Surface Ig (sIg) of follicular lymphoma (FL) is vital for tumor cell survival. We found previously that the Ig in FL is unusual, because the variable region genes carry sequence motifs for N-glycan addition. These are introduced by somatic mutation and are tumor specific. Unexpectedly, added glycans terminate at high mannose, suggesting a potentially important interaction of FL cells with mannose-binding lectins of the innate immune system. We have now identified mannosylated IgM at the surface of primary lymphoma cells. Recombinant lectin domains of the mannose receptor (MR) or DC-SIGN bind mannosylated Igs in vitro and bind to FL cells, signaling sIgM-associated increases in intracellular Ca 2+ . Lectins also bind to normal B cells but fail to signal. In contrast, anti-Ig signaled similarly in both FL and normal B cells. Mannosylation patterns were mimicked by FL Ig-derived single-chain Fvs (scFv), providing probes for potential receptors. Mannosylated scFv bound specifically to the lectin domains of the MR and DC-SIGN and blocked signaling. Mannosylated scFv also bound to DC-SIGN on the surface of dendritic cells. This unique lymphoma-specific interaction of sIg with lectins of innate immunity reveals a potential route for microenvironmental support of tumor cells, mediated via the key B-cell receptor.B cell | B-cell receptor | B-cell lymphoma | immunoglobulin

SCI-25 The B-cell receptor (BCR) is a flexible and variable environmental sensor with no fixed ligand. The central component is surface Ig (sIg), which appears to function at several levels, mediating a “tonic” signal essential for survival and also binding to antigens via its variable (V) regions. Expression of sIg and reliance on BCR signals generally persist in neoplastic B cells. Indolent tumors, including chronic lymphocytic leukemia (CLL), allow insight into the pathogenetic role of the BCR prior to therapy as well as revealing key proteins within these pathways for drug targeting. CLL is heterogeneous, arising at two points of differentiation, generating two major subsets, one with unmutated Ig V genes (U-CLL), another with mutated Ig V genes (M-CLL). U-CLL appears to develop from naïve B cells of the natural antibody repertoire aimed against common pathogens. The clinical behavior of the two subsets differs, with U-CLL being of poorer prognosis. Evidence for antigen drive on both subsets comes from detecting “endocytosis in vivo,” whereby sIgM expression and signal capacity in blood cells are variably downmodulated, but can recover in vitro. Mysteriously, sIgD of the same presumed antigen specificity shows no evidence for endocytic downmodulation in vivo. CLL cells apparently engage antigens via sIgM in tissue sites, leading to proliferation and downmodulation, with reexpression gradually occurring during transit through the blood. Expression of CXCR4 closely follows that of sIgM, and clonal analysis reveals subpopulations of potentially dangerous cells with high sIgM/CXCR4 primed for tissue-based proliferative stimulation. In contrast to normal B cells, this is an iterative process exposing the proliferating CLL cells to further genetic changes. Overall higher sIgM levels and increased signal capacity in U-CLL likely account for more aggressive clinical behavior. BCR-induced membrane-proximal events include LYN-mediated phosphorylation of Iga/b followed by recruitment of the tyrosine kinase Syk. Signal propagation then involves Btk and PLCg2. LYN-dependent phosphorylation of CD19 also recruits the p85 subunit of PI3K, a known survival mechanism in CLL. Downstream events include upregulation of MYC proto-oncoprotein expression and induction of MYC-regulated target genes such as cyclin D2, with both proteins detected in proliferation centers. Pathways to increased cell survival include induction of the antiapoptotic MCL1 protein and inactivation of the proapoptotic activity of BIM(EL/L) via enhanced phosphorylation. The ability to phosphorylate BIM(EL) was highly correlated with mutational status and with requirement for treatment. While these events delineate BCR-activated pathways, they provide only the skeleton. sIgM signaling is highly dependent on the polymeric nature of the antigen, with responses to solid-phase stimulus producing a higher and more prolonged signal than the soluble form. Clearly, CLL cells have to integrate BCR signals with those from other receptors for the multitude of microenvironmental factors. This is a two-way process, since BCR signals operate “inside-out” by modulating the expression of molecules involved in migration and adhesion. The fact that the glycan composition of sIgM is also modulated to a mannosylated form, potentially able to bind to mannose-binding lectins, could contribute to the latter. Clinical effects of Syk, Btk and PI3Kd inhibitors, known to affect BCR signaling and potentially other pathways, are both explicable and exciting. Disclosures: No relevant conflicts of interest to declare.

Key Points• The study reveals that circulating CLL cells contain intraclonal subgroups that differ in the level of signalresponsive surface IgM.• Subgroups with higher surface IgM express more CXCR4, potentially predicting a dangerous ability to migrate to tissue and engage antigen.Chronic lymphocytic leukemia (CLL) is a tumor of circulating B cells, variably stimulated and anergized following exposure to antigen in lymphoid tissues. Down-modulation of surface IgM (sIgM) occurs, but expression and signal capacity can recover in vitro and apparently in vivo during recirculation. We have now dissected individual circulating clones of CLL cases according to sIgM expression level by differential binding to beadbound anti-IgM. Four clear subgroups (SG1-4) with increasing sIgM were identified in 37/37 cases. Engagement of sIgM induced phosphorylation of PLCg2 and ERK1/2 at levels ranging from very low in SG1 to high in SG4. Phosphorylation was suppressed by the BTK inhibitor ibrutinib. Expression of CXCR4 also increased from SG1 to SG4, but markers of previous activation and proliferation were dominant in SG1. Incubation of whole CLL populations in vitro led to striking increases in CXCR4 expression as well as recovery of sIgM. Clonal analysis reveals dynamic SGs following presumed antigen stimulation in tissues. SG4 represents a fully recovered, potentially dangerous population equipped to migrate to tissue and receive a proliferative stimulus. SG1 likely represents a postmitotic unresponsive "resting" population. The effect of ibrutinib on the small SG4 population may be the critical factor in therapeutic success. (Blood. 2013;122(15):2664-2672

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Key Points• Stimulation of the B-cell receptor of chronic lymphocytic leukemia cells results in activation of an unfolded protein response.• Unfolded protein response activation following surface immunoglobulin M stimulation in vitro is dependent on the activity of BTK and SYK.B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined.Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissuebased antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors. (Blood. 2014;124(20):3101-3109) IntroductionChronic lymphocytic leukemia (CLL) provides a unique opportunity to understand how antigen can influence the behavior of malignant lymphocytes. It also acts as a model for the development of novel therapies targeted toward B-cell receptor (BCR) signaling pathways. [1][2][3][4] CLL comprises 2 major subsets with differing levels of somatic hypermutation of tumor IGV genes. CLL with unmutated IGV (U-CLL) derives from naïve CD5 1 CD27 2 B cells of the normal natural antibody repertoire, whereas CLL with mutated IGV genes (M-CLL) may derive from postgerminal center CD5 1 CD27 1 cells. 5,6 Importantly, these subsets have distinct clinical behavior, and U-CLL has a more aggressive clinical course. Antigen signaling is thought to be ongoing in both subsets and, rather than the presence or absence of signaling, it is the balance between distinct types of responses that appears to determine clinical behavior. 1 Anergy, a state of cellular lethargy that is induced following antigen engagement in the absence of T-cell help, 7 is observed in all CLL but is particularly prominent in M-CLL.1 By contrast, positive antigen signaling leading to proliferation and survival appears more evident in U-CLL. The importance of antigen signaling for CLL is emphasized by recent results that have demonstrated the clinical effectivenes...

Our results may be important in the definition of a biologic predictive profile for the development of NPC within our population.

Abstract. Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDSpolyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein. IntroductionMonoclonal antibodies have become an accepted modality of cancer therapy. Recombinant antibodies and antibody-based fusion proteins hold the promise of further extending the therapeutic possibilities of this modality. Single chain variable fragments (scFv) consist of the variable regions of an antibody's heavy and light chains fused together via a flexible linker, whose length determines the quaternary structure. Thus, they carry the complete antigen binding site in a single polypeptide chain of only about 30 kDa. In tumor targeting, scFv have demonstrated excellent tumor penetration, high ratios of tumor to normal tissue concentration, and low background (1,2). This makes them attractive targeting components of bifunctional fusion proteins such as those needed for antibodydirected enzyme-prodrug therapy (ADEPT). In ADEPT, after binding of an antibody-enzyme construct to the cognate tumor antigen, the enzyme component converts a prodrug into a cytotoxic drug, thus generating drug activity specifically in tumor tissue (3,4).Several ADEPT systems have shown promising in vivo efficacy in a number of tumor models (5) and in several xenograft systems in nude mice (4,6-8), demonstrating in principle that ADEPT can target tumor tissue with high selectivity and deliver chemotherapeutic drugs with high intratumoral concentrations.Senter's group first used bacterial cytosine deaminase for ADEPT to catalyze the deamination of 5-fluorocytosine (5-FC), which is non-toxic in mammals, into 5-fluorouracil (5-FU) (9). Clinical studies on ADEPT have pro...

Chemical conjugates of monoclonal antibodies with fluorophores or enzymes have long been used for diagnostic purposes and experimental therapeutic approaches. Recombinant technology allows for the design and expression of tailored genuine fusion proteins, providing defined molecules as to size, molar ratios of the functional components and stability. The production of functional protein, however, is often limited or impossible due to refolding and solubility problems. Here, we report on the production of a soluble recombinant fusion construct, A33scFv-green fluorescent protein (A33scFv::GFP) in Pichia pastoris. A33scFv is a single-chain antibody recognizing the A33 antigen, which is expressed by approximately 95% of colorectal carcinomas and has become a focus of pre-clinical and clinical investigation. The fusion partner GFP was selected both as an experimental tool for functional studies of the A33 antigen and as a potential diagnostic for colon cancer detection and therapy planning. Pichia pastoris yeast strains were transformed with A33scFv::GFP cDNA under the methanol-inducible AOX1 promotor. The construct was properly expressed and secreted into culture supernatants as a soluble protein, which was bifunctional without additional renaturation or solubilization steps. The crude protein solution was purified by affinity chromatography. Surface plasmon resonance, flow cytometry and fluorescence microscopy on sections of normal and cancerous colon tissue revealed specific binding and the applicability of this fusion protein for diagnostic purposes. In addition, the biodistribution of A33scFv::GFP was analyzed in mice bearing A33-positive tumor xenografts, confirming specific tumor targeting.