FGF-2 is an unconventionally secreted lectin that transmits proangiogenic signals through a ternary complex with high-affinity FGF receptors and heparan sulfate proteoglycans (HSPGs). Although FGF-2 signal transduction is understood in great detail, its mechanism of release from cells, which is independent of the classical secretory pathway, remains elusive. To test the hypothesis that FGF-2 secretion is linked to its cell-surface ligands, we studied FGF-2 release using mutants defective for HSPG binding and cells with impaired HSPG biosynthesis. Here, we report that a functional interaction between FGF-2 and HSPGs is required for net export of FGF-2 from mammalian cells. FGF-2 release requires extracellular, membrane-proximal HSPGs. We propose that extracellular HSPGs form a molecular trap that drives FGF-2 translocation across the plasma membrane.

The cytokine macrophage migration inhibitory factor (MIF) is inducibly secreted by immune cells and certain other cell types to critically participate in the regulation of the host immune response. However, MIF does not contain a N-terminal signal sequence and the mechanism of MIF secretion is unknown. Here we show in a model of endotoxin-stimulated THP-1 monocytes that MIF does not enter the endoplasmatic reticulum and that MIF secretion is not inhibited by monensin or brefeldin A, demonstrating that MIF secretion occurs via a non-classical export route. Glyburide and probenicide but not other typical inhibitors of non-classical protein export strongly block MIF secretion, indicating that the export pathway of MIF involves an ABCA1 transporter. ß

Basic fibroblast growth factor (FGF-2) is a secretory protein that lacks a signal peptide. Consistently, FGF-2 has been shown to be secreted by an ER-Golgi-independent mechanism; however, the machinery mediating this process remains to be established at the molecular level. Here we introduce a novel experimental system based on flow cytometry that allows the quantitative assessment of nonclassical FGF-2 secretion in living cells. Stable cell lines have been created by retroviral transduction that express various kinds of FGF-2-GFP fusion proteins in a doxicyclin-dependent manner. Following induction of protein expression, biosynthetic FGF-2-GFP is shown to translocate to the outer surface of the plasma membrane as determined by both fluorescence activated cell sorting (FACS) and confocal microscopy. Both N-and C-terminal GFP tagging of FGF-2 is compatible with FGF-2 export, which is shown to occur in a controlled fashion rather than through unspecific release. The experimental system described has strong implications for the identification of both FGF-2 secretion inhibitors and molecular components involved in FGF-2 secretion.In the second part of this study we made use of the FGF-2 export system described to analyze the fate of biosynthetic FGF-2-GFP following export to the extracellular space. We find that secreted FGF-2 fusion proteins accumulate in large heparan sulfate proteoglycan (HSPG)-containing protein clusters on the extracellular surface of the plasma membrane. These microdomains are shown to be distinct from caveolae-like lipid rafts known to play a role in FGF-2-mediated signal transduction. Since CHO cells lack FGF high-affinity receptors (FGFRs), it can be concluded that FGFRs mediate the targeting of FGF-2 to lipid rafts. Consistently, FGF-2-GFP-secreting CHO cells do not exhibit increased proliferation activity. Externalization and deposition of biosynthetic FGF-2 in HSPG-containing protein clusters are independent processes, as a soluble secreted intermediate was demonstrated. The balance between intracellular FGF-2 and HSPG-bound secreted FGF-2 is shown not to be controlled by the availability of cell surface HSPGs, indicating that the FGF-2 secretion machinery itself is ratelimiting.

CD4+CD25+ regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.

305 Background: CD138 (Syndecan-1) is highly overexpressed in various solid tumors and hematological malignancies and represents one of the most specific target antigens for identification of multiple myeloma (MM) cells. BT062 (Biotest AG Dreieich, Germany) is an antibody-drug conjugate, comprised of the anti-CD138 chimerized MAb (nBT062) and the cytotoxic agent DM4. Once bound to CD138 on a target cell, the conjugate is internalized and releases DM4, leading to target cell death. We performed the first in man study (969) to investigate safety and efficacy of BT062 in MM. Objectives: To determine the maximum tolerated dose (MTD), the dose-limiting toxicities (DLTs), pharmacokinetics (PK) and anti-MM activity of increasing doses of BT062 on a repeated single dose schedule once every three weeks in patients with relapsed and/or refractory MM. Methods: This is a prospective, open label, dose-escalation, multicenter phase I study. Patients aged ≥18 years with relapsed or relapsed/refractory MM who have failed previous treatments including an immunomodulatory agent and a proteasome inhibitor were eligible to participate. Patients with clinical response (or no evidence of progressive disease) and without unacceptable toxicities were eligible for further treatment cycles. Patients were enrolled in cohorts of 3 at each dose level, with DLT in the first cycle triggering cohort expansion. Toxicities were assessed by CTCAE v3 and clinical response was assessed according to the international myeloma working group criteria. Results: A total of 32 patients have been treated with BT062, receiving one of 7 dose levels ranging from 10 mg/m2 to 200 mg/m2. Maximum administered dose has been defined at 200 mg/m2, with mucositis as the dose limiting toxicity (CTC grade III in 2 of the 3 patients in this cohort). Thirteen of 32 patients have been treated in an expanded MTD-cohort at 160 mg/m2. The most frequently reported adverse events to date are mild to moderate and cover primarily events expected for the underlying disease and patient group. A few adverse events have also been observed involving skin and/or mucosa (tissues of epithelial origin with CD138 expressing cells), as well as the eye. CTC grade II/III toxicity involving skin and/or mucosa (e.g. mucositis, stomatitis, hand/foot syndrome) has been observed mainly at the dose levels 160 mg/m2 or higher. Adverse events involving the eye (e.g. blurred vision, dry eye) have also been reported mainly in patients at the dose levels 160 mg/m2 or higher, all restricted to CTC grade I/II. Among the 27 evaluable patients, 3 patients responded including 1 partial response and 2 minor responses, with one patient (minor response) remaining on treatment for more than a year. Stabilization of disease was noted in an additional 11 patients, receiving a median of 5 cycles of therapy (range of 4–10). Thus stable disease or better was noted in 52% of patients. Most patients came off study due to disease progression. Conclusion: Preliminary data from this study demonstrate an acceptable toxicity profile of BT062. Even in this phase I patient population, evidence of clinical activity was observed. Based on the favourable safety profile, the pharmacokinetic data and early signs of clinical activity, a Phase I/IIa study in MM (975) is initiated to further evaluate the safety and anti-MM efficacy of BT062 in a more frequent dosing regimen. To date 13 patients have been treated with BT062 on the intensified multi-dose regimen, receiving one of the first four dose levels. Updated results on safety, PK and anti-MM efficacy of BT062 will be presented. Disclosures: Jagannath: Celgene: Honoraria; Millennium/Takeda Pharma: Honoraria; J&J Family: Honoraria; Onyx: Honoraria; Merck: Honoraria. Heffner:Millennium: Research Funding. Avigan:Genzyme: Consultancy, Research Funding; Celgene: Research Funding; Curetec: Research Funding. Lutz:ImmunoGen, Inc.: Employment. Engling:Biotest AG: Employment. Uherek:Biotest AG: Employment. Osterroth:Biotest AG: Employment. Ruehle:Biotest AG: Employment. Beelitz:Biotest Pharmaceuticals Corporation: Employment. Niemann:Biotest AG: Employment. Wartenberg-Demand:Biotest AG: Employment. Haeder:Biotest AG: Employment. Anderson:Merck: Consultancy; Onyx: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.