mice died of ALT-like lymphoproliferative disorder with attenuated HTLV-1 specific immunoresponses within two to three months of infection. In contrast, when HTLV-1 producing cells were inoculated orally, infection of human T-cells was established but the proviral loads (PVL) was kept constant in small amount (0.2-9.5 copies / 100 PBMCs) without any pathological feature, even after 18 weeks post infection. The amount of antibody against HTLV-1 in the plasma of orally infected mice was equivalent to that of intraperitoneally infected mice at 2-6 weeks post infection. The titer of antibody was maintained throughout lifetime in orally infected mice but not in intraperitoneally infected mice. Furthermore, depletion of CD8 T-cells from orally infected mice by injecting anti-CD8 antibody resulted in transient increase of PVL in PBMCs, indicating the involvement of cytotoxic T-cells in the control of PVL in these mice. Thus, orally infected humanized NOG mice should provide a model system of HTLV-1 carrier to examine factors involved in the development of ATL from carrier state and play a significant role in evaluating preventive vaccine candidates and antiretroviral drugs for HTLV-1 infection.

Inhibition of ATL development in humanized mouse model by AZT/INF treatment

HTLV-1 infection of humanized NOG mice has been demonstrated to recapitulate the development of ATL-like symptoms within several months of infection. Infected human T-cells in these mice start to proliferate vigorously in a couple weeks after infection and the mice die of ALT-like lymphoproliferative disorder. Thus, this mouse model should provide a potent tool to analyze the in vivo effect of various candidates for ATL treatment. Treatment of ATL with the combination of anti-viral agents, zidovudine (AZT) and interferon-alpha (IFN), has been reported to be highly effective, especially to indolent type, but the mechanism of action is totally unknown. We, therefore, examined the efficacy and the in vivo mechanism of AZT/IFN treatment in the humanized mouse system. HTLV-1 infected humanized mice were inoculated daily with AZT and IFN from two to four weeks post infection and the number of infected cells and proviral loads (PVL) were analyzed. Treatment with either AZT or IFN alone attenuated the onset of lymphoproliferative disorder, whereas the combined treatment suppressed the growth of infected T-cells in PBL almost completely and the PVL remained low throughout lifetime. The suppressive effect is infected-cell specific because the number of uninfected human lymphocytes in PBL stayed constant on the administration of drugs. It is suggested that infected cells expressing higher level of viral gene, most provably Tax, should have been selectively eliminated, since a similar suppressive effect has been obtained in HTLV-1 infected humanized mice treated with an Hsp90 inhibitor, 17-DMAG, which enhances the degradation of Tax.

Novel Therapeutic Approach To Graft-Versus-Host Disease With Allele-Specific Anti-HLA Monoclonal Antibody

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) using cord blood or haploidentical donor is a promising alternative option for patients who can not find an HLA-matched donor. However, HLA-mismatch allo-HSCT may be complicated by graft-versus-host disease (GVHD), a major cause of non-relapse mortality mediated by alloreactive T cells. Infusion of anti-thymocyte globulin (ATG) is used both as a treatment for and as a prophylactic against GVHD but ATG, like other immunosuppressive regimens, reacts against cells without distinguishing between donor and recipient cells. ATG is polyclonal and causes many side effects such as allergic reaction and increasing susceptibility to infection due to repression of T cells. Our ultimate goal is to develop a novel therapeutic approach to GVHD with anti-HLA monoclonal antibody specifically targeting the mismatched HLA molecule of donor origin to transiently ablate alloreactive T cells. To generate allele-specific anti-HLA antibody (ASHmAb), we established a rapid and efficient strategy using two different HLA-transgenic mouse strains, HLA-A24 and HLA-A2 and immunized them with HLA-A* 02:01 and HLA-A* 24:02 tetramers loaded with cytomegalovirus PP65 peptide, respectively. Antibodies were screened for HLA allele-specific complement-dependent cytotoxicity in vitro. We successfully established three killing ASHmAbs (kASHmAbs) against HLA-A* 02:01, -A* 02:01/–A* 03:01, and -A* 23:01/-A* 24:02. In vivo cytotoxicity and specificity of a kASHmAb against HLA-A* 02:01 (A* 02:01-kASHmAb) was examined in detail using a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγnull (NOG) mice were injected with either HLA-A2 or HLA-A26 positive human peripheral blood mononuclear cells (PBMCs) from healthy donors (i.e. HLA-A2 mice or HLA-A26 mice, respectively). Administration of A* 02:01-kASHmAb promoted the survival of HLA-A2 mice to 100%, with a mean survival of more than 6 months. In contrast, administration of A* 02:01-kASHmAb to nonreactive HLA was not effective; all HLA-A26 mice died within 2 months (p<0.0001), demonstrating kASHmAb can successfully treat a GVHD mouse model. Next, we examined the cytotoxicity of the kASHmAb on the human graft in bone marrow using humanized NOG mice. Sublethally irradiated NOG mice were reconstituted with CD34 positive hematopoietic stem/progenitor cells from HLA-A2 positive cord blood. These humanized mice showed about 70-90% human T, B and myeloid cells in the peripheral blood. When these mice were administrated with A* 02:01-kASHmAb for five consecutive days (60 mg/day), mature human blood cells disappeared immediately from the mouse peripheral blood. Interestingly, human PBMCs were detectable again in the mouse blood in 2-4 weeks after the last kASHmAb administration, suggesting kASHmAb may have preferential cytotoxic effect on mature PBMCs sparing hematopoietic stem progenitor cells. The mechanism of this preferential killing is not clear, but an optimal dose of kASHmAb may be safely administered to GVHD patients without permanently ablating the graft and necessitating repeated transplantation. This study is the first reported instance of using the cytotoxic effect of HLA antibodies against GVHD. Unlike ATG, kASHmAb specifically killed donor cells and contributed to the survival of the recipients. While this novel therapeutic approach requires a panel of kASHmAbs to different HLA alleles, this may open a new door for treatment of severe GVHD. Furthermore, optimization of the kASHmAb administration protocol may contribute to tolerance induction. Disclosures: No relevant conflicts of interest to declare.

EBV-Encoded Mirnas Facilitate Inflammation and Tumor Formation Through Both Intra- and Inter-Cellular Regulation In Vivo Mouse Model

IntroductionEB virus (EBV) is associated with heterogeneous lymphomas. Hodgkin's lymphoma (HL) cells are embedded in non-neoplastic bystanders: B, T cells, and macrophages. Without these bystander cells, the lymphoma cells are incapable of being engrafted in immunodeficient mice. In this context, the bystanders are tumor-supportive “inflammatory niche”. Recently, EBV-infected cells produce exosomes that contain EBV specifically encoded miRNAs (EBV-miRNAs). The miRNAs are transferred to cells, and involved in tumor metastasis. However, the detailed mechanism is unknown. Accordingly, we hypothesized that exosomal EBV-miRNAs might redirect tumor surrounding immune cells from tumor reactive into tumor-supportive “inflammatory niche”. MethodsWe evaluated the expression of EBV-miRNAs in EBV+HL clinical specimens by in situ hybridization, their functional characterization in vitro, and their effects on persistent infection and tumor development in vivo humanized NOG mice model. Moreover, in order to clarify its sorting mechanism, trans factor and cis factor which determined secreted and non-secreted miRNAs was analyzed by use of mass-spectrograhy and next-generation sequencing. Results and DiscussionThe EBV-miRNAs effects were potent on monocyte/macrophage Mo/Mf in inducing CD69, IL-10, and TNF, suggesting that EBV-miRNAs might polarize Mo/Mf into tumor associated Mf (TAM). EBV-miRNAs suppress tumor cell proliferation in vitro, implying that it works as tumor-suppressor in the tumor cells, while they are required to develop LPD in vivo, which seems contradict to the result in vitro. These results suggest that EBV-miRNAs intra-cellularly regulate the tumor cells to adjust to the surrounding circumstances, for example, to escape from immune surveillance, and inter-cellularly regulate Mo/Mf to support the tumor survival or development. Most importantly, exosomal EBV-miRNAs derived from the tumor cells were transferred to Mf in human EBV+ HL samples.Interestingly, one EBV coded miRNA was not secreted at all, though it abundantly expresses in the cells. The miRNA has been reported to strongly promote cell proliferation in EBV infected tumor cells. It made us hypothesized that the sorting system of secretary and non-secretary miRNAs is critical in the formation of “inflammatory niche”. In order to clarify the mechanism of the sorting, the chimeric miRNA was constructed then, we determined the sequence, which regulates secretion and non-secretion, and purified the protein complex, which specifically bound to the sequence. Mass spectrography and successive knockdown assay, the trans factor which inhibits secretion was identified.Moreover, the next sequencing analysis for the small RNAs revealed that abundant EBV-coded small RNAs occupied RNA-induced silencing complex (RISC), and that non-secreted EBV-miRNA was specifically modified. It is now under investigation whether the modification is involved in the sort mechanism between secretary and non-secretary miRNAs.Taken together, EBV-miRNAs have critical roles in intra- and inter-cellular manner. Especially, the functions as an inter-cellular communicator might be important in the tumor formation and the mechanism needs further investigation. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 1567: Use of humanized NOG mice within the pre-clinical development of new human specific therapies.

Abstract Immune cells within the tumor microenvironment are now well recognized as to be targets of interest for cancer treatment. However there are no available pre-clinical models that will accurately support the pre-clinical development of such therapeutic approaches. Humanized NOG (NOD/shi-scid/IL-2Rγnull) mice, bearing human immune cells with or without human target tumor cells, are relevant models to test various therapeutic strategies (e.g. antibody dependent cell cytotoxicity, Treg targeting antibodies, TLR agonists, vaccines, adoptive T cells transfer, …) in various pathologies (e.g. oncology, autoimmune disease, inflammation, transplantation, …). Humanization of NOG mice was already characterized and validated using freshly collected human PBMCs or hematopoietic stem cells (HSCs) in both newborn and adult NOG mice. The complete characterization of the reconstituted NOG mice was done in peripheral blood as well as central lymphoid organs such as bone marrow, spleen and thymus, using FACS analysis. Moreover, the human immune reconstitution and function in NOG mice was also evidenced with human cytokine and immunoglobulin quantification in mouse plasma samples. The injection of mature human PBMC in adult NOG mice leads to the consistent development of a xenogeneic graft-versus-host disease (GvHD), which mimics human GvHD (modification of CD4/CD8 ratio, expression of T cells activation markers and cytokine production). Growth characteristics of human tumors models on humanized mice will be presented either as subcutaneous or as disseminated intravenous model. Randomization parameters were selected regarding both tumor and immune cell sides. Required readouts to understand immune cell modulation (Cell phenotyping and functionality) and related antitumor efficacy will be described. Finally, such models, i.e. humanized NOG mice bearing or not human tumor cells in the context of evaluation of new therapies will be detailed in various pathologies. Citation Format: Caroline Mignard, Jean-Francois Mirjolet, Holger Kissel, Francis Bichat, Olivier Duchamp. Use of humanized NOG mice within the pre-clinical development of new human specific therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1567. doi:10.1158/1538-7445.AM2013-1567

Recurrence of Primary Sclerosing Cholangitis after Living Donor Liver Transplantation and Roles of B-Cell Depletion

Primary sclerosing cholangitis (PSC) recurs at a high rate after living donor liver transplantation. This high recurrence rate is thought to involve: (1) HLA homology (genetic predisposition of the liver graft) and (2) the mechanism of autoimmune disorder. Seven patients have undergone living donor liver transplantation for PSC at our institution. There has been no PSC recurrence in 2 patients 5 years after they had undergone ABO-incompatible living donor liver transplantation and after 2 years of follow-up. These patients differed from other patients in that they received pre-transplant rituximab (RTX) to avoid humoral rejection after ABO-incompatible transplantation. Subjects: The subjects were 7 patients who had undergone living donor liver transplantation. They were 3 men and 4 women aged 3-42 years at the time of transplantation. In 6 subjects, donors were their parents who matched the recipients at three or more of six HLA loci. The remaining one subject was a woman, matching only 1 HLA locus with the donor who was of fifth degree of kinship to the patient. PSC recurrence was determined by blood biochemical tests, MRCP imaging, and liver biopsy. Immunodeficient mice were engrafted with peripheral blood mononuclear cells from patients who used RTX, and anti-donor antibodies against blood group antigens were measured by ELISA. Results: Five patients had a PSC recurrence 1-3 years after transplantation. RTX was not used in these 5 patients because of blood group compatibility with the donors, who were their parents. In 2 patients who received pre-transplant RTX, there was no PSC recurrence 5 years after transplantation and after 2 years of follow-up. When humanized NOG mice were sensitized to donor blood group antigens, the recipient B cells did not produce antibodies to donor blood group antigens. Discussion: The results of humanized NOG mice indicated that B1 cells responsive to donor blood group antigens (present in donor liver) are deleted before reconstitution of B cells that were depleted with RTX, and immune tolerance is induced. B1 cells are thought to be involved in autoimmune disorders. The results of this study suggest the potential involvement of B1 cell immune tolerance against donor antigens in the prevention of PSC recurrence.