Species-specific inflammatory responses as a primary component for the development of glomerular lesions in mice and monkeys following chronic administration of a second-generation antisense oligonucleotide.

Chronic administration of drisapersen, a 2'-OMe phosphorothioate antisense oligonucleotide (AON) to mice and monkeys resulted in renal tubular accumulation, with secondary tubular degeneration. Glomerulopathy occurred in both species with species-specific characteristics. Glomerular lesions in mice were characterized by progressive hyaline matrix accumulation, accompanied by the presence of renal amyloid and with subsequent papillary necrosis. Early changes involved glomerular endothelial hypertrophy and degeneration, but the chronic glomerular amyloid and hyaline alterations in mice appeared to be species specific. An immune-mediated mechanism for the glomerular lesions in mice was supported by early inflammatory changes including increased expression of inflammatory cytokines and other immunomodulatory genes within the renal cortex, increased stimulation of CD68 protein, and systemic elevation of monocyte chemotactic protein 1. In contrast, kidneys from monkeys given drisapersen chronically showed less severe glomerular changes characterized by increased mesangial and inflammatory cells, endothelial cell hypertrophy, and subepithelial and membranous electron-dense deposits, with ultrastructural and immunohistochemical characteristics of complement and complement-related fragments. Lesions in monkeys resembled typical features of C3 glomerulopathy, a condition described in man and experimental animals to be linked to dysregulation of the alternative complement pathway. Thus, inflammatory/immune mechanisms appear critical to glomerular injury with species-specific sensitivities for mouse and monkey. The lower observed proinflammatory activity in humans as compared to mice and monkeys may reflect a lower risk of glomerular injury in patients receiving AON therapy.

Characterization of a novel potency endpoint for the evaluation of immune checkpoint blockade in humanized mice.

Introduction: Humanized mice are emerging as valuable models to experimentally evaluate the impact of different immunotherapeutics on the human immune system. These immunodeficient mice are engrafted with human cells or tissues, that then mimic the human immune system, offering an alternative and potentially more predictive preclinical model. Immunodeficient NSG mice engrafted with human CD34+ cord blood stem cells develop human T cells educated against murine MHC. However, autoimmune graft versus host disease (GvHD), mediated by T cells, typically develops 1 year post engraftment. Methods: Here, we have used the development of GvHD in NSG mice, using donors with HLA alleles predisposed to autoimmunity (psoriasis) to weight in favor of GvHD, as an endpoint to evaluate the relative potency of monoclonal and BiSpecific antibodies targeting PD-1 and CTLA-4 to break immune tolerance. Results: We found that treatment with either a combination of anti-PD-1 & anti-CTLA-4 mAbs or a quadrivalent anti-PD-1/CTLA-4 BiSpecific (MEDI8500), had enhanced potency compared to treatment with anti-PD-1 or anti-CTLA-4 monotherapies, increasing T cell activity both in vitro and in vivo. This resulted in accelerated development of GvHD and shorter survival of the humanized mice in these treatment groups commensurate with their on target activity. Discussion: Our findings demonstrate the potential of humanized mouse models for preclinical evaluation of different immunotherapies and combinations, using acceleration of GvHD development as a surrogate of aggravated antigenic T-cell response against host.

SARS-CoV-2 Spike Protein Expression In Vitro and Hematologic Effects in Mice Vaccinated With AZD1222 (ChAdOx1 nCoV-19).

Severe COVID-19 can be associated with a prothrombotic state, increasing risk of morbidity and mortality. The SARS-CoV-2 spike glycoprotein is purported to directly promote platelet activation via the S1 subunit and is cleaved from host cells during infection. High plasma concentrations of S1 subunit are associated with disease progression and respiratory failure during severe COVID-19. There is limited evidence on whether COVID-19 vaccine-induced spike protein is similarly cleaved and on the immediate effects of vaccination on host immune responses or hematology parameters. We investigated vaccine-induced S1 subunit cleavage and effects on hematology parameters using AZD1222 (ChAdOx1 nCoV-19), a simian, replication-deficient adenovirus-vectored COVID-19 vaccine. We observed S1 subunit cleavage in vitro following AZD1222 transduction of HEK293x cells. S1 subunit cleavage also occurred in vivo and was detectable in sera 12 hours post intramuscular immunization (1x1010 viral particles) in CD-1 mice. Soluble S1 protein levels decreased within 3 days and were no longer detectable 7-14 days post immunization. Intravenous immunization (1x109 viral particles) produced higher soluble S1 protein levels with similar expression kinetics. Spike protein was undetectable by immunohistochemistry 14 days post intramuscular immunization. Intramuscular immunization resulted in transiently lower platelet (12 hours) and white blood cell (12-24 hours) counts relative to vehicle. Similarly, intravenous immunization resulted in lower platelet (24-72 hours) and white blood cell (12-24 hours) counts, and increased neutrophil (2 hours) counts. The responses observed with either route of immunization represent transient hematologic changes and correspond to expected innate immune responses to adenoviral infection.

Comprehensive mutagenesis identifies the peptide repertoire of a p53 T-cell receptor mimic antibody that displays no toxicity in mice transgenic for human HLA-A*0201.

T-cell receptor mimic (TCRm) antibodies have expanded the repertoire of antigens targetable by monoclonal antibodies, to include peptides derived from intracellular proteins that are presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface. We have previously used this approach to target p53, which represents a valuable target for cancer immunotherapy because of the high frequency of its deregulation by mutation or other mechanisms. The T1-116C TCRm antibody targets the wild type p5365-73 peptide (RMPEAAPPV) presented by HLA-A*0201 (HLA-A2) and exhibited in vivo efficacy against triple receptor negative breast cancer xenografts. Here we report a comprehensive mutational analysis of the p53 RMPEAAPPV peptide to assess the T1-116C epitope and its peptide specificity. Antibody binding absolutely required the N-terminal arginine residue, while amino acids in the center of the peptide contributed little to specificity. Data mining the immune epitope database with the T1-116C binding consensus and validation of peptide recognition using the T2 stabilization assay identified additional tumor antigens targeted by T1-116C, including WT1, gp100, Tyrosinase and NY-ESO-1. Most peptides recognized by T1-116C were conserved in mice and human HLA-A2 transgenic mice showed no toxicity when treated with T1-116C in vivo. We conclude that comprehensive validation of TCRm antibody target specificity is critical for assessing their safety profile.

Developmental and reproductive safety of AZD1222 (ChAdOx1 nCoV-19) in mice.

AZD1222 (ChAdOx1 nCoV-19) is a COVID-19 vaccine that is not yet licensed for use during pregnancy. To support the inclusion of pregnant and breastfeeding people in AZD1222 clinical studies, a non-clinical developmental and reproductive toxicity study was performed to evaluate its effects on fertility and reproductive processes of female CD-1 mice during the embryofetal development phase, and postnatal outcomes during the littering phase. Immunogenicity assessments were also made in dams, fetuses, and pups. There were no vaccine-related unscheduled deaths throughout the study. Furthermore, there were no vaccine-related effects on female reproduction, fetal or pup survival, fetal external, visceral, or skeletal findings, pup physical development, and no abnormal gross pathology findings in pups or dams. Antibody responses raised in dams were maintained throughout gestation and postnatal periods, and seroconversion in fetuses and pups indicate placental and lactational transfer of immunoglobulins. Together with clinical data from non-pregnant people, these results support the inclusion of pregnant and breastfeeding people in AZD1222 clinical studies.

CRISPR/Cas9-Mediated Foxp1 Silencing Restores Immune Surveillance in an Immunocompetent A20 Lymphoma Model.

The interaction of lymphoma cells with their microenvironment has an important role in disease pathogenesis and is being actively pursued therapeutically using immunomodulatory drugs, including immune checkpoint inhibitors. Diffuse large B-cell lymphoma (DLBCL) is an aggressive high-grade disease that remains incurable in ~40% of patients treated with R-CHOP immunochemotherapy. The FOXP1 transcription factor is abundantly expressed in such high-risk DLBCL and we recently identified its regulation of immune response signatures, in particular, its suppression of the cell surface expression of major histocompatibility class II (MHC-II), which has a critical role in antigen presentation to T cells. Using CRISPR/Cas9 genome editing we have depleted Foxp1 expression in the aggressive murine A20 lymphoma cell line. When grown in BALB/c mice, this cell line provides a high-fidelity immunocompetent disseminated lymphoma model that displays many characteristics of human DLBCL. Transient Foxp1-depletion using siRNA, and stable depletion using CRISPR (generated by independently targeting Foxp1 exon six or seven) upregulated cell surface I-Ab (MHC-II) expression without impairing cell viability in vitro. RNA sequencing of Foxp1-depleted A20 clones identified commonly deregulated genes, such as the B-cell marker Cd19, and hallmark DLBCL signatures such as MYC-targets and oxidative phosphorylation. Immunocompetent animals bearing Foxp1-depleted A20 lymphomas showed significantly-improved survival, and 20% did not develop tumors; consistent with modulating immune surveillance, this was not observed in immunodeficient NOD SCIDγ mice. The A20 Foxp1 CRISPR model will help to further characterize the contribution of Foxp1 to lymphoma immune evasion and the potential for Foxp1 targeting to synergize with other immunotherapies.

MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potential.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. Targeting and agonizing GITR in mice promotes antitumor immunity by enhancing the function of effector T cells and inhibiting regulatory T cells. Here, we describe MEDI1873, a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerization domain and the human GITRL extracellular domain (ECD). MEDI1873 was optimized through systematic testing of different trimerization domains, aglycosylation of the GITRL ECD and comparison of different Fc isotypes. MEDI1873 exhibits oligomeric heterogeneity and superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Further, it recapitulates, in vitro, several aspects of GITR targeting described in mice, including modulation of regulatory T-cell suppression and the ability to increase the CD8+:CD4+ T-cell ratio via antibody-dependent T-cell cytotoxicity. To support translation into a therapeutic setting, we demonstrate that MEDI1873 is a potent T-cell agonist in vivo in non-human primates, inducing marked enhancement of humoral and T-cell proliferative responses against protein antigen, and demonstrate the presence of GITR- and FoxP3-expressing infiltrating lymphocytes in a range of human tumors. Overall our data provide compelling evidence that MEDI1873 is a novel, potent GITR agonist with the ability to modulate T-cell responses, and suggest that previously described GITR biology in mice may translate to the human setting, reinforcing the potential of targeting the GITR pathway as a therapeutic approach to cancer.