Dataset Information

CRISPR-Cas9-Edited Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in Transplant Recipients.

ABSTRACT: Viral infections, such as with cytomegalovirus (CMV), remain a major risk factor for mortality and morbidity of transplant recipients because of their requirement for lifelong immunosuppression (IS). Antiviral drugs often cause toxicity and sometimes fail to control disease. Thus, regeneration of the antiviral immune response by adoptive antiviral T cell therapy is an attractive alternative. Our recent data, however, show only short-term efficacy in some solid organ recipients, possibly because of malfunction in transferred T cells caused by ongoing IS. We developed a vector-free clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based good manufacturing practice (GMP)-compliant protocol that efficiently targets and knocks out the gene for the adaptor protein FK506-binding protein 12 (FKBP12), required for the immunosuppressive function of tacrolimus. This was achieved by transient delivery of ribonucleoprotein complexes into CMV-specific T cells by electroporation. We confirmed the tacrolimus resistance of our gene-edited T cell products in vitro and demonstrated performance comparable with non-tacrolimus-treated unmodified T cells. The alternative calcineurin inhibitor cyclosporine A can be administered as a safety switch to shut down tacrolimus-resistant T cell activity in case of adverse effects. Furthermore, we performed safety assessments as a prerequisite for translation to first-in-human applications.

SUBMITTER: Amini L

PROVIDER: S-EPMC7791012 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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CRISPR-Cas9-Edited Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in Transplant Recipients.

Amini Leila L Wagner Dimitrios Laurin DL Rössler Uta U Zarrinrad Ghazaleh G Wagner Livia Felicitas LF Vollmer Tino T Wendering Désirée Jacqueline DJ Kornak Uwe U Volk Hans-Dieter HD Reinke Petra P Schmueck-Henneresse Michael M

Molecular therapy : the journal of the American Society of Gene Therapy 20200908 1

Viral infections, such as with cytomegalovirus (CMV), remain a major risk factor for mortality and morbidity of transplant recipients because of their requirement for lifelong immunosuppression (IS). Antiviral drugs often cause toxicity and sometimes fail to control disease. Thus, regeneration of the antiviral immune response by adoptive antiviral T cell therapy is an attractive alternative. Our recent data, however, show only short-term efficacy in some solid organ recipients, possibly because ...[more]

PMID: 32956624

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Project description:PurposeCancer-related mortality rates among kidney transplant recipients (KTR) are high, but these patients have largely been excluded from trials of immune checkpoint inhibitors because of immunosuppression and risk of treatment-related allograft loss (TRAL). We conducted a prospective clinical trial testing nivolumab (NIVO) + tacrolimus (TACRO) + prednisone (PRED) ± ipilimumab (IPI) in KTR with advanced cutaneous cancers.MethodsAdult KTR with advanced melanoma or basal, cutaneous squamous, or Merkel cell carcinomas were eligible. Immunosuppression was standardized to TACRO (serum trough 2-5 ng/mL) + PRED 5 mg once daily. Patients then received NIVO 480 mg IV once every 4 weeks. The primary composite end point was partial or complete (tumor) response (CR) or stable disease per RECIST v1.1 without allograft loss at 16W. Patients with progressive disease (PD) could receive IPI 1 mg/kg IV + NIVO 3 mg/kg once every 3 weeks × 4 followed by NIVO. Donor-derived cell-free DNA (dd-cfDNA) levels were measured approximately once every 2 weeks as a potential predictor of allograft rejection.ResultsAmong eight evaluable patients, none met the trial's primary end point. All eight patients experienced PD on NIVO + TACRO + PRED; TRAL occurred in one patient. Six patients then received IPI + NIVO + TACRO + PRED. Best overall responses: two CR (one with TRAL) and four PD (one with TRAL). In total, 7 of 8 pre-NIVO tumor biopsies contained a paucity of infiltrating immune cells. In total, 2 of 5 on-NIVO biopsies demonstrated moderate immune infiltrates; both patients later experienced a CR to IPI + NIVO. In 2 of 3 patients with TRAL, dd-cfDNA elevations occurred 10 and 15 days before increases in serum creatinine.ConclusionIn most KTR with advanced skin cancer, TACRO + PRED provides insufficient allograft protection and compromises immune-mediated tumor regression after administration of NIVO ± IPI. Elevated dd-cfDNA levels can signal treatment-related allograft rejection earlier than rises in serum creatinine.

Project description:BackgroundCalcineurin inhibitors, including tacrolimus, remain a cornerstone of immunosuppressive therapy after kidney transplantation. However, the therapeutic window is narrow, and nephrotoxic side effects occur with overdose, while the risk of alloimmunization and graft rejection increases with underdose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows quantification of tacrolimus in biological samples from patients. This study investigates the feasibility of quantifying tacrolimus in scalp hair from kidney transplant (KT) recipients and correlates hair tacrolimus concentrations with tacrolimus dosage and blood trough levels. The aim was to provide proof-of-principle for hair tacrolimus drug monitoring in KT recipients.MethodSingle-center prospective study between September 9, 2021 and December 4, 2021, including KT recipients under tacrolimus. Minors, patients with active skin or hair diseases, and patients with scalp hair shorter than 4 cm were excluded from participation. Scalp hair was collected from the posterior vertex of patients, cut into segments, and analyzed for tacrolimus by LC-MS/MS. Patients filled out a questionnaire on hair treatments and washing habits. In parallel, tacrolimus trough levels were measured in whole blood and correlated with hair tacrolimus concentrations.ResultsIn total, 39 consenting KT recipients were included, and hair samples were collected at 53 visits. Tacrolimus was detected in 98% of hair samples from patients exposed to the drug. Tacrolimus hair levels and whole blood trough levels were correlated with a beta coefficient of 0.42 (95% CI: -0.22-1.1, p = n.s.). Age and dark hair affected hair tacrolimus measurements, while different tacrolimus formulations (immediate release vs. extended release), hair washes, and permanent coloring did not. Longitudinal measurements in a subgroup of patients indicate that long-term measurement of hair tacrolimus levels is feasible.ConclusionMeasuring tacrolimus in hair is a potentially reliable method to monitor drug exposure in KT patients. Rapid wash-in effects and consistent concentrations over time indicate that tacrolimus is incorporated into the hair matrix, allowing temporal resolution in the analysis of recent exposure and exposure history. This method provides a simple and low-risk alternative to regular blood sampling, sparing patients from frequent hospital visits through the self-collection of hair samples.

Project description:IntroductionFew studies have compared within-patient variability measures of tacrolimus trough levels by formulation and assessed within-patient variability on outcomes of kidney transplant recipients.Research questions(1) To compare within-patient variability of trough levels when converting from twice-daily to once-daily tacrolimus using standard deviation, coefficient of variation, and intrapatient variability percent. (2) To use the 3 measures of variability to examine the relationship between tacrolimus once-daily within-patient variability and total graft failure (i.e., return to chronic dialysis, pre-emptive retransplant, death with graft function).DesignIn this observational cohort study, within-patient variability of trough levels pre- and post-conversion from twice-daily to once-daily tacrolimus were compared using Wilcoxon matched-pairs signed-rank test. Graft outcomes were analyzed using Kaplan-Meier curves and multivariable Cox proportional hazards models.ResultsIn 463 patients, within-patient variability differences pre- and post-conversion of median standard deviation, coefficient of variation, and intrapatient variability percent were -0.16 (P = 0.09), -0.01 (P = 0.52), and -1.41 (P = 0.32), respectively. Post-conversion, every 1 unit increase in within-patient variability standard deviation and intrapatient variability percent and every 0.1 unit increase in the coefficient of variation was associated with an increased hazard ratio [1.19 (P = 0.004), 1.02 (P = 0.030), 1.13 (P = 0.001), respectively] of total graft failure. Post-conversion, within-patient variability above cohort medians using standard deviation and coefficient of variation had a significantly higher risk of total graft failure.DiscussionUnder a program-wide conversion, no significant difference was observed in within-patient variability post-conversion from twice-daily to once-daily tacrolimus using the three measures of variability. High within-patient variability was associated with adverse transplant outcomes post-conversion.

Dataset Information

CRISPR-Cas9-Edited Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in Transplant Recipients.

Publications

CRISPR-Cas9-Edited Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in Transplant Recipients.

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