The Independence at Home (IAH) Demonstration Year 1 results have confirmed earlier studies that showed the ability of home‐based primary care (HBPC) to improve care and lower costs for Medicare's ...frailest beneficiaries. The first‐year report showed IAH savings of 7.7% for all programs and 17% for the nine of 17 programs that surpassed the 5% mandatory savings threshold. Using these results as applied to the Medicare 5% claims file, the effect of expanding HBPC to the 2.2 million Medicare beneficiaries who are similar to IAH demonstration participants was projected. Total savings ranged from $12 billion to $53 billion depending on the speed and extent of dissemination of HBPC among this IAH‐like population. Using a fixed growth rate, as hospitalists experienced in their first decade, 35% coverage would be achieved at the end of 10 years, with total 10‐year savings through IAH reaching $37.5 billion and $17.3 billion accruing to the Centers for Medicare and Medicaid Services as a net reduction in overall expenditures, with $12.6 billion from Medicare Parts A and B savings.
Abstract Purpose Black patients with diabetes are at greater risk of underuse of antidepressants even when they have equal access to health insurance. This study aimed to evaluate the impact of ...removing a significant financial barrier to prescription medications (drug caps) on existing black-white disparities in antidepressant treatment rates among patients with diabetes and comorbid depression. Methods We used an interrupted time series with comparison series design and a 5% representative sample of all fee-for-service Medicare and Medicaid dual enrollees to evaluate the removal of drug caps on monthly antidepressant treatment rates. We evaluated the impact of drug cap removal on racial gaps in treatment by modeling the month-to-month white-black difference in use within age strata (younger than 65 years of age or 65 years of age or older). We compared adult dual enrollees with diabetes and comorbid depression living in states with strict drug caps (n = 221) and those without drug caps (n = 1133) before the policy change. Our primary outcome measures were the proportion of patients with any antidepressant use per month and the mean standardized monthly doses (SMDs) of antidepressants per month. Findings The removal of drug caps in strict drug cap states was associated with a sudden increase in the proportion of patients treated for depression (4 percentage points; 95% CI, 0.03–0.05, P < 0.0001) and in the intensity of antidepressant use (SMD: 0.05; 95% CI, 0.03–0.07, P < 0.001). Although antidepressant treatment rates increased for both white and black patients, the white-black treatment gap increased immediately after Part D (0.04 percentage points; 95% CI, 0.01–0.08) and grew over time (0.04 percentage points per month; 95% CI, 0.002–0.01; P < 0.001). Implications Policies that remove financial barriers to medications may increase depression treatment rates among patients with diabetes overall while exacerbating treatment disparities. Tailored outreach may be needed to address nonfinancial barriers to mental health services use among black patients with diabetes.
Background: The use of lipid-lowering agents is suboptimal among dual enrollees, particularly blacks. Objectives: To determine whether the removal of restrictive drug caps under Medicare Part D ...reduced racial differences among dual enrollees with diabetes. Research Design: An interrupted time series with comparison series design (ITS) cohort study. Subjects: A total of 8895 black and white diabetes patients aged 18 years and older drawn from a nationally representative sample of fee-for-service dual enrollees (January 2004–December 2007) in states with and without drug caps before Part D. Measures: We examined the monthly (1) proportion of patients with any use of lipid-lowering therapies; and (2) intensity of use. Stratification measures included age (less than 65, 65 y and older), race (white vs. black), and sex. Results: At baseline, lipid-lowering drug use was higher in no drug cap states (drug cap: 54.0% vs. nondrug cap: 66.8%) and among whites versus blacks (drug cap: 58.5% vs. 44.9%, no drug cap: 68.4% vs. 61.9%). In strict drug cap states only, Part D was associated with an increase in the proportion with any use nonelderly: +0.07 absolute percentage points (95% confidence interval, 0.06–0.09), P<0.001; elderly: +0.08 (0.06–0.10), P<0.001 regardless of race. However, we found no evidence of a change in the white-black gap in the proportion of users despite the removal of a significant financial barrier. Conclusions: Medicare Part D was associated with increased use of lipid-lowering drugs, but racial gaps persisted. Understanding non– coverage-related barriers is critical in maximizing the potential benefits of coverage expansions for disparities reduction.
To evaluate the impact of transitioning from Medicaid to Medicare Part D drug coverage on the use of noncancer treatments among dual enrollees with cancer.
We leveraged a representative 5% national ...sample of all fee-for-service dual enrollees in the United States (2004–2007) to evaluate the impact of the removal of caps on the number of reimbursable prescriptions per month (drug caps) under Part D on 1) prevalence and 2) average days’ supply dispensed for antidepressants, antihypertensives, and lipid-lowering agents overall and by race (white and black).
The removal of drug caps was associated with increased use of lipid-lowering medications (days’ supply 3.63; 95% confidence interval CI 1.57–5.70). Among blacks in capped states, we observed increased use of lipid-lowering therapy (any use 0.08 percentage points; 95% CI 0.05–0.10; and days’ supply 4.01; 95% CI 2.92–5.09) and antidepressants (days’ supply 2.20; 95% CI 0.61–3.78) and increasing trends in antihypertensive use (any use 0.01 percentage points; 95% CI 0.004–0.01; and days’ supply 1.83; 95% CI 1.25–2.41). The white-black gap in the use of lipid-lowering medications was immediately reduced (−0.09 percentage points; 95% CI −0.15 to −0.04). We also observed a reversal in trends toward widening white-black differences in antihypertensive use (level −0.08 percentage points; 95% CI −0.12 to −0.05; and trend −0.01 percentage points; 95% CI −0.02 to −0.01) and antidepressant use (−0.004 percentage points; 95% CI −0.01 to −0.0004).
Our findings suggest that the removal of drug caps under Part D had a modest impact on the treatment of hypercholesterolemia overall and may have reduced white-black gaps in the use of lipid-lowering and antidepressant therapies.
Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies ...directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.
Abstract
Rapid (< 70 min), add-and-read assays for cytokine determination were developed with Lumit™ immunoassay technology. Separate antibodies labeled with complementary SmBiT or LgBiT subunits ...bind to their shared cytokine target, resulting in proximity-driven reconstitution of NanoBiT luciferase and luminescence signal. Lumit immunoassays for human IL-1β, IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α exhibit LLODs < 10 pg/ml and linearity > 3 logs of analyte in 96-well format. Maximal S/B ratios are greater than 1000, and an assay signal T1/2 of 2 h accommodates batch plate processing. 384-well, assay performance (Z′ > 0.7) on treated human peripheral blood mononuclear cells (hPBMC) indicates the assay format is amenable to screening applications.
Assay reagents were added directly to cell wells for multiple culture models. For hPBMC, LPS, R848, or a combination of PMA and ionomycin (CSC) produced dose-and time-dependent release of cytokines with a wide range of responses, all measurable without sample dilution. In a mix of purified CD8+ T cells and target Raji B cells treated with the bispecific T-cell engager Blincyto®, IL-2, IFN-γ, and TNF-α release was observed with an EC50 of ~0.2 ng/ml and max S/B ratios of 82-, 168-, and 31-fold, respectively. For Th2 cells, significant release of IL-4 was observed in response to CSC treatment. An alternative to the no-transfer assay method, split-sample analysis, can be performed to easily determine the levels of multiple cytokines released in the same cell well.
The implementation of this novel detection chemistry will enable simple and rapid assay of cytokine release from cells for both low-and high-throughput applications, including quantitative assessments of T cell activation and differentiation.
Abstract
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 K. Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, 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 Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2179.
Abstract
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.
Abstract
Cytokines play a major role in cancer biology as inflammatory and immunomodulatory agents and are frequently measured in cell culture models and other biological samples. Various methods are ...available for in vitro measurement of cytokines, but they typically require sample transfer, sample dilutions, multiple wash steps, time-consuming protocols, and/or specialized instrumentation. We have utilized NanoLuc® Binary Technology (NanoBiT®) to develop a completely homogeneous and rapid assay method (< 70 min completion time) to measure cytokines released from cells in culture without the need for sample transfer and requiring only a standard, plate-reading luminometer for signal acquisition. In this approach, separate antibodies to a specific cytokine are individually labeled with either the small, 11-amino acid subunit of NanoBiT luciferase (SmBiT) or its 17.6 kDa complementary subunit (LgBiT). When SmBiT- and LgBiT-labeled antibodies converge on the target cytokine, the resultant proximity of SmBiT and LgBiT subunits reconstitutes a bright luciferase that produces light proportional to analyte levels when the substrate furimazine is present. Utilizing this technology, homogeneous bioluminescent immunoassays have been developed for several cytokines, including IL-1β, IL-2, IL-6 and IFN-γ. These assays share excellent sensitivities (LODs typically < 10 pg/ml) and broad linear ranges extending over three or more logs of analyte concentration, significantly mitigating the need for sample dilutions. Following 24-hour treatment of human PBMCs in 96-well plate format with vehicle, LPS, R848, or a combination of PMA and ionomycin, cytokine detection reagents were added directly to the culture wells containing cells and medium. Depending on cell stimulus, maximal signal to background ratios (S/B) achieved for the various cytokines assayed were 347-, 450-, 580- and 655-fold for IL-1β, IL-2, IL-6 and IFN-γ, respectively. In a separate cell model comprised of activated T cells and target Raji B cells induced for 20 hours with increasing concentrations of the bispecific T-cell engager Blincyto®, dose-dependent release of IL-2 and IFN-γ were observed with an EC50 of ~0.2 ng/ml and maximal S/B for IL-2 and IFN-γ release of 82- and 168-fold, respectively. In all cases, a calibration curve of recombinant cytokine enabled straightforward conversion of relative light units (RLU) to concentration of released cytokines. The implementation of this novel detection chemistry will enable rapid “add-and-read” assays for cytokine detection amenable for both low- and high-throughput screening applications.
Citation Format: Dan F. Lazar, Kevin R. Kupcho, Casey A. Sondgeroth, David V. Thompson, Martha A. O'Brien, Julia K. Gilden, Kevin Hsiao, James J. Cali. Rapid and sensitive determination of cytokine release from cells without the need for sample transfer abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5595.
Abstract
We have utilized NanoLuc® Binary Technology (NanoBiT®) to develop a homogeneous and rapid assay method (≤ 70 min completion time) to measure cytokines released from cells in culture without ...the need for sample transfer and requiring only a standard, plate-reading luminometer for signal acquisition. Separate antibodies to a specific cytokine are labeled with the small, 11-amino acid subunit of NanoBiT luciferase (SmBiT) or its 17.6 kDa complementary subunit (LgBiT). When SmBiT- and LgBiT-labeled antibodies converge on the target cytokine, the resultant proximity of SmBiT and LgBiT reconstitutes a bright luciferase that produces light proportional to analyte levels. In this manner, immunoassays have been developed for several cytokines, including IL-1β, IL-2, IL-6 and IFN-γ. In general, these assays share LLODs < 10 pg/ml and linearity over three logs of analyte concentration, mitigating the need for sample dilution. Following 24 h treatment of human PBMCs in 96-well plates with vehicle, LPS, R848, or a combination of PMA and ionomycin, cytokine detection reagents were added directly to the culture wells containing cells and medium. Depending on stimulus, maximal signal to background ratios (S/B) achieved for the cytokines were 347-, 450-, 580- and 655-fold for IL-1β, IL-2, IL-6 and IFN-γ, respectively. In a cell model comprised of activated T cells and target Raji B cells induced for 20 h with increasing bispecific T-cell engager Blincyto®, release of IL-2 and IFN-γ were observed with an EC50 of ~0.2 ng/ml and maximal S/B for IL-2 and IFN-γ of 82- and 168-fold, respectively. The implementation of this novel detection chemistry will enable rapid “add-and-read” assays for cytokine detection for both low- and high-throughput applications.
PDF
![Lumit&[trade] Immunoassays ...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0022-1767/small "Lumit&[trade] Immunoassays for Simple and Rapid Determination of Cytokine Release from Cell Culture Models")
