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Adoptive transfer of T cells genetically modified to express antigen receptor transgenes has shown promise for the treatment of cancer. Both chimeric antigen receptors (CARs) and transgenic T cell receptors (TCRs) have been used to redirect T cells in preclinical and clinical studies. TCR Therapy (TCR-T) can target essentially any tumor antigen via the major histocompatibility (MHC) system. However, the development of novel tumor antigen specific TCRs is hampered by the lack of easy-to-use model systems and sensitive readouts to facilitate candidate screening and development. Particularly, determination of transgenic TCR activation in primary and immortalized T cells is confounded by the presence of endogenous TCR expression which results in mixed alpha/beta TCR pairings and compression of assay readouts. Herein, we describe the development of a novel cell-based TCR knockout (TCR-KO) reporter assay platform for the development and characterization of novel TCR candidates. CRISPR/Cas9 was used to knockout the endogenous TCR chains in a T cell line that stably expresses a luciferase reporter gene responsive to TCR activation. Reintroduction of a transgenic TCR into the TCR-KO reporter cells results in robust antigen-specific reporter activation compared to parental reporter cells. The further development of CD4/CD8 double-positive and double-negative versions enable low and functional high avidity TCR screening with or without MHC bias. Furthermore, stable TCR-expressing reporter cells generated from TCR-KO reporter cells exhibit sufficient sensitivity and robustness for safety and specificity studies of lead TCR candidates. Together, our data demonstrate that TCR-KO reporter cells can be a useful tool for the discovery, characterization, and deployment of T cell immunotherapy. Citation Format: Jamison Grailer, Michael Slater, Jim Hartnett, Frank Fan, Mei Cong. A novel bioluminescent bioassay platform for the discovery and development of engineered T cell therapies for 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 905.

Background We aimed to evaluate whether genotype-guided warfarin dosing is superior to conventional clinical dosing for the outcomes of interest in Chinese patients. Methods - All patients with nonvalvular AF were randomly divided into two groups, genetic group and control group. We included genotypes for CYP2C9 and VKORC1 in the gene group，then doctors and pharmacists used the warfarin dosing algorithm and clinical information to determine patients’ initial dose, while in control group doses were determined by experince. The international normalized ratio (INR) measurement and standard protocols were used for further dose adjustment in both groups. The primary outcome measure was the percentage of time in the therapeutic range (%TTR) of the INR during follow up after initiation of warfarin treatment. Results The average TTR was (68.36 ± 20.57) % vs (48.52 ± 21.56) %, P0.05). Conclusion Genotype-guided dosing could improve the average TTR, improve the safety of treatment, achieve a higher level of TTR in the early anticoagulation period and reduce the risk of ischemic stroke

Redirected T cell therapies represent a new paradigm for cancer treatment. Two main approaches for T-cell-based therapies involve molecular T cell redirection by CD3 bispecific molecules such as bispecific T-cell engagers (BiTE) and cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). BiTEs redirect the cytotoxic activity of endogenous polyclonal T cells by simultaneously engaging CD3 on T cells and tumor antigens on target cells. Hitherto, BiTE potency studies have relied on primary cells, which measure target cell killing through redirected T cell cytotoxicity (RTCC) or cytokine release. However, these primary cell-based assays suffer from high donor-to-donor variability, as well as lengthy and hard to implement protocols. We have recently developed a new NanoBiT-based assay platform-based RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of CD3 bispecific antibodies or BiTEs and similar biologics that induce T Cell-dependent target cell killing and cytokine production. In this assay, preactivated, Thaw-and-Use cytotoxic T cells and target cells stably expressing HaloTag-HiBiT are co-incubated with a BiTE, which results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins then bind to LgBiT in the detection reagent and form functional NanoLuc Luciferase to generate luminescence. The assay is homogenous, highly sensitive, and has a robust assay window. Furthermore, BiTE-induced cytokine production (e.g IL-2 and IFN gamma) from the activated cytotoxic T cells can be quantitatively measured in the homogenous NanoBiT Immunoassays. Use of CAR-T has demonstrated promising results in treating leukemia, while the development of TCR-engineered T cells that can recognize intracellular tumor antigens, is still in early stages. To facilitate the screening and characterization of new transgenic TCRs, we used CRISPR/Cas9 to develop two TCRab-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR a and b chains into TCRab-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRab-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies. Citation Format: Julia Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, Gopal Krishnan, Frank Fan, Mei Cong, Zhijie Jey Cheng. Bioluminescent bioassays for the discovery and development of molecular and cellular T-cell redirecting cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2750.

Redirected T cell therapies represent a new paradigm for cancer treatment. Two main approaches for T-cell-based therapies involve molecular T cell redirection by CD3 bispecific molecules such as bispecific T-cell engagers (BiTE) and cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). BiTEs redirect the cytotoxic activity of endogenous polyclonal T cells by simultaneously engaging CD3 on T cells and tumor antigens on target cells. Hitherto, BiTE potency studies have relied on primary cells, which measure target cell killing through redirected T cell cytotoxicity (RTCC) or cytokine release. However, these primary cell-based assays suffer from high donor-to-donor variability, as well as lengthy and hard to implement protocols. We have recently developed a new RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of BiTEs and similar biologics. In this assay, preactivated, Thaw-and-Use cytotoxic T cells and target cells stably expressing HaloTag-HiBiT are co-incubated with a BiTE, which results in lysis of the target cells and release of HiBiT proteins. These HiBiT proteins then bind to LgBiT in the detection reagent and form functional NanoLuc Luciferase to generate luminescence. The assay is homogenous, highly sensitive, and has a robust assay window. Furthermore, BiTE-induced cytokine production (e.g IL-2 and IFN-γ) from the activated cytotoxic T cells can be quantitatively measured in the homogenous NanoBiT Immunoassays. Use of CAR-T has demonstrated promising results in treating leukemia, while the development of TCR-engineered T cells that can recognize intracellular tumor antigens, is still in early stages. To facilitate the screening and characterization of new transgenic TCRs, we used CRISPR/Cas9 to develop two TCRαβ-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR α and β chains into TCRαβ-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRαβ-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies. Citation Format: Julia Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, Vanessa Ott, Frank Fan, Mei Cong, Zhijie Jey Cheng. Novel bioluminescent bioassays for the discovery and development of molecular and cellular T cell-redirecting cancer therapy [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 334.

Avitourism is one of the faster growing subsectors of ecotourism, recognized for its economic value. Much of our current understanding of the economic value of avitourism revolves around bird festivals, migration events, or well-known birdwatching sites. Birdwatchers are a diverse group, some of whom competitively seek vagrant birds (i.e., birds outside their normal geographic range). The economic value from these unpredictable and transient birdwatching events remains poorly known. Using the travel cost method in a readily-quantifiable environment, we estimated that a vagrant Black-backed Oriole in Pennsylvania, United States of America, stimulated travel activity valued at about $223,000 USD or about $3,000 per day over 67 days. Some birdwatchers value rare birds, contributing significant time and financial resources to their viewing. Identifying such significant real economic value from avitourism can help to evaluate competing costs in debate over human land-use scenarios.

Aims: The treatment of prostate cancer has evolved markedly over the last 40 years, including radiotherapy, notably with escalated dose and targeting. However, the optimal treatment for localised disease has not been established in comparative randomised trials. The aim of this article is to describe the history ofprostate radiotherapy trials, including their quality assurance processes, and to compare these with the ProtecT trial.Materials and methods: The UK ProtecT randomised trial compares external beam conformal radiotherapy, surgery and active monitoring for clinically localized prostate cancer and will report on the primary outcome (disease-specific mortality) in 2016 following recruitment between 1999 and 2009. The embedded quality assurance programme consists of on-site machine dosimetry at the nine trial centres, a retrospective review of outlining and adherence to dose constraints based on the trial protocol in 54 participants (randomly selected, around 10% of the total randomised to radiotherapy, n ¼ 545). These quality assurance processes and results were compared with prostate radiotherapy trials of a comparable era.Results: There has been an increasingly sophisticated quality assurance programme in UK prostate radiotherapy trials over the last 15 years, reflecting dose escalation and treatment complexity. In ProtecT, machine dosimetry results were comparable between trial centres and with the UK RT01 trial. The outliningreview showed that most deviations were clinically acceptable, although three (1.4%) may have been of clinical significance and were related to outlining of theprostate. Seminal vesicle outlining varied, possibly due to several prostate trials running concurrently with different protocols. Adherence to dose constraints inProtecT was considered acceptable, with 80% of randomised participants having two or less deviations and planning target volume coverage was excellent.Conclusion: The ProtecT trial quality assurance results were satisfactory and comparable with trials of its era. Future trials should aim to standardise treatment protocols and quality assurance programmes where possible to reduce complexities for centres involved in multiple trials. 2016 Published by Elsevier Ltd on behalf of The Royal College of Radiologists.