Background Several lines of evidence suggest the possibility that inhalation exposure to multi-walled carbon nanotubes (MWCNT) at occupationally relevant doses can lead to systemic immunotoxicity. To test this hypothesis, we undertook in-depth examination of immune function in mice and rats exposed by inhalation to relatively low levels of 1020 Long Multiwalled Carbon Nanotubes (L-MWNT-1020).Methods Studies were conducted to determine the systemic and pulmonary immunotoxic effects in mice and rats exposed to L-MWNT-1020 following whole-body inhalation for 6 h/day for 5 days/week for 30 (mice and rats) and 90 (mice) days at dose levels of 0, 0.06, 0.2, and 0.6 mg/m3. Additional groups were administered cyclophosphamide (CPS) as a positive control for each cohort. Following exposure, pulmonary macrophage phagocytosis, immunophenotypic analysis of immune cells populations in the spleen, and systemic immune function, including tests for humoral (T-dependent antibody response, TDAR), cell-mediated (cytotoxic T-lymphocyte [CTL] activity), and innate (Natural Killer [NK] cell activity) immunity were conducted.Results While exposure increased pulmonary macrophage activity, no major changes were observed in any of the systemic immune parameters measured in mice exposed for 30 or 90 days. In rats, there was a slight decrease in humoral immunity coinciding with an increase in the number of splenic T cell and NK cell populations.Conclusion Although pulmonary macrophage activity increased in mice following exposure to L-MWNT-1020, systemic immune function for the most part remained unaffected. In contrast, rats demonstrated a slight decrease in humoral immune function as well as an increase in spleen cell numbers, T cell, and NK cell populations suggesting species-specific effects on systemic immunity, however, these effects were small and their biological significance with respect to altering disease susceptibility is unclear.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

In 2005, the World Health Organization (WHO) re-evaluated Toxic Equivalency factors (TEFs) developed for dioxin-like compounds believed to act through the Ah receptor based on an updated database of relative estimated potency (REP)(REP2004 database). This re-evalution identified the need to develop a consistent approach for dose-response modeling. Further, the WHO Panel discussed the significant heterogeneity of experimental datasets and dataset quality underlying the REPs in the database. There is a critical need to develop a quantitative, and quality weighted approach to characterize the TEF for each congener. To address this, a multi-tiered approach that combines Bayesian dose-response fitting and meta-regression with a machine learning model to predict REPS' quality categorizations was developed to predict the most likely relationship between each congener and its reference and derive model-predicted TEF uncertainty distributions. As a proof of concept, this ‘Best-Estimate TEF workflow’ was applied to the REP2004 database to derive TEF point-estimates and characterizations of uncertainty for all congeners. Model-TEFs were similar to the 2005 WHO TEFs, with the data-poor congeners having larger levels of uncertainty. This transparent and reproducible computational workflow incorporates WHO expert panel recommendations and represents a substantial improvement in the TEF methodology.

Background: CD39 catalyzes the conversion of extracellular adenosine triphosphate (ATP) into adenosine monophosphate (AMP), resulting in decreased levels of immunostimulatory ATP and increased levels of immunosuppressive adenosine in the tumor microenvironment (TME). By blocking CD39 in the TME, particularly in combination with immunogenic cell death (ICD)-inducing chemotherapies, local levels of ATP can increase, leading to myeloid cell activation and improved tumor control. AB598 potently binds and inhibits enzymatic activity of human and cynomolgus CD39 but not murine CD39. Human CD39 knock-in (hCD39KI) mice have been employed to determine anti-tumor efficacy of AB598 in animals with competent immune systems. Methods: Binding affinity and enzymatic inhibition of AB598 were determined in primary human immune cells. ATP-mediated activation of monocyte-derived dendritic cells (moDCs) and macrophages were assayed in vitro with and without AB598. Whole blood and tissue-based receptor occupancy (RO) assays were developed using AB598-competitive and non-competitive anti-CD39 reagents. In vitro binding of AB598 was determined in samples from human and cynomolgus donors. CD39 protein expression, target engagement, and enzymatic inhibition were assessed in whole blood and tissue samples collected from AB598-dosed animals. CD39 expression patterns in peripheral immune subsets were confirmed in samples from healthy human donors. CD39 enzymatic activity was evaluated in blood-derived cells and serum from healthy human donors and cancer patients. Results: AB598 binds to CD39-expressing monocytes, B cells, and other immune cell populations in human and cynomolgus whole blood with sub-nanomolar affinity. Following AB598 dosing in preclinical species, decreases in CD39 surface expression on peripheral immune cells were observed, consistent with other anti-CD39 therapeutic antibodies. Target engagement and enzymatic inhibition were evident in tissues collected from AB598-dosed animals. Treatment with AB598/chemotherapy combinations resulted in significant syngeneic tumor control in hCD39KI mouse models. Conclusion: When used in combination with an ICD-inducing chemotherapy agent, AB598 can promote anti-tumor immunity by activation of myeloid cells due to increased local ATP levels. Preclinical characterization of the pharmacodynamic effects of AB598 support our therapeutic rationale and demonstrate target engagement and inhibition both peripherally and intratumorally. Citation Format: Amy E. Anderson, Angelo Kaplan, Urvi Vani, Enzo Stagnaro, Kaustubh Parashar, Julie Clor, Nigel P. Walker, Steve W. Young, Matthew J. Walters, Ester Fernandez-Salas, Christine E. Bowman, Lisa Seitz. Characterization of AB598, a therapeutic anti-human CD39 antibody for the treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5673.

The toxic equivalency factors (TEFs) approach for dioxin-like chemicals (DLCs) is currently based on a qualitative assessment of a heterogeneous data set of relative estimates of potency (REPs) spanning several orders of magnitude with highly variable study quality and relevance. An effort was undertaken to develop a weighting framework to systematically evaluate and quantitatively integrate the quality and relevance for development of more robust TEFs. Six main-study characteristics were identified as most important in characterizing the quality and relevance of an individual REP for human health risk assessment: study type, study model, pharmacokinetics, REP derivation method, REP derivation quality, and endpoint. Subsequently, a computational approach for quantitatively integrating the weighting framework parameters was developed and applied to the REP2004 database. This was accomplished using a machine learning approach which infers a weighted TEF distribution for each congener. The resulting database, weighted for quality and relevance, provides REP distributions from >600 data sets (including in vivo and in vitro studies, a range of endpoints, etc.). This weighted database provides a flexible platform for systematically and objectively characterizing TEFs for use in risk assessment, as well as providing information to characterize uncertainty and variability. Collectively, this information provides risk managers with information for decision making.

Background AB598 is being developed as a novel cancer immunotherapy which potently binds and inhibits CD39 enzymatic activity. CD39 catalyzes the conversion of extracellular adenosine triphosphate (ATP) into adenosine monophosphate (AMP), resulting in decreased amounts of immunostimulatory ATP and increased levels of immunosuppressive adenosine in the tumor microenvironment (TME). By blocking CD39 in the TME, local levels of ATP increase, leading to myeloid cell activation and improved tumor control. AB598 is highly potent and specific, binding and inhibiting human CD39 with sub-nanomolar potency. AB598 binds and inhibits both human and cynomolgus monkey CD39 but not murine CD39, presenting a challenge for studying CD39 inhibition in an immune-competent syngeneic tumor model. Methods Human CD39 knock-in (hCD39KI) mice were employed to examine the preclinical anti-tumor efficacy of AB598 in animals with a fully competent immune system. The use of a murine model with natural expression and distribution of human CD39, targetable by AB598, allowed for a more physiological assessment of CD39 inhibition in solid tumors compared to the alternative use of human cancer cells growing in immuno-deficient mice. Combination of CD39 inhibition with chemotherapy was explored. A murinized version of AB598, ch39_mIgG2a, which contains a murine Fc silent domain was used for in vivo studies. Results Real-time measurement of ATP showed the ability of oxaliplatin to induce ATP release in MC38 tumor cells in vitro. In a hCD39KI mouse MC38 tumor model, ch39_mIgG2a in combination with oxaliplatin significantly inhibited tumor growth compared to treatment with either single agent. The combination was well tolerated and no decreases in body weight were observed. Analysis of cytokines in the periphery resulted in no significant increases after ch39_mIgG2a administration. Analysis of tumors from both MC38 and 4T1 models revealed substantial inhibition of intratumoral CD39 enzymatic activity in ch39_mIgG2a-treated mice. Tumor draining lymph nodes in the MC38 model showed a decrease in cell surface CD39 in ch39_mIgG2a mice, a finding supported by peripheral receptor occupancy studies. Relative percentages of the immune cells in the lymph nodes were unaffected, suggesting internalization or downregulation, not cellular depletion, as the mechanism for the decrease in cell-surface CD39. Conclusions Our results indicate the superb ability of AB598 to inhibit enzymatic activity and tumor growth in vivo and provide a rationale for the combination of CD39 inhibition with ICD-inducing chemotherapy in the clinic.

TIGIT is an inhibitory receptor expressed primarily on NK and T cell subsets, and binding to its cognate receptor ligand, CD155, results in multiple mechanisms of immunosuppression. Blocking the TIGIT-CD155 interaction in the context of cancer promotes anti-tumor immunity. We characterized cellular subsets that TIGIT blockade may impact and the pharmacology of two anti-TIGIT antibodies - representing functional (AB308) and non-functional (AB154) Fc domain classes - undergoing clinical evaluation. Surrogate antibodies were leveraged to interrogate TIGIT biology in mouse syngeneic tumor models. Human tumor-infiltrating lymphocytes from a variety of cancer types expressed appreciable levels of TIGIT on relevant immune populations, including Tregs and CD8+ T cells with tumor reactive or pre-dysfunctional stem-like phenotypes. Both antibodies potently bound TIGIT and blocked the TIGIT-CD155 interaction as well as displayed the predicted phenotypes in terms of Fcγ receptor (FcγR) engagement. In line with FcγRIII binding, AB308 demonstrated a capacity to induce ADCC against TIGIT-expressing target cells. Combination of anti-TIGIT antibodies with other therapeutic approaches that promote T cell activation resulted in enhanced immune responses. In mice, while combining Fc-silent or Fc-enabled anti-mouse TIGIT antibody with anti-PD-1 resulted in greater tumor growth inhibition than with anti-PD-1 alone, the activity of Fc-enabled anti-TIGIT was associated with intratumoral Treg depletion. These data provide a rationale for combination with immune-activating agents and support ongoing clinical evaluation of AB154 and AB308 with biomarker strategies focused on understanding the role of Fc functionality.

BackgroundTIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an inhibitory receptor expressed on natural killer (NK) cells, CD8< sup >+ T cells, CD4< sup >+ T cells and regulatory T cells (T < sub >regs). On the surface of these cells, TIGIT competes with another receptor, CD226, for shared receptor ligands (mainly CD155) that are expressed by cancer and antigen-presenting cells. Binding of CD155 to TIGIT results in immune suppression through multiple mechanisms. When TIGIT is blocked, binding of CD155 to CD226 promotes immune activation and anti-tumor immunity. We describe the preclinical characterization of AB308, a humanized wild-type IgG1 anti-TIGIT antibody that is currently undergoing clinical evaluation.MethodsBinding of AB308 to TIGIT and inhibition of the TIGIT/CD155 interaction were evaluated < i >in vitro. Functional assays were used to evaluate the immunomodulatory activity of AB308 alone or in combination with zimberelimab (anti-PD-1) or etrumadenant (a small molecule A< sub >2aA< sub >2b adenosine receptor antagonist). Surrogate Fc-silent and Fc-enabled antibodies that recognize mouse TIGIT or PD-1 were leveraged to interrogate TIGIT biology in syngeneic mouse tumor models.ResultsHuman tumor-infiltrating lymphocytes from a variety of cancer types expressed appreciable levels of TIGIT on relevant immune populations, including tumor reactive CD39< sup >+CD103< sup >+ CD8< sup >+ T cells and T< sub >regs. AB308 has a high binding affinity for human TIGIT, potently blocks the TIGIT-CD155 interaction, and induces Fcγ receptor (FcγR)-mediated signaling. In line with FcγRIII binding, AB308 also demonstrated the ability to induce NK cell-driven antibody-dependent cell-mediated cytotoxicity against TIGIT-expressing target cells. AB308 significantly increased IL-2 secretion by peripheral blood mononuclear cells activated with superantigen A, an activity that was further enhanced with zimberelimab. Blocking TIGIT with AB308 potently activated CD226 signaling in Jurkat T cells co-cultured with CD155-expressing cells, and combination of AB308 with etrumadenant in this system abrogated adenosine-mediated T cell suppression that occurred even in the presence of checkpoint inhibition. In mice, while combining Fc-silent or Fc-enabled anti-mouse TIGIT antibody with anti-PD-1 resulted in greater tumor growth inhibition than with anti-PD-1 alone, the activity of Fc-enabled anti-TIGIT was associated with intratumoral T< sub >regsdepletion.ConclusionsAB308 is a potent and highly effective anti-TIGIT antibody. Concurrent blockade of multiple immune checkpoints has the potential to confer effective and durable responses in the treatment of cancer. The data presented here support the clinical use of AB308 and provides a rationale for combination with zimberelimab and adenosine pathway blocking agents such as etrumadenant and CD73 small molecule inhibitor, AB680.Ethics ApprovalAnimal experiments were performed at Arcus Biosciences, Inc. in accordance with federal, state and Institutional guidelines and were approved by Arcus’ Institutional Animal Care and Use Committee.

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme's natural substrate, adenosine 5'-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.

Botanical safety science continues to evolve as new tools for risk assessment become available alongside continual desire by consumers for "natural" botanical ingredients in consumer products. Focusing on botanical food/dietary supplements a recent international roundtable meeting brought together scientists to discuss the needs, available tools, and ongoing data gaps in the botanical safety risk assessment process. Participants discussed the key elements of botanical safety evaluations. They provided perspective on the use of a decision tree methodology to conduct a robust risk assessment and concluded with alignment on a series of consensus statements. This discussion highlighted the strengths and vulnerabilities in common assumptions, and the participants shared additional perspective to ensure that this end-to-end safety approach is sufficient, actionable and timely. Critical areas and data gaps were identified as opportunities for future focus. These include, better context on history of use, systematic assessment of weight of evidence, use of in silico approaches, inclusion of threshold of toxicological concern considerations, individual substances/matrix interactions of plant constituents, assessing botanical-drug interactions and adaptations needed to apply to in vitro and in vivo pharmacokinetic modelling of botanical constituents.

Background: Increasing evidence from rodent studies indicates that inhaled multi-walled carbon nanotubes (MWCNTs) have harmful effects on the lungs. In this study, we examined the effects of inhalation exposure to MWCNTs on allergen-induced airway inflammation and fibrosis. We hypothesized that inhalation pre-exposure to MWCNTs would render mice susceptible to developing allergic lung disease induced by house dust mite (HDM) allergen. Methods: Male B6C3F1/N mice were exposed by whole-body inhalation for 6 h a day, 5 d a week, for 30 d to air control or 0.06, 0.2, and 0.6 mg/m(3) of MWCNTs. The exposure atmospheres were agglomerates (1.4-1.8 mu m) composed of MWCNTs (average diameter 16 nm; average length 2.4 mu m; 0.52% Ni). Mice then received 25 mu g of HDM extract by intranasal instillation 6 times over 3 weeks. Necropsy was performed at 3 and 30 d after the final HDM dose to collect serum, bronchoalveolar lavage fluid (BALF), and lung tissue for histopathology. Results: MWCNT exposure at the highest dose inhibited HDM-induced serum IgE levels, IL-13 protein levels in BALF, and airway mucus production. However, perivascular and peribronchiolar inflammatory lesions were observed in the lungs of mice at 3 d with MWCNT and HDM, but not MWCNT or HDM alone. Moreover, combined HDM and MWCNT exposure increased airway fibrosis in the lungs of mice. Conclusions: Inhalation pre-exposure to MWCNTs inhibited HDM-induced TH2 immune responses, yet this combined exposure resulted in vascular inflammation and airway fibrosis, indicating that MWCNT pre-exposure alters the immune response to allergens.

Introduction: Recent nationwide surveys found that natural products, including botanical dietary supplements, are used by ∼18% of adults. In many cases, there is a paucity of toxicological data available for these substances to allow for confident evaluations of product safety. The National Toxicology Program (NTP) has received numerous nominations from the public and federal agencies to study the toxicological effects of botanical dietary supplements. The NTP sought to evaluate the utility of in vitro quantitative high-throughput screening (qHTS) assays for toxicological assessment of botanical and dietary supplements. Materials and Methods: In brief, concentration-response assessments of 90 test substances, including 13 distinct botanical species, and individual purported active constituents were evaluated using a subset of the Tox21 qHTS testing panel. The screen included 20 different endpoints that covered a broad range of biologically relevant signaling pathways to detect test article effects upon endocrine activity, nuclear receptor signaling, stress response signaling, genotoxicity, and cell death signaling. Results and Discussion: Botanical dietary supplement extracts induced measurable and diverse activity. Elevated biological activity profiles were observed following treatments with individual chemical constituents relative to their associated botanical extract. The overall distribution of activity was comparable to activities exhibited by compounds present in the Tox21 10K chemical library. Conclusion: Botanical supplements did not exhibit minimal or idiosyncratic activities that would preclude the use of qHTS platforms as a feasible method to screen this class of compounds. However, there are still many considerations and further development required when attempting to use in vitro qHTS methods to characterize the safety profile of botanical/dietary supplements.

The current/traditional human health risk assessment paradigm is challenged by recent scientific and technical advances, and ethical demands. The current approach is considered too resource intensive, is not always reliable, can raise issues of reproducibility, is mostly animal based and does not necessarily provide an understanding of the underlying mechanisms of toxicity. From an ethical and scientific viewpoint, a paradigm shift is required to deliver testing strategies that enable reliable, animal-free hazard and risk assessments, which are based on a mechanistic understanding of chemical toxicity and make use of exposure science and epidemiological data. This shift will require a new philosophy, new data, multidisciplinary expertise and more flexible regulations. Re-engineering of available data is also deemed necessary as data should be accessible, readable, interpretable and usable. Dedicated training to build the capacity in terms of expertise is necessary, together with practical resources allocated to education. The dialogue between risk assessors, risk managers, academia and stakeholders should be promoted further to understand scientific and societal needs. Genuine interest in taking risk assessment forward should drive the change and should be supported by flexible funding. This publication builds upon presentations made and discussions held during the break-out session 'Advancing risk assessment science - Human health' at EFSA's third Scientific Conference 'Science, Food and Society' (Parma, Italy, 18-21 September 2018).

There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.

The National Toxicology Program's (NTP) mission is to evaluate agents of public health concern, by developing and applying the tools of modern toxicology and molecular biology. Botanical dietary supplements (BDS) represent agents of public health concern due to widespread exposure to high doses, a lack of safety data for most products, variable quality, and reports of adverse events. This commentary will address lessons learned in NTP testing activities with BDS and recommendations for moving forward.