The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.nanobody | CXCR4 antagonist | chemotaxis | HIV | stem cell mobilization

Cervical cancer results from persistent infection with high-risk human papillomavirus (hrHPV). Common genetic aberrations in cervical (pre)cancers encompass large genomic regions with numerous genes, hampering identification of driver genes. This study aimed to identify genes functionally involved in HPV-mediated transformation by analysis of focal aberrations (<3 Mb) in high-grade cervical intraepithelial neoplasia (hgCIN). Focal chromosomal aberrations were determined in high-resolution array comparative genomic hybridization data of 60 hgCIN. Genes located within focal aberrations were validated using 2 external gene expression datasets or qRT-PCR. Functional roles of candidate genes EYA2 (20q13) and hsa-miR-375 (2q35) were studied by siRNA-mediated knock-down and overexpression, respectively, in hrHPV-containing cell lines. We identified 74 focal aberrations encoding 305 genes. Concurrent altered expression in hgCIN and/or cervical carcinomas compared with normal cervical samples was shown for ATP13A3, HES1, OPA1, HRASLS, EYA2, ZMYND8, APOBEC2, and NCR2. Gene silencing of EYA2 significantly reduced viability, migratory capacity, and anchorage-independent growth of HPV16-transformed keratinocytes. For hsa-miR-375, a direct correlation between a (focal) loss and significantly reduced expression was found. Downregulation of hsa-miR-375 expression was confirmed in an independent series of cervical tissues. Ectopic expression of hsa-miR-375 in 2 cervical carcinoma cell lines reduced cellular viability. Our data provide a proof of concept that chromosomal aberrations are actively contributing to HPV-induced carcinogenesis and identify EYA2 and hsa-miR-375 as oncogene and tumor suppressor gene, respectively.

The chemokine receptor CXCR3 is involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis, and allograft rejection in transplantation patients. The CXCR3 ligands CXCL9, CXCL10, and CXCL11 are expressed at sites of inflammation, and they attract CXCR3-bearing lymphocytes, thus contributing to the inflammatory process. In this study, we characterize five nonpeptidergic compounds of different chemical classes that block the action of CXCL10 and CXCL11 at the human CXCR3, i.e., the 3H-pyrido[2,3-d]pyrimidin-4-one derivatives N-1R- [3-(4-et-

The chemokine receptor CXCR2 is involved in different inflammatory diseases, like chronic obstructive pulmonary disease, psoriasis, rheumatoid arthritis, and ulcerative colitis; therefore, it is considered an attractive drug target. Different classes of small CXCR2 antagonists have been developed. In this study, we selected seven CXCR2 antagonists from the diarylurea, imidazolylpyrimide, and thiazolopyrimidine class and studied their mechanisms of action at human CXCR2. All compounds are able to displace 125

bEpidemiological studies identified 12 high-risk HPV (hrHPV) types and 8 probable/possible hrHPV types that display different cancer risks. Functional studies on transforming properties of hrHPV are mainly limited to HPV16 and -18, which induce immortalization of human foreskin keratinocytes (HFKs) by successive bypass of two proliferative life span barriers, senescence and crisis. Here, we systematically compared the in vitro immortalization capacities, as well as influences on p53, pRb, hTERT, growth behavior, and differentiation capacity, of nine hrHPV types (HPV16, -18, -31, -33, -35, -45, -51, -52, and -59), and two probable hrHPV types (HPV66 and -70). By retroviral transduction, the respective E6/E7 coding sequences were expressed in HFKs from two or three independent donors. Reduced p53 levels and low-level hTERT expression in early-passage cells, as seen in HPV16-, -31-, -33-, and -35-, and to a lesser extent HPV18-transduced HFKs, was associated with continuous growth and an increased immortalization capacity. Less frequent immortalization by HPV45 and -51 and immortalization by HPV66 and -70 was preceded by an intervening period of strongly reduced growth (crisis) without prior increase in hTERT expression. Immortalization by HPV59 was also preceded by a period crisis, despite the onset of low hTERT expression at early passage. HPV52 triggered an extended life span but failed to induce immortality. Variations in p53 and pRb levels were not correlated with differences in alternative E6/E7 mRNA splicing in all hrHPV-transduced HFKs. On collagen rafts, transductants showed disturbed differentiation reminiscent of precancerous lesions. In conclusion, in vitro oncogenic capacities differ between the established hrHPV types, and both some established and probable hrHPV types display weak or moderate immortalization potential. C ervical cancer, the third most common cancer among women worldwide, is caused by a persistent infection with certain types of human papillomavirus (HPV) (1, 2). Most HPV infections are transient and are cleared within 1 to 2 years. However, a fraction of infections eventually give rise to either squamous cell carcinoma (SCC) or adenocarcinoma (AdCA) of the cervix. SCCs develop from so-called cervical intraepithelial neoplasia (CIN) precursor lesions. Low-grade CIN lesions (CIN1) mostly reflect a productive infection in which viral replication and virion production are linked to the differentiation program of the epithelium. High-grade CIN lesions (CIN2/3) mainly represent transforming infections and are characterized by deregulated expression of the early viral E6 and E7 genes in the proliferating basal cells of the epithelium (3, 4).HPV types belonging to the alpha (␣) genus can infect the cervical mucosa (5) and are classified into low-risk (lrHPV) and high-risk (hrHPV) HPV based on their association with malignancy (6). Infections with lrHPV types, e.g., HPV type 6 (HPV6) and HPV11, are associated with benign warts or low-grade lesions, whereas infections with hrHPV can give rise ...