Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia.

Tisagenlecleucel (CTL019) is an investigational immunotherapy that involves reprogramming a patient's own T cells with a transgene encoding a chimeric antigen receptor to identify and eliminate CD19-expressing cells. We previously reported that CTL019 achieved impressive clinical efficacy in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), including the expansion and persistence of CTL019 cells, which correlates with response to therapy. Here, we performed formal cellular kinetic analyses of CTL019 in a larger cohort of 103 patients treated with CTL019 in 2 different diseases (ALL and CLL). CTL019 was measured in peripheral blood and bone marrow, using quantitative polymerase chain reaction and flow cytometry. CTL019 levels in peripheral blood typically peaked at 10 to 14 days postinfusion and then declined slowly over time. Patients with complete response (CR)/CR with incomplete count recovery had higher levels of CTL019 in peripheral blood, with greater maximal concentration and area under the curve values compared with nonresponding patients (P < .0001 for each). CTL019 transgene levels were measurable up to 780 days in peripheral blood. CTL019 trafficking and persistence were observed in bone marrow and cerebrospinal fluid. CTL019 expansion correlated with severity of cytokine release syndrome (CRS) and preinfusion tumor burden in pediatric ALL. The results described here are the first detailed formal presentation of cellular kinetics across 2 diseases and highlight the importance of the application of in vivo cellular kinetic analyses to characterize clinical efficacy and CRS severity associated with CTL019 therapy.

Panobinostat PK/PD profile in combination with bortezomib and dexamethasone in patients with relapsed and relapsed/refractory multiple myeloma.

PURPOSE: Panobinostat, a potent pan-deacetylase inhibitor, improved progression-free survival (PFS) in patients with relapsed and refractory multiple myeloma when combined with bortezomib and dexamethasone in a phase 3 trial, PANORAMA-1. This study aims to explore exposure-response relationship for panobinostat in this combination in a phase 1 trial, B2207 and contrast with data from historical single-agent studies. METHODS: Panobinostat plasma concentration-time profiles were obtained in patients from PANORAMA-1 (n = 12) and B2207 (n = 12) trials. Overall response rates (ORR) and major adverse events (AE) by panobinostat exposure were investigated in the B2207 trial. Panobinostat PK data from combination trials were contrasted with data from single-agent studies. RESULTS: At maximum tolerated dose (MTD), the geometric mean of panobinostat area under curve from 0 to 24 h (AUC0-24) was 47.5 ng h/mL (77 % CV), and maximum plasma concentration (Cmax) was 8.1 ng/mL (90 % CV). These values were comparable with exposure data obtained in PANORAMA-1, but were 20 % lower than those without dexamethasone, and ∼ 50 % lower from single-agent trials, likely due to enzyme induction by dexamethasone. Higher levels of panobinostat exposure were associated with higher response rates and higher incidences of diarrhea and thrombocytopenia. CONCLUSIONS: Apparent panobinostat exposure-AE and exposure-ORR relationships were observed when combined with bortezomib and dexamethasone in the treatment of patients with relapsed and refractory multiple myeloma. The addition of dexamethasone facilitated best response even though plasma exposure of panobinostat was reduced. Combination with a strong enzyme inducer should be avoided in future trials to prevent further reduction of panobinostat exposure.

Next-Generation Sequencing of Minimal Residual Disease for Predicting Relapse after Tisagenlecleucel in Children and Young Adults with Acute Lymphoblastic Leukemia.

We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection >0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03-9.97; P < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18-10.8; P < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44-7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87-50; P < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33-4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19- (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion. SIGNIFICANCE: Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies.See related commentary by Ghorashian and Bartram, p. 2.This article is highlighted in the In This Issue feature, p. 1.

Tisagenlecleucel immunogenicity in relapsed/refractory acute lymphoblastic leukemia and diffuse large B-cell lymphoma.

Tisagenlecleucel is indicated for pediatric and young adult patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) and adult patients with r/r diffuse large B-cell lymphoma (DLBCL). The tisagenlecleucel chimeric antigen receptor (CAR) contains a murine single-chain variable fragment domain; we examined the effects of humoral and cellular immune responses to tisagenlecleucel on clinical outcomes using 2 validated assays. Data were pooled from the ELIANA (registered at www.clinicaltrials.gov as #NCT02435849) and ENSIGN (#NCT02228096) trials in r/r B-ALL (N = 143) and the JULIET trial (#NCT02445248) in r/r DLBCL (N = 115). Humoral responses were determined by flow cytometric measurement of anti-murine CAR19 (mCAR19) antibodies in serum. Cellular responses were determined using T-cell production of interferon-γ in response to 2 different pools of mCAR19 peptides. Pretreatment anti-mCAR19 antibodies were detected in 81% of patients with r/r B-ALL and 94% of patients with r/r DLBCL. Posttreatment anti-mCAR19 antibodies were higher than patient-specific baseline in 42% of r/r B-ALL and 9% of r/r DLBCL patients. Pretreatment and posttreatment anti-mCAR19 antibodies did not affect tisagenlecleucel cellular kinetics, including maximum concentration and persistence (r2 < 0.05), clinical response (day-28 response, duration of response, and event-free survival), and safety. T-cell responses were consistent over time, with net responses <1% at baseline and posttreatment time points in a majority of patients and no effect on transgene expansion or persistence or outcomes. Presence of baseline and/or posttreatment anti-mCAR19 antibodies or T-cell responses did not alter the activity of tisagenlecleucel in patients with r/r B-ALL or r/r DLBCL.

Tisagenlecleucel cellular kinetics, dose, and immunogenicity in relation to clinical factors in relapsed/refractory DLBCL.

The anti-CD19 chimeric antigen receptor (CAR)-T cell therapy tisagenlecleucel was evaluated in the global, phase 2 JULIET study in adult patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). We correlated tisagenlecleucel cellular kinetics with clinical/product parameters in 111 patients treated in JULIET. Tisagenlecleucel persistence in responders and nonresponders, respectively, was demonstrated for 554 and 400 days maximum by flow cytometry and for 693 and 374 days maximum by quantitative polymerase chain reaction (qPCR). No relationships were identified between cellular kinetics (qPCR) and product characteristics, intrinsic/extrinsic factors, dose, or immunogenicity. Most patients with 3-month response had detectable transgene at time of response and continued persistence for ≥6 months. Expansion (maximal expansion of transgene/CAR-positive T-cell levels in vivo postinfusion [Cmax]) was potentially associated with response duration but this did not reach statistical significance (hazard ratio for a twofold increase in Cmax, 0.79; 95% confidence interval, 0.61-1.01). Tisagenlecleucel expansion was associated with cytokine-release syndrome (CRS) severity and tocilizumab use; no relationships were observed with neurologic events. Transgene levels were associated with B-cell levels. Dose was associated with CRS severity, but this was not statistically significant after adjusting for baseline tumor burden. In contrast to the results from B-cell precursor acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia, similar exposure was observed in DLBCL in this study regardless of response and expansion was lower in DLBCL than B-ALL, likely from differences in cancer location and/or T-cell intrinsic factors. Relationships between expansion and CRS severity, and lack of relationships between dose and exposure, were similar between DLBCL and B-ALL. Tisagenlecleucel cellular kinetics in adult relapsed/refractory DLBCL improve current understanding of in vivo expansion and its relationships with safety/efficacy endpoints. This trial was registered at www.clinicaltrials.gov as #NCT02445248.

Clinical Pharmacology of Tisagenlecleucel in B-cell Acute Lymphoblastic Leukemia.

PURPOSE: Tisagenlecleucel is an anti-CD19 chimeric antigen receptor (CAR19) T-cell therapy approved for the treatment of children and young adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). PATIENTS AND METHODS: We evaluated the cellular kinetics of tisagenlecleucel, the effect of patient factors, humoral immunogenicity, and manufacturing attributes on its kinetics, and exposure-response analysis for efficacy, safety and pharmacodynamic endpoints in 79 patients across two studies in pediatric B-ALL (ELIANA and ENSIGN). RESULTS: Using quantitative polymerase chain reaction to quantify levels of tisagenlecleucel transgene, responders (N = 62) had ≈2-fold higher tisagenlecleucel expansion in peripheral blood than nonresponders (N = 8; 74% and 104% higher geometric mean Cmax and AUC0-28d, respectively) with persistence measurable beyond 2 years in responding patients. Cmax increased with occurrence and severity of cytokine release syndrome (CRS). Tisagenlecleucel continued to expand and persist following tocilizumab, used to manage CRS. Patients with B-cell recovery within 6 months had earlier loss of the transgene compared with patients with sustained clinical response. Clinical responses were seen across the entire dose range evaluated (patients ≤50 kg: 0.2 to 5.0 × 106/kg; patients >50 kg: 0.1 to 2.5 × 108 CAR-positive viable T cells) with no relationship between dose and safety. Neither preexisting nor treatment-induced antimurine CAR19 antibodies affected the persistence or clinical response. CONCLUSIONS: Response to tisagenlecleucel was associated with increased expansion across a wide dose range. These results highlight the importance of cellular kinetics in understanding determinants of response to chimeric antigen receptor T-cell therapy.

A phase-1, open-label, single-dose study of the pharmacokinetics of buparlisib in subjects with mild to severe hepatic impairment.

The pharmacokinetics (PK) and safety of single-dose buparlisib (30 mg) were assessed in subjects with mild to severe hepatic impairment (n = 6 each) relative to healthy controls (n = 13). Blood samples were collected until 336 hours postdose and evaluated by liquid chromatography tandem mass spectrometry. PK parameters (including area under the curve [AUC∞ ] and Cmax ) were derived using noncompartmental analysis. Buparlisib was rapidly absorbed in all groups (median Tmax 1.0-1.3 h). Buparlisib exposure (AUC∞ ) was moderately increased in subjects with mild (geometric mean ratio [GMR] 1.16; 90%CI 0.81, 1.65), moderate (GMR 1.14; 90%CI 0.80, 1.63), or severe (GMR 1.20; 90%CI 0.84, 1.72) hepatic impairment, relative to healthy controls. Apparent oral clearance was similar across groups. Due to a higher unbound fraction in the severe group (0.21) than all other groups (0.17), subjects with severe hepatic impairment had greater exposure to unbound buparlisib (GMR relative to healthy controls: AUC∞ 1.52; 90%CI 1.09, 2.13; Cmax 1.83; 90%CI 1.42, 2.36). The results indicate that a buparlisib dose adjustment may not be necessary for patients with mild to moderate hepatic impairment. The safety and therapeutic indices should be considered before determining if a dose adjustment is appropriate for patients with severe hepatic impairment.

Establishment of the integrity of lysosomes in a glycoprotein-rich matrix. Distribution pattern of seven lysosomal enzymes in gastric mucosa.

Gel-forming mucosal glycoproteins strongly interfere with standard methods of cell fractionation. Thus, acid hydrolase-bound particles imbedded in the gel, sediment on centrifugation, in the nuclear fraction of homogenates of canine antral mucosa. These particles can be cleared by direct solubilization of the gel; however, the viscosity of the solution obtained prevents sedimentation of some of the latent hydrolases, even at very high speeds. The use of a new step-wise scheme of centrifugation and dilution successfully isolates lysosomal particles containing acid hydrolases from mucin-rich mucosa. All of the enzymes investigated, including acid phosphatase, cathepsin D, alpha- and beta-galactosidase, beta-B-acetylhexosaminidases, but with the exception of alpha-fucosidase, were found to be particle bound, exhibiting high degrees of latency. However, active mucosal particles are polydisperase in size and density, sedimenting under different centrifugal forces.

A phase I, open-label, multicenter study to evaluate the pharmacokinetics and safety of oral panobinostat in patients with advanced solid tumors and varying degrees of renal function.

PURPOSE: This study assessed the pharmacokinetics and safety of oral panobinostat and its metabolite BJB432 in patients with advanced solid tumors and normal to severely impaired renal function. METHODS: Patients with varying degrees of renal impairment, defined by their 24-h baseline urine creatinine clearance (as normal, mild, moderate or severe), received a single oral dose of 30 mg panobinostat. Serial plasma samples were collected pre-dose and up to 96-h post-dose. Serial urine samples were collected for 24-h post-dose. Following the serial PK sampling, patients received 30 mg oral panobinostat thrice weekly for as long as the patient had benefit. Pharmacokinetic parameters were derived using non-compartmental analysis. RESULTS: Thirty-seven patients were enrolled, and median age was 64 (range 40-81) years. Eleven patients had normal renal function; 10, 10, and 6 patients had mild, moderate, and severe renal impairment, respectively. Geometric means of AUC(0-∞) in the normal, mild, moderate, and severe groups were 224.5, 144.3, 223.1, and 131.7 ng h/mL, respectively. Geometric mean ratio of BJB432 to parent drug plasma AUC(0-∞) was 0.64 in the normal group and increased to 0.81, 1.13, and 1.20 in the mild, moderate, and severe groups, respectively. The fraction excreted as unchanged panobinostat was small (<2 %), with a large variability. The renal clearance of panobinostat and tolerability was similar across all four groups. CONCLUSION: Systemic exposure to panobinostat did not increase with severity of renal impairment, and the drug was tolerated equally; thus, patients with renal impairment do not require starting dose adjustments.

The turnover of mucin glycoprotein in the stomach.

It is proposed that the gastric mucosa, during early stages of fasting, accumulates secreted mucin on the luminal surface of the tissue. The accumulated mucin is subsequently released from its adherent state, on precipitation by freshly secreted acid from the stimulated parietal cells, or, as an alternate mechanism, simply by the passage of ingested food. The gastric mucosa on stimulation by ulcerogenic aspirin, on the other hand, responds immediately to the insult by rapid shedding of its adherent mucin, then, within 5 minutes by an almost complete shut-down of respiration and biosynthesis, not only of glycoproteins but also of proteins and nucleic acids. However, oxygen consumption and biosynthesis quickly revive, in an endeavour to restore the mucosal tissue which has survived, with the result that the turnover of intracellular contents is augmented very much more than its normal rapid rate, illustrated in the gastric epithelial cell by Bennett in 1971 and in the non-secreting duodenal columnar cell by Bennett and Leblond (1970). 14C-Fucose was found in the Golgi apparatus, 2 minutes after injection into the animal, while within 20 minutes it had reached the surface membranes before extrusion.

A phase I, open-label, multicenter study to evaluate the pharmacokinetics and safety of oral panobinostat in patients with advanced solid tumors and various degrees of hepatic function.

PURPOSE: To evaluate the pharmacokinetics and safety of oral panobinostat in patients with advanced solid tumors and varying degrees of hepatic function. METHODS: Patients with advanced solid malignancies, acceptable bone marrow and renal function, and normal or impaired hepatic function, per NCI-ODWG criteria, were eligible. Initially patients received a single oral dose of 30 mg panobinostat for a 1-week pharmacokinetic study (core phase). Subsequently, patients received thrice-weekly panobinostat for as long as beneficial (extension phase safety assessment). Core phase serial blood samples for panobinostat and metabolite BJB432 assay were collected pre-dose and up to 96 h post-dose. RESULTS: Twenty-five patients were enrolled, median age 58 years (range 45-76). Fifteen patients had hepatic dysfunction (8 mild, 6 moderate, and 1 severe). Reductions in panobinostat plasma clearance were 30 and 51 %, with concomitant 43 and 105 % increase in exposure, for patients with mild and moderate hepatic dysfunction, respectively. Median peak plasma concentrations were 1.4-(mild) and 1.8-(moderate) fold higher than the normal group. Hepatic impairment did not alter panobinostat absorption with Tmax unchanged at 2 h. Geometric mean ratios of BJB432 to panobinostat plasma AUC0-∞ were similar in patients with normal, mild, or moderate hepatic impairment. Safety data were consistent with known safety profile of panobinostat in patients with advanced cancers and normal liver function. CONCLUSION: Despite increased plasma exposure, patients with mild or moderate hepatic dysfunction could be safely treated with the same starting dose of panobinostat as patients with normal hepatic function, with careful monitoring and dose adjustments as required.

The mucolytic activity of amides: a new approach to mucus dispersion.

A method which will reduce significantly the viscosity of epithelial mucus is essential to the physiological mechanisms involved in the mobilization and removal of such secretions. The life expectancy of patients with chronic pulmonary conditions and cystic fibrosis has been considerably increased and consequently the problem of liquefying mucin acquires new importance. In view of these considerations, as well as to facilitate research into the structural relationship of the glycoprotein macromolecule, a systematic investigation of mucolysis was undertaken using gastric mucin. Three amides, carbamide, acetamide and formamide, were found to dissolve gastric gel mucin with minimal degradation, and rapidly disperse the viscous secretions produced in pathological conditions of the tracheobronchial tree. Their effect on secretions from patients with cystic fibrosis and bronchiectasis is dramatic, and within five minutes of adding the reagent the flow time was reduced by at least 95%. Clinical studies were carried out with carbamide (urea in anhydrous, lyophilized, sterile powder form) in 32 patients with a variety of bronchial conditions, including chronic bronchitis, cystic fibrosis, asthma, bronchiectasis and emphysema. With the concentrations used, no irritant, bronchospastic or other reactions were observed.IT IS CONCLUDED THAT AMIDES OF THIS TYPE HAVE AT LEAST TWO ACTIONS ON THE EPITHELIAL MUCOUS SECRETION: (1) breakage of the three-dimensional gel structure and (2) a slower reduction in viscosity followed by solution of the solid material.

In vitro action of gastric mucosal lysosomal enzymes on intracellular gastric glycoproteins.

The results show that incubation of gastric mucosal cells from rat at pH approximately 4.5 or in the presence of aspirin is associated with a specific increase in the activity of some acid-hydrolases. Intracellular glycoproteins, isolated by non-degrative techniques from rat or dog fundic mucosal cells, were found to be potential bio-substrates for these acid-hydrolyses. This may suggest that cleavage of the carbohydrate moieties of the intracellular and mucosal cell wall glycoproteins is a fundamental step in the development of gastric ulceration. A model for gastric lesions is proposed and discussed in the light of the results obtained.

Isolation and characterization of human and canine gastric mucosal glycoproteins and their degradation by proteases and acid hydrolases.

High molecular weight glycoproteins were isolated and purified from canine antral and fundic mucosal tissue by means of non-degrading techniques. The results disclosed the advantage of urea extraction technique over the culture method in isolating the native glycoproteins. The glycoproteins were susceptible to degradation by protease, thus yielding low molecular weight glycopeptides. Chemical analysis of these glycopeptides and their parent macromolecules revealed that the oligosaccharide residues are attached to threonine, serine and proline residues of the protein chains. Similarly, high molecular weight glycoproteins isolated from human gastric gel mucin showed the same characteristics of canine gastric glycoproteins. Canine fundic glycoprotein or glycopeptide released their prosthetic carbohydrate groups under the lytic effect of fundic acid hydrolases.

Acid hydrolases. Assay of activity and latency in the varied mixed cell populations of canine gastric mucosa.

The specific enzymic properties, membrane or particle binding capacities, and the total activities of certain acid hydrolases, including cathepsin D, acid phosphatase, arylsulfatase, and five acid glycosidases have been compared in normal canine antral and fundic mucosae and in liver. The two major regions of the gastric mucosa, whose cell populations are comparable in type but have very distinct functions, also differ in many properties of their lysosomal enzymes. These differences necessitate several major modification in their method of assay. Using optimal conditions, the activities of most of these enzymes were found to differ: levels in the antrum, in spite of its high water and mucin-glycoprotein content, were significantly greater, suggesting that the high lysosomal hydrolytic activity may be associated with the rapid autophagic processes of normal turnover of its surface epithelial and mucous neck cells. Lysosomal membrane stability or latency is also greater in the antrum; this may account, in part at least, for antral resistance to erosions brought about by stress.

Effect of bile on lysosomal stability in the mucosa of the canine gastric antrum.

The bound and free acid hydrolases from bile and bile- then acid-treated canine antral mucosa were compared with control tissue, using explants held in a series of Lucite chambers. Normal levels of activity of bound mucosal enzymes decreased as a result of bile acid insult, but "free" activity in the cytosol increased markedly. Organelle membrane stability was "tested" by submitting isolated particulate matter containing lysosomes, to hypoosmotic shock and to mechanical stress. Release of cathepsin D, only, was observed after bile treatment in vivo. Bile, followed by acid, however, damaged organelle integrity causing release or "leakage" of seven acid hydrolases.

Increased prothrombin time and metabolic changes with high serum aluminum levels following long-term exposure to Bayer-process alumina.

Serum aluminum levels twice to three times those in controls were found in 30 of 36 workers in a factory in an atmosphere of alumina dust. Inorganic phosphate and total alkaline phosphatase levels were within normal range, with no clinical evidence of phosphate depletion syndrome. Serum intestinal alkaline phosphatase, acid phosphatase and adenosine triphosphate levels were significantly reduced in the group with high levels of aluminum. The finding that the mean prothrombin time was significantly prolonged is of particular interest with respect to beneficial antithrombogenic effect.