Presence Of Human Immune Cells Research Articles

Abstract 3994: N glycosylation inhibition hinders immunosuppressive tumor microenvironment cells improving CAR T cell efficacy

Abstract Adoptive transfer of CAR T cells demonstrated impressive results against B-cell malignancies, but still limited efficacy against solid tumors. In this context, multiple challenges need to be overcome, including poor tumor recognition and strong immunosuppression within the tumor microenvironment (TME). Our Unit has recently reported that pharmacological inhibition of N-glycan synthesis in cancer cells increases CAR T cell efficacy by improving tumor recognition and preventing T cell exhaustion. In this project, we investigated the role of N-glycosylation blockade on TME cells in the context of colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC)-derived liver metastases, exploiting CEA-specific CAR T cell therapy. To understand the effect of N-glycosylation blockade on TME cells (both M2-like macrophages, M2-M and Hepatic stellate cells, HepSCs), we analyzed the phenotypic and transcriptional profile and we performed in vitro functional assays, such as tripartite co-cultures, suppressive assays and released-cytokines analysis. In vitro studies revealed that N-glycosylation inhibition abolishes the ability of both TME cells to restrain T cell proliferation and increases the elimination of cancer cell lines and patient-derived tumor organoids (PDOs from CRC-liver metastases). Interestingly, these effects were associated with profound phenotypic and transcriptional changes in M2-M and HepSCs. In particular, the treatment was able to inhibit M2-polarization in terms of surface markers expression, IL-10 secretion and gene expression profile, and was shown to hinder the activation of HepSCs. Moreover, to evaluate the effect of N-glycosylation inhibition on TME cells in vivo, we exploited immunodeficient mice reconstituted with a human immune system (huSGM3), engrafted intra-liver with tumor cells and treated with CEA CAR T cells. Interestingly, in this in vivo model, the presence of human immune cells supports CAR T cell responses and helps recreate an immune TME more representative of the human disease. Importantly, using these mice we observed that N-glycosylation inhibition improves CEA CAR T cells fitness, enhancing their antitumor activity in terms of survival. This effect is associated with a modulation of human immune TME populations and in particular with the downregulation of immunosuppressive genes in tumor-infiltrating human immune cells (from scRNAseq and Nanostring analysis). Overall, these data suggest that blocking N-glycosylation can help overcome multiple barriers that currently limit CAR T cell efficacy in solid tumors, acting not only on tumor cells, but also on immunosuppressive tumor microenvironment cells. Citation Format: Camilla Sirini, Beatrice Greco, Chiara Balestrieri, Barbara Camisa, Laura Falcone, Rita El Khoury, Oronzina Botrugno, Giulio Giovannoni, Giovanni Tonon, Emanuela Brunetto, Maria Giulia Cangi, Federica Pedica, Alice Bergamini, Fabio Ciceri, Chiara Bonini, Monica Casucci. N glycosylation inhibition hinders immunosuppressive tumor microenvironment cells improving CAR T cell efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3994.

Abstract 3104: Inverse correlation of circulating CD8+ T cells with tumor burden from immune checkpoint treatment in humanized orthotopic model of renal cell carcinoma

Abstract Background: Renal cell carcinoma (RCC) is a highly heterogeneous and metastatic disease with a widely varying prognosis. RCC is the first solid tumor treated with immune checkpoint blockade and approved by the FDA for showing improvement of clinical outcomes. Immune checkpoints physiologically regulate immune response via appropriate signals to T cells and maintain homeostasis, while pathologically they can be co-opted by cancer cells to evade the immune system. Here we developed a humanized (Hu)-mice model with adaptive transferred human immune cells to test the role of ICB [anti-PD-1/PD-L1, or anti-CTLA-4 antibody (Ab)] in an orthotopic human RCC tumor-bearing xenograft mouse model and analyzed the correlation of innate immune cells with tumor burden. Methods: Luciferase-tagged RCC cell line 786-O were injected intra-kidney into immune-deficient Rag2 mice. After tumor formation, mice were humanized by intraperitoneal injection of donor PBMCs (20×106/per mouse). Treatment groups included control, anti-PD-1/PD-L1 Abs or anti-CTLA-4 Ab (all Abs 200 µg/mouse, iv) weekly for 4 weeks. Tumor growth was measured by weekly bioluminescent imaging (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes was confirmed by flow cytometry and immunohistochemistry staining. The significance of the differences between groups was determined by student t test. The Pearson correlation coefficient was obtained by analysis of correlation between different study parameters. Results: BLI level showed a steady xenograft tumor progression for nine weeks. Anti-PD-1/PD-L1 Abs treatment trended toward reducing tumor BLI level while anti-CTLA-4 Ab significantly decreased tumor BLI level (p<0.01). Furthermore, anti-PD-1/PD-L1 Abs reduced lung metastasis with a significant decrease of BLI level compared to control group (p<0.05). Meanwhile, humanization was evidenced by robust, elevated levels of human CD45+ lymphocytes in the peripheral blood compared to non-reconstituted Rag2 mice. Infiltrated human CD3+CD8+ cells were found in mouse blood and spleen. Granzyme B+ cells, CD31+ cells, and pan-cytokeratin+ cells were found in tumors. Interestingly, we observed inverse correlation between circulating human CD8+ T cells and mouse RCC tumor burden. Conclusion: We successfully generated a humanized RCC mouse model which allows the RCC xenograft to grow steadily within the human immune system reconstituted Rag2 mice. Circulating CD8+ T cells are inversely correlated to tumor growth in this model. The PD-1/PD-L1 or CTLA-4-targeted immunotherapies showed distinct therapeutic effects in our humanized orthotopic xenograft model, which may lead to personalized treatment targeting different immune checkpoints to reinvigorate anti-tumor responses in RCC patients. Citation Format: Xin Zhang, Amita Bhattarai, Ravan Moret, Grace Maresh, Alicia Nicole Ray, David Woods, Rachel Graham, Maria Latis, Stephen Bardot, Li Li. Inverse correlation of circulating CD8+ T cells with tumor burden from immune checkpoint treatment in humanized orthotopic model of renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3104.

Abstract 6705: An integrated in vitro and in vivo approach providing insights in immune modulation

Abstract In order to exploit the complex tumour microenvironment, immunoreactivity in a multitude of cell-based assays is required to predict mode of action (MOA), pharmacodynamics, and efficacy downstream. We have developed an integrated approach starting with single cell and co-culture cell-based assays in 2D and 3D formats, followed by an in vivo set-up based on humanized mice to monitor an immunotherapeutic's interaction with specific cell types such as T cells including CAR-T cells, natural killer cells, macrophages, dendritic cells, neutrophils, and fibroblasts. Rituximab, a monoclonal antibody against CD20, is mainstay in the therapy for a broad variety of B-cell malignancies. Its MOA is not fully understood as direct signalling, complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity (ADCC) all seem to have an impact. To elucidate the share and effect each of those mechanism has, we tested Rituximab in our immuno-oncology translational platform. Furthermore, our immuno-oncology platform was utilized to assess efficacy, bystander effects and off-target toxicity of a CD19 CAR-T cell therapy. In vitro assays for efficacy and bystander effect involved co-culture of the CAR-T cells with a mixture of non-target expressing cells and mid to high target antigen expressing cells, where T cell activation and cell death of both target cell types was quantified. Beyond efficacy the assay platforms were used to examine the influence of the therapy, antibodies or CAR-T cells, on normal human cells. These in vitro assays utilizing primary human cells from healthy tissue and/or differentiated iPCS-derived cells were used to assess cytotoxicity. The primary tissues were selected based on their potential safety risk by either expressing low levels of the target antigen or being major organs to de-risk for off-target effects. The results from our in vitro screen were validated in a xenograft in vivo system in the presence and absence of human immune cells. Four cell line derived xenografts were tested in vivo in immunocompromised mice subcutaneously and disseminated. The presence of human immune cells enhanced the antitumoral activity of Rituximab markedly, although the targeted effect of Rituximab induced already a significant prolongation in the overall survival (p< 0.005; Log-rank (Mantel-Cox) test).Over the last 30 years IO has progressed considerably with approvals for the use of various IO therapeutics including vaccines, cytokines, tumor-directed monoclonal antibodies, immune checkpoint inhibitors as well as Chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cell therapies. Comprehensive characterization of novel IO compounds using a multitude of such in vitro and in vivo assays is essential to provide insights and understanding of the detailed mechanism of action and identify potential toxicity issues to ultimately improve translation to the clinic. Citation Format: Shilina Roman, Julia Schüler, Sanne Holt, Jeroen DeGroot, Omar Aziz, Ian Waddell. An integrated in vitro and in vivo approach providing insights in immune modulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6705.

Humanized Rag2 mice as patient-derived orthotopic mouse models for immune checkpoint blockade and conventional chemotherapeutic drug treatment for human solid tumors

Abstract To evaluate therapeutic efficacy of immune checkpoint blockade (ICB) and conventional chemotherapy in pre-clinical models, we developed humanized patient-derived orthotopic xenograft (PDOX) models for colorectal cancer (CRC) and renal cell carcinoma (RCC). Rag2 mice were humanized by donor peripheral blood mononuclear cells (PBMC) transfer. Luciferase-tagged CRC and RCC tumor cells were injected intra-rectally and intra renal subcapsular, respectively. Groups of mice (n=5–10) received ICB, anti-PD-1 and anti-PD-L1 antibodies (Nivolumab and Atezolizumab, 200 μg each/mouse) for 4 weeks, and conventional therapy of 5FU (200 mg/kg, for CRC) alone or in combination. Tumor growth was measured by weekly bioluminescent image (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes were confirmed by flow cytometry and immunohistochemistry staining. Humanization was evidenced by >45% HLA-ABC+CD45+ circulating human cells in both CRC and RCC models. CRC tumor weight showed significant reduction in group treated with combination of ICB and 5FU compared with untreated controls or 5FU monotherapy group (p<0.05) using patient tumor cell CoCa302. The lung/liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively. For RCC, tumor size was reduced by >30% from controls in ICB treated group using patient tumor cell KiCa118 and cell line SN12K1. The later had 20% reduction of liver metastasis. The combination of ICB and conventional chemotherapies for CRC and RCC can be tested in our humanized PDOX mouse models. The effect of the combination therapy using MHC matched donors or patient autologous PBMC will be further investigated.

Abstract B85: Combination therapy using the programmed death receptor-1 and the programmed death-ligand 1 inhibitors and Fluorouracil in human colorectal carcinoma in a humanized patient-derived orthotopic mouse model

Abstract Background: One of the recently understood mechanisms associated with the progression of cancer is the immune checkpoint pathway. Many tumors can stimulate the expression of immune checkpoint molecules, resulting in a phenotype of exhausted T cells that cannot restrain tumor progression. One such inhibitory ligand and receptor pair in solid tumors are the programmed death-ligand 1 (PD-1) and programmed death receptor-1 (PD-L1). They prevent the killing of cancer cells by cytotoxic T-lymphocytes. PD-1 receptor is expressed by activated T cells among other cells, while PD-L1 is overexpressed on many tumor types including colorectal carcinoma (CRC). We have developed a humanized patient-derived orthotopic xenograft (hPDOX) model for CRC. Here, we investigate the potential efficacy of combination of immune checkpoint inhibitors (ICIs) and fluorouracil (5FU) in this CRC hPDOX model. Methods: Humanized mice were established by IP injection of donor peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged patient tumor CoCaPt302 cells were injected intrarectally into Rag2 mice. Groups of mice (n=7-10) received ICIs, anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 μg/mouse and atezolizumab, 200 μg/mouse), and 5FU (200 mg/kg) once a week for 4 weeks, IV injection alone or in combination. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, tumor weights were measured. Human HLA-ABC+CD45+ hematopoietic cells and CD4+ or CD8+ T cells were detected in peritoneal lavage, blood, and spleen by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating lymphocytes was confirmed by immunohistochemistry staining. Results: Humanization was evidenced by mouse blood containing more than 45% HLA-ABC+CD45+ human cells when tested by flow cytometry. Tumor weight showed significant reduction in group treated with combination of ICIs and 5FU compared with untreated controls (p=.0038). Tumors were also significantly smaller than ICIs (p=.0123) or 5FU (p=.0427) monotherapy groups. In addition, the lung and liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively, and exhibited better antitumor response as well as less development of metastases. CD3+ T cells, especially CD4+, CD8+ T cells in blood and spleen, were reduced in the ICIs only and combination treatment groups, but not in 5FU group compared to the controls. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as a hPDOX model for CRC; and treatment efficacy including inhibiting tumor growth and distant organ metastasis was significantly enhanced through ICI and 5FU combination therapy. The effect of the combination therapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated. Citation Format: Xin Zhang, Grace Maresh, Linh Hellmers, Henry Yip, Lara McKean Baste, Heather Green, David Margolin, Li Li. Combination therapy using the programmed death receptor-1 and the programmed death-ligand 1 inhibitors and Fluorouracil in human colorectal carcinoma in a humanized patient-derived orthotopic mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B85.

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease's pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

Development and characterization of a humanized mouse model of osteoarthritis

Purpose: Normal 0 21 false false false FR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tableau Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman",serif; border:none;} <Osteoarthritis (OA) is mainly characterized by cartilage breakdown and synovial inflammation. While OA is one of the most common rheumatic disorders, no efficient disease-modifying drugs still exist today. Mesenchymal stem cells (MSC) have generated significant medical considerations since they exhibit tissue-regenerative properties through the secretion of bioactive factors. Phase I-II clinical trials have shown that intra-articular (IA) injection of MSC is well-tolerated and exhibited promising clinical results. However, MSC display a poor survival and engraftment rate in vivo. Using encapsulating systems could protect MSC and maintain their bioactivity after intra-articular injection. Considering the pivotal role of inflammation and immune system in OA and with regards to the differences between MSC-mediated immunomodulation in human and mice (12), the development of sophisticated animal models that could more closely mimic the human immune system, could be instrumental. Amongst these models, immunologically humanized mice may represent a preclinical bridge between in vitro studies and the in vivo evaluation of human stem cells in OA. Our objective is therefore to develop an experimental model of osteoarthritis by destabilizing the medial meniscus (DMM) in humanized mice.> Methods: Normal 0 21 false false false FR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tableau Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman",serif; border:none;} <4 weeks-old NOD/LtSz-scid IL2Rγnull (NSG) mice were humanized as follow. After irradiation (1,5Gy), 5.106 CD34+ human hematopoietic stem cells were injected intravenously. 120 days after the humanization procedure, the level of human immune cells CD45+ (leukocytes), CD3+ (T lymphocytes), CD19+ (B lymphocytes), CD56+ (NK cells) and CD14+ (macrophages) were evaluated by flow cytometry. Humanized NSG mice then had undergone a DMM or a SHAM surgery. 6 and 12 weeks after OA induction the development of osteoarthritis was assessed histologically by safranin-o staining, OA score and histomorphometric analysis of the knees. In addition, to evidence the presence of human immune cells in the joint, Immunohistochemicals stainings for human CD45+, CD3+, CD19+ et CD56+ and CD14+ cells have also been performed.> Results: Normal 0 21 false false false FR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tableau Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman",serif; border:none;} <Our results showed that 120 days after the humanization procedure, NSG mice exhibit engraftment level (hCD45+) from 10% to 75 % and the presence of human T and B lymphocytes as well as macrophages and NK cells in blood and spleen. Our histological results show that the DMM procedure induce a significant increase in OA score at 12 weeks compared to sham mice. Our histomorphometric results also evidence a significant increase in bone volume of the anterior medial meniscus. However, no significant differences have been observed between DMM and Sham mice on the OA score at 6 weeks and on the subchondral bone morphometry at 6 and 12 weeks’ post-surgery. Immunohistochemical analyses have however revealed the presence of human B lymphocytes and macrophages in the subchondral bone marrow but not in the synovial membrane at 6 and 12 weeks post-surgery.> Conclusions: Normal 0 21 false false false FR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tableau Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman",serif; border:none;} <Finally, using 2 methods of analysis (histology and histomorphometry), we have demonstrated that humanized NSG mice could develop post-traumatic OA after a DMM surgery. The presence of human immune cell in the spleen and subchondral bone marrow confirms the success of the hematopoietic transplant. However, we failed to detect any immune cells in the synovial membrane or articular cartilage although human immune cells were observed near the joint in the subchondral bone marrow. The search for these immune cells in the synovial membrane would be more appropriate during the inflammatory burst, meaning within the first 2 weeks following the DMM surgery. Our results therefore evidence the feasibility to induce OA by DMM in humanized mice. This model could constitute a useful tool to study the therapeutic efficacy of encapsulated human mesenchymal stem cells.>

Abstract B027: Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model

Abstract Background: The immune system plays an important role in tumor immune surveillance and progression. Immune checkpoint blockade is a new approach for cancer immunotherapy. The programmed death receptor-1 (PD-1) and the programmed death-ligand 1 (PD-L1) are immune checkpoint molecules and their expression results in negative regulation of T-cells primarily within the tumor microenvironment by preventing the killing of cancer cells by cytotoxic T-lymphocytes. Antibody blocking of the PD-1/PD-L1 signal exhibits promising therapeutic effects in non-small cell lung cancer and melanoma in patients. Due to its early success, more trials have been conducted to evaluate their efficacy for different tumors. We have developed a patient-derived orthotopic xenograft mouse model for colorectal carcinoma (CRC). Here, we investigate the potential efficacy of PD-1/PD-L1 blockade in our humanized orthotopic mouse models for human CRC. Methods: All studies were conducted under approved guidelines of the Institutional Animal Care and Use Committee and the Investigative Review Board of Ochsner Clinic Foundation. Humanized mice were established by intraperitoneal injection of donor human peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged CRC cells were injected intra-rectally into Rag2 mice. One group of mice received a combination of anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 µg/mouse and atezolizumab, 200 µg/mouse, intravenous injection) once a week for 3 weeks. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, the CRC tumor weights were measured. Human CD45+ hematopoietic cells, CD4+ and CD8+ T-cells, and CD20+ B cells were detected in peritoneal lavage, blood, and tumor by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating human lymphocytes was further confirmed by immunohistochemistry staining on paraffin-embedded tissue slides of mouse spleen and tumor, respectively. Results: Blood from mice receiving human PBMCs contained on average 31.4% CD45+ human cells (n=9) when tested by FACS analysis. Smaller tumor growth was observed in mice given PBMCs. This may be due to alloreactivity based on the recognition of MHC alloantigens in the transplanted tumor cells. However, mice further treated with the combination therapy of anti-PD-1 and anti-PD-L1 antibodies exhibited better antitumor response as well as less development of lung metastases compared to untreated controls. On average, tumor weight was reduced by 36% and lung metastases measured by ex-vivo BLI was reduced by 82% (n=5). In addition, circulating CD326+ tumor cells were reduced by 21% and CD3, CD4, CD8 and CD20 positive human lymphocytes were also reduced in the combination treatment group. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as an orthotopic xenograft model for CRC; and treatment benefit for CRC through targeting immune checkpoint molecules PD-1 and PD-L1. The effect of immune checkpoint blockade in combination with conventional chemotherapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated. Citation Format: Li Li, Xin Zhang, Grace Maresh, Linh Hellmers, Avi Patel, Ravan Moret, Sarah Cohen, David Margolin. Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model [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 B027.

Human immune cell engraftment does not alter development of severe acute Rift Valley fever in mice.

Rift Valley fever (RVF) in humans is usually mild, but, in a subset of cases, can progress to severe hepatic and neurological disease. Rodent models of RVF generally develop acute severe clinical disease. Here, we inoculated humanized NSG-SGM3 mice with Rift Valley fever virus (RVFV) to investigate whether the presence of human immune cells in mice would alter the progression of RVFV infection to more closely model human disease. Despite increased human cytokine expression, including responses mirroring those seen in human disease, and decreased hepatic viral RNA levels at terminal euthanasia, both high- and low-dose RVFV inoculation resulted in lethal disease in all mice with comparable time-to-death as unengrafted mice.

Abstract LB-363: Characterization of in vitro and in vivo models generated from disseminated tumor cells of patients without manifest metastasis

Abstract We recently showed that dissemination of few melanoma cells to the lymph nodes is a quantitative risk factor for death from melanoma. However, the analysis of disseminated tumor cells (DTC) is limited by their extremely low frequency, resulting in a lack of in vitro/in vivo models for mechanistic studies of early systemic disease. Therefore, the objective of this study was to develop protocols to expand single DTC from lymph nodes of melanoma patients to establish better preclinical models for adjuvant cancer therapies. Sentinel lymph nodes from melanoma patients were cut in halves, disaggregated and analyzed for the presence of gp100-positive DTC. The other half was used for diagnostic assessment by histopathology. Single DTC within the lymph node-derived single cell suspension were then propagated under specific sphere forming conditions and expanded by transplantation in immunodeficient mice. DTC based pre-clinical in vitro/in vivo models were then characterized on molecular level and were used for functional analyses. To test drugs on DTC in presence of a human immune system we additionally generated humanized xenograft models. We successfully established cell lines and xenografts from DTCs of 17 melanoma patients. We confirmed the origin of the cells by genomic fingerprint and analyzed the genomic profile of the expanded cells over various culture and animal passages. DTC cell lines were used for functional analysis and for testing the efficacy of targeted therapies. In addition, we set up a novel preclinical mouse model based on expanded melanoma DTC. For this, we established a human immune system in immunodeficient mice, which were then transplanted with melanoma DTC-derived cells. Interestingly, the presence of a human immune system significantly induced tumor formation, as well as dissemination of melanoma cells and improved the response to a targeted therapy compared to mice without a humanized immune system. The development of DTC based pre-clinical in vitro/in vivo models enables mechanistic studies on DTCs and drug tests against the target cells of adjuvant therapies in the presence of human immune cells. These models may help to identify candidate adjuvant therapies targeting DTCs and to understand mechanisms of drug resistance of currently applied targeted therapies. Citation Format: Christian Werno, Kathrin Weidele, Steffi Treitschke, Catherine Botteron, Sebastian Scheitler, Bernhard Polzer, Melanie Werner-Klein, Christoph Andreas Klein. Characterization of in vitro and in vivo models generated from disseminated tumor cells of patients without manifest 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 LB-363.

PO-440 Development and characterisation of pre-clinical in vitro/in vivo models from disseminated tumour cells of melanoma patients

IntroductionWe recently showed that dissemination of few melanoma cells to the lymph nodes is a quantitative risk factor for death from melanoma. However, the analysis of disseminated tumour cells (DTC) is limited by their extremely low frequency, resulting in a lack of in vitro/in vivo models for mechanistic studies of early systemic disease. Therefore, the objective of this study was to develop protocols to expand single DTC from lymph nodes of melanoma patients to establish better preclinical models for adjuvant cancer therapies.Material and methodsSentinel lymph nodes from melanoma patients were cut in halves, disaggregated and analysed for the presence of gp100-positive DTC. The other half was used for diagnostic assessment by histopathology. Single DTC within the lymph node-derived single cell suspension were then propagated under specific sphere forming conditions and expanded by transplantation in immunodeficient mice. DTC based pre-clinical in vitro/in vivo models were then characterised on molecular level and were used for functional analyses. To test drugs on DTC in presence of a human immune system we additionally generated humanised xenograft models.Results and discussionsWe successfully established cell lines and xenografts from DTCs of 17 melanoma patients. We confirmed the origin of the cells by genomic fingerprint and analysed the genomic profile of the expanded cells over various culture and animal passages. DTC cell lines were used for functional analysis and for testing the efficacy of targeted therapies. In addition, we set up a novel preclinical mouse model based on expanded melanoma DTC. For this, we established a human immune system in immunodeficient mice, which were then transplanted with melanoma DTC-derived cells. Interestingly, the presence of a human immune system significantly induced tumour formation, as well as dissemination of melanoma cells and improved the response to a targeted therapy compared to mice without a humanised immune system.ConclusionThe development of DTC based pre-clinical in vitro/in vivo models enables mechanistic studies on DTCs and drug tests against the target cells of adjuvant therapies in the presence of human immune cells. These models may help to identify candidate adjuvant therapies targeting DTCs and to understand mechanisms of drug resistance of currently applied targeted therapies.

Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice.

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis.This article has an associated First Person interview with the first author of the paper.

A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

A Model Method for Studying Norovirus

Norovirus is an extremely contagious positive sense RNA virus responsible for gastroenteritis. It has gained media attention due to its presence and impact on food and drinking water, and in the cruise ship industry. Researchers studying Norovirus have faced many challenges in their field with a limited number of reagents available for detecting the virus and growing it in culture, and no dependable small animal model. This research highlight focuses on the work of Taube et al. in the development of a mouse model for human Norovirus. This study used BALB/c mice deficient in recombination-activating genes 1 and 2 (Rag) and common gamma chain (γc) to demonstrate infection and replication of human Norovirus within the mice macrophages of the liver and spleen. They further showed the presence of human immune cells was not necessary to establish infection, but that the background strain of the mouse determined susceptibility. This discovery provides a huge step forward for basic and clinical applications of Norovirus research.

A Mouse Model for Human Norovirus

ABSTRACTHuman noroviruses (HuNoVs) cause significant morbidity and mortality worldwide. However, despite substantial efforts, a small-animal model for HuNoV has not been described to date. Since “humanized” mice have been successfully used to study human-tropic pathogens in the past, we challenged BALB/c mice deficient in recombination activation gene (Rag) 1 or 2 and common gamma chain (γc) (Rag-γc) engrafted with human CD34+ hematopoietic stem cells, nonengrafted siblings, and immunocompetent wild-type controls with pooled stool isolates from patients positive for HuNoV. Surprisingly, both humanized and nonhumanized BALB/c Rag-γc-deficient mice supported replication of a GII.4 strain of HuNoV, as indicated by increased viral loads over input. In contrast, immunocompetent wild-type BALB/c mice were not infected. An intraperitoneal route of infection and the BALB/c genetic background were important for facilitating a subclinical HuNoV infection of Rag-γc-deficient mice. Expression of structural and nonstructural proteins was detected in cells with macrophage-like morphology in the spleens and livers of BALB/c Rag-γc-deficient mice, confirming the ability of HuNoV to replicate in a mouse model. In summary, HuNoV replication in BALB/c Rag-γc-deficient mice is dependent on the immune-deficient status of the host but not on the presence of human immune cells and provides the first genetically manipulable small-animal model for studying HuNoV infection.

Humanized nonobese diabetic-scid IL2rγnullmice are susceptible to lethalSalmonellaTyphi infection

Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S. Typhi can replicate in vivo in nonobese diabetic (NOD)-scid IL2rgamma(null) mice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast, S. Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknown Salmonella virulence determinants, including Salmonella Pathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication of S. Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights into S. Typhi pathogenesis and devise strategies for the prevention of typhoid fever.

CD200 Expression on Tumor Cells Suppresses Antitumor Immunity: New Approaches to Cancer Immunotherapy

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.

Distribution of parasite stages in tissues of Toxoplasma gondii infected SCID mice and human peripheral blood lymphocyte-transplanted SCID mice.

The establishment of Toxoplasma gondii infection in the tissues of SCID mice and SCID mice transplanted with human peripheral blood lymphocytes (PBL) was investigated. The presence of bradyzoites and tachyzoites was analysed in hu-PBL SCID mice using Southern blot of reverse transcriptase-polymerase chain reaction products for the expression of B1, BAG1 and SAG1 of T. gondii. BAG1 was present by week 1 in brain, lung, liver and spleen of some animals; by week 3, BAG1 was present in all animals and in all of these tissues. In contrast, SAG1 was rarely detected until week 2 (mainly in the lung and brain) and by week 3, some animals still did not have detectable SAG1 in brain, lung, liver and spleen. SAG1 expression was increased in the lungs of animals transplanted with human PBL compared to nontransplanted SCID mice. Human PBL engraftment was demonstrated, initially in uninfected mice, by the presence of human CD3+ T cells in the spleen (3.1 x 10(5) positive cells) and peritoneal cavity (3.4 x 10(5) cells) 4 weeks after transplantation. The final outcome of infection was not influenced by the presence of human PBL, with similar mortality in human PBL transplanted and nontransplanted mice. These studies provide a detailed analysis of the kinetics and distribution of both the cyst and tachyzoite stage of T. gondii. This system has been established to allow evaluation of therapies against T. gondii immunodeficient mice in the presence of human immune cells.