T cell-dependent bispecific antibodies alter organ-specific endothelial cell-T cell interaction.

Preclinical and clinical studies demonstrate that T cell-dependent bispecific antibodies (TDBs) induce systemic changes in addition to tumor killing, leading to adverse events. Here, we report an in-depth characterization of acute responses to TDBs in tumor-bearing mice. Contrary to modest changes in tumors, rapid and substantial lymphocyte accumulation and endothelial cell (EC) activation occur around large blood vessels in normal organs including the liver. We hypothesize that organ-specific ECs may account for the differential responses in normal tissues and tumors, and we identify a list of genes selectively upregulated by TDB in large liver vessels. Using one of the genes as an example, we demonstrate that CD9 facilitates ICAM-1 to support T cell-EC interaction in response to soluble factors released from a TDB-mediated cytotoxic reaction. Our results suggest that multiple factors may cooperatively promote T cell infiltration into normal organs as a secondary response to TDB-mediated tumor killing. These data shed light on how different vascular beds respond to cancer immunotherapy and may help improve their safety and efficacy.

Automated tumor immunophenotyping predicts clinical benefit from anti-PD-L1 immunotherapy.

Cancer immunotherapy has transformed the clinical approach to patients with malignancies, as profound benefits can be seen in a subset of patients. To identify this subset, biomarker analyses increasingly focus on phenotypic and functional evaluation of the tumor microenvironment to determine if density, spatial distribution, and cellular composition of immune cell infiltrates can provide prognostic and/or predictive information. Attempts have been made to develop standardized methods to evaluate immune infiltrates in the routine assessment of certain tumor types; however, broad adoption of this approach in clinical decision-making is still missing. We developed approaches to categorize solid tumors into 'desert', 'excluded', and 'inflamed' types according to the spatial distribution of CD8+ immune effector cells to determine the prognostic and/or predictive implications of such labels. To overcome the limitations of this subjective approach, we incrementally developed four automated analysis pipelines of increasing granularity and complexity for density and pattern assessment of immune effector cells. We show that categorization based on 'manual' observation is predictive for clinical benefit from anti-programmed death ligand 1 therapy in two large cohorts of patients with non-small cell lung cancer or triple-negative breast cancer. For the automated analysis we demonstrate that a combined approach outperforms individual pipelines and successfully relates spatial features to pathologist-based readouts and the patient's response to therapy. Our findings suggest that tumor immunophenotype generated by automated analysis pipelines should be evaluated further as potential predictive biomarkers for cancer immunotherapy. © 2024 The Pathological Society of Great Britain and Ireland.

Assessment of Color Reproducibility and Mitigation of Color Variation in Whole Slide Image Scanners for Toxicologic Pathology.

Digital pathology workflows in toxicologic pathology rely on whole slide images (WSIs) from histopathology slides. Inconsistent color reproduction by WSI scanners of different models and from different manufacturers can result in different color representations and inter-scanner color variation in the WSIs. Although pathologists can accommodate a range of color variation during their evaluation of WSIs, color variability can degrade the performance of computational applications in digital pathology. In particular, color variability can compromise the generalization of artificial intelligence applications to large volumes of data from diverse sources. To address these challenges, we developed a process that includes two modules: (1) assessing the color reproducibility of our scanners and the color variation among them and (2) applying color correction to WSIs to minimize the color deviation and variation. Our process ensures consistent color reproduction across WSI scanners and enhances color homogeneity in WSIs, and its flexibility enables easy integration as a post-processing step following scanning by WSI scanners of different models and from different manufacturers.

Obinutuzumab Effectively Depletes Key B-cell Subsets in Blood and Tissue in End-stage Renal Disease Patients.

The THEORY study evaluated the effects of single and multiple doses of obinutuzumab, a type 2 anti-CD20 antibody that induces antibody-dependent cell-mediated cytotoxicity and direct cell death, in combination with standard of care in patients with end-stage renal disease. Methods: We measured B-cell subsets and protein biomarkers of B-cell activity in peripheral blood before and after obinutuzumab administration in THEORY patients, and B-cell subsets in lymph nodes in THEORY patients and an untreated comparator cohort. Results: Obinutuzumab treatment resulted in a rapid loss of B-cell subsets (including naive B, memory B, double-negative, immunoglobulin D+ transitional cells, and plasmablasts/plasma cells) in peripheral blood and tissue. This loss of B cells was associated with increased B cell-activating factor and decreased CXCL13 levels in circulation. Conclusions: Our data further characterize the mechanistic profile of obinutuzumab and suggest that it may elicit greater efficacy in indications such as lupus where B-cell targeting therapeutics are limited by the resistance of pathogenic tissue B cells to depletion.

Population-wide gene disruption in the murine lung epithelium via AAV-mediated delivery of CRISPR-Cas9 components.

With the aim of expediting drug target discovery and validation for respiratory diseases, we developed an optimized method for in situ somatic gene disruption in murine lung epithelial cells via AAV6-mediated CRISPR-Cas9 delivery. Efficient gene editing was observed in lung type II alveolar epithelial cells and distal airway cells following assessment of single- or dual-guide AAV vector formats, Cas9 variants, and a sequential dosing strategy with combinatorial guide RNA expression cassettes. In particular, we were able to demonstrate population-wide gene disruption within distinct epithelial cell types for separate targets in Cas9 transgenic animals, with minimal to no associated inflammation. We also observed and characterized AAV vector integration events that occurred within directed double-stranded DNA break sites in lung cells, highlighting a complicating factor with AAV-mediated delivery of DNA nucleases. Taken together, we demonstrate a uniquely effective approach for somatic engineering of the murine lung, which will greatly facilitate the modeling of disease and therapeutic intervention.

Distinct resistance mechanisms arise to allosteric vs. ATP-competitive AKT inhibitors.

The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition.

Whole-body CD8+ T cell visualization before and during cancer immunotherapy: a phase 1/2 trial.

Immune checkpoint inhibitors (ICIs), by reinvigorating CD8+ T cell mediated immunity, have revolutionized cancer therapy. Yet, the systemic CD8+ T cell distribution, a potential biomarker of ICI response, remains poorly characterized. We assessed safety, imaging dose and timing, pharmacokinetics and immunogenicity of zirconium-89-labeled, CD8-specific, one-armed antibody positron emission tomography tracer 89ZED88082A in patients with solid tumors before and ~30 days after starting ICI therapy (NCT04029181). No tracer-related side effects occurred. Positron emission tomography imaging with 10 mg antibody revealed 89ZED88082A uptake in normal lymphoid tissues, and tumor lesions across the body varying within and between patients two days after tracer injection (n = 38, median patient maximum standard uptake value (SUVmax) 5.2, IQI 4.0-7.4). Higher SUVmax was associated with mismatch repair deficiency and longer overall survival. Uptake was higher in lesions with stromal/inflamed than desert immunophenotype. Tissue radioactivity was localized to areas with immunohistochemically confirmed CD8 expression. Re-imaging patients on treatment showed no change in average (geometric mean) tumor tracer uptake compared to baseline, but individual lesions showed diverse changes independent of tumor response. The imaging data suggest enormous heterogeneity in CD8+ T cell distribution and pharmacodynamics within and between patients. In conclusion, 89ZED88082A can characterize the complex dynamics of CD8+ T cells in the context of ICIs, and may inform immunotherapeutic treatments.

The peptide symporter SLC15a4 is essential for the development of systemic lupus erythematosus in murine models.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease representing a serious unmet medical need. The disease is associated with the loss of self-tolerance and exaggerated B cell activation, resulting in autoantibody production and the formation of immune complexes that accumulate in the kidney, causing glomerulonephritis. TLR7, an important mediator of the innate immune response, drives the expression of type-1 interferon (IFN), which leads to expression of type-1 IFN induced genes and aggravates lupus pathology. Because the lysosomal peptide symporter slc15a4 is critically required for type-1 interferon production by pDC, and for certain B cell functions in response to TLR7 and TLR9 signals, we considered it as a potential target for pharmacological intervention in SLE. We deleted the slc15a4 gene in C57BL/6, NZB, and NZW mice and found that pristane-challenged slc15a4-/- mice in the C57BL/6 background and lupus prone slc15a4-/- NZB/W F1 mice were both completely protected from lupus like disease. In the NZB/W F1 model, protection persisted even when disease development was accelerated with an adenovirus encoding IFNα, emphasizing a broad role of slc15a4 in disease initiation. Our results establish a non-redundant function of slc15a4 in regulating both innate and adaptive components of the immune response in SLE pathobiology and suggest that it may be an attractive drug target.

Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.