QF-Pro®

Quantitative Functional PROteomics platform

Unveiling Protein Interactions And PTM In Pathology

The use of two-site assays in diagnostics is desirable and commonly employed in simple capture-detection systems but has not been exploited in tissue biopsies. FASTBASE uses a proprietary two-site detection platform to drive specificity of tissue-based analyses, providing functional proteomics information relevant to disease and disease treatment. The underlying technology adapts the property of FRET, visualised through lifetime changes, to detect readout proximity in the ≤10nm range. This drives specificity of detection in for example protein phosphorylation readouts, and also enables the detection of protein complexes.

This pathology data obtained is for in situ, undisrupted samples, providing a balance of specificity, sensitivity, dynamic range and tissue information not achievable with any other available approach.

Mechanism of Action

Providing evidence on the critical molecular mechanisms operating in pathological settings is the driver for our biomarker programmes. These mechanistic insights typically represent changes in the function of regulatory proteins responsible for determining cellular behavior – an inflammatory response, inappropriate cell proliferation, etc. Such changes are manifest in post-translational modifications (including phosphorylation, acetylation) and in complex assembly (for example ligand-receptor binding, intracellular signaling assemblies). The power of the FASTBASE platform is exemplified by its ability to recognize receptor ligand engagement spanning two cells – this powerful capability lies at the heart of our immune-oncology portfolio.

PD-1 interaction with its ligand PD-L1 occurs between T-Cells and an antigen presenting cell (eg a tumour cell) causing a suppression of the T-Cell response. Interventions employed to block this type of immune evasion are widely employed in cancer. FASTBASE has developed a molecular readout of this complex formation in patient biopsies employing the QF-Pro® platform to monitor the ≤10nm proximity of ligand-receptor pairs engaged between cells.

Novel Biomarkers That Improve Personalised Medicine

Delivering the right therapeutic treatment to the right patient requires accurate tools to evaluate whether they are likely to respond to the treatment. This relies on accurate evaluation of biomarkers either in blood or tissue biopsies – informing on cells causing the disease (e.g. cancer cells) and/or cells combating the disease (e.g. immune cells). A major failing of many medicines and of many drugs in development is the lack of adequate predictive biomarkers that enables selection of patients most likely to benefit from a particular treatment.

Current biomarker approaches, observing the expression of genes and proteins, do not provide the functional data reflective of the complex pathways involved in disease(s). This limits the ability of delivering on the promise of precision medicine.

Our “functional proteomics” approach provides clinicians, pharmaceutical companies and academia with the resolution demanded by personalised medicine.

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A Case Study In Immune-oncology

Today, when a patient is diagnosed with non-small cell lung cancer, the predictive biomarker used to decide treatment with anti-PD1 is the level of PD-L1 expressed in the tumour biopsy. Based on an expression score, the patient will receive first line treatment with immunotherapy or with a traditional chemotherapy.

The application of these potentially life-threatening treatments has engendered a strong impetus towards more informative stratification biomarkers.

The immunotherapy treatment is designed to block PD1-PD-L1 interaction and the extent to which this is actually occurring in patients is at the heart of the ability to respond to its blockade. If there is no interaction because the tumour evades the immune system by another mechanism, then there is little chance of blockade of this event triggering an immune response to clear the tumour. Similarly, there are situations in which low PD-L1 levels preclude current treatment options but, nevertheless, retain high complex formation. These patients may not be receiving optimal treatments.

The QF-Pro® technology from FASTBASE allows pathologists to access the extent of complex formation in tumour biopsies, from routine tumour sections, without disrupting the organization of the tissue. This is a profound advance that is enabling not only to this specific checkpoint blockade but others in use and in the pipeline.

Papers

A Complex Interplay of Anionic Phospholipid Binding Regulates 3′-Phosphoinositide-Dependent-Kinase-1 Homodimer Activation

Gloria de las Heras-Martínez*, Véronique Calleja*, Remy Bailly, Jean Dessolin, Banafshé Larijani**, Jose Requejo-Isidro** (2019) A Complex Interplay of Anionic Phospholipid Binding Regulates 3′-Phosphoinositide-Dependent-Kinase-1 Homodimer Activation. Scientific Reports.

Time resolved amplified FRET identifies protein kinase B activation state as a marker for poor prognosis in clear cell renal cell  carcinoma

James Miles, Christopher J. Applebee, Pierre Leboucher, SoniaLopez-Fernandez, Dae-Jin Lee, Rosa Guarch, Stephen Ward, Peter J. Parker, Jose I. López, Banafshé Larijani (2017) Time resolved amplified FRET identifies protein kinase B activation state as a marker for poor prognosis in clear cell renal cell  carcinoma. BBACLI 161.

Uncoupling TORC2 from AGC kinases inhibits tumour growth

Angus J M Cameron, Selvaraju Veeriah, Jacqueline T Marshall, Banafshé Larijani and Peter J Parker (2017) Uncoupling TORC2 from AGC kinases inhibits tumour growth. Oncotarget.

Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET

Gloria de las Heras-Martı´nez, Josu Andrieu, Banafshé Larijani* and Jose Requejo-Isidro* (2017) Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET Journal of Biophotonics

A Small Molecule Inhibitor of PDK1/PLCγ1 Interaction Blocks Breast and Melanoma Cancer Cell Invasion

Claudio Raimondi Veronique Calleja, Riccardo Ferro, Alessandro Fantin, Andrew M. Riley, Barry V. L.  Potter, Caroline H. Brennan, Tania Maffucci, Banafshé Larijani & Marco Falasca (2016) A Small Molecule Inhibitor of PDK1/PLCγ1 Interaction Blocks Breast and Melanoma Cancer Cell Invasion. Scientific Reports

Endomembrane PtdIns(3,4,5)P3 activates the PI3K/Akt pathway

Jethwa N, Chung GH, Lete MG, Alonso A, Byrne RD, Calleja V, Larijani B (2015) Endomembrane PtdIns(3,4,5)P3 activates the PI3K/Akt pathway. J Cell Sci

Akt/PKB activation in human primary breast carcinoma is a poor prognostic marker: determined by high throughput coincidence amplified-FRET.

Selvaraju Veeriah, Pierre Leboucher, Julien de Naurois, Nirmal Jethwa, Emma Nye, Tamara Bunting, Richard Stone, Véronique Calleja, Gordon Stamp, Stefanie Jeffrey, Peter J Parker,Banafshé Larijani (2014) Akt/PKB activation in human primary breast carcinoma is a poor prognostic marker: determined by high throughput coincidence amplified-FRET. Cancer Research

Patient-derived xenografts of triple negative breast cancer recapitulate biomarker and PIK3CA mutation status of patients’ tumors and respond to mTOR inhibition

Haiyu Zhang, Adam L Cohen, Sujatha Krishnakumar, Irene L Wapnir, Selvaraju Veeriah, Glenn Deng, Marc A Coram, Caroline M. Piskun, Teri A. Longacre, Michael Herrler, Daniel O Frimannsson, Melinda L Telli, Frederick M Dirbas, A C Matin, Shanaz H Dairkee, Banafshé Larijani, Andrea H Bild, Gennadi V Glinsky, Stefanie S Jeffrey (2014) Patient-derived xenografts of triple negative breast cancer recapitulate biomarker and PIK3CA mutation status of patients’ tumors and respond to mTOR inhibition Breast Cancer Research

Restricted state selection in fluorescent protein FRET

Thomas A Masters, Richard J.Marsh, Daven A. Armoogum, Nick Nicolaou, Banafshé Larijani * and Angus Bain* (2013) Restricted state selection in fluorescent protein FRET. Journal of American Chemical Society

Regulation of 3-phosphoinositide dependent protein kinase 1 activity by homodimerization in live cells

Thomas A. Masters, Véronique Calleja, Daven A. Armoogum, Richard J. Marsh, Christopher J. Applebee, Michel Laguerre, Angus J. Bain, Banafshé Larijani (2010) Regulation of 3-phosphoinositide dependent protein kinase 1 activity by homodimerization in live cells Science Signaling