Summary Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-β1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-β1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-β are described, including prostaglandin E 2 receptor downmodulation, TGF-β insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

Transcription factors of the Myc/Max/Mad network affect multiple aspects of cellular behavior, including proliferation, differentiation, and apoptosis. Recent studies have shown that Mad proteins can inhibit cellular growth and transformation and thus antagonize the function of Myc proteins. To define further the contribution of these proteins to cellular growth control, we have studied the expression of the respective genes and proteins in 3T3-L1 cells, both upon serum stimulation of quiescent cells and during adipocytic differentiation in response to insulin, dexamethasone, and isobutylmethylxanthine. We found distinct expression patterns for the mad genes. Mad4 was induced when cells exit the cell cycle and, together with mad1, during the late phase of differentiation. In contrast, mad3 expression was associated with progression through S phase and the proliferative burst of differentiating preadipocytes, overlapping in part c-myc expression. DNA binding analyses revealed that the most prominent network complex both in cycling and in differentiating cells was Mnt/Max, whereas c-Myc/Max complexes were detectable only during peak c-Myc expression periods. Ectopic expression of Mad1 in preadipocytes resulted in the inhibition of S phase and the proliferation associated with the proliferative burst; as a consequence, adipocytic differentiation was significantly inhibited. Our findings suggest that the precise temporal regulation of Myc/Max/Mad network proteins is critical for determining cellular behavior.

FGFR2 is a transmembrane tyrosine kinase receptor, consisting of three extracellular N-terminal immunoglobulin-like domains which are involved in ligand-binding as well as in receptor dimerization. Ligand-independent activation of FGFR2 signaling either via genomic amplification, gene fusion events, mRNA overexpression, or mutations has been observed e.g. in gastric cancer, colorectal cancer (CRC), and triple-negative breast cancer (TNBC). As such, FGFR2 has been described to be involved in cancer progression, promotion of oncogenesis, neoangiogenesis, as well as resistance to targeted therapies. Overexpression of FGFR2 and relatively low levels of cell surface expression of FGFR2 in normal human tissues renders FGFR2 an attractive candidate to explore targeted alpha therapy (TAT). We describe the generation of a high energy, alpha-particle emitting FGFR2 targeted thorium-227 conjugate (FGFR2-TTC). The FGFR2-TTC consists of a fully human FGFR2 binding IgG1 antibody (BAY 1179470) cross-reactive with mouse FGFR2, covalently linked via an amide bond to a chelator moiety (3,2 HOPO), enabling radiolabeling with the alpha particle emitting thorium-227 (227Th). In vitro cytotoxicity experiments with FGFR2-TTC demonstrated potency in the sub-picomolar range compared to a non-targeting control-TTC and a correlation between decrease in cell viability and increasing number of anti-FGFR2 antibodies bound per cell (ABC counts) in a panel of FGFR2-positive cancer cell lines. Upon treatment of cells with FGFR2-TTC, the DNA damage response marker protein γH2AX was up-regulated indicating that the mode-of-action involves induction of DNA double strand breaks. Furthermore, induction of the immunogenic cell death marker calreticulin was observed Biodistribution studies of the FGFR2-TTC in mouse models, evaluated by whole body autoradiography and acquisition of gamma-spectra specific for thorium-227, demonstrated specific accumulation of thorium-227 in FGFR2-positive tumors and very limited signal in murine organs and tissues. FGFR2-TTC exhibited in vivo tumor growth inhibition after a single dose in mouse xenograft models of CRC (NCI-H716) and gastric cancer (SNU-16). In addition, FGFR2-TTC showed anti-tumor activity in the aggressive murine syngeneic orthotopic 4T1 TNBC model. In summary, FGFR2-TTC has been established as a promising targeted alpha therapy (TAT) for efficacious and selective delivery of alpha emitter-based radiotherapy in several FGFR2-positive cancer indications. Further exploration for cancer therapy may thus be of interest. Citation Format: Urs B. Hagemann, Anette Sommer, Alexander Kristian, Ellen Wang, Åsmund Larsen, Uta Wirnitzer, Heidrun Ellinger-Ziegelbauer, Steffen Sandmann, Thorsten Poethko, Jenny Karlsson, Roger M. Bjerke, Lars Linden, Bertolt Kreft, Hanno Wild, Alan S. Cuthbertson. Preclinical activity of the FGFR2-targeted thorium-227 conjugate in preclinical models of colorectal, gastric and triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2017-5199

We are developing a novel fibroblast growth factor receptor 2 (FGFR2) directed antibody (BAY 1179470) as well as an FGFR2-antibody drug conjugate (FGFR2-ADC) for cancer therapy. FGFR2 is expressed in a range of tumor types such as gastric, breast, HNSCC, pancreatic and ovarian cancers. FGFR2 has been described to promote tumor growth and survival and play a role in chemotherapy resistance. In this study, we aim to identify stratification and pharmacodynamic biomarkers for two antibody-based anti-FGFR2 therapies in preclinical models. As potential biomarkers, FGFR2 antigen expression levels were investigated in tumors by FGFR2 immunohistochemistry (IHC), FGFR2 gene amplification by FISH, FGFR2 mRNA by RNAscope, and FGFR2 protein by mass spectrometry. Furthermore, FGFR2 levels in xenograft tumors were compared to those in clinical samples of human tumors. Efficacy of BAY 1179470 in gastric cancer tumor models with high FGFR2 expression levels was generally higher compared to models with low FGFR2 expression. SNU-16 and GC10-0608, which are characterized by high expression levels of FGFR2, had T/C values of 0.13 and 0.55 when treated with 5 mg/kg BAY 1179470. Tumor growth was not inhibited by BAY 1179470 in MKN-45 and T47D, which had no detectable FGFR2 expression. MFM-223 and NCI-H716 with high FGFR2 expression levels did not respond to BAY1179470 suggesting that they were not dependent on FGFR2 signaling. In contrast, all three established tumor models SNU-16, MFM-223, and NCI-H716 were highly responsive to FGFR2-ADC. Analysis and scoring of FGFR2 expression by IHC using human tumor samples (gastric, breast, HNSCC, pancreatic and ovarian cancer, respectively) demonstrated that tumor cell FGFR2 expression varied both within and between tumor types. IHC scoring revealed that 54% of gastric cancers, 73% of triple negative breast cancers, 90% of HNSCC, 43% of pancreatic cancers, and 43% of ovarian cancer investigated were FGFR2 positive. Within the tumor indications investigated patients showed FGFR2 expression of score 0, 1, 2 or 3 respectively.In the SNU-16 gastric cancer xenograft model, total and phospho-FGFR2 were reduced upon treatment with BAY 1179470. Depending on dose, total FGFR2 levels showed the lowest level 1-4 days after treatment and returned to baseline after 21 days. Suppression of the downstream signaling pathway component phospho-RPS6 signaling was also shown. In summary, these results demonstrate that FGFR2-directed therapies are effective in xenograft models with high FGFR2 expression. FGFR2 expression as assessed by IHC and FGFR2 gene amplifications are therefore candidate biomarkers for stratification of the FGFR2-directed studies. The anti-FGFR2 antibody BAY 1179470 is currently in Phase I testing (NCT01881217). Citation Format: Christoph A. Schatz, Charlotte Kopitz, Sabine Wittemer-Rump, Anette Sommer, Lars Lindbom, Motonobu Osada, Hiroshi Yamanouchi, Hung Huynh, Thomas Krahn, Khusru Asadullah. Pharmacodynamic and stratification biomarker for the anti-FGFR2 antibody (BAY1179470) and the FGFR2-ADC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4766. doi:10.1158/1538-7445.AM2014-4766