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Targeted Tumor Therapies
In tumor therapy, tumors that cannot be completely removed by local treatment pose a significant problem, since complete, durable remissions are achievable by chemotherapy in only a small proportion of tumor entities. Therefore, immunological therapeutic approaches are of greatest interest.
With the discovery of monoclonal antibodies, the idea was born to specifically inhibit tumor cell growth by coupling tumor cell-specific antibodies to cytotoxic substances while leaving normally differentiated cells unharmed. Instead of antibodies, ligands for tumor cell-specific receptors such as growth factors or interleukins are also used. In the development of such target cell-directed toxins, it became clear that the main problems are due to three reasons:
- The antigen specificity of the antibodies or the tumor cell specificity of the antigen is not sufficient, so that binding to normally differentiated cells also occurs.
- The toxins themselves bind to the surfaces of normally differentiated cells and reach their cytosol via toxin-specific or cellular membrane transfer mechanisms.
- The targeted toxins reach the target cells, but there is insufficient uptake of the toxin into the cytosol.
In addition, there are other reasons that may cause the targeted toxin to be ineffective in individual cases, such as intense shedding of the antigen and the resulting appearance of a soluble competitor.
In the following project descriptions, we demonstrate approaches to increase uptake into the cytosol by designing targeted toxins as well as by using glycosylated triterpenoids to address the aforementioned problems.
Current Projects
The calcitonin receptor as target for the treatment of glioblastoma by glycan-based, site-specific coupling of nanobodies to toxins
A co-operative project with the University of Melbourne, Australia.
Glioblastoma is a lethal brain tumor that kills 50% of patients within 15 to 17 months of diagnosis. There is no effective treatment, representing an unmet need in global healthcare.
Peter Wookey's group in Melbourne found that most glioblastomas (78–88%) express the calcitonin receptor (CTR). As a result, the antibody mAb2C4, which targets CTR, was developed in his group. Interestingly, cancer stem cells that drive the spread of brain tumors are also targeted. This means that mAb2C4 or the recently derived smaller nanobodies that allow deep penetration into solid tumors may be an effective means of targeting malignant cells of solid brain tumors. Furthermore, it is important to consider that access to the tumor microenvironment is compromised by the blood-brain barrier.
Linking the nanobody to a toxin provides an opportunity to improve the therapeutic effect on cancer cells; however, the inefficient endosomal escape of the internalized toxin and the site-specific homogeneous modification of the nanobody still pose challenges. Our group, together with European partners, has developed the ENDOSCAPE technology (see Endosomal Escape Enhancer and ENDOSCAPE project), which enables effective transfer of the drug into the cytosol. Furthermore, our group has developed advanced technologies for site-specific modification of proteins to obtain precisely defined and homogeneous coupling products. Together with the Melbourne group, we have confirmed the efficacy of mAb2C4 immunotoxins for the treatment of brain tumor stem cells in a joint publication. Initial animal studies have confirmed that the prototype immunotoxin can be effective in a mouse model with glioblastoma.
The project is currently developing and producing different nanobody toxin variants using a novel, site-specific, glycan-based coupling technology. The immunotoxins will then be used for empirical testing in cell culture and mouse xenograft models, both subcutaneous and intracranial orthotopic models.
The use of suicide genes for tumor therapy
Instead of toxins, it is also possible to deliver the genes of these toxins into the tumor cells. Although this has the disadvantage that the target cell-specific introduction of DNA into the tumor cells is even more difficult than that of proteins, it has the advantage of amplification, because any number of toxic proteins can be translated from just one gene. Since in this way the cell produces the lethal toxins itself, one also speaks of suicide genes. Since this is a gene therapy approach, further information can be found on the pages on controlled gene transfer.
Completed Projects (Selection)
Improved therapy of B-cell non-Hodgkin lymphoma by obinutuzumab-dianthin conjugates in combination with the endosomal escape enhancer SO1861
Immunotoxins not only bind to cancer-specific receptors to mediate the elimination of tumor cells by the innate immune system, but also enhance cytotoxicity through the activity of the toxin. In previous publications, our group has shown that the plant glycoside SO1861 enhances endosomal escape of targeted toxins in non-hematopoietic cells, thereby increasing their cytotoxicity several-fold (e.g., Bharghava et al., von Mallinckrodt et al.).
Here, we tested this technology for the first time in an in vivo model of lymphoma. First, the therapeutic CD20 antibody obinutuzumab was chemically conjugated to the ribosome-inactivating protein dianthin. The cytotoxicity of obinutuzumab-dianthin (ObiDi) was evaluated in human B lymphocytes (Burkitt's lymphoma cells, Raji) and compared with human T-cell leukemia cells (Jurkat) as off-target cells. When combined with SO1861, cytotoxicity was 131-fold higher for the target cells than for the off-target cells.
The in vivo study in a mouse xenograft model of B-cell lymphoma showed that ObiDi/SO1861 effectively prevented tumor growth (51.4% response rate) compared with monotherapy with ObiDi (25.9%) and unconjugated obinutuzumab (20.7%). Tumor volume reduction and survival were also improved. Overall, our results contribute significantly to the development of a combination therapy with SO1861 as a platform technology to improve the efficacy of therapeutic antibody-toxin conjugates in lymphoma and leukemia (Panjideh et al.).
A cleavable peptide adapter improves the efficacy of targeted toxins in combination with the glycosidic endosomal escape enhancer SO1861
Treatment with targeted tumor toxins attempts to overcome the drawbacks of conventional cancer therapies by targeting the cytotoxic effect of a drug to cancer cells. However, success with targeted toxins is hampered by the fact that the constructs generally remain bound outside the cell or are either transported back to the cell surface or degraded in lysosomes after receptor-mediated endocytosis. Therefore, solutions for effective endosomal escape are an urgent need in the treatment with targeted toxins.
In this work, a molecular adapter consisting of a cell-penetrating peptide and two cleavable peptides was inserted into a targeted toxin between the ribosome-inactivating protein dianthin and epidermal growth factor. Using cytotoxicity assays, this study investigated whether addition of the adaptor further enhances endosomal escape, which is already enhanced up to more than 1000-fold by the glycosylated triterpenoid SO1861. Introduction of the peptide adapter into the targeted toxin resulted in an approximately 12-fold increase in cytotoxicity to target cells, while SO1861 provided a 420-fold increase. The combination of adapter and glycosylated triterpenoid resulted in more than 3960-fold enhancement and an additional 47-fold increase in specificity. This indicates that the cleavable peptide adapter is a promising technology to further enhance glycosylated triterpenoid-mediated endosomal escape.
Dianthin-EGF in combination with the endosomal escape enhancer SO1861 is a potential powerful targeted therapy for non-small cell lung cancer
Lung cancer is one of the most malignant cancers with the highest incidence and mortality rate worldwide. The development of new targeted drugs is therefore of particular importance. In our study, we expressed and purified both the ribosome-inactivating protein dianthin and a fusion protein of dianthin and epidermal growth factor (EGF) targeting EGF receptor (EGFR). A cytotoxicity assay was used to demonstrate the efficacy of dianthin and dianthin-EGF in cell cultures with or without the support of the glycosylated triterpenoid SO1861, which mediates endosomal escape. The results of in vitro cytotoxicity assays showed that dianthin-EGF specifically targets the lung cancer cell line PC9 with high EGFR expression. In addition, SO1861 enhanced the endosomal escape of dianthin-EGF by 55,000-fold.
The combined therapy was applied in xenograft mouse models, and different treatments (dianthin-EGF ± SO1861, placebo) were compared. After eight treatment cycles, blood, organs, and tumor were collected from the mice for further analysis. The combination of dianthin-EGF and SO1861 reduced the final tumor size in mice by more than 70%. At the same time, no side effects of dianthin-EGF and SO1861 on the organs and blood of mice were observed. Thus, dianthin-EGF is a promising targeted toxin that can greatly limit the proliferation of tumor cells using SO1861.