Produktion und Evaluierung von oral applizierbaren Impfstoffen aus Pflanzen

Advances in the development of plant-based oral vaccines are generally hampered by insufficient and varying antigen accumulation as well as the low immunogenicity of most subunit vaccines. The latter is on the one hand caused by the sensitivity of soluble antigens to proteolytic degradation in the gastro-intestinal tract (GIT), and on the other hand by the inability to activate mechanisms of innate immunity and reduced uptake by intestinal immune cells. The aim of this study is to increase protein accumulation in the plant model systems N. tabacum cv. Petit Havana SR1 and Nft51 as well as the transcytotic antigen uptake by intestinal M cells. This should be realized by membrane anchoring of the model antigens HVR1 and MPT64 of Hepatitis C virus and Mycobacterium tuberculosis by genetic fusion to truncated versions of the constant region of the human T-cell receptor beta-chain (TCRbeta). N-terminal fusion of the cholera toxin B subunit (CTB), a high affinity ligand of the ubiquitous mammalian cell surface receptor ganglioside GM1, should mediate a selective adhesion and antigen uptake by cells of the intestinal epithelium. The influence of TCRbeta-based membrane anchor variants, differing with respect to the length of the constant extracellular domain, on plant productivity was compared to the secreted soluble antigen. These include three versions, one with the extracellular domain of the TCRbeta removed completely (TCR), and a second version comprising the complete hinge region (TCRCys). A final version without the N-terminal cysteine residue (TCRhinge), which is involved in the formation of an intermolecular disulfide bridge between the alpha- and beta-chain of the authentic TCR complex, should allow the evaluation of the influence of a potential homo-dimerization of two truncated beta-chains on antigen yield. Heterologous targeting to the plant cell membrane led to a positive effect in both a transient and a transgenic approach. Quantification by GM1-ELISA demonstrated a 185.4- or 25-fold increase in accumulation respectively after fusion of CTB-HVR1 to TCRCys, and a correct assembly of the CTB domains into the functional pentameric GM1-binding form. In comparison to CTB-HVR1 a less stabilizing effect was observed for CTB-MPT64, with a 1.6- or 19.7-fold increase in accumulation. After direct comparison of the variants TCRhinge and TCRCys, it could be concluded that the stabilizing effect was significantly enhanced by the homo-dimerization of two truncated TCRbeta-chains. The functionality of TCRCys in tobacco was confirmed by laser scanning confocal microscopy, comparative analyses of the cellular release of soluble and TCRCys-membrane-anchored antigens into the culture supernatants of transgenic BY- 2 suspension cells, and membrane isolation, making use of the C-terminal reporter protein GFP. The generated data gave some indication of a relatively unstable membrane anchoring, potentially due to the positively charged lysine residue in the transmembrane domain, which is neutralized by the opposite charged amino acid residues of the invariant CD3 chains in the native TCR complex. In spite of sufficient length of the transmembrane domain of TCRbeta for anchoring the antigens in the cell membrane, results from confocal microscopy couldnt clearly discriminate between a temporary or final localization in membranes of the ER and the Golgi apparatus. Transgenic leaf material of the CTB-HVR1-TCRCys-producing line N. tabacum cv. Petit Havana SR1, possessing the best relationship between nicotine content and antigen concentration, was applied orally to mice in the form of feed pellets containing leaf material processed to different degrees. Independent of the preparation variant and the intranasal co-administration of the mucosal adjuvants BPPCysPEG (modified MALP-2) or PQS-PEG specific antibody responses against CTB and HVR1 could neither be detected on local (sIgA), nor on systemic level (IgG). The following reasons for the absence of specific immune responses are discussed: Inefficient uptake of CTB-HVR1-TCRCys across the intestinal epithelium, inability of monomeric CTB fusions to assemble into functional pentamers in the microenvironment of plant membranes, insufficient antigen dose, proteolytic degradation in the GIT, inaccessibility of relevant epitopes, induction of oral tolerance, suppression of immune function by nicotine, and the ineffectiveness of the mucosal adjuvants, because of an application in distinct mucosal tissues. In this study it was demonstrated that a heterologous targeting of model antigens to the plant cell membrane by fusion to the membrane anchor variant TCRCys is capable of increasing antigen yields in tobacco to levels, which are in principle adequate for an oral administration of unprocessed plant material. The development of an effective plant-based oral vaccine however, requires not only the selection of a suitable production system but also the optimization of the vaccination strategies

Produktion und Evaluierung von oral applizierbaren Impfstoffen aus Pflanzen

Advances in the development of plant-based oral vaccines are generally hampered by insufficient and varying antigen accumulation as well as the low immunogenicity of most subunit vaccines. The latter is on the one hand caused by the sensitivity of soluble antigens to proteolytic degradation in the gastro-intestinal tract (GIT), and on the other hand by the inability to activate mechanisms of innate immunity and reduced uptake by intestinal immune cells. The aim of this study is to increase protein accumulation in the plant model systems N. tabacum cv. Petit Havana SR1 and Nft51 as well as the transcytotic antigen uptake by intestinal M cells. This should be realized by membrane anchoring of the model antigens HVR1 and MPT64 of Hepatitis C virus and Mycobacterium tuberculosis by genetic fusion to truncated versions of the constant region of the human T-cell receptor beta-chain (TCRbeta). N-terminal fusion of the cholera toxin B subunit (CTB), a high affinity ligand of the ubiquitous mammalian cell surface receptor ganglioside GM1, should mediate a selective adhesion and antigen uptake by cells of the intestinal epithelium. The influence of TCRbeta-based membrane anchor variants, differing with respect to the length of the constant extracellular domain, on plant productivity was compared to the secreted soluble antigen. These include three versions, one with the extracellular domain of the TCRbeta removed completely (TCR), and a second version comprising the complete hinge region (TCRCys). A final version without the N-terminal cysteine residue (TCRhinge), which is involved in the formation of an intermolecular disulfide bridge between the alpha- and beta-chain of the authentic TCR complex, should allow the evaluation of the influence of a potential homo-dimerization of two truncated beta-chains on antigen yield. Heterologous targeting to the plant cell membrane led to a positive effect in both a transient and a transgenic approach. Quantification by GM1-ELISA demonstrated a 185.4- or 25-fold increase in accumulation respectively after fusion of CTB-HVR1 to TCRCys, and a correct assembly of the CTB domains into the functional pentameric GM1-binding form. In comparison to CTB-HVR1 a less stabilizing effect was observed for CTB-MPT64, with a 1.6- or 19.7-fold increase in accumulation. After direct comparison of the variants TCRhinge and TCRCys, it could be concluded that the stabilizing effect was significantly enhanced by the homo-dimerization of two truncated TCRbeta-chains. The functionality of TCRCys in tobacco was confirmed by laser scanning confocal microscopy, comparative analyses of the cellular release of soluble and TCRCys-membrane-anchored antigens into the culture supernatants of transgenic BY- 2 suspension cells, and membrane isolation, making use of the C-terminal reporter protein GFP. The generated data gave some indication of a relatively unstable membrane anchoring, potentially due to the positively charged lysine residue in the transmembrane domain, which is neutralized by the opposite charged amino acid residues of the invariant CD3 chains in the native TCR complex. In spite of sufficient length of the transmembrane domain of TCRbeta for anchoring the antigens in the cell membrane, results from confocal microscopy couldnt clearly discriminate between a temporary or final localization in membranes of the ER and the Golgi apparatus. Transgenic leaf material of the CTB-HVR1-TCRCys-producing line N. tabacum cv. Petit Havana SR1, possessing the best relationship between nicotine content and antigen concentration, was applied orally to mice in the form of feed pellets containing leaf material processed to different degrees. Independent of the preparation variant and the intranasal co-administration of the mucosal adjuvants BPPCysPEG (modified MALP-2) or PQS-PEG specific antibody responses against CTB and HVR1 could neither be detected on local (sIgA), nor on systemic level (IgG). The following reasons for the absence of specific immune responses are discussed: Inefficient uptake of CTB-HVR1-TCRCys across the intestinal epithelium, inability of monomeric CTB fusions to assemble into functional pentamers in the microenvironment of plant membranes, insufficient antigen dose, proteolytic degradation in the GIT, inaccessibility of relevant epitopes, induction of oral tolerance, suppression of immune function by nicotine, and the ineffectiveness of the mucosal adjuvants, because of an application in distinct mucosal tissues. In this study it was demonstrated that a heterologous targeting of model antigens to the plant cell membrane by fusion to the membrane anchor variant TCRCys is capable of increasing antigen yields in tobacco to levels, which are in principle adequate for an oral administration of unprocessed plant material. The development of an effective plant-based oral vaccine however, requires not only the selection of a suitable production system but also the optimization of the vaccination strategies

Human Cytolytic Fusion Proteins: Modified Versions of Human Granzyme B and Angiogenin Have the Potential to Replace Bacterial Toxins in Targeted Therapies against CD64+ Diseases

Targeted therapies for the treatment of cancer, but also inflammation and autoimmune diseases will reduce major side effects accompanied with conventional treatment modalities. The immunotoxin concept uses bacterial or plant toxins, coupled to antibodies or natural ligands targeting cancer cells. Initially, immunotoxins suffered from drawbacks like nonspecific cytotoxicity. Even the third generation of immunotoxins comprised of truncated antibodies and modified effector molecules experienced clinical set-backs due to immune responses. Long-term treatment of cancer and non-life-threatening chronic inflammatory diseases requires their complete ‘humanization’. This lead to evaluating human cytolytic fusion proteins (hCFPs), based on human apoptosis-inducing proteins. Lacking an endogenous translocation domain dramatically reduces the cell-death inducing capacity of such proteins. Here, we report on optimizing hCFPs, based on the anti-CD64 single chain variable fragment H22(scFv), specifically eliminating CD64+ macrophages and malignant progenitor cells. We replaced the bacterial toxin in H22(scFv)-ETA' with the pro-apoptotic human granzyme B or angiogenin. Translocation was promoted by a sophisticated adapter containing a membrane transfer peptide (MTD) flanked by endosomal and cytosolic cleavable peptides, thus achieving in vitro cytotoxic activity comparable to bacterial immunotoxins. We demonstrate for the first time that optimized hCFPs, based on granzyme B or angiogenin, can compete with classical ETA-based immunotoxins

Improving the Therapeutic Potential of Human Granzyme B for Targeted Cancer Therapy

Conventional cancer treatments lack specificity and often cause severe side effects. Targeted therapeutic approaches are therefore preferred, including the use of immunotoxins (ITs) that comprise cell-binding and cell death-inducing components to allow the direct and specific delivery of pro-apoptotic agents into malignant cells. The first generation of ITs consisted of toxins derived from bacteria or plants, making them immunogenic in humans. The recent development of human cytolytic fusion proteins (hCFP) consisting of human effector enzymes offers the prospect of highly-effective targeted therapies with minimal side effects. One of the most promising candidates is granzyme B (GrB) and this enzyme has already demonstrated its potential for targeted cancer therapy. However, the clinical application of GrB may be limited because it is inactivated by the overexpression in tumors of its specific inhibitor serpin B9 (PI-9). It is also highly charged, which means it can bind non-specifically to the surface of non-target cells. Furthermore, human enzymes generally lack an endogenous translocation domain, thus the endosomal release of GrB following receptor-mediated endocytosis can be inefficient. In this review we provide a detailed overview of these challenges and introduce promising solutions to increase the cytotoxic potency of GrB for clinical applications

Targeted ex vivo reduction of CD64-positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B-based cytolytic fusion proteins

CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A