Targeted proteinase inhibitors in the study of antigen processing

Lead Research Organisation: University College London
Department Name: Immunology and Molecular Pathology


Vertebrates respond to infections with viruses, bacteria or other microorganisms by very specific immunological responses, which act to prevent the invading organisms from multiplying and causing disease. These responses are the basis on which all vaccines work. The responses are carried out by cells called lymphocytes, which represent about 20% of all white blood cells. The most well known type of lymphocyte are called B cells and make antibody, molecules which attach tightly to the surface of individual viral or bacterial components, and stop them working, or target them for destruction by phagocytes. Another type of lymphocyte, however, the CD4 T lymphocyte, is also essential for most immune responses, because of its role in organising the co-operation between different white blood cells. The vital importance of this cell type has been highlighted by AIDS, a disease in which HIV virus leads to the destruction of the CD4 lymphocyte, leaving the body susceptible to all sorts of infections. CD4 T lymphocytes are able to recognise a bewildering number of different infections. However, their recognition system is such that it can only recognise pieces of viral or bacterial proteins if the proteins are first cut up into small sections by enzymes called proteinases. The aim of this study is to find out which type of proteinase is important in this process, and how inhibiting their activity may be used to change the immunological response. For this purpose, we will use chemicals which are known to inhibit various types of proteinases (inhibitors) and test them on various immunological responses, including the immune response to the parasite malaria. The inhibitors we have available work well with isolated proteinases, but they are often insoluble in water or blood and not good at reaching the right cells of the immune system or penetrating into the right cellular compartment. We will therefore make better versions of these inhibitors, by attaching them to larger 'carrier' molecules which will be able to carry them efficiently and selectively to the right cell within the body. The results of these studies will add to our understanding of how the immune system works, and thus ultimately help to develop better ways to prevent infection.

Technical Summary

Limited proteolysis within antigen presenting cells is an important step in the presentation of protein antigens to CD4 T cells. This proposal investigates the role of various class of proteinase in this process. Although many small molecular weight proteinase inhibitors have been described, the application of most of these to this problem has been limited by factors including poor solubility and bioaccessability in physiological solutions. Previous work from the applicants' laboratories has shown that it is possible to improve the solubility and cell targeting of a potent microbial proteinase inhibitor, pepstatin, by coupling to mannosylated bovine albumin carrier. This project will extend these studies. Specific aims will include using the available pepstatin conjugate to further explore the role of aspartic proteinases in other antigen systems, and with other antigen presenting cells. Radioactive and fluorescence versions of the conjugated inhibitor will also be synthesised to track intracellular distribution and metabolism. Conjugates with improved properties for in vivo use will also be synthesised coupling pepstatin to a hexavalent mannose carrier or to antigen presenting cell specific antibodies. In the later phases of the project, the same approach will be extended to other classes of proteinase, synthesising conjugates with available inhibitors of asparaginyl endopeptidase, and the cysteine proteinase cathepsin S. The project will use small molecular weight inhibitors to provide insights into a biological process which is central to an understanding of adaptive immunity, and which despite intensive study, has remained poorly understood. In addition, the development of inhibitor/conjugate chemistry will provide a generic platform for the study of cellular proteinases in other biological phenomena.
Description 1. Targeted delivery of an aspartic proteinase inhibitor pepstatin to the antigen processing compartment of dendritic cells , resulting in selective block of processing and presentation of ovalbumin and malaria epitopes in vitro. Validated by functional antigen processing assays. (Objectives 1 and 10). In parallel demonstrated that aspartic proteinases were not absolutely required for invariant chain processing in dendritic cells.

2. Targeted delivery of pepstatin to the endocytic pathway of dendritic cells and macrophages cells in vitro and in vivo, via mannose binding receptor. Validated by confocal microscopy and flow cytometry. (Objective 4 and 5). In particular, BSA-mannose-pepstatin conjugates showed selective access to macrophages and dendritic cells at sites of inflammation.

3. Demonstrate that the basic synthetic strategy can be used as a generic delivery system of inhibitors to dendritic cells and macrophages, by synthesis of a new set of targeted cathepsin S inhibitors using similar strategy. (Objective 6)
Exploitation Route The strategy of targeting small molecules to macrophages via targeting to sugars is of general application to inflammation research
Sectors Pharmaceuticals and Medical Biotechnology

Description Collaboration to study repertoire of cathepsin E deficient mice 
Organisation Weizmann Institute of Science
Country Israel 
Sector Academic/University 
PI Contribution A collaboration with Weizmann Institute, Israel to study the repertoire of cathepsin E deficient mice using high throughput seqeuncing
Start Year 2009