400 Persistence and tissue distribution of agent-797 – a native allogeneic iNKT cell-therapy drug product

BackgroundInvariant Natural Killer T (iNKT) cells are key effectors and regulators of immune responses, making them an ideal immunotherapy. There is a paucity of evidence describing the persistence and trafficking of these cells in humans to inform the optimal clinical application. Here, we describe the development of a murine Xenograft model for the study of an unmodified human iNKT cell therapy (Agent-797) and present data on the persistence and tissue distribution of human iNKT cells in this model. We further describe the development and validation of a digital PCR-based methodology to track unmodified allogeneic human iNKT cells in blood and tissue and present exploratory clinical data on iNKT cell persistence in patients with cancer and viral ARDS treated with Agent-797.MethodsPersistence and tissue distribution of ex-vivo expanded human iNKT cells was investigated in immune compromised mice (NOG), as well as in NOG mice expressing human IL15 (NOG-hIL15), a key cytokine promoting iNKT cell survival. Persistence of iNKT cells was determined over a 35-day period, with takedowns on day 1, 7, 14, 21 and 35. iNKT cells were phenotyped for activation markers by flow cytometry. An assay based on Imegen Quimera digital PCR technology was developed and validated to quantify human iNKT in an allogeneic setting. We employed this assay to measure persistence of Agent-797 drug product in patients participating in clinical trials using iNKT cell-based immunotherapy in viral ARDS (NCT04582201) or multiple myeloma (NCT04754100).ResultsHuman IL15 was essential for the engraftment and persistence of human iNKT cells in NOG mice. Following injection, iNKT cells located to the blood, lung, liver, spleen, and bone marrow. iNKT cells persisted most prominently in bone marrow, where they demonstrated an activated phenotype. In mice challenged with hematological tumor cells (ALL cell line NALM6 expressing CD1d) persistence of iNKT cells in blood was prolonged. Initial data from human trials confirmed rapid translocation from peripheral blood of this tissue resident immune cell population following infusion of Agent-797.ConclusionsWe established a murine xenograft model and digital PCR-based methodology to characterize the persistence, trafficking, and efficacy of native allogeneic human iNKT cell-based products. Our models recapitulated the human iNKT distribution and demonstrated iNKTs induced preclinical efficacy in a tumor model. We further successfully developed a validated methodology to track unmodified allogeneic iNKT cells in humans.Trial RegistrationNCT04582201 and NCT04754100Ethics ApprovalAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards

The Flow Cytometry Marker for Early Detection of Irradiation Injury in NOG Mice Bone Marrow: A Short Report

Background Radiation is used for myeloablation in mice's bone marrow (BM) for hematopoietic stem cells (HSC) engraftment experiments. To assess post-radiation damage to the BM, an accessible and convenient marker is needed. This study comparatively evaluated the flow cytometer's effectiveness in detecting the ROS and Annexin V-PI level. Methods We divided 30 NOG mice between irradiated (n = 20) and control groups (n = 10) for each time point. After sacrificing BM samples were collected, the percentage of annexin V, PI, and ROS were investigated at two different time points (Day 2 and 14 after exposure). Results At the first timepoint, the level of ROS was higher in the irradiated group than in the control group, and this difference was statistically significant (P < 0.05). Also, at the second timepoint, the mean differences of all markers in the irradiated group were significant compared to the control group (P < 0.05). Conclusions Thus, in this mice strain to assess the BM irradiation-induced injury, measurement of ROS level is helpful.

Ex Vivo Expanded and Activated Natural Killer Cells Prolong the Overall Survival of Mice with Glioblastoma-like Cell-Derived Tumors

Glioblastoma (GBM) is the leading malignant intracranial tumor and is associated with a poor prognosis. Highly purified, activated natural killer (NK) cells, designated as genuine induced NK cells (GiNKs), represent a promising immunotherapy for GBM. We evaluated the anti-tumor effect of GiNKs in association with the programmed death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint pathway. We determined the level of PD-1 expression, a receptor known to down-regulate the immune response against malignancy, on GiNKs. PD-L1 expression on glioma cell lines (GBM-like cell line U87MG, and GBM cell line T98G) was also determined. To evaluate the anti-tumor activity of GiNKs in vivo, we used a xenograft model of subcutaneously implanted U87MG cells in immunocompromised NOG mice. The GiNKs expressed very low levels of PD-1. Although PD-L1 was expressed on U87MG and T98G cells, the expression levels were highly variable. Our xenograft model revealed that the retro-orbital administration of GiNKs and interleukin-2 (IL-2) prolonged the survival of NOG mice bearing subcutaneous U87MG-derived tumors. PD-1 blocking antibodies did not have an additive effect with GiNKs for prolonging survival. GiNKs may represent a promising cell-based immunotherapy for patients with GBM and are minimally affected by the PD-1/PD-L1 immune evasion axis in GBM.

HER2-antigen-specific humoral immune response in breast cancer lymphocytes transplanted in hu-PBL hIL-4 NOG mice

The status of humoral immunity of cancer patients is not clear compared to cellular immunity because the ability of specific antibody production is difficult to analyze in vitro. We previously developed a humanized mouse model to evaluate antigen-specific antibody production by transplanting human peripheral blood mononuclear cells (PBMCs) into NOG-hIL-4-Tg mice (hu-PBL hIL-4 NOG). In this study, these mice were transplanted with PBMCs derived from breast cancer patients (BC) and immunized with a human epidermal growth factor receptor 2 (HER2) peptide, CH401MAP, to analyze humoral immunity of BCs. The hu-PBL hIL-4 NOG mice recapitulated immune environment of BCs as the ratio of CD8+/CD4+T cells was lower and that of PD-1 + T cells was higher compared to healthy donors (HDs). Diverse clusters were detected in BC-mouse (BC-M) plasma components involving immunoglobulins and complements unlike HD-M, and there was a significant diversity in CH401MAP-specific IgG titers in BC-M. The number of B cell clones producing high CH401MAP-specific IgG was not increased by immunization in BC-M unlike HD-M. These results demonstrated that the humoral immunity of BCs appeared as diverse phenotypes different from HDs in hu-PBL hIL-4 NOG mice, which may provide important information for the study of personalized medicine.

Human Microglia Extensively Reconstitute in Humanized-BLT Mice With Human Interleukin-34 Transgene and Support HIV-1 Brain Infection

Humanized bone marrow-liver-thymic (hu-BLT) mice develop a functional immune system in periphery, nevertheless, have a limited reconstitution of human myeloid cells, especially microglia, in CNS. Further, whether bone marrow derived hematopoietic stem and progenitor cells (HSPCs) can enter the brain and differentiate into microglia in adults remains controversial. To close these gaps, in this study we unambiguously demonstrated that human microglia in CNS were extensively reconstituted in adult NOG mice with human interleukin-34 transgene (hIL34 Tg) from circulating CD34+ HSPCs, nonetheless not in hu-BLT NOG mice, providing strong evidence that human CD34+ HSPCs can enter adult brain and differentiate into microglia in CNS in the presence of hIL34. Further, the human microglia in the CNS of hu-BLT-hIL34 NOG mice robustly supported HIV-1 infection reenforcing the notion that microglia are the most important target cells of HIV-1 in CNS and demonstrating its great potential as an in vivo model for studying HIV-1 pathogenesis and evaluating curative therapeutics in both periphery and CNS compartments.

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.

HER2-antigen-specific humoral immune response in breast cancer lymphocytes transplanted in hu-PBL hIL-4 NOG mice

The status of humoral immunity of cancer patients is not clear compared to cellular immunity because the ability of specific antibody production is difficult to analyze in vitro. We previously developed a humanized mouse model to evaluate antigen-specific antibody production by transplanting human peripheral blood mononuclear cells (PBMCs) into NOG-hIL-4-Tg mice (hu-PBL hIL-4 NOG). In this study, these mice were transplanted with PBMCs derived from breast cancer patients (BC) and immunized with a human epidermal growth factor receptor 2 (HER2) peptide, CH401MAP, to analyze humoral immunity of BCs. The hu-PBL hIL-4 NOG mice recapitulated immune environment of BCs as the ratio of CD8+/CD4 + T cells was lower and that of PD-1 + T cells were higher compared to healthy donor (HD). Diverse clusters were detected in BC-mouse (BC-M) plasma components involving immunoglobulins and complements unlike HD-M, and there was a significant diversity in CH401MAP-specific IgG titers in BC-M. The number of B cell clones producing high CH401MAP-specific IgG was not increased by immunization in BC-M unlike HD-M. These results demonstrated that the humoral immunity of BCs appeared as diverse phenotypes different from HDs in hu-PBL hIL-4 NOG mice, which may provide important information for the study of personalized medicine. (198)

Establishment of a Patient-Derived Xenograft Model of Colorectal Cancer in CIEA NOG Mice and Exploring Smartfish Liquid Diet as a Source of Omega-3 Fatty Acids

Cancer patient-derived xenografts (PDXs) better preserve tumor characteristics and microenvironment than traditional cancer cell line derived xenografts and are becoming a valuable model in translational cancer research and personalized medicine. We have established a PDX model for colorectal cancer (CRC) in CIEA NOG mice with a 50% engraftment rate. Tumor fragments from patients with CRC (n = 5) were engrafted in four mice per tumor (n = 20). Mice with established PDXs received a liquid diet enriched with fish oil or placebo, and fatty acid profiling was performed to measure fatty acid content in whole blood. Moreover, a biobank consisting of tissue and blood samples from patients was established. Histology, immunohistochemistry and in situ hybridization procedures were used for staining of tumor and xenograft tissue slides. Results demonstrate that key histological characteristics of the patients’ tumors were retained in the established PDXs, and the liquid diets were consumed as intended by the mice. Some of the older mice developed lymphomas that originated from human Ki67+, CD45+, and EBV+ lymphoid cells. We present a detailed description of the process and methodology, as well as possible issues that may arise, to refine the method and improve PDX engraftment rate for future studies. The established PDX model for CRC can be used for exploring different cancer treatment regimes, and liquid diets enriched with fish oil may be successfully delivered to the mice through the drinking flasks.

Human microglia extensively reconstitute in humanized BLT mice with human interleukin-34 transgene and support HIV-1 brain infection

Humanized bone marrow-liver-thymic (hu-BLT) mice develop a functional immune system in periphery but have a limited reconstitution of human myeloid cells, especially microglia, in CNS. Further, whether bone marrow derived hematopoietic stem and progenitor cells (HSPCs) can enter the brain and differentiate into microglia in adults remains controversial. To close these gaps, in this study we unambiguously demonstrated that human microglia in CNS were extensively reconstituted in adult NOG mice with human interleukin-34 transgene (hIL34 Tg) from circulating CD34+ HSPCs but no in hu-BLT NOG mice, providing strong evidence that human CD34+ HSPCs can enter adult brain and differentiate into microglia in CNS in the presence of hIL34. Further, the human microglia in the CNS of hu-BLT-hIL34 NOG mice robustly supported HIV-1 infection reenforcing the notion that microglia are the most important target cells of HIV-1 in CNS and demonstrating its great potential as an in vivo model for studying HIV-1 pathogenesis and evaluating curative therapeutics in both periphery and CNS compartments.