Differences in genotype and virulence among four multidrug-resistant <i>Streptococcus pneumoniae</i> isolates belonging to the PMEN1 clone

We report on the comparative genomics and characterization of the virulence phenotypes of four &lt;i&gt;S. pneumoniae&lt;/i&gt; strains that belong to the multidrug resistant clone PMEN1 (Spain&lt;sup&gt;23F&lt;/sup&gt; ST81). Strains SV35-T23 and SV36-T3 were recovered in 1996 from the nasopharynx of patients at an AIDS hospice in New York. Strain SV36-T3 expressed capsule type 3 which is unusual for this clone and represents the product of an in vivo capsular switch event. A third PMEN1 isolate - PN4595-T23 - was recovered in 1996 from the nasopharynx of a child attending day care in Portugal, and a fourth strain - ATCC700669 - was originally isolated from a patient with pneumococcal disease in Spain in 1984. We compared the genomes among four PMEN1 strains and 47 previously sequenced pneumococcal isolates for gene possession differences and allelic variations within core genes. In contrast to the 47 strains - representing a variety of clonal types - the four PMEN1 strains grouped closely together, demonstrating high genomic conservation within this lineage relative to the rest of the species. In the four PMEN1 strains allelic and gene possession differences were clustered into 18 genomic regions including the capsule, the blp bacteriocins, erythromycin resistance, the MM1-2008 prophage and multiple cell wall anchored proteins. In spite of their genomic similarity, the high resolution chinchilla model was able to detect variations in virulence properties of the PMEN1 strains highlighting how small genic or allelic variation can lead to significant changes in pathogenicity and making this set of strains ideal for the identification of novel virulence determinant

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins

Mechanisms of Acquired Resistance to Trastuzumab Emtansine in Breast Cancer Cells

AbstractThe receptor tyrosine kinase HER2 is overexpressed in approximately 20% of breast cancer, and its amplification is associated with reduced survival. Trastuzumab emtansine (Kadcyla, T-DM1), an antibody–drug conjugate that is comprised of trastuzumab covalently linked to the antimitotic agent DM1 through a stable linker, was designed to selectively deliver DM1 to HER2-overexpressing tumor cells. T-DM1 is approved for the treatment of patients with HER2-positive metastatic breast cancer following progression on trastuzumab and a taxane. Despite the improvement in clinical outcome, many patients who initially respond to T-DM1 treatment eventually develop progressive disease. The mechanisms that contribute to T-DM1 resistance are not fully understood. To this end, we developed T-DM1–resistant in vitro models to examine the mechanisms of acquired T-DM1 resistance. We demonstrate that decreased HER2 and upregulation of MDR1 contribute to T-DM1 resistance in KPL-4 T-DM1–resistant cells. In contrast, both loss of SLC46A3 and PTEN deficiency play a role in conferring resistance in BT-474M1 T-DM1–resistant cells. Our data suggest that these two cell lines acquire resistance through distinct mechanisms. Furthermore, we show that the KPL-4 T-DM1 resistance can be overcome by treatment with an inhibitor of MDR1, whereas a PI3K inhibitor can rescue PTEN loss–induced resistance in T-DM1–resistant BT-474M1 cells. Our results provide a rationale for developing therapeutic strategies to enhance T-DM1 clinical efficacy by combining T-DM1 and other inhibitors that target signaling transduction or resistance pathways. Mol Cancer Ther; 17(7); 1441–53. ©2018 AACR.</jats:p

Potential Mechanisms for Thrombocytopenia Development with Trastuzumab Emtansine (T-DM1)

Abstract Purpose: Trastuzumab-emtansine (T-DM1) is an antibody–drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1–induced thrombocytopenia. Experimental Design: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H3]-DM1 and 125I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR. Results: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure. Conclusions: These data support the hypothesis that T-DM1–induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs. Clin Cancer Res; 21(1); 123–33. ©2014 AACR.</jats:p

Supplemental Figures and Legends from Mechanisms of Acquired Resistance to Trastuzumab Emtansine in Breast Cancer Cells

Supplemental Figure 1. Upregulated BHLHE41 in KPL-4 TR and BT-474M1 TR cells does not confer resistance to T-DM1. Supplemental Figure 2. Flow cytometry analysis of HER2 and MDR1 expression in KPL-4 P and TR cells, and tumor derived KPL-4 TR cells. Supplemental Figure 3. qRT-PCR analysis of ErbB receptors and ligands in KPL-4 and BT-474M1 P and TR cells. Supplemental Figure 4. Flow cytometry analysis of HER2 expression in BT-474M1 P and TR cells. Supplemental Figure 5. Immunofluorescence detection of cell surface HER2 on BT-474M1 and T-DM1 resistant cells. Supplemental Figure 6. Analysis of T-DM1 uptake in BT-474M1 P and TR, and KPL-4 P and TR cells using radiolabeled T-DM1. Supplemental Figure 7. Treatment of KPL-4 TR cells with anti-IGF-1R antibody 10H5 or the selective c-met inhibitor PHA665725 does not restore sensitivity to T-DM1. Supplemental Figure 8. Upregulated EGFR does not mediate resistance to T-DM1 in KPL-4 TR cells. Supplemental Figure 9. Potential autocrine receptor-ligand interactions for T-DM1 resistance. Supplemental Figure 10. Comparable shRNA knockdown of PTEN in BT-474M1 cells in 2 different shRNA clones, #1 and #3. Supplemental Figure 11. Molecular alterations in BT-474M1 TR cells that did not confer T-DM1 resistance. Supplemental Figure 12. Expression of drug resistance transporters, assessed by qRT-PCR, in KPL-4 and BT-474M1 parental and TR cells. Supplemental Figure 13. The MDR1 inhibitor XR9051 and the BCRP inhibitor Ko143 do not affect cell viability Supplemental Figure 14. MRP4 siRNA in BT-474M1 TR does not reverse T-DM1 resistance. Supplemental Figure 15. MRP4 siRNA in KPL-4 TR does not reverse T-DM1 resistance. Supplemental Figure 16. Expression of SLC46A3 in KPL-4 parental and TR cells assessed by qRT-PCR. Supplemental Figure 17. SLC46A3 siRNA in BT-474M1 TR cells significantly reverses T-DM1 resistance.</p

Supplemental Table 3 from Mechanisms of Acquired Resistance to Trastuzumab Emtansine in Breast Cancer Cells

Differentially expressed genes in T-DM1-resistant BT-474M1 cells with respect to the parental cell line (fold change > 2 fold and P < 0.05 of individual probes for a given gene in the microarray)</p

Supplemental Table 4 from Mechanisms of Acquired Resistance to Trastuzumab Emtansine in Breast Cancer Cells

Differentially expressed genes in T-DM1-resistant KPL-4 cells with respect to the parental cell line (fold change > 2-fold and P < 0.05 of individual probes for a given gene in the microarray)</p

Supplemental Table 1 from Mechanisms of Acquired Resistance to Trastuzumab Emtansine in Breast Cancer Cells

BT-474M1 TR vs. Parental cells, gene expression changes >5-fold</p