Strategies to Target Tumor Immunosuppression

The tumor microenvironment is currently in the spotlight of cancer immunology research as a key factor impacting tumor development and progression. While antigen-specific immune responses play a crucial role in tumor rejection, the tumor hampers these immune responses by creating an immunosuppressive microenvironment. Recently, major progress has been achieved in the field of cancer immunotherapy, and several groundbreaking clinical trials demonstrated the potency of such therapeutic interventions in patients. Yet, the responses greatly vary among individuals. This calls for the rational design of more efficacious cancer immunotherapeutic interventions that take into consideration the “immune signature” of the tumor. Multimodality treatment regimens that aim to enhance intratumoral homing and activation of antigen-specific immune effector cells, while simultaneously targeting tumor immunosuppression, are pivotal for potent antitumor immunity

Bispecific Radioimmunoconjugates for Molecular Imaging and Radioimmunotherapy of HER2 Overexpressing Breast Cancer

The HER2 receptor is overexpressed in approximately 20% of breast cancer (BC) cases, and is the target for multiple therapies. One challenge in treating HER2+ BC is that a large proportion of patients demonstrate resistance to therapy. Upregulation of other members of the HER family, plays an important role in resistance to HER2-targeted therapies. The hypothesis was that bispecific radioimmunoconjugates (bsRICs) developed against HER2 and another member of the HER receptor family would be able to successfully target cells expressing one or more receptor for molecular imaging and radioimmunotherapy of BC. Trastuzumab Fab fragments were conjugated to heregulin (HRG) or epidermal growth factor (EGF) with a 24-mer polyethylene glycol (PEG) linker, and labeled with 111In or 177Lu. 111In-DTPA-Fab-PEG24-HRG and 111In-DTPA-Fab-PEG24-EGF demonstrated the ability to specifically bind to one or both receptors on cancer cell lines, and demonstrated higher binding than monospecific agents recognizing HER2, HER3 or EGFR. SPECT imaging was performed on mice bearing tumour xenografts, and optimal tumour uptake was found 48 hr after injection. Biodistribution studies showed tumour: blood uptake ratios of 6-8. The cytotoxicity of 111In-DTPA-Fab-PEG24-EGF and 177Lu- DOTA-Fab-PEG24-EGF was evaluated through clonogenic assays which revealed specific cell killing. Radiation absorbed doses to tumours and normal tissues were estimated and compared for 111In and 177Lu-labeled bsRICs. The maximum injected amount of 177Lu-DOTA-Fab-PEG24-EGF which caused no observable adverse effects (NOAEL) was identified to be 11.1 MBq. Mice bearing trastuzumab-sensitive or resistant tumours were treated with 111In and 177Lu-labeled bsRICs and 177Lu-bsRICs were found to inhibit tumour growth more effectively than 111In-bsRICs due to a 9.3-fold higher radiation absorbed dose (55.0 vs. 5.9 Gy, respectively). These results suggest that bsRICs may be useful for imaging and radioimmunotherapy of HER2-positive BC co-expressing HER3 or EGFR. Co-expression of these receptors in HER2+ tumours is associated with resistance to HER2-targeted therapies.Ph.D

Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species

Mass cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in mass cytometry, called mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO4 and RuO4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO4 and RuO4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO4 and RuO4 were used in a 20-sample barcoding protocol together with palladium isotopes. As mass channels occupied by osmium and ruthenium are not used for antibody detection the number of masses effectively utilized in a single experiment is expanded. OsO4 and RuO4 can therefore be used as MCB reagents for a wide range of mass cytometry workflows. © 2016 International Society for Advancement of Cytometry

Development and validation of a NANOGold™ immunoassay for the detection of vascular endothelial growth factor (VEGF) in human serum using inductively coupled plasma mass spectrometry

This work aimed to develop and validate a NANOGold based assay, quantified using inductively coupled plasma mass spectrometry (ICP-MS), for the detection of human vascular endothelial growth factor (hVEGF) in serum. The initial assay range based on calibration standards was 62.5-2000 pg/mL with a detection limit of approximately 30 pg/mL. After validation using spiked validation controls, a quantification range between 175 and 1928 pg/mL was obtained. The inter-assay precision was between 2.3 and 18.9% with accuracy between -8.8 and -3.1%. Additional performance parameters, including dilutional linearity, matrix specificity and time-factored drift, were within +/-20%, as defined by the validation acceptance criteria for the validation of macromolecule immunoassays used within our clinical environment. Serum samples from healthy donors were analysed to determine the endogenous levels of VEGF present; these ranged from 164 to 580 pg/mL with a mean of 273 pg/mL. The intra- and inter-assay precision obtained from the healthy donor samples were 1.3-10.7% and 4.2-17.5%, respectively. This demonstration of a validated immunoassay opens further possibilities, utilising the simultaneous detection capabilities of ICP-MS for the detection of multiple analytes in a single validated immunoassay, for routine use within a clinical environment