The transcription factor FOXP3 plays a key role in CD4⁺CD25⁺ regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4⁺CD25⁺ cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti‐FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow‐cytometric analysis showed that FOXP3 was expressed by the majority of CD4⁺CD25^high T cells in peripheral blood. By contrast, less than half of the CD4⁺CD25^int population were FOXP3⁺, providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4⁺CD25^high pool. Although FOXP3 expression was primarily restricted to CD4⁺CD25⁺ cells, it was induced following activation of both CD4⁺ and CD8⁺ T cell clones. These findings indicate that the frequency of FOXP3⁺ cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4⁺ and CD8⁺ T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use. See accompanying Commentary http://dx.doi.org/10.1002/eji.200526303

Genetically engineered ‘lymphoreporter’ mouse strains are used to track melanoma dissemination in vivo, identifying midkine as a tumour-secreted factor that acts at a distance, preparing pre-metastatic niches and serving as an indicator of poor prognosis in patients. Lymph nodes are a common metastatic site of many tumour types, including melanomas. The contribution of lymphangiogenesis to metastatic progression is not well defined, as removing the sentinel lymph node does not always improve the survival chances of melanoma patients. Here the authors develop a 'lymphoreporter' mouse model that allows them to track lymphatic vessels with a VEGFR3 reporter, and use it to visualize metastatic routes during relapse. In addition, the authors identify a mediator of lymphangiogenesis, MIDKINE, that contributes to metastasis and indicates a poor prognosis for patients with melanoma. The authors suggest that VEGFR3 reporters could be useful in the search for further drivers and inhibitors of metastasis. Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis1,2,3. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma4,5. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress6,7. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma8,9,10,11,12,13,14. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear2,14. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches15. Injectable lymphatic tracers have been developed7, but their limited diffusion precludes whole-body imaging at visceral sites16. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive ‘lymphoreporter’17 because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer17,18. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a ‘MetAlert’ discovery platform for drivers and inhibitors of metastasis.

There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8^N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8^N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8^N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

Glioblastomas (GBM) are aggressive and therapy-resistant brain tumours, which contain a subpopulation of tumour-propagating glioblastoma stem-like cells (GSC) thought to drive progression and recurrence. Diffuse invasion of the brain parenchyma, including along preexisting blood vessels, is a leading cause of therapeutic resistance, but the mechanisms remain unclear. Here, we show that ephrin-B2 mediates GSC perivascular invasion. Intravital imaging, coupled with mechanistic studies in murine GBM models and patient-derived GSC, revealed that endothelial ephrin-B2 compartmentalises non-tumourigenic cells. In contrast, upregulation of the same ephrin-B2 ligand in GSC enabled perivascular migration through homotypic forward signalling. Surprisingly, ephrin-B2 reverse signalling also promoted tumourigenesis cell-autonomously, by mediating anchorage-independent cytokinesis via RhoA. In human GSC-derived orthotopic xenografts, EFNB2 knock-down blocked tumour initiation and treatment of established tumours with ephrin-B2-blocking antibodies suppressed progression. Thus, our results indicate that targeting ephrin-B2 may be an effective strategy for the simultaneous inhibition of invasion and proliferation in GBM.

Purpose: Previous gene expression studies have shown that fibroblast growth factor receptor 3 (FGFR3) is overexpressed in early stages of bladder cancer. To study the potential use of therapeutic antibodies against FGFR3, we have produced a collection of human single-chain Fv (scFv) antibody fragments by using phage display libraries. Experimental Design: Two “naïve” semi-synthetic human scFv libraries were used to select antibodies against the extracellular domain of FGFR3α(IIIc). The reactivity of the selected scFvs with a recombinant FGFR3 was characterized by an enzyme immunoassay and surface plasmon resonance analysis and with RT112 bladder carcinoma cells by a fluorescence-activated cell sorter. The capacity of the selected scFvs to block RT112 cell proliferation was determined. Results: We have isolated six human scFv antibody fragments directed against FGFR3. These human scFvs specifically bound FGFR3, but not the homologous molecule FGFR1. Biacore analysis was used to determine the affinity constants, which ranged from 12 to 40 nmol/L. Competition analysis showed that the FGF9 ligand was able to block the binding of two scFvs, 3C and 7D, to FGFR3, whereas FGF1 only blocked 7D. Immunoprecipitation and flow cytometric analysis confirmed the specificity of the antibodies to native membrane FGFR3. Two scFvs, 3C and 7D, gave an strong immunofluorescence staining of RT112 cells. Moreover, they recognized equally well wild-type and mutant FGFR3 containing the activating mutation S249C. Furthermore, they blocked proliferation of RT112 cells in a dose- and FGF-dependent manner. Conclusion: Our results suggest that these human anti-FGFR3 scFv antibodies may have potential applications as antitumoral agents in bladder cancer.