Reducing target binding affinity improves the therapeutic index of anti-MET antibody-drug conjugate in tumor bearing animals.

Many oncology antibody-drug conjugates (ADCs) have failed to demonstrate efficacy in clinic because of dose-limiting toxicity caused by uptake into healthy tissues. We developed an approach that harnesses ADC affinity to broaden the therapeutic index (TI) using two anti-mesenchymal-epithelial transition factor (MET) monoclonal antibodies (mAbs) with high affinity (HAV) or low affinity (LAV) conjugated to monomethyl auristatin E (MMAE). The estimated TI for LAV-ADC was at least 3 times greater than the HAV-ADC. The LAV- and HAV-ADCs showed similar levels of anti-tumor activity in the xenograft model, while the 111In-DTPA studies showed similar amounts of the ADCs in HT29 tumors. Although the LAV-ADC has ~2-fold slower blood clearance than the HAV-ADC, higher liver toxicity was observed with HAV-ADC. While the SPECT/CT 111In- and 124I- DTPA findings showed HAV-ADC has higher accumulation and rapid clearance in normal tissues, intravital microscopy (IVM) studies confirmed HAV mAb accumulates within hepatic sinusoidal endothelial cells while the LAV mAb does not. These results demonstrated that lowering the MET binding affinity provides a larger TI for MET-ADC. Decreasing the affinity of the ADC reduces the target mediated drug disposition (TMDD) to MET expressed in normal tissues while maintaining uptake/delivery to the tumor. This approach can be applied to multiple ADCs to improve the clinical outcomes.

Influence of physiochemical properties on the subcutaneous absorption and bioavailability of monoclonal antibodies.

Many therapeutic monoclonal antibodies (mAbs) were initially developed for intravenous (IV) administration. As a means to improve mAb drug-ability and the patient experience, subcutaneous (SC) administration is an increasingly important delivery route for mAbs. Unlike IV administration, bioavailability limitations for antibodies have been reported following SC injection and can dictate whether a mAb is administered via this parenteral route. The SC bioavailability of antibodies has been difficult to predict, and it can be variable and partial, with values ranging from ~50% to 100%. The mechanisms leading to the incomplete bioavailability of some mAbs relative to others are not well understood. There are some limited data that suggest the physiochemical properties inherent to a mAb can contribute to its SC absorption, bioavailability, and in vivo fate. In this study, we evaluated the integrated influence of multiple mAb physiochemical factors on the SC absorption and bioavailability of six humanized mAbs in both rats and cynomolgus monkeys. We demonstrate the physiochemical properties of mAbs are critical to their rate and extent of SC absorption. The combination of high positive charge and hydrophobic interaction significantly reduced the rate of the evaluated mAb's SC absorption and bioavailability. Reduction or balancing of both these attributes via re-engineering the mAbs restored desirable properties of the molecules assessed. This included reduced association with SC tissue, improvements in mAb absorption from the SC space and overall SC bioavailability. Our findings point to the importance of evaluating the relative balance between various physiochemical factors, including charge, hydrophobicity, and stability, to improve the SC drug-ability of mAbs for selecting or engineering mAbs with enhanced in vivo absorption and bioavailability following SC administration.

The Properties of Cysteine-Conjugated Antibody-Drug Conjugates Are Impacted by the IgG Subclass.

Among the numerous antibody-drug conjugate (ADC) clinical candidates, one of the most prevalent types utilizes the interchain cysteines in antibodies to conjugate auristatin via a maleimide-containing linker. In this class of ADCs, there are a paucity of systematic studies characterizing how IgG subclass influences the biophysical properties and in vivo pharmacokinetics of the ADC molecules. In the current investigation, we studied cysteine-conjugated ADCs using a model system consisting of human IgG1, IgG2, and IgG4 antibodies with the same variable region. Our findings identified some unforeseen differences among the three ADCs. Drug conjugation profiling by LC-MS revealed that 50% of inter heavy-light chain disulfide bonds are disrupted to conjugate drugs in IgG1 antibody while only 10% in IgG2 antibody and 20% in IgG4 antibody. The solution behavior of the ADCs was interrogated in concentrating experiments and diffusion interaction parameter measurements. We found that drug conjugation affected the solution property of the three antibodies differently, with the IgG2-based ADC having the most increased propensity to aggregate. Rat PK studies using a sensitive LC-MS-based bioanalytical method showed that the IgG1-based ADC has poor peripheral linker-payload stability while the IgG2- and IgG4-based ADCs are stable. The conjugate stability of the IgG2-based ADC was further confirmed in a cynomolgus monkey PK study. Overall, the IgG2-based ADC exhibited the best PK/conjugate stability but also the most deterioration in stability among the three ADCs. Our findings provide important information and present multifactorial considerations for the selection of IgG subclass during ADC drug discovery when employing stochastic cysteine conjugation.

Antihemostatic activity of human granzyme B mediated by cleavage of von Willebrand factor.

The cytotoxic lymphocyte protease granzyme B (GrB) is elevated in the plasma of individuals with diseases that elicit a cytotoxic lymphocyte-mediated immune response. Given the recently recognized ability of GrB to cleave extracellular matrix proteins, we examined the effect of GrB on the pro-hemostatic molecule von Willebrand factor (VWF). GrB delays ristocetin-induced platelet aggregation and inhibits platelet adhesion and spreading on immobilized VWF under static conditions. It efficiently cleaves VWF at two sites within the A1-3 domains that are essential for the VWF-platelet interaction. Like the VWF regulatory proteinase ADAMTS-13, GrB-mediated cleavage is dependent upon VWF conformation. In vitro, GrB cannot cleave the VWF conformer found in solution, but cleavage is induced when VWF is artificially unfolded or presented as a matrix. GrB cleaves VWF with comparable efficiency to ADAMTS-13 and rapidly processes ultra-large VWF multimers released from activated endothelial cells under physiological shear. GrB also cleaves the matrix form of fibrinogen at several sites. These studies suggest extracellular GrB may help control localized coagulation during inflammation.

Magnesium maintains endothelial integrity, up-regulates proteolysis of ultra-large von Willebrand factor, and reduces platelet aggregation under flow conditions.

Mg (++) regulates endothelial functions and has anti-inflammatory effects. Its effects on thrombosis have been demonstrated, but the mechanism remains poorly understood. We investigated the roles of MgSO(4) in regulating the release and cleavage of the prothrombotic ultra-large (UL) von Willebrand factor (VWF) and VWF-mediated platelet adhesion and aggregation. Washed platelets were perfused over cultured endothelial cells from human umbilical cord veins under a shear stress of 2.5 dyn/cm(2). Release and cleavage of ULVWF by ADAMTS-13 was measured in the absence or presence of physiological or therapeutic levels of MgSO(4). Whole blood or plasma-free reconstituted blood was perfused over immobilized collagen to measure the effect of MgSO(4) on platelet adhesion and aggregation. Also studied were the effects of MgSO(4) on ristocetin-induced platelet aggregation andVWF-collagen interaction. Maintenance of endothelial integrity required physiological levels of MgSO(4), but exogenous MgSO(4) showed no additional benefits. Exogenous MgSO(4) significantly enhanced the cleavage of the newly released ULVWF strings by ADAMTS-13 and markedly reduced platelet aggregation on immobilized collagen under flow conditions. This effect is likely to be mediated through VWF as Mg(++) partially inhibited ristocetin-induced platelet aggregation and VWF binding to collagen. MgSO(4) is critical for maintaining endothelial integrity and regulates ULVWF proteolysis and aggregation under flow conditions. These results provide a new insight into additional mechanisms involved with magnesium therapy.

Shear-induced disulfide bond formation regulates adhesion activity of von Willebrand factor.

von Willebrand factor (VWF) is the largest multimeric adhesion ligand circulating in blood. Its adhesion activity is related to multimer size, with the ultra-large forms freshly released from the activated endothelial cells being most active, capable of spontaneously binding to platelets. In comparison, smaller plasma forms circulating in blood bind platelets only under high fluid shear stress or induced by modulators. The structure-function relationships that distinguish the two types of VWF multimers are not known. In this study, we demonstrate that some of the plasma VWF multimers contain surface-exposed free thiols. Physiological and pathological levels of shear stresses (50 and 100 dynes/cm(2)) promote the formation of disulfide bonds utilizing these free thiols. The shear-induced thiol-disulfide exchange increases VWF binding to platelets. The thiol-disulfide exchange involves some or all of nine cysteine residues (Cys(889), Cys(898), Cys(2448), Cys(2451), Cys(2490), Cys(2491), Cys(2453), Cys(2528), and Cys(2533)) in the D3 and C domains as determined by mass spectrometry of the tryptic VWF peptides. These results suggest that the thiol-disulfide state may serve as an important structural determinant of VWF adhesion activity and can be modified by fluid shear stress.

Acquired ADAMTS-13 deficiency in pediatric patients with severe sepsis.

We studied the state of ultra-large von Willebrand factor (ULVWF) proteolysis in 21 pediatric patients with severe sepsis and found that the overall group of patients had moderately reduced ADAMTS-13 activity, but 31% had severe enzymatic deficiency. The severe deficiency correlated with greater adhesion activity of von Willebrand factor, severity of thrombocytopenia and plasma levels of interleukin-6. It also correlated clinically with severity of illness and organ dysfunction. These results suggest that ULVWF proteolysis is insufficient in septic patients and severely deficient in a subgroup of patients. The deficiency may contribute to the development of thrombocytopenia and ischemic organ failure associated with sepsis.