Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma.

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.

Effect of attachment site on stability of cleavable antibody drug conjugates.

The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

Effectiveness of the anaesthetic MS-222 in gilthead seabream, Sparus aurata: effect of feeding time and day-night variations in plasma MS-222 concentration and GST activity.

Feeding time is a potent zeitgeber capable of synchronising behavioural and physiological daily rhythms in fish. However, the effect of feeding time on the daily rhythm of drugs toxicity and/or effectiveness remains unexplored to date. In this paper we investigated the day/night variations in the effectiveness of an anaesthetic commonly used in fish (Tricaine, MS-222) in a teleost of great chronobiological and aquaculture interest (gilthead seabream). To this end, fish were kept under LD 12:12 and fed at mid-light (ML), mid-darkness (MD) or random times (RD). The time needed to induce anaesthesia (reduction of locomotor activity) during MS-222 exposure (65 mg/L) as well as the recovery period were investigated at ML and MD in the three experimental groups using specialised video tracking software. In addition, daily rhythms of GST activity in the liver (as an indicator of detoxification processes) and plasma MS-222 concentration (related to uptake) were determined. The results revealed that MS-222 effectiveness in the ML group was higher during the day than at night (significant reduction of activity after 3 min vs. 5 min) whereas in the MD group, the daily variation of MS-222 effectiveness was inverted (significant reduction of activity after 7 min at ML vs. 2 min at MD), suggesting that feeding time can shift the day-night variations in the effectiveness of MS-222. Hepatic GST also seemed to be affected by feeding time: in fish fed at MD or RD this enzyme activity showed significant differences during the day, and the highest levels were found at different times of the day in each group. Plasma MS-222 concentrations were higher at ML (142.4±12.8 ng/ml) than at MD (96.3±10.9 ng/ml) (t-Student, p<0.05). These results suggest that the daily variation in MS-222 concentration following exposure might be involved, among other factors, in the existence of day-night variations in the effectiveness of this anaesthetic. Furthermore, manipulation of the feeding schedule can be used to modify the daily variations in MS-222 effectiveness, which has basic as well applied implications for optimising anaesthesia protocols in fish aquaculture.

Diagnostic value of plasma PABA level in pancreatic diseases.

NBT-PABA test was performed in 72 subjects with simultaneous determination of the plasma PABA time-concentration curve. The curve in 25 controls after oral administration of N-benzoyl-L-tyrosyl-P-aminobenzoic acid (NBT-PABA) peaked (3.88 +/- 0.78 micrograms/ml, M +/- SD) at the third hour. In patients with chronic pancreatitis (24) and pancreatic carcinoma (6), the curves were flattened, and peaked at the fifth hour (2.7 +/- 1.4 micrograms/ml, M +/- SD), and fourth hour (3.14 +/- 2.26 micrograms/ml, M +/- SD), respectively. The discrimination between the controls and patients with chronic pancreatitis was most significant in the third-hour plasma PABA concentration (P less than 0.0001). This study shows that determination of the third-hour plasma PABA concentration is better in sensitivity and specificity than the 6-hour urinary excretion of PABA, suggesting that the third-hour plasma PABA concentration after oral administration of NBT-PABA might be a sensitive index of exocrine pancreatic function test.

Evaluation of two forms of N-benzoyl-L-tyrosyl p-aminobenzoic acid in pancreatic function testing of dogs.

Plasma para-aminobenzoic acid (PABA) concentrations were compared in 12 dogs after oral administration of either a powdered suspension or a solution of N-benzoyl-L-tyrosyl-PABA. Peak PABA plasma concentrations were significantly higher at 30, 60 and 90 minutes after administration of the solution (P less than 0.05). As the solution may now be used as a clinical test, interpretation of the results by comparison with normal absorption curves obtained after administration of the suspension could contribute to a failure to diagnose canine exocrine pancreatic insufficiency.

Pancreatic function testing: serum PABA measurement is a reliable and accurate measurement of exocrine function.

A comparison between the NBT-PABA/14C-PABA test (NBT-PABA, n-benzoyl-tyrosyl para-aminobenzoic acid) using the PABA excretion index (PEI) and serum PABA estimation at 90 minutes has been made in 42 consecutive subjects attending for investigation of possible pancreatic disease to a District General Hospital (DGH). The PEI was unobtainable or incorrect on 38% of occasions compared with 9% for the serum test. Sensitivity, specificity, and efficiency for the PEI (n = 33 valid results) were 71%, 88%, and 79% respectively and for the serum PABA (n = 41 valid results), 95%, 90%, and 93% respectively. These results confirm that measurement of serum PABA is a simpler, more reliable, and a more accurate method of assessing pancreatic function.

Liquid-chromatographic measurement of p-aminobenzoic acid and its metabolites in serum.

This is a high-performance liquid-chromatographic method for measuring p-aminobenzoic acid (PABA) and its metabolites in plasma or serum. Samples are deproteinized, then extracted with organic solvents before chromatography. For quantification, the peak height of the individual compound is compared with that of the internal standard. Analytical recoveries ranged from 41% to 100%, depending on the compound studied. Comparison of patients' samples after oral administration of either N-benzoyl-L-tyrosyl-p-aminobenzoic acid or free PABA revealed that PABA is extensively metabolized and conjugated to either p-acetamidobenzoic acid, p-aminohippuric acid, or p-acetamidohippuric acid. PABA concentrations in serum as measured with the Bratton-Marshall ultraviolet spectrophotometric procedure would appear predominantly to reflect measurements of metabolites, with only a minor contribution from PABA itself.

[Specificity and sensitivity of the 4-N-acetyl-L-tyrosyl-p- aminobenzoic acid test in childhood with serum p-aminobenzoic acid determination]. / Spezifität und Sensitivität des ALTAB-Testes im Kindesalter bei Serum-Paba-Bestimmung.

Specificity and sensitivity of ALTAB test have been estimated in 40 healthy children and 13 cystic fibrosis patients. The test has been carried out in the modification as 2-hour-serum PABA-test. The found specificity was 90%, the sensitivity 100% respectively. Therefore this test is suitable for evaluation of exocrine pancreas function.

The usefulness of serum PABA measurement after BT-PABA administration in the diagnosis of chronic pancreatitis.

We examined the maximal serum PABA concentration within 3 hrs (MS-PABA) and the 6 hr urinary PABA recovery (6 hr U-PABA) after BT-PABA administration, PABA excretion index, and chymotrypsin secretory response to caerulein-secretin stimulation in ten control subjects and fifteen patients with chronic pancreatitis diagnosed on the basis of pancreatograms. The results suggested that MS-PABA can distinguish the patients with definite irregular dilatation of the main pancreatic duct from the controls, but not those with localized irregular dilatations of the side branches from the controls. MS-PABA showed a significant correlation with 6 hr U-PABA, PABA excretion index, chymotrypsin output and bicarbonate output. This modified method was shown to be useful in the diagnosis of chronic pancreatitis with unequivocally abnormal pancreatograms and/or markedly decreased chymotrypsin secretion. This modification will serve to simplify the BT-PABA test by eliminating urinary collection and shortening the procedure time.

Colorimetric plasma assay for the bentiromide test (BT-PABA) for exocrine pancreatic insufficiency.

Bentiromide is a synthetic peptide, N-benzoyl-L-tyrosyl-p-aminobenzoic acid, which has been used as a test for exocrine pancreatic function. Following oral administration, bentiromide is hydrolyzed by chymotrypsin to yield free p-aminobenzoic acid (PABA) which is absorbed, conjugated and excreted in the urine. The PABA conjugates reach their peak levels in blood in 90-120 min. Healthy individuals have higher levels of PABA than patients with pancreatic insufficiency. A simple, accurate, and precise method for the determination of PABA in blood has been developed and validated. The plasma (1 ml) is deproteinized by perchloric acid. The conjugates are hydrolyzed and the total PABA is determined colorimetrically by the Bratton-Marshall test. The standard curve in plasma is linear up to 8 micrograms/ml of PABA. A similar semimicro method using 200 microliter of plasma suitable for pediatric samples shows comparable results. Average analytical recovery is 97% and precision studies of pooled within-run and total between-run showed CV% of 5.0 and 5.7%, respectively.

Serum PABA and fluorescein in the course of Bz-Ty-PABA and pancreolauryl test as an index of exocrine pancreatic insufficiency.

Forty-six subjects (20 chronic pancreatitis, 7 chronic liver disease, 7 recovered from acute pancreatitis, 2 Crohn's disease, and 10 healthy controls) classified by S-C test as having normal pancreatic function (26 subjects), or moderate (10 subjects) and severe (10 cases) pancreatic insufficiency, were given, on different days, 1 g of oral PABA or 348 mg of oral fluorescein dilaurate. At the 1st, 2nd, and 4th hours (PABA) and the 2nd, 4th, and 6th hours (fluorescein) serum samples were taken for assay. In the presence of severe exocrine pancreatic insufficiency, the sensitivity of the fluorescein serum levels was higher than that observed for the PABA (100% and 80%, respectively), and quite similar to that shown by the urinary tests (100% and 70%, respectively). On the contrary, in presence of moderate pancreatic insufficiency, both the urinary test (pancreolauryl and (PABA) give a sensitivity higher than that found in the serum tests (30-40% and 10-30%, respectively). The parallel combination of both the serum or urinary tests does not significantly improve the sensitivity of the single test. These results suggest that the serum PABA and serum fluorescein tests can be valid choice when a prolonged urinary collection is difficult, i.e., in children and in elderly patients. However, the slight diagnostic gain does not justify the routine use of both urinary and serum tests.

Value of serum PABA as a pancreatic function test.

In a total of 71 subjects (19 controls, 24 patients with non-pancreatic gastrointestinal disease, and 27 patients with pancreatic disease) an oral pancreatic function test using N-benzoyl-L-tyrosyl-PABA (BT-PABA) was performed with simultaneous determination of the serum para-aminobenzoic acid (PABA). Urinary excretion of PABA was significantly less (p less than 0.001) in patients with chronic pancreatitis (n = 12) and pancreatic carcinoma (n = 10) than in controls and in patients with non-pancreatic disease. The serum concentration curve in patients with chronic pancreatitis was significantly flattened (p less than 0.001) compared with that of the control group and the patients with non-pancreatic gastrointestinal disease. The discrimination between the controls and the patients with chronic pancreatitis was best at 120 minutes after administration of BT-PABA (lower limit of normal: 2.8 micrograms/ml). The results of our study show that determination of PABA serum concentration two hours after administration of BT-PABA is as valuable an index of pancreatic function as the urinary excretion of PABA.

BT-PABA test with plasma PABA measurements: evaluation of sensitivity and specificity.

Urinary recovery and plasma time curve of PABA were determined in 15 control subjects and 65 patients during a six hour period after ingestion of a meal containing 1 g BT-PABA (2.333 mmol PABA). In controls, the plasma PABA time curve registered a peak at two hours followed by a rapid decrease to reach its lowest value at six hours. In patients with chronic pancreatitis (n=32), the peak was lower and was followed by a slow decrease; furthermore, it was delayed in those with severe pancreatic insufficiency. The best discrimination between controls and patients with chronic pancreatitis was obtained by using the maximal value of plasma PABA (MPPABA) at two or three hours. In 56 subjects a hyperbolic relationship between MPPABA and duodenal lipase output stimulated by a meal was mathematically demonstrated and it was calculated that the lower limit of normal MPPABA (mean -2 SD) corresponded to a lipase output equal to 20.6% of the mean value of normal subjects (lower limit of normal lipase output = 40%). By comparison, steatorrhoea occurred when lipase output was less than 10%. Consequently, MPPABA was low not only in all patients with steatorrhoea but also in some who had pancreatic deficiency but normal daily faecal fat. With lipase output as the reference, 12 subjects who had normal MPPABA were proved to have falsely abnormal urinary results. Urinary PABA excretion after oral administration of 2.333 mmol free PABA was also determined in 27 subjects. The PABA excretion index (PEI) was calculated: PEI = PABA urinary excretion after BT-PABA/PABA urinary excretion after free PABA. Seven patients with normal MPPABA but low urinary excretion after BT-PABA had a normal PABA excretion index. It was also observed that, in five patients with intestinal disease, free PABA absorption was not impaired. In conclusion, BTPABA test with MPPABA determination used as an index of exocrine function is (1) less sensitive than Lundh's test, but more sensitive than steatorrhoea; (2) more specific than the test with urinary recovery determination alone, and as specific as PABA excretion index, which requires a double test.