Engineered cysteine antibodies: an improved antibody-drug conjugate platform with a novel mechanism of drug-linker stability.

Antibody-drug conjugates (ADCs) are fulfilling the promise of targeted therapy with meaningful clinical success. An intense research effort is directed towards improving pharmacokinetic profiles, toxicity and chemical stability of ADCs. The majority of ADCs use amide and thioether chemistry to link potent cytotoxic agents to antibodies via endogenous lysine and cysteine residues. While maleimide-cysteine conjugation is used for many clinical stage ADC programs, maleimides have been shown to exhibit some degree of post-conjugation instability. Previous research with site-directed mutagenic incorporation of cysteine residues for conjugation revealed that the stability of the drug-antibody linkage depends on the site of conjugation. Here we report on a collection of engineered cysteine antibodies (S239C, E269C, K326C and A327C) that can be site-specifically conjugated to potent cytotoxic agents to produce homogenous 2-loaded ADCs. These ADCs confirm that site of conjugation impacts maleimide stability and present a novel mechanism of thioether stabilization, effectively unlinking stability from either local chemical environment or calculated solvent accessibility and expanding the current paradigm for ADC drug-linker stability. These ADCs show potent in vitro and in vivo activity while delivering half of the molar equivalent dose of drug per antibody when compared to an average 4-loaded ADC. In addition, our lead engineered site shields highly hydrophobic drugs, enabling conjugation, formulation and clinical use of otherwise intractable chemotypes.

Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex.

The heterotrimeric kinesin-II holoenzyme purified from sea urchin (Strongylocentrotus purpuratus) eggs is assembled from two heterodimerized kinesin-related motor subunits of known sequence, together with a third, previously uncharacterized 115-kD subunit, SpKAP115. Using monospecific anti-SpKAP115 antibodies we have accomplished the molecular cloning and sequencing of the SpKAP115 subunit. The deduced sequence predicts a globular 95-kD non-motor "accessory" polypeptide rich in alpha-helical segments that are generally not predicted to form coiled coils. Electron microscopy of individual rotary shadowed kinesin-II holoenzymes also suggests that SpKAP115 is globular, with a somewhat asymmetric morphology. Moreover, the SpKAP115 subunit lies at one end of the 51-nm-long kinesin-II complex, being separated from the two presumptive motor domains by a approximately 26-nm-long rod, in a manner similar to the light chains (KLCs) of kinesin itself. This indicates that SpKAP115 and the KLCs may have analogous functions, yet SpKAP115 does not display significant sequence similarity with the KLCs. The results show that kinesin and kinesin-II are assembled from highly divergent accessory polypeptides together with kinesin related motor subunits (KRPs) containing conserved motor domains linked to divergent tails. Despite the lack of sequence conservation outside the motor domains, there is striking conservation of the ultrastructure of the kinesin and kinesin-II holoenzymes.

Laparoscopy without pneumoperitoneum. Effects of abdominal wall retraction versus carbon dioxide insufflation on hemodynamics and gas exchange in pigs.

Laparoscopic surgery with CO2 insufflation is associated with adverse effects on hemodynamics and gas exchange. The abdominal wall retractor (AWR) is an alternative for pneumoperitoneum. Hemodynamics and gas exchange during the use of an AWR were compared to those of CO2 pneumoperitoneum. In eight pigs subjected to 1 h of CO2 pneumoperitoneum or abdominal wall retraction, hemodynamics, gas exchange, and oxygen transport were studied in a randomized cross-over study design. The only change observed during abdominal wall retraction was mild respiratory alkalosis. In contrast, during CO2 pneumoperitoneum mean arterial blood pressure increased 13%, central filling pressures doubled, and a small increase in cardiac output was observed. Peak airway pressures increased 50%, end-tidal CO2 increased 20%, and respiratory acidosis was induced (arterial pH from 7.46 +/- 0.07 to 7.31 +/- 0.06 and pCO2 from 33 +/- 3 mmHg to 53 +/- 4 mmHg). Arterial PO2 decreased but mixed venous oxygen saturation and oxygen consumption were unaffected. In contrast with CO2 pneumoperitoneum, laparoscopy using abdominal wall retraction was not associated with adverse effects on hemodynamics or gas exchange.

Effects of pneumoperitoneum with helium on hemodynamics and oxygen transport: a comparison with carbon dioxide.

Pneumoperitoneum with CO2 is associated with adverse effects, such as hypercarbia, arrhythmias, and circulatory depression, which may limit its use in patients with underlying disease. Some of these effects may be caused by CO2 absorption resulting in acid-base disturbances. Laparoscopic insufflation with helium may be a good alternative for CO2, since it is chemically inert. Because there are few data on the use of helium for laparoscopy, we studied hemodynamics and gas exchange during insufflation with CO2 or helium in 8 pigs at 10, 15, and 20 mm Hg intraabdominal pressure. Heart rate did not change significantly with both gases. Arterial blood pressure increased with CO2 (p < 0.05) but not with helium. Cardiac output, mixed venous oxygen saturation, and oxygen consumption did not decrease, whereas central venous filling pressures increased during insufflation with either gas. Insufflation with CO2 resulted in mild increases in arterial, central venous, and end-expiratory CO2. The results suggest that pneumoperitoneum with helium will not be associated with profound circulatory depression or oxygen transport abnormalities. In addition, the use of helium is not associated with acid-base disturbances, although central venous filling pressures are similarly increased as with the use of CO2 pneumoperitoneum.

Does transient blocking of axonal conduction occur in uraemic neuropathy?

In uraemic and other patients with neuropathies, motor unit spike intervals were analysed using a special computer program to detect prolonged intervals of twice the normal duration, so-called "double intervals." These may have different causes--trigger failure, double discharge, or a transient fall in the firing rate of coactive motor units. There was not sufficient evidence that transient conduction blockings will occur in the main axon in uraemic neuropathies.

Serological response of multiple sclerosis patients and controls to 6/94-parainfluenza virus.

The serological responses of 195 multiple sclerosis (MS) patients and 251 controls were tested against 6/94-parainfluenza virus, which was previously isolated from brain tissue of two patients with MS. The hemagglutination-inhibition titers of greater than or equal to 1: 128 were found more frequently in MS patients (21.5%) than in controls (14.0%). However, the geometric mean titers did not differ between these two groups. The present study concludes that a causal relationship of 6/94-virus to MS, based on a specific immune response, is improbable, although it does not exclude the possibility of a pathogenetic significance of the agent in the cases from which the autopsy material was derived.