These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA'-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

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Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.

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.The clonal hematopoietic stem cell disorder CMML (chronic myelomonocytic leukemia), is a highly aggressive and resistant form of leukemia. It is characterized by persistent monocytosis in the peripheral blood, at least one dysplasia component in the bone marrow, and <20% promonocytes and blasts in peripheral blood and bone marrow. 1 CMML can be classified further according to blast numbers, that is, CMML1 (<5% blasts in peripheral blood and <10% blasts in bone marrow) and CMML2 (<20% blasts in peripheral blood and 10-19% blasts in bone marrow).2,3 The disease occurs mainly in elderly people with a median survival of 15-20 months and leukemic transformation rates of 15-30%, both factors depending on the disease subtype. 4 Other diagnostic criteria include immunophenotyping based on the overlapping antigen expression patterns in CMML and acute myelomonocytic leukemia (AMML). The expression of CD56 combined with reductions in the levels of myeloid markers such as CD15 and CD13 is a unique signature of CMML monocytes.5 Furthermore, CD14 is expressed at higher levels on bone marrow monocytes in CMML compared with reactive monocytosis and normal marrow samples. 5 High levels of CD33 have also been reported. 6 Clonal cytogenetic abnormalities are found in 20-40% of CMML patients, but these do not appear to be specific. [7][8][9] The close relationship between CMML and other myelodysplastic (MDS)/MPNs makes correct diagnosis and treatment a significant challenge. Novel molecular markers described more recently include specific alleles of TET2 and CBL, but these

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.