Human Transgenic Mice Research Articles

Bone marrow derived macrophages from human CETP transgenic mice exhibit reduced phenotypic polarization to M1.

Background and Aims: Macrophages are key participants in atherosclerosis is a chronic progressive inflammation course. In general, macrophages can be classified as M1 or M2 activation phenotypes. Recently, M2 macrophages have been found associated with atherosclerosis regression. The M2 phenotype can secrete anti-inflammatory factors such as IL-10, promote tissue remodeling and repairing. We showed that huCETP expression enhances the mouse survival rate in an experimental systemic inflammation model. However, the role of CETP in directly influencing macrophage function is poorly understood.

TIGIT/CD155 blockade enhances anti-PD-L1 therapy in head and neck squamous cell carcinoma by targeting myeloid-derived suppressor cells

ObjectivesAnti-PD-1/PD-L1 therapy has recently been approved for head and neck squamous cell carcinoma (HNSCC). However, given that large numbers of patients with HNSCC do not respond to PD-1/PD-L1 antibodies, combination strategies for elevating the response rate need to be further investigated. The goal of this study was to explore the possibility of dual-targeting CD155/TIGIT and PD-1/PD-L1 signalling in HNSCC. Materials and methodsMultiplex flow cytometry was performed to determine the co-expression of CD155 and PD-L1 in human HNSCC and transgenic HNSCC mouse models. The combined application of TIGIT mAb and PD-L1 mAb in a mouse model was used to explore the therapeutic effect. ResultsCD155 and PD-L1 were highly co-expressed on myeloid-derived suppressor cells (MDSCs) derived from patients with HNSCC and were inversely associated with the percentage of tumour CD3+ T and effector memory T cells. CD155+PD-L1+ MDSCs in the mouse model were gradually enriched in the tumour microenvironment in the middle and late stages of tumour progression. Anti-PD-L1 treatment alone upregulated the expression of CD155 on MDSCs and while anti-TIGIT treatment upregulated the expression of PD-L1 on MDSCs in mice. The combined blockade of TIGIT/CD155 and PD-1/PD-L1 signalling in mice significantly inhibited tumour growth, enhanced the percentages of effector T cells and cytokine secretion and elicited immune memory effects. ConclusionOur study indicated that CD155+PD-L1+ MDSCs are enriched in the tumour microenvironment and blocking TIGIT/CD155 can effectively enhance the response rate of HNSCC to PD-L1 mAb therapy, which provides the clinical potential of co-targeting TIGIT/CD155 and PD-1/PD-L1 signalling.

Benzo(a)pyrene exposure in utero exacerbates Parkinson's Disease (PD)-like α-synucleinopathy in A53T human alpha-synuclein transgenic mice

BackgroundPrevious work indicated that benzo[a]pyrene (B(a)P) exposure in utero might adversely affect neurodevelopment and cause Parkinson's Disease (PD)-like symptoms. However, the effect of utero exposure to B(a)P on PD-like α-synucleinopathy and the mechanism under are unclear. ObjectiveThe A53T human alpha-synuclein (α-syn) transgenic mice (M83+/−) were used in this study to gain insights into the role of B(a)P exposure in utero in the onset of α-syn pathology and neuronal damage. MethodTimed-pregnant M83+/− dams were exposed to 1) corn oil (vehicle) or 2) 5 mg/kg bw/d B(a)P or 3) 20 mg/kg bw/d B(a)P at gestational day 10–17 by oral gavage and then the SNCA transcription, α-syn accumulation and aggregation, neuroinflammation and nigral dopaminergic neurodegeneration of 60-day-old pups were evaluated. ResultSNCA mRNA and α-syn protein expression in the midbrain of 60 days adult mice were found to be remarkably elevated after B(a)P exposure in utero, the protein degradation capacity was injured (in 20 mg/kg dose group) and α-syn aggregation could be observed in the substantia nigra (SN); Enhanced Iba1 expression in the midbrain and microglial activation (in 20 mg/kg dose group) in the SN were also figured out; Besides, dopaminergic neurons in the SN of 60 days adult mice were significantly decreased. ConclusionsOur findings demonstrated that B(a)P exposure in utero could exacerbate α-syn pathology and induce activation of microglia which might further lead to dopaminergic neuronal loss in the SN.

Novel Humanized Mesothelin-Expressing Genetically Engineered Mouse Models Underscore Challenges in Delivery of Complex Therapeutics to Pancreatic Cancers.

Antibody-based therapies designed for human use frequently fail to cross-react with the murine isoform of their target. Because of this problem, preclinical studies of antibody-based mesothelin (Msl)-targeted therapeutics in immunocompetent systems have been limited by the lack of suitable mouse models. Here, we describe two immunocompetent humanized mesothelin transgenic mouse lines that can act as tolerant hosts for C57Bl/6-syngeneic cell lines expressing the human isoform of mesothelin. Thyroid peroxidase (TPO) mice have thyroid-restricted human mesothelin expression. Mesothelin (Msl) mice express human mesothelin in the typical serosal membrane distribution and can additionally be utilized to assess on-target, off-tumor toxicity of human mesothelin-targeted therapeutics. Both transgenic strains shed human mesothelin into the serum like human mesothelioma and patients with ovarian cancer, and serum human mesothelin can be used as a blood-based surrogate of tumor burden. Using these models, we examined the on-target toxicity and antitumor activity of human mesothelin-targeted recombinant immunotoxins. We found that immunotoxin treatment causes acute and chronic histologic changes to serosal membranes in Msl mice, while human mesothelin-expressing thyroid follicular cells in TPO mice are resistant to immunotoxin despite excellent drug delivery. Furthermore, poor delivery of immunotoxin to syngeneic orthotopic human mesothelin-expressing pancreatic adenocarcinoma limits antitumor activity both alone and in combination with immune checkpoint inhibition. In summary, we have developed two high-fidelity, immunocompetent murine models for human cancer that allow for rigorous preclinical evaluation of human mesothelin-targeted therapeutics.

Half-life extension of efficiently produced DARPin serum albumin fusions as a function of FcRn affinity and recycling

Serum albumin shows slow clearance from circulation due to neonatal Fc receptor (FcRn)-mediated recycling and has been used for half-life extension. We report here fusions to a high-affinity DARPin, binding to Epithelial Cell Adhesion Molecule (EpCAM). We developed a novel, efficient expression system for such fusion proteins in Pichia pastoris with titers above 300 mg/L of lab-scale shake-flask culture. Since human serum albumin (HSA) does not bind to the murine FcRn, half-lives of therapeutic candidates are frequently measured in human FcRn transgenic mice, limiting useable tumor models. Additionally, serum albumins with extended half-life have been designed. We tested HSA7, motivated by its previously claimed extraordinarily long half-life in mice, which we could not confirm. Instead, we determined a half-life of only 29 h for HSA7, comparable to MSA. The fusion of HSA7 to a DARPin showed a similar half-life. To rationalize these findings, we measured binding kinetics and affinities to murine and human FcRn. Briefly, HSA7 showed affinity to murine FcRn only in the micromolar range, comparable to MSA to its cognate murine FcRn, and an affinity in the nanomolar range only to the human FcRn. This explains the comparable half-life of MSA and HSA7 in mice, while wild-type-HSA has a half-life of only 21 h, as it does not bind the murine FcRn and is not recycled. Thus, HSA-fusions with improved FcRn-affinity, such as HSA7, can be used for preclinical experiments in mice when FcRn transgenes cannot be used, as they reflect better the complex FcRn-mediated recycling and distribution mechanisms.

In Vitro and In Vivo Evaluation of GSK-3 Radioligands in Alzheimer's Disease: Preliminary Evidence of Sex Differences.

Glycogen synthase kinase-3 (GSK-3) is a positron emission tomography (PET) imaging target with implications in the pathogenesis of Alzheimer's disease (AD). This preliminary study evaluates human AD and transgenic P301L mouse brain tissues using the GSK-3-targeting radiotracers [3H]PF-367 and [3H]OCM-44 in radioligand binding assays. A saturation analysis showed decreased GSK-3 density in female human AD compared to a normal healthy brain. Equivalence in density (B max), affinity (K d), and apparent affinity (K i) of both radiotracers was demonstrated to enable their interchangeability for in vitro evaluations of GSK-3 expression. An evaluation of P301L mouse brain by [3H]/[11C]OCM-44 delineated differences in the B max of GSK-3 between the control and transgenic mice within male subjects. PET imaging showed similar trends to those observed in vitro. Sex differences are revealed as a potential parameter to consider in the development of GSK-3-targeted diagnostics and therapeutics and could guide recruitment for clinical studies.

Accelerated antibody discovery targeting the SARS-CoV-2 spike protein for COVID-19 therapeutic potential.

BackgroundRapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in aggressive timelines.MethodsIn this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the severe acute respiratory syndrome coronavirus-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb™ mouse model for increased diversity.ResultsThe panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation.ConclusionsBy combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months.

Myelocytomatosis-Protein Arginine N-Methyltransferase 5 Axis Defines the Tumorigenesis and Immune Response in Hepatocellular Carcinoma.

HCC is a leading cause of cancer-related deaths globally with poor outcome and limited therapeutic options. Although the myelocytomatosis (MYC) oncogene is frequently dysregulated in HCC, it is thought to be undruggable. Thus, the current study aimed to identify the critical downstream metabolic network of MYC and develop therapies for MYC-driven HCC. Liver cancer was induced in mice with hepatocyte-specific disruption of Myc and control mice by administration of diethylnitrosamine. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses revealed that urinary dimethylarginine, especially symmetric dimethylarginine (SDMA), was increased in the HCC mouse model in an MYC-dependent manner. Analyses of human samples demonstrated a similar induction of SDMA in the urines from patients with HCC. Mechanistically, Prmt5, encoding protein arginine N-methyltransferase 5, which catalyzes SDMA formation from arginine, was highly induced in HCC and identified as a direct MYC target gene. Moreover, GSK3326595, a PRMT5 inhibitor, suppressed the growth of liver tumors in human MYC-overexpressing transgenic mice that spontaneously develop HCC. Inhibition of PRMT5 exhibited antiproliferative activity through up-regulation of the tumor suppressor gene Cdkn1b/p27, encoding cyclin-dependent kinase inhibitor 1B. In addition, GSK3326595 induced lymphocyte infiltration and major histocompatibility complex class II expression, which might contribute to the enhanced antitumor immune response. Combination of GSK3326595 with anti-programed cell death protein 1 (PD-1) immune checkpoint therapy (ICT) improved therapeutic efficacy in HCC. This study reveals that PRMT5 is an epigenetic executer of MYC, leading to repression of the transcriptional regulation of downstream genes that promote hepatocellular carcinogenesis, highlights a mechanism-based therapeutic strategy for MYC-driven HCC by PRMT5 inhibition through synergistically suppressed proliferation and enhanced antitumor immunity, and finally provides an opportunity to mitigate the resistance of "immune-cold" tumor to ICT.

The role of IL-32 in Bacillus Calmette-Guérin (BCG)-induced trained immunity in infections caused by different Leishmania spp.

BackgroundCells of the innate immune system undergo long-term functional reprogramming in response to Bacillus Calmette-Guérin (BCG) exposure via a process called trained immunity, conferring nonspecific protection to unrelated infections. Here, we investigate whether BCG-induced trained immunity is able to protect against infections caused by different Leishmania spp., protozoa that cause cutaneous and mucosal or visceral lesions. MethodsWe used training models of human monocytes with BCG and subsequent infection by L. braziliensis, L. amazonensis and L. infantum, and the vaccination of wild-type and transgenic mice for IL-32γ before in vivo challenge with parasites. ResultsWe demonstrated that monocytes trained with BCG presented enhanced ability to kill L. braziliensis, L. amazonensis and L. infantum through increased production of reactive oxygen species. Interleukin (IL)-32 appears to play an essential role in the development of trained immunity. Indeed, BCG exposure induced IL-32 production in human primary monocytes, both mRNA and protein. We have used a human IL-32γ transgenic mouse model (IL-32γTg) to study the effect of BCG vaccination in different Leishmania infection models. BCG vaccination decreased lesion size and parasite load in infections caused by L. braziliensis and reduced the spread of L. amazonensis to other organs in both infected wild-type (WT) and IL-32γTg mice. In addition, BCG reduced the parasite load in the spleen, liver and bone marrow of both WT and IL-32γTg mice infected with L. infantum. BCG vaccination increased inflammatory infiltrate in infected tissues caused by different Leishmania spp. In all infections, the presence of IL-32γ was not mandatory, but it increased the protective and inflammatory effects of BCG-induced training. ConclusionsBCG's ability to train innate immune cells, providing protection against leishmaniasis, as well as the participation of IL-32γ in this process, pave the way for new treatment strategies for this neglected infectious disease.

Abstract LB143: BNZ-2, a dual specific IL15/IL21 inhibitor, rescues humanized NOG-IL15 transgenic mice from intestinal acute graft versus host disease without disrupting NK and CD8 T cell engraftment

Abstract Advances in adoptive transfer immunotherapy have found beneficial effects for Interleukin-15 (IL-15) in the graft versus tumor (GvT) activity of NK and cytotoxic T cells. However, the benefits of this activity must be weighed against the increased risk of acute graft versus host disease (GvHD) in patients. We propose that BNZ-2, a peptide antagonist of IL-15 and IL-21 signaling, as a therapeutic option for GvHD that may relieve intestinal pathologies while retaining adoptive transfer activity. As a novel model of intestinal inflammatory disease, we have found that humanization with peripheral blood mononuclear cells (PBMCs) of NOG mice with transgenic expression of human IL-15 (NOG-IL15) consistently catalyzes the onset of intestinal GvHD within twenty days of engraftment. To investigate whether BNZ-2 can inhibit IL-15-catalized intestinal GvHD we treated NOG-IL15 mice and assessed the degree of intestinal GvHD and the immunophenotype of engrafted cells following twenty days of humanization with four million unmanipulated PBMCs. BNZ-2 treatment inhibited with equal efficacy to anti-IL-15 the localization of immune cells to the intestinal lamina propria protecting the tissue from inflammation-induced loss of tissue integrity. However, BNZ-2 treatment retained the systemic engraftment of the blood and spleen, including NK and CD8 cytotoxic T-cells. These data support BNZ-2 as a therapeutic candidate for GvHD treatment, describe a new model of intestinal GvHD, and suggest that a therapeutic window exists that separates intestinal GvHD pathologies from the benefits of adoptive cell transfer. Citation Format: Kevin R. Kipp, Nick Doerr, Laith Q. Al-Mawsawi, Woo Jae Kim, Adrian J. Giovannone, Nazli Azimi. BNZ-2, a dual specific IL15/IL21 inhibitor, rescues humanized NOG-IL15 transgenic mice from intestinal acute graft versus host disease without disrupting NK and CD8 T cell engraftment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB143.

Abstract 1629: IO-202, a first-in-class LILRB4 antagonist antibody, activates dendritic cells and inhibits solid tumor growth in preclinical studies

Abstract LILRB4, also known as ILT3, is an immune inhibitory transmembrane protein found on monocytes and antigen presenting cells, including dendritic cells (DCs). LILRB4 upregulation in DCs induces a tolerogenic phenotype that facilitates the generation of antigen-specific T regulatory cells. LILRB4 is also expressed on acute myeloid leukemia (AML) with monocytic differentiation, in which it promotes T cell suppression and tumor infiltration. IO-202 is a first-in-class LILRB4 antagonist antibody that is being evaluated in a Phase I trial (NCT04372433) for the treatment of AML and chronic myelomonocytic leukemia (CMML). RNA-seq data from TCGA database indicates that LILRB4 expression is upregulated in many solid tumor types. Therefore, the therapeutic potential of IO-202 in solid tumors was investigated in this study. Using computational biology approaches, we found that high LILRB4 expression in solid tumors from TCGA database is associated with macrophage infiltration in the tumor microenvironment. Flow cytometric analysis of cancer patients' blood and tumor samples confirmed LILRB4 expression on tumor-associated macrophages (TAMs) and on monocytic myeloid-derived suppressor cells (M-MDSCs) and DCs in tumor and periphery. These data raised the hypothesis that LILRB4 functions as a myeloid checkpoint and contributes to tumor immune evasion, not only in AML, but also in many solid tumor types. LILRB4 is not present in rodents. As such, the functional activity and anti-tumor efficacy of IO-202 were evaluated using human primary immune cells and immunocompetent LILRB4 transgenic mice, respectively. In healthy donor monocyte-derived DCs, IO-202 treatment promoted DC maturation, activation and an antigen presenting phenotype, as well as enhanced their ability to activate allogeneic T cells. In the radiation therapy (RT)-resistant Lewis lung carcinoma (LLC) syngeneic model, IO-202 in combination with RT resulted in tumor growth inhibition, which was associated with changes in the relative abundance of tumor-infiltrating immune cells, evidenced by a reduction in M-MDSCs and an increase in T cells, along with increased T cell activation. In conclusion, our data suggest that LILRB4 functions as a myeloid checkpoint in multiple cancer types and that IO-202, the first-in-class LILRB4 antagonist antibody, enhances dendritic cell function and T cell activation in vitro and anti-tumor immunity in a solid tumor model in vivo. These data demonstrate the therapeutic potential of IO-202 as a myeloid checkpoint inhibitor for solid tumors. Citation Format: Maria José Costa, Tao Huang, Jing-Tyan Ma, Caroline Bonnans, Azita Tabrizi, Xiaoling C. Liao, Paul Woodard, Benjamin P. Chen, Ching-Cheng Hsu, Mi Deng, Cheng Cheng Zhang, An Song. IO-202, a first-in-class LILRB4 antagonist antibody, activates dendritic cells and inhibits solid tumor growth in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1629.

Abstract 1871: A differentiated CD73 blocking antibody with a unique intra-dimer binding mechanism for cancer immunotherapy

Abstract Introduction: CD73 is an emerging target for cancer immunotherapy owing to its critical role for the generation of an adenosine-enriched tumor microenvironment. Unlike other CD73 antibodies which adopt bivalent binding to two separate CD73 dimers at the N-terminal domain (termed inter-dimer binding), typically resulting in a “hook effect”, we describe a differentiated CD73 antibody, TJD5, that achieves full inhibition through monovalent binding to the C-terminal domain of a single CD73 dimer (termed intra-dimer binding), leading to a more favorable pharmacologic profile. Methods: TJD5 was discovered through a series of screening assays, including CD73 binding and enzyme activity inhibition. The critical residues for antibody binding were identified by a shotgun mutagenesis library and structure modeling. We are further analyzing the structure of TJD5 Fab and CD73 dimer complex by cryogenic electron microscopy (cryoEM). The inhibitory potency of TJD5 was compared with other antibodies in both in vitro enzymatic assay and in situ histochemical assay. We also profiled the immuno-modulatory properties of TJD5 via in vitro T cell and B cell activation assays as well as anti-tumor activities in human CD73 transgenic mouse and tumor xenograft model. Results: Comparing with other CD73 antibodies with epitopes in the N-terminal region which reportedly adopt an inter-dimer binding mode to separate CD73 dimers, TJD5 binds to the outside edge of the CD73 dimer interaction surface at the C-terminal domain. While we are analyzing the TJD5:CD73 complex by cryoEM, modeling data indicate an intra-dimer binding of TJD5 to a single CD73 dimer which confers the antibody with two unique features. First, TJD5 showed a stronger potency with the ability to completely inhibit CD73 enzyme as opposed to other antibodies exhibited a “hook effect” as manifested by a U-shaped inhibition curve. Second, TJD5 Fab could also inhibit CD73 activity, indicating a monovalent interaction is sufficient for the inhibition whereas others require a bivalent interaction. Consistently, a complete inhibition of intra-tumor CD73 activity by TJD5 could be demonstrated in vivo. Immunologically, in addition to the reversal of adenosine mediated T cell suppression, TJD5 directly increased B cell activation and antigen presentation as evidenced by upregulation of CD69, CD83, CD86 and HLA-DR that was unaffected by the addition of adenosine. Combination of TJD5 with a PD-(L)1 antibody showed synergistic anti-tumor activities both in vitro and in vivo. Conclusion: TJD5 is a humanized anti-CD73 antibody with a unique C-terminal domain epitope that enables a novel intra-dimer binding mode, which strongly differentiates it from other CD73 antibodies both in terms of mechanism of action and inhibitory potency. TJD5 is now in phase I clinical development both in the US and China to validate these differentiated properties and anti-tumor activity. Citation Format: Zhengyi Wang, Wei Cao, Yanni Zhang, Cong Xu, Yuan Meng, Taylor B. Guo. A differentiated CD73 blocking antibody with a unique intra-dimer binding mechanism for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1871.

Pentraxin 3 is a stromally-derived biomarker for detection of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC), characterized by dense desmoplastic stroma laid down by pancreatic stellate cells (PSC), has no reliable diagnostic biomarkers for timely detection. A multi-center cohort of PDAC patients and controls (chronic pancreatitis, intra-ductal papillary neoplasms, gallstones and otherwise healthy) donated serum in an ethically approved manner. Serum PTX3 above 4.34 ng/mL has a higher sensitivity (86%, 95% confidence interval (CI): 65–97%) and specificity (86%, 95% CI: 79–91%), positive predictive value (97%) and likelihood ratio (6.05), and is superior when compared to serum CA19-9 and CEA for detection of PDAC. In vitro and ex vivo analyses of PTX3, in human PDAC samples, PSCs, cell lines and transgenic mouse model for PDAC, suggest that PTX3 originates from stromal cells, mainly PSC. In activated PSC, PTX3 secretion could be downregulated by rendering PSC quiescent using all-trans-retinoic acid (ATRA). PTX3 organizes hyaluronan in conjunction with tumor necrosis factor-stimulated gene 6 (TSG-6) and facilitates stellate and cancer cell invasion. In SCALOP clinical trial (ISRCTN96169987) testing chemo-radiotherapy without stromal targeting, PTX3 had no prognostic or predictive role. However, in STARPAC clinical trial (NCT03307148), stromal modulation by ATRA even at first dose is accompanied with serum PTX3 response in patients who later go on to demonstrate disease control but not those in whom the disease progresses. PTX3 is a putative stromally-derived biomarker for PDAC which warrants further testing in prospective, larger, multi-center cohorts and within clinical trials targeting stroma.

Pathological tau drives ectopic nuclear speckle scaffold protein SRRM2 accumulation in neuron cytoplasm in Alzheimer\u2019s disease

Several conserved nuclear RNA binding proteins (sut-1, sut-2, and parn-2) control tau aggregation and toxicity in C. elegans, mice, and human cells. MSUT2 protein normally resides in nuclear speckles, membraneless organelles composed of phase-separated RNAs and RNA-binding proteins that mediate critical steps in mRNA processing including mRNA splicing. We used human pathological tissue and transgenic mice to identify Alzheimer’s disease-specific cellular changes related to nuclear speckles. We observed that nuclear speckle constituent scaffold protein SRRM2 is mislocalized and accumulates in cytoplasmic lesions in AD brain tissue. Furthermore, progression of tauopathy in transgenic mice is accompanied by increasing mislocalization of SRRM2 from the neuronal nucleus to the soma. In AD brain tissue, SRRM2 mislocalization associates with increased severity of pathological tau deposition. These findings suggest potential mechanisms by which pathological tau impacts nuclear speckle function in diverse organisms ranging from C. elegans to mice to humans. Future translational studies aimed at restoring nuclear speckle homeostasis may provide novel candidate therapeutic targets for pharmacological intervention.

Evaluation of ROR1 Targeted Antibody Drug Conjugates in Acute and Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Introduction: Receptor tyrosine kinase like orphan receptor 1 (ROR1) is a developmentally restricted receptor tyrosine kinase that is expressed on the surface of multiple hematological and solid malignancies but not on normal adult tissues. Recently, there has been an increased interest in ROR1 as a potential therapeutic target in multiple ROR1 positive leukemia and lymphomas. In this study, we present novel anti-ROR1 monoclonal antibody drug conjugates (ADCs) based on chimeric rabbit/human mAb “XBR1-402” or its humanized version “huXBR1-402”, that show both in vitro and in vivo efficacy at eliminating ROR1 positive chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and pre B cell acute lymphocytic leukemia (pre B cell ALL) leukemic cells.Methods: From our studies, we saw no difference in activity between XBR1-402-PNU and huXBR1-402-PNU. XBR1-402-PNU was used for our studies in pre B cell ALL and huXBR1-402-PNU was used for our studies in MCL and CLL. For ALL in vitro experiments, we monitored cytotoxicity in Kasumi2 and 697 cell lines. For MCL in vitro experiments we monitored cytotoxicity in Jeko and Mino cell lines. For CLL in vitro experiments, we monitored cytotoxicity in primary CLL samples. For ALL in vivo experiments we monitored overall survival in a murine disseminated 697 model. For CLL in vivo experiments we monitored ROR1+ leukemic cell depletion in human ROR1 transgenic mice engrafted with human ROR1+TCL1 leukemia.Results: We observed a decrease in viability for all XBR1-402-PNU and huXBR1-402-PNU treated ROR1 positive leukemic cell lines and primary cells in vitro . In the in vitro model of pre B cell ALL we observed a significant decrease in viability of the ROR1 positive pre B cell ALL cell lines 697 and Kasumi-2 when treated with XBR1-402-PNU(EC50 = 77 and 1 ng/mL, respectively) when compared to treatment with the isotype-matched control ADC Trastuzumab-PNU (Tras-PNU) (EC50 = 2987 and 5330 ng/mL, respectively). In the in vitro model of MCL, we observed a decrease in viability for huXBR1-402-PNU treated ROR1 positive Jeko and Mino cell lines but not ROR1 negative Mec-1 and Jurkat cell lines when compared to the unconjugated huXBR1-402 antibody or the control Tras-PNU. Additionally, we tested primary CLL samples confirmed to be positive for ROR1 and found a significant decrease in viability over 96 hours when treated with the antibody-drug-conjugate huXBR1-402 (78.6 + 6.1%)as compared to the unconjugated antibody (100% + 2.4; p=0.022, N=7) and the control Tras-PNU (87.4% +/- 6.1; p=0.038). We have also detected promising in vivo activity with XBR1-402-PNU and huXBR1-402-PNU in ALL and CLL models, respectively. In vivo studies on a murine disseminated 697 ALL model showed that treatment with XBR1-402-PNU increased overall survival when compared to treatment with Tras-PNU control (Median survival of 32 and 23 days after implantation respectively, p=0.021, n=6). In addition, in preliminarily experiments, we have tested depletion of human ROR1+ leukemic cells and normal B cells in vivo using human ROR1 transgenic mice engrafted with human ROR+TCL1 leukemia and observed a preferential depletion of ROR1+TCL1 leukemic cells upon treatment with huXBR1-402-PNU compared to control Tras-PNU.Conclusion: Our results suggest that anti-ROR1 ADC huXBR1-402-PNU is an effective and promising targeted cytotoxic therapy for ROR1 positive leukemic cells of CLL, MCL, and pre B ALL and warrants further evaluation for clinical consideration in patients with ROR1 positive leukemia and lymphomas. Current long-term studies are ongoing to evaluate the in vivo cytotoxic effects of huXBR1-402-PNU on total tumor burden, disease progression, and overall survival.(EH is supported by Graduate Pelotonia Fellowship) DisclosuresWaldmeier:NBE-Therapeutics Ltd: Employment. Beerli:NBE-Therapeutics Ltd: Employment. Grawunder:NBE-Therapeutics Ltd: Employment. Byrd:The Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding.

Blockade of IL-2 and IL-15 Gamma Chain Receptor Signaling Decreases Leukemic Cell Viability in T-Cell Large Granular Lymphocyte Leukemia and Adult T-Cell Leukemia

IntroductionInterleukin-15 (IL-15) and IL-2 are important cytokines that drive the progression of many T-cell malignancies such as T-cell Large Granular Lymphocyte Leukemia (T-LGLL) and HTLV-1 derived Adult T-cell Leukemia (ATL).T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with a constitutively active JAK/STAT pathway controlled by cytokines including IL-15 and IL-2. A computational systems approach identified IL-15 as a master regulator of T-LGLL survival and patients have increased IL-15/IL-15Ra serum levels. Cytokine signaling leads to increased phosphorylation and activation of STAT proteins. T-LGLL cells exhibit STAT3 or STAT5 mutational activation in some cases and constitutive activation in all patients. T-LGLL treatments are not selective or curative, and the commonly utilized immunosuppressive agents are not effective in all patients.ATL is an aggressive T-cell disorder that also exhibits aberrant IL-2 and IL-15 cytokine signaling. Tax, identified as an essential oncogene in the disease, is associated with increased cytokine and cytokine receptor expression. ATL patients have high IL-2Ra gene expression and PBMCs from chronic/smoldering ATL patients can depend on autocrine production of cytokines for proliferation. Patients with ATL have poor prognosis and current cytokine-targeting therapies have not proven to be efficacious.The gc receptor is shared by the gc-family cytokines including IL-2, -4, -7, -9, -15 and -21. BNZ-1 (formerly known as BNZ132-1-40) is a pegylated peptide designed to specifically bind the gc receptor to block IL-2, IL-15 and, to a lesser extent, IL-9 cytokine signaling. Because these cytokines are vital to the survival of T cells in T-LGLL and ATL, these diseases represent ideal models to test the hypothesis that treatment of leukemic cells with BNZ-1 will result in reduced proliferation and viability.MethodsThese studies utilized a human T-LGL cell line (TL1), PBMCs from T-LGLL patients and ATL patients, and human IL-15 transgenic NSG mice injected with the ED40515(+) ATL cell line expressing luciferase. In vitro assays were performed after an overnight cytokine withdrawal and 20-minute pretreatment with BNZ-1 prior to addition of cytokine. Leukemic mice were divided into 3 treatment groups; BNZ-1 (40 mg/kg i.v. 2x per week for 4 weeks), JAK1/2 inhibitor Ruxolitinib (Ruxo) (50 mg/kg/day s.c. by pump for 28 days) and PBS control. Tumor burden was measured through bioluminescence imaging (Xenogen IVIS).ResultsOur results demonstrate that in vitro treatment of TL1 cells with BNZ-1 inhibits IL-2 and IL-15 mediated expansion and viability. BNZ-1 impaired the growth of these cytokine-dependent cells to the level observed in cells without exogenous cytokine supplementation. In addition, BNZ-1 was able to drastically block IL-15 and IL-2 induced phosphorylation events, specifically STAT1, STAT3, STAT5, ERK and Akt. The compound blocked IL-15 mediated viability and signaling more effectively than that mediated by IL-2.The BNZ-1 mediated decreased viability of PBMCs from T-LGLL patients was observed in the presence of either IL-2 or IL-15 stimulation and regardless of STAT3 mutational status. A potent reduction of IL-2 and IL-15 mediated STAT1, STAT3, STAT5 and ERK phosphorylation was also observed in T-LGLL PBMCs with BNZ-1 treatment. Proliferation of PBMCs from ATL patients was inhibited by 58 to 80 percent by the addition of either the antiIL-2Ra antibody daclizumab or and the combination treatment of daclizumab, anti-IL-15 and anti-IL-9. BNZ-1 at 10 µM inhibitedproliferation by 60 to 80 percent.Leukemic burden in the ATL tumor bearing mice was reduced by both BNZ-1 and Ruxo when compared to controls. The BNZ-1 treatment group showed comparable efficacy to that observed in the Ruxo group. Furthermore, BNZ-1 therapy was associated with lower levels of IL-2Ra, which reflects reduced tumor burden at 4 and 7 weeks when compared to that observed with the PBS group.In summary, BNZ-1 shows promise as a potential therapy for T-LGLL and ATL. Results from this study support its advancement in clinical trials in the treatment of T-LGLL and ATL patients. DisclosuresTagaya:Bioniz Therapeutics: Employment. Azimi:Bioniz Therapeutics: Employment.

Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys.

The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.

Contribution of SARS-CoV-2 Accessory Proteins to Viral Pathogenicity in K18 Human ACE2 Transgenic Mice

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University’s COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, −6, −7a, −7b, and −8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19.IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/β-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy

An amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3P209L-eGFP mice. The mutation renders hBAG3P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption.