Developing and investigating the molecular mechanisms involved in fluorescence-guided surgery specif

Developing and investigating the molecular mechanisms involved in fluorescence-guided surgery specific for paediatric brain cancers.

Background: Brain tumours are the most common solid tumours that develop in children. Children of any age can be affected and about 400 children in the UK develop brain tumours each year. Surgery to remove brain tumours is challenging and it remains one of the pillar in their treatment and it is associated with survival.

Fluorescence-guided surgery (FGS) is based on the principle of directly labelling tumour cells with fluorescence leading to a clear visualisation and differentiation of viable tumour from neighbouring structures during surgery. FGS is an innovative technique to help the surgeons to remove cancer more effectively and safer.

Aims/Objectives:

AIM 1: To develop and test the effectiveness of fluorescence-guided surgery (FGS) in better visualising paediatric brain tumours, using novel injectable fluorescent probes specific for B7H3, in preclinical models.

Objective 1: To identify and develop the optimal fluorescently labelled probes (anti-B7H3mAb-IRdye800), selected from different affinity B7H3 monoclonal antibodies (mAbs), for sensitive and specific visualisation of brain tumours in vitro and in vivo.

Objective 2: To define pharmacokinetic (PK) and pharmacodynamic (PD) properties of the lead anti-B7H3-mAb-IRdye800 probes in different cancer mouse models.

AIM 2: To discover new mechanism of actions in FGS to better understand and predict effective translation into humans.

Objective 1: To define mechanisms of intracellular/extracellular degradation and off-target binding of the anti-B7H3mAb-IRdye800 in vitro and in vivo.

Objective 2: To develop and study the properties of smaller probes, called mini-bodies (‘mini’-anti-B7H3mAb-IRdye800) for better penetration to the core of solid cancers and to cross the blood-brain barrier (BBB).

AIM 3: To prove that image-guided surgery, using anti-B7H3mAb-IRdye800, is more effective than traditional surgery in achieving tumour-free survival in a randomised trial in an animal model.

Methods:

We will conjugate and test in vitro anti-B7H3mAb and IRdye800 (fluorescent probe). Duration of staining will be evaluated in time courses following washing off antibody, whilst internalisation will be evaluated using pHRodo-labelled antibody. Then the staining properties of the anti-B7H3mAbs-IRdye800 will be correlated with affinity using Biacore biosensor technology (real-time surface plasmon resonance, GE Healthcare) A dose-finding experiment of anti-B7-H3mAb-IRdye800 will be tested in subcutaneous and orthotopic models of brain tumours. We will inject two tumours (physiological expression and B7H3-negative, respectively) per mouse to reduce the number of animals so that each mouse is its own control. In vivo imaging with bioluminescence imaging (BLI), NIR-I (Elevision IR platform, Medtronic), SWIR (Nirvana 640 camera, Teledyne). Then, we will generate minibodies from the lead ScFv using standard molecular cloning. Purified minibodies will be compared with full-length mAb for visualisation of orthotopic brain tumour using the Med8A medulloblastoma model, which Prof Anderson has already optimised for B7H3 therapeutic targeting. We will measure uptake of minibodies into 3D blood-brain barrier (BBB) spheroids. Finally, we will assess the potential utility of anti-B7-H3mAb-IRdye800 probe to facilitate tumour resection and improve tumour-free survival. We will compare surgical resection of brain tumour by white light ‘traditional surgery’ alone (group A) vs combined white light plus FGS (group B) in a randomised control trial.

Timeline (if applicable):

Each aim will require about one year to be completed

References:

1. Paraboschi I et al. Fluorescence imaging in pediatric surgery: State-of-the-art and future perspectives. J Pediatr Surg. 2021;56(4):655-662
2. Mieog JSD et al. Fundamentals and developments in fluorescence-guided cancer surgery. Nat Rev Clin Oncol. 2022;19(1):9-22
3. Majzner RG et al. CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors. Clin Cancer Res Off J Am Assoc Cancer Res. 2019;25(8):2560-2574.