Acquired ADAMTS-13 deficiency in pediatric patients with severe sepsis

We studied the state of ultralarge 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

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

Distribution of DL488 fluorescently labeled HAV and LAV in the liver of living rats.

Multiphoton fluorescence excitation images of the livers of living Munich-Wistar-Fromter rats were collected 2 hours after intravenous injection of 6 mg/kg DL488-HAV (Panel A) or DL488-LAV (Panel B). Indicated regions are reproduced at 3X magnification in insets. Similar to the results obtained at 24 hours, punctate fluorescence is observed in rats injected with DL488-HAV but not DL488-LAV. (C) Time series of images collected from the same region of the liver prior to (left panel) and then again 1 and 2 hours after intravenous injection of DL488-HAV (middle and right panels, respectively). In this study, DL488-HAV accumulation was particularly pronounced, becoming apparent within one hour, and continuing to accumulate over the next hour. Neither construct is detectibly associated with hepatocytes, as identified by their brown autofluorescence, arranged in linear “cords”. Bright white punctate fluorescence reflects broad-spectrum autofluorescence of vitamin A in stellate cells. Blue signal derives from second harmonic generation, likely from collagen. Scale bars represent 200 microns in panels A and B, and 50 microns in panel C. All images are collected using identical microscope settings and contrast enhanced identically. (TIF)</p

Representative immunofluorescence detection images.

LAV-ADC (A1, A2 and A3), HAV-ADC (B1, B2 and B3) and isotype control ADC (C1 and C2) in HT29 tumors following a single 10 mg/kg IV administration of each molecule 24 hours post dose. Nuclei were stained with DAPI. Data are shown from a representative HT-29 tumor section from a single rat for each compound. Colocalization shows the overlap of the ADC and MET target. The scale bars represent 100 microns.</p

Mean (+/- SD) concentrations (%ID/g) of the radiolabeled LAV and HAV MET-ADCs following a single IV administration of ~6 mg/kg (~10 mCi/animal) in male HT29-tumor bearing rats.

124I-labeled LAV and HAV MET-ADCs in (A1) blood, (B1) liver, (C1) kidneys and (D1) tumor or 111In-DTPA-labeled LAV and HAV MET-ADCs in (A2) blood, (B2) liver, (C2) kidneys and (D2) tumor. Data are N = 3/timepoint.</p

Tumor regression profiles of LAV- and HAV-ADCs in HT29 tumor bearing rats.

Data are the mean (+/-SD) concentrations for six animals/timepoint for both molecules following a single 3-, 6- or 10-mg/kg IV administration of each molecule. The assessment of the tumor regression activity of an isotype control ADC was included following a single 10 mg/kg IV administration.</p

Representative SPECT/CT images from a single animal.

LAV (A1 and A2) and HAV (B1 and B2) 124I- (A1 and B1) or 111In-DTPA- (A2 and B2) labeled MET-ADCs following a single IV administration of ~6 mg/kg (~10 mCi/animal) in male HT29-tumor bearing rats. Images scaled to “fixed percent injected dose (%ID)” are scaled so that the maximum voxel value in the image is equal to a fixed percentage of the dose injected into the animal in mCi (decay corrected to the time of imaging). Voxel values between 0 and the fixed max are scaled linearly. Images are thus quantitatively comparable across animals, because similar color values are representative of similar radioactivity levels. Arrows show the location of the HT29 tumor xenografts on the flank of rats.</p

Rat mean (± SD) AST and ALT values for the HAV- and LAV-ADCs over time.

Rat mean (± SD) AST and ALT values for the HAV- and LAV-ADCs over time.</p

Distribution of DL488 fluorescently-labeled HAV and LAV in the liver and kidney of the same living rats.

Multiphoton fluorescence excitation images of the kidneys of living Munich-Wistar-Fromter rats were collected 24 hours after intravenous injection of 6 mg/kg DL488-HAV or DL488-LAV. The livers of these same rats were then presented for intravital microscopy of the liver. (A) DL488-HAV in the liver. (B) DL488-LAV in the liver. (C) DL488-HAV in the kidney of the same rat as shown in panel A. (D) DL488-LAV in the kidney of the same rat as shown in panel B. Indicated regions are reproduced at 2x magnification in the center column. Brown fluorescence in the liver results from hepatocyte autofluorescence, and yellow-orange fluorescence in the kidney results from proximal tubule autofluorescence. Arrows indicate distal tubules (identified as segments lacking autofluorescence) with low levels of probe fluorescence, indicating modest amounts of filtration. Scale bars represent 200 microns length. All images are collected using identical microscope settings. Images of each organ are contrast enhanced identically.</p

Pharmacokinetic profiles in of the LAV and HAV ADCs in normal Sprague Dawley rats.

Data are the mean (+/-SD) concentrations for three animals/timepoint for both molecules. The total human IgG (A and B) and payload conjugated IgG (C and D) kinetics of the ADCs were characterized following a single 3-, 6- or 10-mg/kg IV administration of each molecule.</p