Efficacy of artificial imprinted antibodies in driving unwarranted immune responses

Why did we fund this project?

This award aims to use molecular imprinting technology to create artificial antibodies that do not need in vivo batch-to-batch efficacy studies.

Antibodies are used in a wide range of applications, including immunotherapies where monoclonal antibodies are generated to target protein receptors on cancer cells. Research to determine which receptors and cancer types respond to immunotherapy are typically performed using animal-derived antibodies. The animal is immunised and bled to harvest the antibodies of interest. This may be repeated a number of times over an animal’s lifetime. However, the antibodies produced vary between batches often requiring laboratories to use further animals to confirm in vivo efficacy ahead of experiments. Molecular imprinting technology can be used to synthesise artificial antibodies termed molecularly imprinted polymers (MIPs) where every batch produced is identical. MIPs can be produced to target many different proteins and receptors for use in research to assess a patient’s response to immunotherapy ahead of treatment and to identify novel targets for immunotherapy development. MIPs could also be used in place of commercial biological antibodies for in vitro diagnostic experiments.

The student, with Dr Shoba Amarnath, will develop MIPs to target the PD-1 receptor, an immune checkpoint that has previously had success in cancer immunotherapy. They will use the crystallographic structure of PD-1 to develop batches of MIPs to increase or decrease PD-1 downstream signalling. The student will then use in vitro assays to demonstrate the applicability of MIPs in in vitro diagnostic assays, where there is currently limited use of the technology. The student will develop skills in atomistic computational modelling, T-cell culture, and antibody-based assays such as ELISAs and flow cytometry in order to check the function of MIPs and compare them to commercially available antibodies.

Biologically derived antibodies are the primary reagents that are utilised for most immunological studies with recombinant or phage derived antibody technologies yet to become commonly used. The generation of agonistic and antagonistic antibodies against immune cell receptors are mostly performed using polyclonal antibody technology.

Artificial antibodies can be synthesised by molecular imprinting technology, a process where a target molecule (or a derivative thereof) acts as a template around which interacting and crosslinking monomers are arranged and copolymerised to form a cast-like shell. After removal of the template, high affinity binding sites are exposed that are complementary to the target molecule in size, shape and chemical functionality. The inherent advantages of molecularly imprinted polymers (MIPs) include robustness, high specificity, refractory to biological degradation and the incorporation of (fluorescent) probes to monitor interactions in in-vivo assays.

Antibodies targeting PD-1 immune-checkpoints has shown remarkable efficacy only in 30% of cancer patients and the lack of efficacy in non-responders is unknown. One way to stratify patients for these therapies would involve testing the response rate of the immune system to these antibodies in in vitro diagnostic assays. However, in vitro assays with biological monoclonal antibodies although can guarantee specificity, considerable batch variability occurs thereby decreasing the feasibility of such an approach. A novel method of overcoming this biological disparity and heterogeneity of antibodies would be to develop a stream-lined methodology that would result in identical antibody products which is possible using artificial molecular imprinting.

This studentship will aim to establish a proof of principle model to generate MIPs that can act as agonists or antagonists against PD-1 receptor on human immune cells. Specific objectives are outlined below:

Objective-1:Generate models and design agonists and antagonist antibodies against the PD-1 receptor

The structural information of immune checkpoint proteins known as PD-1 and its interaction with its ligands PDL-1 has been resolved. The interaction of PD1 with PDL1 in crystallography studies show both proteins interact using large hydrophobic surfaces of the respective Ig-like V-type domains. Antibodies to PD-1 has been recently published with most of them targeting the two sites within PD-1: the C’/D loop (site 1) and the C,C’ and F strands (site II). In nature, site II is predominantly utilised by its ligands.

Within this objective we will,

1. Design MIPs that can incorporate site 1 (to increase antibody affinity) and site II with an innovative solid-phase synthesis strategy.

2. A combination of monomers, including a fluorescent monomer to monitor nanoMIP attachment, will be polymerised around the target. We will build atomistic models to evaluate the binding enthalpy and complement experimental work, which will lead to production of high affinity MIPs.

Objective-2: Testing whether MIPs are agonistic or antagonistic against PD-1 receptor

1. MIPs generated will be tested for their efficacy in controlling or enhancing human T cell proliferation in response to antiCD3 and anti-CD28 stimulation.

2. MIP mode of action will be determined by using assays that will include apoptosis assays, cytokine production capacity of T cells, using functional ELISAs and intracellular flow cytometry.

3. MIP function will be tested in antigen specific proliferation assays (HLA-A2 restricted Influenza epitopes will be used) in order to identify the efficacy of MIPs in enhancing antigen specific CD8+T cell proliferation.

This study will establish a new area of health care technology research that will allow us to generate plastic antibodies that structurally mimic biologics and have the potential to be used in diagnostic assays.