Using MHC class I peptides to modulate NK cell activity, as a basis for immunotherapy

Natural killer (NK) cells are cells of the immune system that can fight infections and cancer. Their responses are controlled by a number of cell surface receptors including the killer cell immunoglobulin-like receptors (KIR). There are many different KIR but extensive studies have shown that NK cells and specific KIR genes can be important factors in determining the outcome of both infections, such as HIV and hepatitis C, and also of cancers, including leukaemia and liver cancer. To date harnessing specific sub-populations of NK cells for therapeutic benefit has been difficult because of a lack of understanding of NK cell biology. This has meant that NK cell based therapies are relatively crude and do not take account of the fact that different NK cells express different cell-surface receptors that may be relevant only in specific diseases. For instance the gene KIR2DL3 is associated with protection against hepatitis C, and the gene KIR2DS2 is associated with a better response to treatment in non-small cell lung cancer.
Work in our group has pioneered how NK cell reactivity can be altered by small peptides expressed on the surface of cells. The aim of this project is to develop our ideas so that a therapeutic reagent can be obtained. We will do this by targeting both the KIR2DL3 and KIR2DS2 genes, and identifying peptides that specifically bind these receptors to activate NK cells. We will also look for peptides that can activate KIR3DS1-positive NK cells. This is important because KIR3DS1 is associated with protection against HIV infection and also against the development of liver cancer. Whilst much activity has been focused on HIV, liver cancer continues to be a major health problem worldwide (it is the 6th commonest cancer), and alarmingly is on the increase in the UK. Importantly it is difficult to treat, and there is only one chemotherapeutic reagent currently licensed for this condition.
Having identified key peptides that we can use to activate NK cell, we will then use them in vitro to activate NK cells. We will devise optimal strategies for expressing the identified peptides and then optimising the culture conditions necessary for growing large numbers of NK cells expressing the relevant receptor. By the end of the project we hope to have a reagent suitable for therapeutic use, which depending on the results of the investigation maybe directly suitable for phase I studies.

We will use transporter associated with antigen processing (TAP) deficient cell lines for peptide loading experiments to identify specific peptides that bind HLA and also either bind the activating receptor KIR2DS2 or function as peptide antagonists for KIR2DL2/3. Peptides will be designed based on our on-going experiments and the current literature.
We have identified an HCV derived peptide involved in the direct recognition of virus-expressing cells by KIR2DS2. We have used this to synthesize related peptides that induce KIR2DS2-binding and these experiments will inform the design of additional peptides that will engage KIR2DS2 to stimulate NK cells in vitro.
KIR3DS1 is a receptor associated with protective responses to HIV and hepatocellular carcinoma. We have identified that KIR3DS1 is also protective against chronic HCV infection. We will use HLA-B*2705 as an allele with which to perform a screen for peptides that may bind KIR3DS1, taking a motif-based approach and going on to perform a large scale screen if the initial approach is unsuccessful.
To investigate inhibitory receptors, we will make use of our published and pilot data to identify antagonist peptides for KIR2DL2/3. This will inform the design and testing of peptides that may antagonize inhibition by other HLA-C alleles.
To translate these into a potential therapeutic reagent we will set up assays of endogenous peptide presentation using minigenes and vector constructs to express the peptide with or without its cognate MHC class I ligands on the cell surface. Cell lines expressing these constructs, and exosomes derived from these cell lines, will be tested for their potential to activate and proliferate NK cells expressing specific KIR. We will also express our peptides transiently in monocyte derived dendritic cells in cultures with autologous NK cells so that HLA class I is completely matched as would be the case for in vivo therapy.

We anticipate that the work will impact academia, patients, the pharmaceutical industry, charities and could have wider global health implications.

Academia
Immunotherapy was highlighted by Science magazine as one of the breakthroughs of 2013. Therefore we feel that the work has an opportunity to benefit the academic community including researchers in immunology, NK cell biology, cancer medicine and infectious diseases.

Patients
The work proposed has a strong translational component with a broad aim to develop new NK cell based therapeutics. Therefore there is potential for a large clinical community to benefit. These could include patients with a variety of different malignancies and patients with viral infection. Given the expertise at Southampton in cancer immunotherapy we feel that there is a potential for a rapid translation into phase I studies through collaboration with the Experimental Cancer Medicine Centre at Southampton, which has experience of DNA vaccines. Thuse there is an opportunity for patient benefit within the timespan of 3-5 years.

Pharmaceutical industry
The work should also generate patentable materials eg peptides that specifically activate NK cells. As NK cell therapy is in its infancy and these ideas are novel they could from the basis for a first-in-class therapeutic, which could be attractive to the pharmaceutical industry. We would anticipate commercializing any reagent in partnership with pharma, giving them the opportunity to benefit from this research in collaboration with the University of Southampton. If developed as a first-in-class therapy this would have the benefit that the UK could be world-leading in targeted NK cell therapy.

Charities
We feel that our work will be of value to cancer and liver charities. Such organisations will include CRUK, Leukemia and Lymphoma Research, The British Liver Trust and The Brian Mercer Charitable Trust, which has a specific interest in treatment and prevention of primary liver cancer. These may stimulate an interest in the charities in developing NK cell based therapeutics. Particularly important is the need to highlight the difficulties and lack of therapeutic options that patients with liver cancer currently have, and so it is anticipated that this work will improve this.

Global opportunities
With the recent knowledge that NK cells have memory properties one long-term goal could be the use of an NK cell vaccine as a prophylaxis against cancer or viral infections. Such infections could include both hepatitis C and HIV, both of which have protective associations with KIR genes. Therefore there is a potential for this work to have a global benefit. If a DNA vaccine could be produced this could be a low cost therapeutic or prophylactic vaccine for use in developing countries. This type of therapy would require additional studies to demonstrate in vivo generation of memory type NK cells but anticipate it would be possible within 5 years.