Acceptance of Different Self-sampling Methods for Semiweekly SARS-CoV-2 Testing in Asymptomatic Children and Childcare Workers at German Day Care Centers: A Nonrandomized Controlled Trial.

Importance: Closure of day care centers (DCCs) to contain the COVID-19 pandemic has been associated with negative effects on children's health and well-being. Objective: To investigate the acceptance of self-sampling methods for continuous SARS-CoV-2 surveillance among asymptomatic children and childcare workers (CCWs) in DCCs. Design, Setting, and Participants: This nonrandomized pilot study included children and CCWs at 9 DCCs in Wuerzburg, Germany, from May to July 2021. Interventions: Twice weekly testing for SARS-CoV-2 was conducted by self-sampled mouth-rinsing fluid (saliva sampling [SAL], with subsequent pooled polymerase chain reaction test) plus nasal rapid antigen self-test (RAgT) (group 1), SAL only (group 2), or RAgT only (group 3) in children and CCWs. Main Outcomes and Measures: Main outcomes were rates for initial acceptance and successful (≥60% of scheduled samples) long-term participation. The probability of SARS-CoV-2 introduction into DCCs was modeled as a function of age-adjusted background incidence and DCC size. Results: Of 836 eligible children, 452 (54.1%; 95% CI, 50.7%-57.4%) participated (median [IQR] age: 4 [3-5] years; 213 [47.1%] girls), including 215 (47.6%) in group 1, 172 (38.1%) in group 2, and 65 (14.4%) in group 3. Of 190 CCWs, 139 (73.2%; 95% CI, 66.4%-79.0%) participated (median [IQR] age: 30 [25-46] years; 128 [92.1%] women), including 96 (69.1%) in group 1, 29 (20.9%) in group 2, and 14 (10.1%) in group 3. Overall, SARS-CoV-2 PCR tests on 5306 SAL samples and 2896 RAgTs were performed in children, with 1 asymptomatic child detected by PCR from SAL. Successful long-term participation was highest in group 2 (SAL only; children: 111 of 172 [64.5%]; CCWs: 18 of 29 [62.1%]). Weekly participation rates in children ranged from 54.0% to 83.8% for SAL and from 44.6% to 61.4% for RAgT. Participation rates decreased during the study course (P < .001). The probability of SARS-CoV-2 introduction into a DCC with 50 children was estimated to reach at most 5% for an age-adjusted SARS-CoV-2 incidence below 143. Conclusions and Relevance: Self-sampling for continuous SARS-CoV-2 testing was well accepted, with SAL being the preferred method. Given the high number of negative tests, thresholds for initiating continuous testing should be established based on age-adjusted SARS-CoV-2 incidence rates. Trial Registration: German Registry for Clinical Trials Identifier: DRKS00025546.

Immunocompetent cancer-on-chip models to assess immuno-oncology therapy.

The advances in cancer immunotherapy come with several obstacles, limiting its widespread use and benefits so far only to a small subset of patients. One of the underlying challenges remains to be the lack of representative nonclinical models that translate to human immunity and are able to predict clinical efficacy and safety outcomes. In recent years, immunocompetent Cancer-on-Chip models emerge as an alternative human-based platform that enables the integration and manipulation of complex tumor microenvironment. In this review, we discuss novel opportunities offered by Cancer-on-Chip models to advance (mechanistic) immuno-oncology research, ranging from design flexibility to multimodal analysis approaches. We then exemplify their (potential) applications for the research and development of adoptive cell therapy, immune checkpoint therapy, cytokine therapy, oncolytic virus, and cancer vaccines.

Inhibition of TGF-ß-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer.

BACKGROUND: Immunotherapy with chimeric antigen receptor (CAR)-engineered T-cells is effective in some hematologic tumors. In solid tumors, however, sustained antitumor responses after CAR T-cell therapy remain to be demonstrated both in the pre-clinical and clinical setting. A perceived barrier to the efficacy of CAR T-cell therapy in solid tumors is the hostile tumor microenvironment where immunosuppressive soluble factors like transforming growth factor (TGF)-ß are thought to inhibit the cellular immune response. Here, we analyzed whether CAR T-cells specific for the receptor tyrosine kinase-like orphan receptor 1 (ROR1) antigen, that is frequently expressed in triple-negative breast cancer (TNBC), are susceptible to inhibition by TGF-ß and evaluated TGF-ß-receptor signaling blockade as a way of neutralizing the inhibitory effect of this cytokine. METHODS: CD8+ and CD4+ ROR1-CAR T-cells were prepared from healthy donors and their antitumor function analyzed using the TNBC cell line MDA-MB-231 in vitro and in a microphysiologic 3D tumor model. Analyses were performed in co-culture assays of ROR1-CAR T-cells and MDA-MB-231 cells with addition of exogenous TGF-ß. RESULTS: The data show that exposure to TGF-ß engages TGF-ß-receptor signaling in CD8+ and CD4+ ROR1-CAR T-cells as evidenced by phosphorylation of small mothers against decapentaplegic homolog 2. In the presence of TGF-ß, the cytolytic activity, cytokine production and proliferation of ROR1-CAR T-cells in co-culture with MDA-MB-231 TNBC cells were markedly impaired, and the viability of ROR1-CAR T-cells reduced. Blockade of TGF-ß-receptor signaling with the specific kinase inhibitor SD-208 was able to protect CD8+ and CD4+ ROR1-CAR T-cells from the inhibitory effect of TGF-ß, and sustained their antitumor function in vitro and in the microphysiologic 3D tumor model. Combination treatment with SD-208 also led to increased viability and lower expression of PD-1 on ROR1-CAR T-cells at the end of the antitumor response. CONCLUSION: We demonstrate the TGF-ß suppresses the antitumor function of ROR1-CAR T-cells against TNBC in preclinical models. Our study supports the continued preclinical development and the clinical evaluation of combination treatments that shield CAR T-cells from TGF-ß, as exemplified by the TGF-ß-receptor kinase inhibitor SD-208 in this study.

ROR1-CAR T cells are effective against lung and breast cancer in advanced microphysiologic 3D tumor models.

Solid tumors impose immunologic and physical barriers to the efficacy of chimeric antigen receptor (CAR) T cell therapy that are not reflected in conventional preclinical testing against singularized tumor cells in 2-dimensional culture. Here, we established microphysiologic three-dimensional (3D) lung and breast cancer models that resemble architectural and phenotypical features of primary tumors and evaluated the antitumor function of receptor tyrosine kinase-like orphan receptor 1-specific (ROR1-specific) CAR T cells. 3D tumors were established from A549 (non-small cell lung cancer) and MDA-MB-231 (triple-negative breast cancer) cell lines on a biological scaffold with intact basement membrane (BM) under static and dynamic culture conditions, which resulted in progressively increasing cell mass and invasive growth phenotype (dynamic > static; MDA-MB-231 > A549). Treatment with ROR1-CAR T cells conferred potent antitumor effects. In dynamic culture, CAR T cells actively entered arterial medium flow and adhered to and infiltrated the tumor mass. ROR1-CAR T cells penetrated deep into tumor tissue and eliminated multiple layers of tumor cells located above and below the BM. The microphysiologic 3D tumor models developed in this study are standardized, scalable test systems that can be used either in conjunction with or in lieu of animal testing to interrogate the antitumor function of CAR T cells and to obtain proof of concept for their safety and efficacy before clinical application.

CAR T cells targeting αvß3 integrin are effective against advanced cancer in preclinical models.

OBJECTIVE: Integrins are heterodimeric receptors that convey cell-to-cell and cell-to-matrix interactions. Integrin αvß3 is expressed in several tumour entities including melanoma, glioblastoma, breast, pancreatic and prostate cancer, where it promotes tumour cell survival and metastasis. Here, we generated αvß3-specific chimeric antigen receptor (CAR) T-cells and analysed their antitumour function in pre-clinical models in vitro and in vivo. METHODS: αvß3-CARs comprising a super-humanised hLM609 targeting domain with either high or low affinity (hLM609v7, K d = 3 nM vs. hLM609v11, K d = 160 nM) and equipped with either a long or a short IgG4-Fc extracellular spacer (229 vs. 12 amino acids) were expressed in CD8+ and CD4+ T-cells through lentiviral transduction. RESULTS: αvß3-CAR T-cells eliminated αvß3-positive tumour cells rapidly and specifically, produced IFN-γ and IL-2 (CD4+ > CD8+) and exhibited productive proliferation. In vitro, we observed the strongest reactivity with the higher-affinity hLM609v7 αvß3-CAR in the short spacer configuration, consistent with the tumour membrane-distal localization of the hLM609 epitope. In a murine xenograft model of metastatic A-375 melanoma, the strongest antitumour effect was mediated by the lower-affinity hLM609v11 αvß3-CAR. Notably, a single administration of hLM609v11 αvß3-CAR T-cells was able to induce complete elimination of melanoma lesions, leading to long-term tumour-free survival. CONCLUSIONS: These data establish αvß3 integrin as a novel target for CAR T-cell immunotherapy, and affirm our previous notion that binding domain affinity and spacer length can be calibrated to augment CAR reactivity. CLINICAL IMPLICATIONS: αvß3-CAR T-cells have therapeutic potential in several prevalent solid tumours, including melanoma and triple-negative breast cancer.