Novel Antibody Drug Conjugates Targeting Tumor-Associated Receptor Tyrosine Kinase ROR2 by Functional Screening of Fully Human Antibody Libraries Using Transpo-mAb Display on Progenitor B Cells

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes (“Transpo-mAb Display”) for ROR2 binding. Individual cellular “Transpo-mAb” clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs

Epithelial to mesenchymal transition in breast cancer : a novel murine model system and the regulatory role of tead transcription factors

Cancer is a leading cause of death worldwide, accounting for 7.6 million, or ~13% of all deaths in 2008. The majority of cancers arise from epithelia. Breast cancer, originating from the mammary epithelium, is the most frequent cancer in women worldwide. Breast cancer detection and treatment at early stages is an effective measure of counteracting the number of deaths, however at later stages, cancer cells may spread from the primary tumor to secondary organs, a multistage process called metastasis. This process involves the dissemination of cancer cells from the primary tumor, entrance into the vascular system, extravasation and re-growth (colonization) in the target organ. Metastasis is the actual cause of death in 90% of cancer patients. Breast cancer treatment is complicated by the existence of substantial biological heterogeneity between and within tumors: At least five different subtypes of breast cancer with variable response to treatment and outcome have been proposed. The biological differences between these tumor subtypes are mainly determined by the nature of the oncogenic hit(s) and the cell type in which transformation originally occurred. In addition to different tumor types, progressing tumors (and also their metastatic outgrowth) consist of individual tumor cells with varying features, which can be evoked by acquisition of cumulative genetic/epigenetic alterations and/or by differential stimulation by components of the nearby tumor microenvironment. These circumstances call for a better understanding of the underlying mechanisms that provide cancer cells with malignant features, such as the acquisition of a metastatic behavior and treatment resistance. One mechanism that endows cancer cells with several pro-metastatic features and treatment resistance is the epithelial-mesenchymal transition (EMT). EMT is a latent embryonic program that can be aberrantly reactivated in epithelial tumor cells during tumor progression. Activation of EMT-like programs in tumor cells leads to dissolution of cell-cell adhesions, a loss of polarity and an acquisition of migratory, invasive and stem-cell-like traits. Studies investigating the role of EMT in cancer have mainly employed a combination of different model systems for in vitro and in vivo experiments. Due to the lack of model systems that allow the study of breast cancer associated EMT in vitro and in vivo using the same cell line, I have established a stable cell line (Py2T) from a breast tumor of an MMTV-PyMT transgenic mouse. I show here that this epithelial cell line undergoes bona fide EMT under cell culture conditions when stimulated with the well-known EMT-inducer transforming growth factor β (TGFβ). TGFβ treatment of Py2T cells leads to downregulated expression of the epithelial marker E-cadherin and an upregulation of mesenchymal markers, concomitant with the induction of migratory and invasive properties. When orthotopically injected into mice, Py2T cells generate tumors that are highly invasive and display a mesenchymal phenotype characterized by the absence of E-cadherin expression, suggesting that these cells undergo spontaneous EMT-like changes in vivo. Interestingly, Py2T cells overexpressing a dominant-negative TGFβ-receptor, leading to a block of TGFβ responsiveness, also form tumors upon fat-pad transplantation, yet in these tumors a partial re-expression of E-cadherin can be observed, suggesting that TGFβ signaling contributes to the EMT phenotype in vivo. Together, my results show that the Py2T model system is a versatile tool to study EMT both in vitro and in vivo. The second project presented in this thesis aimed at the identification of critical transcription factors (TFs) that mediate the widespread changes in gene expression observed during EMT. A genome-wide bioinformatics analysis has uncovered that the DNA-binding motif of Tead transcription factors (MCAT motif) is present in a large number of promoters of EMT-regulated genes. Here I show that Tead transcriptional activity is increased during EMT. Moreover, the expression levels of several Tead family members are also upregulated during EMT, and my results demonstrate that elevated transcriptional activity of Tead2 is sufficient to induce EMT. Furthermore, inhibition or depletion of Teads attenuates the EMT process. Moreover, Tead2 levels also can control the subcellular localization of the Tead co-activators Yap and Taz, a mechanism that possibly contributes to the increased Tead activity observed during EMT. I further demonstrate that Zyxin, a focal adhesion component and regulator of actin remodeling, which has previously been shown to be required for EMT-induced migration, is a direct target gene of Tead2. Collectively, these results demonstrate an important regulatory role of Tead transcription factors in the EMT process

Tead2 expression levels control the subcellular distribution of Yap and Taz, zyxin expression and epithelial-mesenchymal transition

The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellular distribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis

Early morphological changes and junction disassembly can be attributed to non-canonical TGFβ signaling pathways.

<p>(<b>A</b>) Smad-mediated canonical TGFβ signaling is dispensable for early changes in morphology and junction disruption. Cells were transfected with a pool of siRNAs against Smad4 or a non-targeting pool and were then treated or not with TGFβ for 1 day as indicated. Fixed cells were stained for the adherens junction components E-cadherin and β-catenin, or for E-cadherin and the tight junction component ZO-1. Note the relocalization of β-catenin from adherens junctions to the cytoplasm upon TGFβ-treatment. Scale bars, 50 µm. (<b>B</b>) Immunoblot analysis of lysates from the experiment described in (A) to control for Smad4 knockdown efficiency. (<b>C</b>) Requirement of non-canonical TGFβ signaling pathways on early morphological changes and junction disassembly. Cells were pre-treated for 4 hours with chemical inhibitors of the kinases indicated, and were then treated with TGFβ for 1 day and analyzed as described in (A). Scale bars, 50 µm. (<b>D</b>) RhoA expression levels during the early stages of EMT. Cells were treated or not with TGFβ for 1 day and RhoA expression levels were analysed by immunoblotting. (<b>E</b>) Importance of RhoA levels for tight- and adherens junction integrity. Epithelial Py2T cells were separately transfected with two different siRNAs targeting RhoA to achieve expression levels comparable to those observed in Py2T cells treated with TGFβ (see D). Cells were stained for the adherens junction components E-cadherin and β-catenin, or for E-cadherin and the tight junction component ZO-1. (<b>F</b>) Immunoblotting analysis to determine the RhoA knockdown efficiency in the experiment described in (E).</p

Kinetics and reversibility of TGFβ-induced EMT in Py2T cells.

<p>(<b>A</b>) Morphological changes of Py2T cells during a time-course of TGFβ-treatment. Cells were cultured in growth medium containing TGFβ (2 ng/ml) and phase-contrast microscopy pictures were taken at the indicated times. (<b>B</b>) Immunoblotting analysis of lysates prepared from Py2T cells treated as in (A). The expression of epithelial (E-cadherin), mesenchymal (N-cadherin, fibronectin), luminal (CK8/18) and basal (CK14) markers was analyzed. (<b>C</b>) Changes in the expression of EMT markers during TGFβ-induced EMT of Py2T cells. Py2T cells were treated for 10 days with TGFβ as described in (A). RNA was extracted at the indicated time points of TGFβ-treatment and quantitative RT-PCR was performed with primers specific for the EMT markers indicated. Expression levels are shown as mean fold difference of untreated cells (0d) ± S.E.M of 5 independent experiments. (<b>D–E</b>) Reversibility of TGFβ-induced EMT. Py2T cells were treated with TGFβ for 30 days to induce EMT and were then further cultured without TGFβ for additional 30 days. Phase-contrast microscopy images were taken at the indicated time points (E). E-cadherin expression levels were analyzed throughout the experiment by immunoblotting (F).</p

Tumors of TGFβ-resistant Py2T cells contain areas with a more epithelial phenotype.

<p>(<b>A</b>) Morphology of tumors generated from Py2T cells stably overexpressing a dominant-negative TGFβRII (Py2T TBRDN) or empty vector control cells (Py2T). 1×10⁶ cells were injected into fat pads of nude mice and tumors were grown for 24 days. Paraffin sections were stained with H&E. Note the appearance of more differentiated epithelial areas in Py2T TBRDN tumors. <i>Top</i>: Epithelial (E) and mesenchymal (M) regions are separated by the dashed line (Scale bar, 200 µm). Bottom panels show larger magnification (Scale bar, 50 µm). (<b>B</b>) Expression of EMT and lineage markers in Py2T tumors and in the more epithelial areas of Py2T TBRDN tumors. Immunohistochemical staining of paraffin sections was performed using the specified antibodies. White squares show higher magnification. Scale bar, 100 µm. (<b>C</b>) Immunofluorescence staining of frozen sections of GFP-labeled Py2T and Py2T TBRDN tumors as described in (A) with antibodies against E-cadherin <i>(red)</i> and Py2T tumor cells <i>(green)</i>. Scale bar, 20 µm. (<b>D</b>) Immunoblotting analysis of epithelial and cytokeratin lineage markers in a series of Py2T and Py2T TBRDN tumors as indicated. (<b>E</b>) Immunofluorescence staining of frozen sections of GFP-labeled Py2T and Py2T TBRDN tumors as described in (A) with antibodies against vimentin <i>(red)</i> and Py2T tumor cells <i>(green)</i>. Scale bar, 20 µm.</p

Changes of migratory and invasive properties of Py2T cells before, during and after TGFβ-induced EMT.

<p>(<b>A</b>) Boyden chamber migration and invasion assay. Cells were treated with TGFβ for the indicated times (LT = long term treatment, as described in Fig. 1F). 25'000 cells were seeded into migration or invasion chambers in duplicate in the absence or presence of TGFβ and allowed to pass through the membrane pores for 24 hours along an FBS gradient. Invasion chambers were pre-coated with growth-factor reduced Matrigel (BD BioCoat chambers). Cells that passed through the membrane pores were stained with crystal violet and photographed (<i>bottom panels</i>) and then counted (<i>top graphs</i>). Results are expressed as mean ± S.E.M of three independent experiments. (<b>B</b>) Scratch wound healing assay. Cells pre-treated with TGFβ or not as indicated were starved over night and scratch wounds were introduced into confluent monolayers. Scratch wound closure was monitored by an IncuCyte™ live cell imaging system. Black masking represents initial gap width at 0 hours. Note the collective, sheet-like wound closure by untreated Py2T cells in contrast to single cell wound infiltration of TGFβ-treated cells (also see Movies S1 and S2 for live imaging data of this experiment). (<b>C</b>) Morphology of epithelial Py2T cells and mesenchymal Py2T LT cells grown on plastic tissue culture dishes (2D) and in Matrigel (4 mg/ml; 3D). Structures were grown for 6 days, and stained directly in Matrigel with antibodies against epithelial E-cadherin and ZO-1 or against mesenchymal vimentin and fibronectin. Immunofluorescence images were acquired by confocal microscopy. Scale bars, 25 µm. (<b>D</b>) Three-dimensional reconstruction of confocal imaging stacks from cells grown in Matrigel as described in (A) (See also Movies S5 and S6 for rotating 3D models). Scale bars, 25 µm.</p

Orthotopic transplantation of Py2T cells into syngeneic mice results in the formation of invasive tumors.

<p>(<b>A</b>) H&E staining of histological sections from tumors of MMTV-PyMT transgenic mice and from transplanted Py2T tumors. 1×10⁶ Py2T cells were transplanted into the fat pad of 8 weeks old female FVB/N mice and allowed to grow tumors for 27 days. Late-stage MMTV-PyMT tumors were from 12 weeks old female mice. <i>Bottom panels</i>: enlarged regions indicated by the white squares in the top panels. Note the typical pushing borders in MMTV-PyMT tumors in contrast to stream-like invasion of fat tissue in Py2T tumors. Scale bars, 200 µm. (<b>B</b>) Polyoma-middle-T (PyMT) expression in MMTV-PyMT and Py2T tumors. Paraffin sections were stained with an antibody against PyMT. Immunohistochemical staining in the absence of primary antibody (1°) was used as negative control. Scale bar, 100 µm. (<b>C</b>) Immunoblotting analysis for EMT markers in tumor lysates of MMTV-PyMT and Py2T tumors. Lysate from cultured Py2T cells is included as a control. Note the loss of E-cadherin expression and upregulation of mesenchymal markers (N-cadherin, fibronectin) in Py2T tumors.</p

Establishment of a murine breast cancer cell line undergoing TGFβ-induced EMT.

<p>(<b>A</b>) Primary tumor cells were isolated from an advanced breast tumor of a MMTV-PyMT transgenic female mouse and were cultured for at least 2 months prior to further experimentation, resulting in a novel cell line termed Py2T. (<b>B</b>) Py2T cells maintain the MMTV-PyMT transgene. The MMTV-PyMT transgene was detected by PCR and agarose gel electrophoresis. DNA from an MMTV-PyMT tumor and from normal murine mammary gland (NMuMG) cells served as positive and negative controls, respectively. (<b>C</b>) Py2T cells lost the expression of the MMTV-PyMT transgene. Immunoblotting for the PyMT protein was performed on lysates of Py2T cells untreated or treated with 0.1 µM Dexamethasone for up to 72 h to induce the MMTV promoter. Lysates of an MMTV-PyMT tumor and NMuMG cells served as positive and negative controls, respectively. (<b>D</b>) Treatment of Py2T cells with known EMT inducers. Cells were continuously treated with the indicated growth factors and cytokines for 10 days (2 ng/mL TGFβ1; 50 ng/mL EGF; 10 ng/mL IGF-I; 50 ng/mL HGF; 20 ng/mL FGF-2; 20 ng/mL PDGF-BB; 50 ng/mL IL-6). Potential morphological changes were analyzed by phase-contrast microscopy. (<b>E</b>) Expression of epithelial (E-cadherin) and mesenchymal (N-cadherin, fibronectin) markers were analyzed by immunoblotting of the lysates of cells treated in (D). (<b>F</b>) Immunoblotting analysis of EMT marker expression in Py2T and Py2T LT cells. The mesenchymal subline Py2T LT (long-term) was generated by TGFβ-treatment of Py2T cells for at least 20 days, and was subsequently maintained in TGFβ containing growth medium. (<b>G</b>) Analysis of markers for EMT and breast cell type before and after TGFβ-induced EMT. Immunofluorescence staining was performed with antibodies against E-Cadherin (epithelial marker), vimentin (mesenchymal marker), estrogen receptor alpha (ERα), cytokeratin 8/18 (luminal markers) and cytokeratin 14 (basal marker). Scale bar, 20 µm.</p