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Anti-tumour Immunity through GP-100-TLR Agonist Conjugation

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Enhancing anti-tumour immunity through gp-100-TLR agonist conjugation.


Soluble cancer vaccines remain an area of high interest to researchers with the ability to enhance immune responses against present cancers and induce protective immunity against future cases. In developing new vaccines finding ways to increase the immunogenicity of cancer antigens is a major challenge(1-3). The addition of Toll-like receptor (TLR) agonists is one strategy which can successfully boost immune cell activation and response to cancer antigens. By stimulating TLRs, these agonists increase expression of several co-stimulatory molecules on antigen presenting cells (APCs) such as CD80/86 and CD40(4-6).They also increase tumour peptide loading onto type 1 & 2 Major histocompatibility complex (MHC) proteins. Together this leads to greater activation of tumour-specific effector immune cells such as CD4+ and CD8+ T-cells resulting in increased tumour clearance via their cytotoxic activity. Vaccine formulations which have included antigen and TLR agonists as a mixture have had promising results with many in clinical trials(4, 7, 8). Despite this, few have assessed the effect of chemically conjugating these constituents, a strategy which could increase efficiency of both TLR activation and peptide loading onto MHC(9-12). Many conjugation strategies that do exist today capitalise on the use of pH and redox sensitive linkers. Differences in pH and redox environments intracellularly enable triggerable release of these vaccines whilst protecting antigen and agonist from degradation extracellularly where they are administered. Research into the use of Glutathione-sensitive disulphide linkers has demonstrated that the immune response to model antigen 'Ovalbumin'(OVA) could be increased through linkage to the TLR agonist, CpG oligodeoxynucleotide (ODN)(10, 11). Our research aims to repeat this using both stable and reversible linkers as well as a more clinically relevant, tumour associated antigen (TAA) called 'gp-100' expressed on melanomas. In addition, we aim to assess the effectivity of different TLR agonists within conjugates including Polyinosinic polycytidylic acid (Poly I:C) and two different classes of CpG ODNs, B and C respectively. Each of these agonists activate different signalling pathways within antigen presenting cells leading to unique cytokine profiles and T-cell responses. Poly I:C for example, is a potent activator of TLR3 which activates the TRIF pathway inducing release of type 1 interferons such as IFN Beta(6, 13, 14). This increases MHC-I expression and stimulates a Th1 type immune response which favours cell-mediated immunity including CD8+ T-cell activation. In comparison, CpG class B and C stimulate TLR 9 activating the MYD88 pathway and release of proinflammatory cytokines IL-6 and IL-17. This results in enhanced CD4+ and CD8+ T-cell responses, B cell activation and antibody production(10, 11, 15). Both types of response have potential to give clinical benefit in different ways highlighting the potential of these conjugates in tumour treatment. Finally, we will also assess how the composition of the TAA effects its presentation on MHC. To assess this, a smaller Gp-100 peptide which does not require intracellular processing will be compared to a longer peptide requiring processing.

This project will assess which conjugates enhance anti-tumour responses in mice and how they achieve this looking specifically at Dendritic cell activation and CD8+ T-cell proliferation and cytokine production.


  1. In terms of CD8+ responses, T cell proliferation and cytokine release, I hypothesize that Poly I:C reversibly linked to processed gp-100 will be the most effective inducing a strong Th1 response and IFN-B cytokine release. This is because Poly I:C stimulates several intracellular signals in addition to TLR3 including RIG-1 and MDA-5. This would lead to increased release of proinflammatory cytokines including IL-6, IL-12, IL-1B and IFN-B. Specifically the release of IFN-B would induce a strong anti-viral-like Th1 T cell response through increased expression of MHC-1 molecules on APCs and inducing release of IFN-Y, TNF-A and IL-2 from CD4+ T cells. As well as this factor, TLR3 is exclusively expressed on myeloid dendritic cells, the most effective dendritic cell subset in presenting antigen, and not expressed on plasmacytoid dendritic cells.
  2. Secondly, I Hypothesize that CpG class C will induce the most cytokine production in dendritic cells including the cytokines IL-6, IL-12, IFN-A and IL-1B. CpG molecules stimulate TLR9 which is expressed in the endolysosomal compartment of plasmacytoid dendritic cells exclusively. As a dendritic cell subset, plasmacytoid DCs are well known for their proinflammatory cytokine production at levels much higher than other DC subsets. CpG class C in particular stimulates the release of IFN-a in addition to IL-6, IL-12 etc. stimulating both a Th1 response and a B cell response.

Aims and objectives


  1. Produce gp-100-CpG ODN and gp-100-Poly I:C reversible and stable conjugates with either processed or non-processed Gp-100 peptides
  2. Measure dendritic cell subset activation through expression of MHC-II, CD40 and CD86 molecules and cytokine release (IL-12, IL-6, IL-1B, IFN-A and IFN-B )
  3. Measure Tumour specific T cell activation (CD4+ and CD8+), proliferation using carboxyfluorecein succinimidyl ester (CSFE) and cytokine release (IFN-Y, IL-2,TNF-A)


The proposed project for the year will focus on three main objectives 1) Produce gp-100-CpG ODN and gp-100-Poly I:C reversible and stable conjugates with either processed or non-processed Gp-100 peptides. First, we will modify free amino groups on the lysine residues of each gp-100 peptide (processed amino acid sequence: KVPRNQDWL vs unprocessed: CAVGALKVPRNQDWLGVPRQL) and TLR agonists (suspended in a modification buffer ph. 8). Then we will link these together with either the stable linker (HYN) or the reversible linker (HYN-SS) in a ph. 6 conjugation buffer separately. Product concentration after each individual modification step will be measured using Nanodrop1000 at 280 m after desalting excess product using vivspin 500 filter. Final product conjugation will be confirmed using the reversed phase liquid chromatograph at the School of Pharmacy which will allow us to visualise each individual product according to their differing polarities, and quantify their ratio.

Our second objective is to Measure dendritic cell subset activation through expression of MHC-II, CD40 and CD86 molecules and cytokine release (IL-12, IL-6, IL-1B, IFN-B, IFN-A). To achieve this, we will isolate bone marrow cells from C57BL/6 mice and treat with GM-CSF to produce CD11c+ dendritic cells. These will then be treated with either individual TLR agonists, TLR agonist-gp100 mixtures or TLR agonist-gp100 conjugates (reversible or non-reversible). After 24hrs of treatment these cells with be stained with fluorescent antibodies for CD80, CD40, CD11C, and MHC-II, viewed on the Gallios flow cytometer in Pathology and analysed using Kaluza software. This experiment will be repeated at least three times to enable statistical analysis, which will be performed using Graph Pad prism software. Cytokine release from these cells will be measured using an enzyme linked immunosorbent assay (ELISA) for IL-6, IL-12, IFN-B and IFN-a.

Our third objective is to Measure Tumour specific CD3+ T cell: activation (CD8+), proliferation (CSFE) and cytokine release (IFN-Y, IL-2). This will be achieved through isolation of splenocytes from Pmel (T-cells specific to gp-100) transgenic mice and sorting of CD8+ cells using the Automacs machine at Pathology. These cells will then be stained using CSFE and co-cultured separately with C57BL/6 BMDCs treated according to objective 2. After 72hrs cells will be analysed using the Gallios flow cytometer to measure T-cell activation (CD3+) and proliferation (CSFE). To measure cytokine release, cell cultures will undergo an ELISA for IFN-Y and IL-2.

Proposed Budget


C57BL/6 x 10 @ $50 each$500

PMEL x 10 @ $50 each$500









Cell culture reagents

IMDM Media$400

Foetal calf serum$500

Cytokine detection

Cytokine detection kit$2000

Conjugation reagents

S4FB Linker$450

S-SS-4FB Linker$350

S-HYNIC cross linker$850



CpG class B$500

CpG class C$500

Poly I:C$500

Vivspin filters$200



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