The oncogenic effects of epidermal growth factor (EGF) have long been established. EGF receptor (EGFr) is overexpressed in many types of tumors and constitutes a target for cancer treatment. The pituitary gland is a target of EGF action and it is very likely that EGFr plays a role in pituitary tumor formation and progression. However, there is a controversy in the literature concerning EGFr expression in the different types of pituitary adenomas. In the present study we investigated the expression pattern of the wild type EGFr (EGFrWT) and the constitutively active variant III (EGFrvIII) at the mRNA and protein levels in a large series of pituitary tumors. EGFrWT was found in a high percentage of hormone-secreting tumors, but only in a small fraction of non-functioning pituitary adenomas, while no expression of the EGFrvIII could be detected by nested RT-PCR in any tumor. Among the hormone-secreting adenomas, the highest incidence of EGFr expression was found in Cushing's pituitary adenomas. Furthermore, immunohistochemistry for the phosphorylated EGFr revealed the presence of activated EGFr in most Cushing's adenomas, compared with most pituitary adenomas. Taking into account that downregulation of p27/Kip1 plays a significant role in corticotrope tumorigenesis and that EGFr mitogenic signaling results in decreased p27/Kip1, we searched for a correlation between EGFr expression and p27/Kip1 levels in corticotropinomas. Low p27/Kip1 immunoreactivity was observed in corticotropinomas expressing EGFr. On the other hand, somatotropinomas expressing EGFr had high p27/Kip1 immunoreactivity. These data suggest a corticotrope-specific phenomenon and indicate that EGFr may have a role in the unbalanced growth of corticotrope tumoral cells.

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Thyrotrophin (TSH) synthesis and secretion is under the positive control of thyrotrophin releasing hormone and under the negative control of the thyroid hormones. However, it is hypothesised that TSH has a direct effect on the regulation of its own synthesis through an intrapituitary loop mediated by pituitary TSH receptors (TSH-R). The aim of this investigation was to study the expression of TSH-R in normal human pituitary at mRNA and protein levels, and to compare the pattern of protein expression between different pituitary adenomas. Using RT-PCR we were able to detect TSH-R mRNA in the normal pituitary, and immunohistochemical studies showed TSH-R protein expression in distinct areas of the anterior pituitary. Double immunostaining with antibodies against each of the intrapituitary hormones and S100 revealed that TSH-R protein is present in thyrotrophs and folliculostellate cells. Examination of 58 pituitary adenomas, including two clinically active and two clinically inactive thyrotroph adenomas, revealed TSH-R immunopositivity in only the two clinically inactive thyrotroph adenomas.This study shows, for the first time, the presence of TSH-R protein in the normal anterior pituitary and in a subset of thyrotroph adenomas. The expression of TSH-R in the thyrotroph and folliculostellate cell subpopulations provides preliminary evidence of a role for TSH in autocrine and paracrine regulatory pathways within the anterior pituitary gland.

Purpose: The medical need due to high mortality in acute myeloid leukemia (AML) remains high, and the treatment of relapsed or refractory AML continues to be therapeutically challenging. MYLOTARG, the only approved anti-CD33 antibody drug conjugate (ADC), has provided proof-of-concept for targeted immunotherapies in AML. Currently, a plethora of ADCs and T-cell engager (TCE) therapies have entered clinical development in AML, but those therapies are often accompanied by dose limiting toxicities, preventing dose escalation to desired anti-tumor efficacy. The biggest challenges seem to be limited target specificity and hyperstimulation of the immune system leading to e.g. myelotoxicities and cytokine release syndrome, respectively. Therefore, more selective therapies are needed to allow for robust anti-tumor activity with a more acceptable safety profile. Experimental design: To address the selectivity challenge, we have generated multi-specific T-cell engaging DARPin® molecules, targeting two different tumor associated antigens (TAAs) with optimized affinity for their targets. In order to find the right target combination, the optimal affinity to increase tumor specificity via avidity, as well as the best molecular architecture, we took advantage of our unique modular DARPin® platform and screened 1000s of combinations of multi-specific DARPin® molecules, binding simultaneously to multiple TAAs in conjunction with our CD3-binding DARPin® molecule. Results: We constructed multi-specific TCEs targeting two different AML antigens with optimized affinity leading to a substantial avidity gain when both targets are co-expressed on tumor cells. The avidity gain resulted in strongly enhanced in vitro potency as shown by activation of both CD8+ and CD4+ T cells and subsequent killing of AML tumor cells, with bioactivities in the range of established TCE benchmark formats (e.g. BiTE® and DART®). In contrast, in an ex vivo whole blood assay the multi-specific DARPin® constructs induced profoundly less cytokine release as compared to benchmark molecules indicating an improved therapeutic window. Finally, we also demonstrated tumor regression in PMBC humanized mouse models bearing MOLM-13 tumors, using both half-life extended (HLE) and non-HLE lead constructs. In conclusion, we have generated TCEs based on multi-specific DARPin® constructs with high potency, selectivity and ultimately with the potential for an improved therapeutic window for the treatment of AML. Citation Format: Nina Reschke, Thamar Looser, Jennifer Krieg, Matteo Bianchi, Patricia Schildknecht, Nicole Bassler, Yvonne Gruebler, Sebastian Grimm, Laura Jeanbart, Tanja Hospodarsch, Alexandra Neculcea, Daniel Steiner, Bernd Schlereth, Christian Reichen. Novel DARPin multi-specific T-cell engager with an improved therapeutic window to overcome dose limiting toxicities in AML therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 525.