Bispecific mAb2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab.

Inhibition of complement activation via the overexpression of complement-regulatory proteins (CRPs), most notably CD46, CD55 and CD59, is an efficient mechanism of disguise of cancer cells from a host immune system. This phenomenon extends to counteract the potency of therapeutic antibodies that could lyse target cells by eliciting complement cascade. The manifold functions and ubiquitous expression of CRPs preclude their systemic specific inhibition. We selected CD59-specific Fc fragments with a novel antigen binding site (Fcabs) from yeast display libraries using recombinant antigens expressed in bacterial or mammalian cells. To produce a bispecific antibody, we endowed rituximab, a clinically applied anti-CD20 antibody, used for therapy of various lymphoid malignancies, with an anti-CD59 Fcab. This bispecific antibody was able to induce more potent complement-dependent cytotoxicity for CD20 and CD59 expressing Raji cell line measured with lactate dehydrogenase-release assay, but had no effect on the cells with lower levels of the primary CD20 antigen or CD20-negative cells. Such molecules are promising candidates for future therapeutic development as they elicit a higher specific cytotoxicity at a lower concentration and hence cause a lower exhaustion of complement components.

Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting.

Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal stabilization of the human CD81 large extracellular loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown to exhibit a positive shift in the melting temperature (Tm) of up to 25 °C. The combination of two most potent disulfide bonds connecting different strands of the protein resulted in a mutant with a Tm of 109 °C, 43 °C over the Tm of the wild-type hCD81 LEL. A peptide sequence binding to the human transferrin receptor (hTfr) was engrafted into the D-segment of the hCD81 LEL, resulting in a mutant that still exhibited a compact fold. Grafting of the same peptide sequence between helices A and B resulted in a molecule with an aberrant profile in size exclusion chromatography (SEC), which could be improved by a de novo cysteine bond connecting both helices. Both peptide-grafted proteins showed an enhanced internalization into the cell line SK-BR3, which strongly overexpresses hTfr. In summary, the tetraspan LEL fold could be stabilized to enhance its amenability for engineering into a more versatile protein scaffold.

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

Vascular endothelial growth factor A as predictive marker for mTOR inhibition in relapsing high-grade serous ovarian cancer.

BACKGROUND: Development of resistance against first line drug therapy including cisplatin and paclitaxel in high-grade serous ovarian cancer (HGSOC) presents a major challenge. Identifying drug candidates breaking resistance, ideally combined with predictive biomarkers allowing precision use are needed for prolonging progression free survival of ovarian cancer patients. Modeling of molecular processes driving drug resistance in tumor tissue further combined with mechanism of action of drugs provides a strategy for identification of candidate drugs and associated predictive biomarkers. RESULTS: Consolidation of transcriptomics profiles and biomedical literature mining results provides 1242 proteins linked with ovarian cancer drug resistance. Integrating this set on a protein interaction network followed by graph segmentation results in a molecular process model representation of drug resistant HGSOC embedding 409 proteins in 24 molecular processes. Utilizing independent transcriptomics profiles with follow-up data on progression free survival allows deriving molecular biomarker-based classifiers for predicting recurrence under first line therapy. Biomarkers of specific relevance are identified in a molecular process encapsulating TGF-beta, mTOR, Jak-STAT and Neurotrophin signaling. Mechanism of action molecular model representations of cisplatin and paclitaxel embed the very same signaling components, and specifically proteins afflicted with the activation status of the mTOR pathway become evident, including VEGFA. Analyzing mechanism of action interference of the mTOR inhibitor sirolimus shows specific impact on the drug resistance signature imposed by cisplatin and paclitaxel, further holding evidence for a synthetic lethal interaction to paclitaxel mechanism of action involving cyclin D1. CONCLUSIONS: Stratifying drug resistant high grade serous ovarian cancer via VEGFA, and specifically treating with mTOR inhibitors in case of activation of the pathway may allow adding precision for overcoming resistance to first line therapy.

Regulation of the MDR1 promoter by E2F1 and EAPP.

Multidrug resistance (MDR), one of the main reasons for diminishing efficacy of prolonged chemotherapy, is frequently caused by the elevated expression of the ABCB1/MDR1 gene encoding PGP (P-glycoprotein). EAPP (E2F Associated PhosphoProtein) is a frequently overexpressed protein in human tumor cells. It inhibits apoptosis in a p21-dependent manner. We show here that EAPP stimulates the MDR1 promoter resulting in higher PGP levels. Independently of EAPP, E2F1 also increases the activity of the MDR1 promoter. Co-expression of pRb inhibits E2F1-, but not EAPP-dependent promoter activation. The upregulation of PGP might contribute to the survival of tumor cells during chemotherapy and worsen the prognosis for the patient.

EAPP modulates the activity of p21 and Chk2.

Genomic instability is thought to be critical for the development of cancer. Among its causes microsatellite instability (MIN) and chromosomal instability (CIN) have attracted the most attention. Cell cycle checkpoints and DNA repair mechanisms are the first line of defense against DNA damage. Among the most dangerous DNA lesions are double-strand breaks. The response to DNA double strand breaks is regulated mainly by the serine/threonine kinases ATM and Chk2 and their downstream target the tumor suppressor p53, which in turn stimulates the expression of growth-inhibitory genes like p21 or pro-apoptotic genes like Bax. The balance between these gene products determines the fate of a cell. EAPP is a nuclear phosphoprotein that is frequently upregulated in human tumors. We have recently shown that EAPP levels are critical for cellular homeostasis. DNA damage elevates EAPP levels and its overexpression results in G1 arrest and impairs apoptosis in a p21-dependent manner. EAPP binds to the p21 promoter, stimulates its activity and seems to be essential for transcription initiation. In the present work we show that EAPP also regulates the phosphorylation status and thus the activity of Chk2. EAPP binding seems to trigger the dephosphorylation of P-Chk2 resulting in its inactivation. A newly described function of Chk2 in mitosis that secures genomic integrity might also be affected by EAPP overexpression. This might explain the abundance of EAPP in aneuploid tumor cells.

Regulation of the E2F-associated phosphoprotein promoter by GC-box binding proteins.

The E2F-associated phosphoprotein (EAPP) is a ubiquitous nuclear protein that interacts with the activating members of the E2F family of transcription factors and increases the activity of several cell-cycle regulated promoters in an E2F-dependent manner. Our previous studies also showed that EAPP levels are elevated in most transformed human cells. To examine the molecular basis of this increase of EAPP we isolated and studied the nucleotide sequence at the 5' end of the EAPP gene. In silico analysis revealed a TATA-less promoter with several putative binding sites for transcription factors, the most probable ones being Sp1, Sp3 and Egr-1. We could confirm the binding of these factors in vitro by electrophoretic mobility shift assays, supershift experiments and competition assays. Additionally we could validate the binding in vivo by chromatin-immunoprecipitation assays. To analyse the function of these transcription factors in the expression of EAPP, we performed reporter-assays with the promoter and truncations thereof. We found that Sp1 and Egr-1 stimulate the EAPP promoter, whereas Sp3 acts as a repressor that could even overcome the positive effect of the activators. Increasing the amounts of Sp3 also caused a strong reduction of EAPP, but the overexpression of Sp1 or Egr-1 resulted in only marginally higher EAPP levels. Our results suggest that the elevated EAPP levels in transformed cells can be caused by reduced Sp3 activity, but higher Sp1 activity might also play a role.

EAPP, a novel E2F binding protein that modulates E2F-dependent transcription.

E2F transcription factors play an essential role in cell proliferation and apoptosis and their activity is frequently deregulated in human cancers. In a yeast two-hybrid screen we identified a novel E2F-binding protein. Due to its strong phosphorylation we named it EAPP (e2F-associated phosphoprotein). EAPP is localized in the nucleus and interacts with E2F-1, E2F-2, and E2F-3, but not with E2F-4. Examination of a number of human cell lines revealed that EAPP levels are elevated in most transformed cells. Moreover, EAPP mRNA was detected in all investigated human tissues in varying amounts. EAPP is present throughout the cell cycle but disappears during mitosis. In transfection assays with reporters controlled by either an artificial E2F-dependent promoter or the murine thymidine kinase promoter, EAPP increased the activation caused by E2F-1 but not by E2F-4. Surprisingly, the promoter of the p14(ARF) gene, which was also activated by E2F-1, became repressed by EAPP. Overexpression of EAPP in U2OS cells resulted in a significant increase of cells in S-phase, whereas RNAi-mediated knock down of EAPP reduced the fraction of cells in S-phase. Taken together, these data suggest that EAPP modulates E2F-regulated transcription, stimulates proliferation, and may be involved in the malignant transformation of cells.