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Team:Stockholm/Planning
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Planning
Here follows the ongoing discussion amongst the group members about the project we are putting together. In addition we include theoretical support from scientific articles for what we want to accomplish.
Cell penetrating peptide (CPP)
Protein A
Protein A would be secreted from the bacteria, penetrating the skin and bind IgG antibodies - that in vitiligo patients bind melanocytes - so that the igG antibodies can not induce an immune response.
"It binds proteins from many of mammalian species, most notably IgGs" - http://en.wikipedia.org/wiki/Protein_A (Find better source)
Johan: Good as IgGs are the main antibody in excess for vitiligo
“It binds with the Fc region of immunoglobulins through interaction with the heavy chain. The result of this type of interaction is that, in serum, the bacteria will bind IgG molecules in the wrong orientation (in relation to normal antibody function) on their surface which disrupts opsonization and phagocytosis.” - http://en.wikipedia.org/wiki/Protein_A
Johan: The disruption is what we want (are there more disruptions to think of, other than opsonization and phagocytosis?)
“It binds with high affinity to human IgG1 and IgG2 as well as mouse IgG2a and IgG2b. Protein A binds with moderate affinity to human IgM, IgA and IgE as well as to mouse IgG3 and IgG1.[1] It does not react with human IgG3 or IgD, nor will it react to mouse IgM, IgA or IgE.” - http://en.wikipedia.org/wiki/Protein_A
Johan: See if the antibody in excess in vitiligo hopefully is IgG1/IgG2
“... possesses several properties which makes it suitable as a partner in fusion proteins: (a) SpA enables purification of the fusion protein by affinity chromatography due to its specific binding to the Fc part of immunoglobulins, (b) it is competent for secretion in E. coli, (c) it is monomeric and relatively small, (d) it has been reported to be extremely soluble in water solutions (Samuels- son et al., 1991), (e) it is proteolytically very stable intracellularly (Nilsson et al., 1985a) and in the periplasm of E. coli (Nilsson and Abrahms6n, 1990). Fusions to the IgG-binding domains of SpA have successfully been applied for high-level production of peptide hormones (Abrahms6n et al., 1986; Moks et al., 1987a, b; Nilsson et al., 1991, 1985b) and as a tool for the production of specific antibodies against gene products (L6wenadler et al., 1987, 1986).” - Fusions to the 5' end of a gene encoding a two-domain analogue of staphylococcal protein A, pp 270-271
Johan: There are shortened versions of protein A. These might have some advantages. E.g. they dont have the domain that normally binds to the cell membrane, instead they are secreted extracellularly.
http://johan.nordholm.se/Skola/igem/Z.pdf Interesting information about the Z domain of Protein A, text from the doctoral thesis "Interaction-Engineered Three-Helix Bundle Domains for Protein Recovery and Detection" by Tove Alm from 2010 at KTH
Andreas: How will we prevent protein A from entering the blood stream? If it is able to penetrate the skin cells to the spot where antibodies are present, it is also likely to ”exit” the skin into the blood stream. If this happens, protein A will not only cause a severe immune shock, it will also interfere with any type of antibody in the blood, not just the ones targeting vitiligo. This is especially true if Protein A is proteolytically very stable, as stated in (e) above.
Are there any examples mammalian in vivo Protein A studies? It would be a great leap forward if there are any such examples, showing how Protein A interacts with the mammalian immune system. - I found a website at the US National Cancer Insitute, where they mention two clinical trials aiming at using Staphylococcus aureus Protein A as a cancer treatment. The clinical trials made it to phase 2 (out of the usual 4, if I remember correctly), but were then closed. I haven’t been able to find any statements, links or articles on why they were closed, but here is a link to the NCI website: http://ncit.nci.nih.gov/ncitbrowser/ConceptReport.jsp?dictionary=NCI%20Thesaurus&code=C17166 - Now I found an article from the mid-80s on Protein A. Not sure if it is connected to the clinical trials at NCI, but here’s a PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/6379115 - There are also several articles mentioning ex vivo methods with Protein A for cancer treatments.
IgG protease
IgG antibodies are enriched in vitiligo patients. IgG proteases could partially solve this problem. Also, IgG protease could be fused with Protein A to get more activity from the protease.
"Actinobacillus actinomycetemcomitans produces a protease to human immunoglobulin G that is an important evasion mechanism […] The results showed that A. actinomycetemcomitans produced a protease to human immunoglobulin G in the culture supernatant […] The molecular mass of the protease active fraction was from 43 to 150 kDa." - IMMUNOGLOBULIN G PROTEOLYTIC ACTIVITY OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS
Johan: Their IgG protease eluted in fraction 13-17. +150 kDa eluted in fraction 9, and 43 kDa eluted in fraction 19. So at least it seems that the IgG protease is closer to 43 kDa than to +150 kDa.. (I wouldnt want to be a CPP carrying a cargo +150 kDa...)
"IdeS, a proteinase from Streptococcus pyogenes, cleaves immunoglobulin (Ig)G antibodies with a unique degree of specificity. Pathogenic IgG antibodies constitute an important clinical problem contributing to the pathogenesis of a number of autoimmune conditions and acute transplant rejection. To be able to effectively remove such antibodies is therefore an important clinical challenge." - IdeS: A Bacterial Proteolytic Enzyme with Therapeutic Potential
