Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells.

BACKGROUND: Chimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study. METHODS: A third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests. RESULTS: Up to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process. DISCUSSION: Cryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.

Determination and Quantitation of Cytotoxic T Cell-Mediated Cell Death.

Cytotoxic T cell-induced cell death is well documented. Cytotoxic T cell releases various cytolytic proteins. The cytolytic proteins induce target cell death. T cell-induced cell death can be measured by the lytic assay. One of the well-known lytic assays uses radioactive tracer, Chromium-51 (51Cr), and detects the amount of 51Cr released from target cells. This assay can detect cell death and the efficiency of the T cell-induced cell death by coculture effector cells (T cells) and target cells. This assay can determine the kinetics of the cell lysis. The issue of this approach is the use of radioactive material. This chapter describes measuring T cell-induced cell death by determining the epigenetic remodeling and the release of cytolytic proteins. Determine the efficiency of T cell-induced cell death by using a flow cytometry-based detection method.

In vivo tracking of transfused platelets for recovery and survival studies: an appraisal of labeling methods.

The measurement of recovery and survival of platelets is an important decisive factor when 'new' platelet products have been developed. Recovery and survival measurements are mostly performed with radioactive-labeled platelets in healthy volunteers. This approach is required by the FDA for acceptance of platelet products that differ substantially in production or storage conditions from standard methods. However, due to regulatory obstacles, such radiolabeling studies are only carried out in designated institutes. Many countries do not require radioactive labeling studies in volunteers prior to accepting new products, and rather rely on surrogate tests. Also, the European guide to the preparation of blood components does not require this step. This paper reviews alternative, non-radioactive methods, which includes biotinylation of platelets, and discrimination of transfused platelets based on HLA discrepancy. The benefits and disadvantages of these methods will be discussed.

Uptake studies of environmentally hazardous (51)Cr in Mung beans.

Attempt has been made to study the accumulation behaviour of a common plant, Mung bean (Vigna radiata) towards Cr(III) and Cr(VI) to have an insight on the migration and bio-magnification of Cr. For this purpose healthy germinated Mung bean seeds were sown in the sand in the presence of Hoagland's nutrient solution containing measured amount of K(2)(51)Cr(2)O(7) and (51)Cr(NO(3))(3).9H(2)O. Growth rate was also studied in the presence and absence of phosphate salts in the medium. It has been found that the transfer of chromium from soil to plant is significantly low (maximum 5% for both Cr(III) and Cr(VI)). Maximum accumulation of Cr occurs in the root with respect to the total chromium accumulation by the plant. Other parts of the Mung bean plant, e.g. cotyledons, shoot and leaves, show negligible accumulation. Therefore, the chance of direct intake of Cr through food as well as through the grazing animals to human body is less.

A novel telomerase-based approach to detect natural cell-mediated cytotoxic activity against tumor cells in vitro.

This study was designed to develop a novel technical approach based on tumor-associated telomerase activity to detect cytotoxic activity of effector cells of the natural immune system against neoplastic cells. Human K562, DAUDI or Raji leukemia cells were co-cultured with NK or LAK effector cells at 37 degrees C for 4 h. Target cell killing was evaluated by 51Cr-release assay (CRA) or reduction of telomerase activity (R-TRAPCTX) of the target after exposure to effector cells. NK and LAK effector cells tested against K562 target cells at effector/target ratio of 50:1 showed cytotoxicity of 65% and 78%, respectively, with CRA and 51% and 74%, respectively, with R-TRAPCTX. Incorrect results were obtained with CRA when target cells were admixed with normal fibroblasts, whereas R-TRAPCTX was not influenced by the presence of normal cells. Control experiments performed with telomerase-negative cells showed that telomerase activity of effector cells was not altered during the cytolytic reaction. Moreover, supernatants obtained from effector-target cell co-cultures did not influence telomerase activity of targets. This novel R-TRAPCTX method to assay anti-tumor natural and possibly antigen-dependent cell-mediated cytotoxicity appears to provide sensible advantages over classical CRA or gamma-interferon release by effector cells in presence of target cells (ELISPOT), since (a) it furnishes reliable data on effector cell killing against neoplastic cells, even when malignant cells are admixed with normal cells, as frequently occurs in tumor biopsies, not manageable with CRA; (b) it provides an actual measure of target cell killing, not furnished by ELISPOT technique.

Chemical stabilization of chromate in blast furnace slag mixed cementitious materials.

Cement waste form (CWF) technology is among the leading approaches to disposing of metals and liquid low-level nuclear waste in the United States. One such material, saltstone, includes slag, fly ash and Portland cement to enhance the immobilization of contaminants (e.g., Cr, (99)Tc) in alkaline liquid wastes. To evaluate the stability of such redox sensitive contaminants in saltstone, the effects of slag as a source of reductant on Cr immobilization was evaluated in aged (<300 d) saltstone monoliths. Specifically, we investigated the effects of artificial cement pore waters on the Cr release and the spatially resolved Cr chemical state analysis using synchrotron based microfocused X-ray microprobe analysis. The microprobe analysis indicated the heterogeneous distribution of insoluble Cr(III)-species in saltstone. Although at most of 20% Crtotal was leached at the top few (2-3) millimeter depth, the release of Cr(VI) was small (<5%) at 5-30 mm with slight changes, indirectly suggesting that Cr is likely present as insoluble Cr(III) species throughout the depths. The study suggests that this saltstone formulation can effectively retain/immobilize Cr under the oxic field condition after ⩽300 d of aging time.

Enhancement of human lymphocyte natural killer activity by somatostatin.

The effect of somatostatin 1-14 (SS) on the natural killer (NK) activity of human peripheral blood lymphocytes was investigated. The NK activity was estimated by means of radioactive chromium (51Cr) assay with the use of human leukemia cells K 562 as targets. The previous exposure of lymphocytes obtained from healthy donors to somatostatin in the concentration of 10(-8) and 10(-6) M resulted in the enhancement of NK activity. The finding provides a further evidence of the immunomodulating somatostatin action.

The measurement of the depth of a point source of a radioisotope from gamma ray spectra.

The spectrum of the scattered radiation from a 'point' source of 99Tcm located within a slab of tissue-like material has been studied with a 19-phototube gamma camera, a 7.5 cm x 7.5 cm sodium iodide detector and a Ge(Li) counter. Both backscatter and forward scattered radiation contribute to the spectra observed; 'infinite' backscatter is achieved at a thickness of 8 cm. The scattered radiation can be used to estimate the thickness of the overlying material. The scatter from 133Xe and 51Cr was also measured. The depth of a source could usually be determined to within a few millimetres from the proportion of scatter in the spectrum; it was more accurate with the 51Cr (320 keV) than with the lower-energy photopeaks of the other two isotopes. The variation in the spectrum as the source moved across the field of the gamma camera was recorded.

Injury-specific cytotoxic response of tumor cells and endothelial cells.

Cytotoxicity indicated by increased release of prelabeled 51chromium (51Cr) and lactate dehydrogenase (LDH) was studied in human prostate cancer and melanoma cells in cell culture following irradiation or exposure to several injurious substances. These changes were compared to those observed in bovine aortic endothelial cells (BAEC) subjected to identical treatments. Further, the effect of irradiation on plasminogen activator (PA) secretion from prostate cancer cells, and the effect of glycine on radiation-induced cytotoxicity in BAEC were also investigated. Radiation, lipopolysaccharide and xanthine/xanthine oxidase stimulated no release of 51Cr or LDH from tumor cells, while these treatments induced a dose- and time-related loss of those cytotoxic indicators from BAEC. Protease, elastase and Triton X-100 incited loss of 51Cr and LDH from all three cell types. Radiation, lipopolysaccharide and xanthine/xanthine oxidase have been shown to cause cell injury via a common pathogenic pathway of oxidant generation. Tumor cells appear quite resistant to oxidant stress. Cell damage precipitated by protease, elastase and Triton probably involves hydrolysis of proteins and phospholipids in the cell membrane, leading to an increased leakage of intracellular proteins such as LDH and those bound with 51Cr. Radiation caused a dose- and time-related reduction in the secretion of PA from prostate cancer cells. PA is alleged to play a role in tumor metastasis; the reduced secretion could be another beneficial effect of radiation, in addition to interruption of cell proliferation, in the impediment of tumor growth and spread. Glycine diminished cytotoxic injury of BAEC inflicted by radiation. This amino acid may prove useful in offering a degree of protection of normal tissue against radiation associated side-effects.

A review of the secretion of radioactivity in human breast milk: data, quantitative analysis and recommendations.

A general method is given to quantify the radioactivity ingested by a breast-fed infant following maternal radiopharmaceutical administration, and to derive the period to interrupt feeding which reduces the corresponding effective dose equivalent to below 1 mSv. Results are presented from applying the method to all available measurements of radioactivity secreted in breast milk. This review includes some hitherto unpublished data, takes more account of the scarcity of the available data, and has resulted in modifications to the guidance for interrupting breast feeding. Recommendations for interrupting feeding with mature milk have been expanded into four categories: (1) interruption not essential; (2) interruption for a fixed period of time; (3) interruption until measurements indicate feeding can be resumed; and (4) cease breast feeding. Changes to the earlier guidance are that 99Tcm-pertechnetate, (99Tcm) erythrocytes, 125I-OIH and 131I-OIH have been moved to the third category, and that recommendations have been included for 99Tcm-DMSA, 99Tcm-glucoheptonate, 99Tcm-HDP, 99Tcm-HMDP, 123I-iodide and 123I-OIH. Before recommendations (1) and (2) can be issued to a mother, quality control measurements must be made of the radiopharmaceutical administered. Recommendations (3) or (4) only should be issued to mothers secreting colostrum. If there are changes in the recommended dosimetry in the future, the calculated fractional ingested activities can be used as a basis to refine the recommendations.

Hematocrit and blood content in the rabbit eye.

The red blood cell volume and the apparent plasma volume in the ocular tissues of the rabbit were measured using 51Cr labelled red blood cells and 125I labelled human serum albumin. On this basis, the hematocrit of the circulating blood in the eye and the blood content in the ocular tissues were estimated. In the retinal circulation, the hematocrit was 26, which was 70% of the hematocrit of the cardiac blood. This value was thought to be also applicable in the uveal tissue, although the extravascular leakage of the tracer albumin did not allow an exact determination of the hematocrit of the circulating blood in this tissue. The blood content per 1 gram tissue was calculated from the red blood cell volume and the hematocrit determined. It was about 63 microliter in the iris, 59 microliter in the ciliary body, 198 microliter in the choroid and 38 microliter in the retina. The value obtained in the iris or ciliary body was similar to that in the cardiac muscle or intestine, and the value in the choroid was similar to that in the kidney, lung or liver. The value in the retina was greater than that reported in the brain.

Piroxicam-beta-cyclodextrin: effects on gastrointestinal blood loss and gastric mucosal appearance in healthy men.

Thirty-six healthy men aged 20-31 years took part in a randomized, double-blind, double-dummy, parallel-group study to compare the effects of repeated doses of piroxicam-beta-cyclodextrin, piroxicam and placebo on faecal blood loss and the endoscopic appearances of gastric and duodenal mucosa. After an initial endoscopy, subjects received on day 0 autologous erythrocytes labelled with 51Cr. Complete daily faecal collections were then made from days 6 to 12. From days 13 to 40, subjects received daily by mouth either piroxicam-beta-cyclodextrin (containing 20 mg piroxicam), piroxicam 20 mg, or placebo. Complete faecal collections were made daily from days 13 to 41; blood samples for red-cell 51Cr activity were taken weekly. Endoscopy was repeated 16-20 h after the last dose of medication. Faecal blood loss was calculated from 51Cr activity of blood and faeces. Compliance with medication was confirmed by blood sampling on days 20, 27 and 34. General tolerability of the medication was good, although 1 subject was withdrawn from piroxicam-beta-cyclodextrin and 1 from piroxicam treatment because of abdominal pain. There were no clinically significant changes in haematology, biochemistry or urinalysis results in any subject. Endoscopic appearances deteriorated moderately in 3 subjects receiving piroxicam and 3 receiving piroxicam-beta-cyclodextrin, but did not deteriorate in any subject receiving placebo. There was a trend for cumulative blood loss to be higher for piroxicam than for the other treatments in the last 12 days of dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Hybrid radioassay of multiple radionuclide mixtures in waste solutions by using liquid and NaI(Tl) scintillation monitors.

A new analytical technique for radioactive waste solutions has been developed by using a combination of a liquid and a NaI(Tl) scintillation monitor, which enables beta-emitter mixtures to be radioassayed using one calculation process with the method of least squares. This hybrid system can facilitate the analysis of beta-emitter mixtures with very similar liquid scintillation pulse height distributions, such as 3H, 51Cr, and 125I. All that is required for the technique are sets of quench standards of the nuclides to be analyzed and calibration standards for the gamma-emitters. Detection limits for seven nuclides were estimated to be about 0.005 Bq mL(-1), which are sufficiently low compared with the values of authorized safety guidelines.

Thermal neutron fluence from ultra low-level gamma-ray spectrometry of spoons activated during the JCO criticality accident at Tokai-mura in 1999.

During the JCO-accident in Tokai-mura in 1999, the surrounding village was irradiated by an uncontrolled neutron flux. At some locations in that village, the thermal neutron flux was determined retrospectively by measurement of the very low activity of 51Cr and 60Co in stainless-steel spoons using gamma-ray spectrometry in underground laboratories. Activities determined in the HADES underground facility are presented here, together with calibrations performed using a well-defined thermal neutron flux to directly estimate the fluence of thermal neutrons independent of most assumptions. The results show measurable 51Cr in three samples and 60Co in four samples taken from locations at distances of up to 430m from the accident location despite the elapse of 4 half-lives of 51Cr before measurement. Effects of air transport of the samples were considered and shown to be negligible.

Measurements of 60Co in spoons activated by neutrons during the JCO criticality accident at Tokai-mura in 1999.

Neutron activated items from the vicinity of the place where the JCO criticality accident occurred have been used to determine the fluence of neutrons around the facility and in nearby residential areas. By using underground laboratories for measuring the activation products, it is possible to extend the study to also cover radionuclides with very low activities from long-lived radionuclides. The present study describes gamma-ray spectrometry measurements undertaken in a range of underground laboratories for the purpose of measuring (60)Co more than 2 years after the criticality event. The measurements show that neutron fluence determined from (60)Co activity is in agreement with previous measurements using the short-lived radionuclides (51)Cr and (59)Fe. Limits on contamination of the samples with (60)Co are evaluated and shown to not greatly affect the utility of neutron fluence determinations using (60)Co activation.

Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays.

Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in (51)Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16(+)CD56(dim) NK cells and CD14(+) monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56(dim) NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a (51)Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.

Comparing individual NK cell activity in vitro.

Natural killer (NK) cells play an important role in the innate immune system by eliminating infected and mutated cells. Their cytotoxic capacities vary markedly among individuals. The cytotoxic activity can be measured in peripheral blood mononuclear cells (PBMCs) using the NK cell-specific target cell line K562. In this chapter, we present a protocol for the standardization and normalization of cell preparation and NK cell cytotoxicity measurement in a (51)Cr-release assay. By following these protocols, it is possible to compare the NK cell activity of numerous-if necessary selected-individuals in vitro.

On the stochastic dependence between photomultipliers in the TDCR method.

The TDCR method (Triple to Double Coincidence Ratio) is widely implemented in National Metrology Institutes for activity primary measurements based on liquid scintillation counting. The detection efficiency and thereby the activity are determined using a statistical and physical model. In this article, we propose to revisit the application of the classical TDCR model and its validity by introducing a prerequisite of stochastic independence between photomultiplier counting. In order to support the need for this condition, the demonstration is carried out by considering the simple case of a monoenergetic deposition in the scintillation cocktail. Simulations of triple and double coincidence counting are presented in order to point out the existence of stochastic dependence between photomultipliers that can be significant in the case of low-energy deposition in the scintillator. It is demonstrated that a problem of time dependence arises when the coincidence resolving time is shorter than the time distribution of scintillation photons; in addition, it is shown that this effect is at the origin of a bias in the detection efficiency calculation encountered for the standardization of (3)H. This investigation is extended to the study of geometric dependence between photomultipliers related to the position of light emission inside the scintillation vial (the volume of the vial is not considered in the classical TDCR model). In that case, triple and double coincidences are calculated using a stochastic TDCR model based on the Monte-Carlo simulation code Geant4. This stochastic approach is also applied to the standardization of (51)Cr by liquid scintillation; the difference observed in detection efficiencies calculated using the standard and stochastic models can be explained by such an effect of geometric dependence between photomultiplier channels.

One-step simple assay to determine antigen-specific cytotoxic activities by single-color flow cytometry.

Assays for cytotoxicity of CTLs in vivo using a fluorescent-based dye, 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE), have been established and widely used. On the basis of this experience, we applied it to in vitro assay system and established a simpe, highly sensitive flow cytometric assay for CTL activity. In our assay, specific activities of CTLs could be detected by a reduction in sensitive target cell numbers on single-color histogram plot analysis. By using this assay, we could determine the changes in cytotoxic activity by single amino acid substitution within an epitope peptide. Adherent cells were also used as target cells in this assay by treatment with excess EDTA and trypsin reagents after incubation with effector CTLs. Furthermore, when fluorescent calibration beads were used as a control, we could determine the cytotoxicity of CTLs against tumor cells. The results obtained from our assay were almost consistent with those from the conventional ( 51)Cr-release assay.Because our assay uses only a stable non-radioactive reagent, CFSE, this assay is safe, inexpensive and extremely easy. These results indicated that this new assay (FACS-CTL assay) would be sufficiently acceptable alternative to classical (51)Cr-release assay.

Assessment of permeability barriers to macromolecules in the rodent endometrium at the onset of implantation.

In rodents, embryo implantation is an invasive process, which begins with its attachment to the uterine wall and culminates in the formation of the definitive placenta several days later. It is critical that the endometrium provide a supportive environment for the implanting embryo during this process, as the placenta is not yet established. The concept of changing permeability barriers to macromolecules between different extracellular compartments in the rodent uterus at the onset of implantation has been established. This chapter provides protocols that can be used to assess this changing permeability barrier and the associated redistribution of macromolecules during the early phases of implantation in rodents. An increased permeability of the endometrial vasculature to plasma proteins occurs in areas adjacent to the implanting blastocyst. In addition, alterations in the extracellular matrix enhance the accumulation of fluid and extravasated macromolecules. We describe several protocols proven to be effective in studying and quantifying early vascular and extravascular responses to natural and artificial "implantation stimuli." The first three protocols represent qualitative and quantitative methods to assess the early endometrial "vascular permeability" response. On the contrary, the fourth protocol addresses the onset of decidualization and the arising permeability barrier, which restricts the movement of macromolecules through the extracellular space. This barrier is believed to provide transient protection for the implanting embryo against potentially harmful maternal serum proteins. This protocol describes assessment of resistance of the primary decidual zone to the movement of macromolecules across the compartments of the extracellular space.