Activation of neuronal FLT3 promotes exaggerated sensorial and emotional pain-related behaviors facilitating the transition from acute to chronic pain.

Acute pain has been associated with persistent pain sensitization of nociceptive pathways increasing the risk of transition from acute to chronic pain. We demonstrated the critical role of the FLT3- tyrosine kinase receptor, expressed in sensory neurons, in pain chronification after peripheral nerve injury. However, it is unclear whether injury-induced pain sensitization can also promote long-term mood disorders. Here, we evaluated the emotional and sensorial components of pain after a single (SI) or double paw incision (DI) and the implication of FLT3. DI mice showed an anxiodepressive-like phenotype associated with extended mechanical pain hypersensitivity and spontaneous pain when compared to SI mice. Behavioral exaggeration was associated with peripheral and spinal changes including increased microglia activation after DI versus SI. Intrathecal microglial inhibitors not only eliminated the exaggerated pain hypersensitivity produced by DI but also prevented anxiodepressive-related behaviors. Behavioral and cellular changes produced by DI were blocked in Flt3 knock-out animals and recapitulated by repeated intrathecal FL injections in naive animals. Finally, humanized antibodies against FLT3 reduced DI-induced behavioral and microglia changes. Altogether our results show that the repetition of peripheral lesions facilitate not only exaggerated nociceptive behaviors but also induced anxiodepressive disorders supported by spinal central changes that can be blocked by targeting peripheral FLT3.

Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival.

Anti­Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets in ovarian carcinoma. Conversely, the role of the three AMH type I receptors (AMHRIs), namely activin receptor­like kinase (ALK)2, ALK3 and ALK6, in ovarian cancer remains to be clarified. To determine the respective roles of these three AMHRIs, the present study used four ovarian cancer cell lines (COV434­AMHRII, SKOV3­AMHRII, OVCAR8, KGN) and primary cells isolated from tumor ascites from patients with ovarian cancer. The results demonstrated that ALK2 and ALK3 may be the two main AMHRIs involved in AMH signaling at physiological endogenous and supraphysiological exogenous AMH concentrations, respectively. Supraphysiological AMH concentrations (25 nM recombinant AMH) were associated with apoptosis in all four cell lines and decreased clonogenic survival in COV434­AMHRII and SKOV3­AMHRII cells. These biological effects were induced via ALK3 recruitment by AMHRII, as ALK3­AMHRII dimerization was favored at increasing AMH concentrations. By contrast, ALK2 was associated with AMHRII at physiological endogenous concentrations of AMH (10 pM). Based on these results, tetravalent IgG1­like bispecific antibodies (BsAbs) against AMHRII and ALK2, and against AMHRII and ALK3 were designed and evaluated. In vivo, COV434­AMHRII tumor cell xenograft growth was significantly reduced in all BsAb­treated groups compared with that in the vehicle group (P=0.018 for BsAb 12G4­3D7; P=0.001 for all other BsAbs). However, the growth of COV434­AMHRII tumor cell xenografts was slower in mice treated with the anti­AMRII­ALK2 BsAb 12G4­2F9 compared with that in animals that received a control BsAb that targeted AMHRII and CD5 (P=0.048). These results provide new insights into type I receptor specificity in AMH signaling pathways and may lead to an innovative therapeutic approach to modulate AMH signaling using anti­AMHRII/anti­AMHRI BsAbs.

Anti-Müllerian hormone (AMH) autocrine signaling promotes survival and proliferation of ovarian cancer cells.

In ovarian carcinoma, anti-Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets. Here, AMH dose-dependent effect on signaling and proliferation was analyzed in four ovarian cancer cell lines, including sex cord stromal/granulosa cell tumors and high grade serous adenocarcinomas (COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN). As previously shown, incubation with exogenous AMH at concentrations above the physiological range (12.5-25 nM) decreased cell viability. Conversely, physiological concentrations of endogenous AMH improved cancer cell viability. Partial AMH depletion by siRNAs was sufficient to reduce cell viability in all four cell lines, by 20% (OVCAR8 cells) to 40% (COV434-AMHRII cells). In the presence of AMH concentrations within the physiological range (5 to 15 pM), the newly developed anti-AMH B10 antibody decreased by 25% (OVCAR8) to 50% (KGN) cell viability at concentrations ranging between 3 and 333 nM. At 70 nM, B10 reduced clonogenic survival by 57.5%, 57.1%, 64.7% and 37.5% in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN cells, respectively. In the four cell lines, B10 reduced AKT phosphorylation, and increased PARP and caspase 3 cleavage. These results were confirmed in ovarian cancer cells isolated from patients' ascites, demonstrating the translational potential of these results. Furthermore, B10 reduced COV434-MISRII tumor growth in vivo and significantly enhanced the median survival time compared with vehicle (69 vs 60 days; p = 0.0173). Our data provide evidence for a novel pro-survival autocrine role of AMH in the context of ovarian cancer, which was targeted therapeutically using an anti-AMH antibody to successfully repress tumor growth.

Therapeutic Activity of Anti-AXL Antibody against Triple-Negative Breast Cancer Patient-Derived Xenografts and Metastasis.

Purpose: AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC).Experimental Design: We evaluate the antitumor efficacy of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and decipher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with AXL expression are enriched in EMT, migration, and invasion signaling pathways.Results: Treatment with 20G7-D9 inhibited tumor growth and bone metastasis formation in AXL-positive TNBC cell xenografts or PDX, but not in AXL-negative PDX, highlighting AXL role in cancer growth and invasion. In vitro stimulation of AXL-positive cancer cells by its ligand GAS6 induced the expression of several EMT-associated genes (SNAIL, SLUG, and VIM) through an intracellular signaling implicating the transcription factor FRA-1, important in cell invasion and plasticity, and increased their migration/invasion capacity. 20G7-D9 induced AXL degradation and inhibited all AXL/GAS6-dependent cell signaling implicated in EMT and in cell migration/invasion.Conclusions: The anti-AXL antibody 20G7-D9 represents a promising therapeutic strategy in TNBC with mesenchymal features by inhibiting AXL-dependent EMT, tumor growth, and metastasis formation. Clin Cancer Res; 23(11); 2806-16. ©2016 AACR.

Examination of HER3 targeting in cancer using monoclonal antibodies.

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells' ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma-tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.

Possible protective effect of membrane lipid rafts against interleukin-1ß-mediated anti-proliferative effect in INS-1 cells.

We recently reported that pancreatic islets from pre-diabetic rats undergo an inflammatory process in which IL-1ß takes part and controls ß-cell function. In the present study, using the INS-1 rat pancreatic ß-cell line, we investigated the potential involvement of membrane-associated cholesterol-enriched lipid rafts in IL-1ß signaling and biological effects on insulin secretion, ß-cell proliferation and apoptosis. We show that, INS-1 cells exposure to increasing concentrations of IL-1ß leads to a progressive inhibition of insulin release, an increase in the number of apoptotic cells and a dose-dependent decrease in pancreatic ß-cell proliferation. Disruption of membrane lipid rafts markedly reduced glucose-stimulated insulin secretion but did not affect either cell apoptosis or proliferation rate, demonstrating that membrane lipid raft integrity is essential for ß-cell secretory function. In the same conditions, IL-1ß treatment of INS-1 cells led to a slight further decrease in insulin secretion for low concentrations of the cytokine, and a more marked one, similar to that observed in normal cells for higher concentrations. These effects occurred together with an increase in iNOS expression and surprisingly with an upregulation of tryptophane hydroxylase and protein Kinase C in membrane lipid rafts suggesting that compensatory mechanisms develop to counteract IL-1ß inhibitory effects. We also demonstrate that disruption of membrane lipid rafts did not prevent cytokine-induced cell death recorded after exposure to high IL-1ß concentrations. Finally, concerning cell proliferation, we bring strong evidence that membrane lipid rafts exert a protective effect against IL-1ß anti-proliferative effect, possibly mediated at least partly by modifications in ERK and PKB expression/activities. Our results 1) demonstrate that IL-1ß deleterious effects do not require a cholesterol-dependent plasma membrane compartmentalization of IL-1R1 signaling and 2) confer to membrane lipid rafts integrity a possible protective function that deserves to be considered in the context of inflammation and especially T2D pathogenesis.

Excessive food intake, obesity and inflammation process in Zucker fa/fa rat pancreatic islets.

Inappropriate food intake-related obesity and more importantly, visceral adiposity, are major risk factors for the onset of type 2 diabetes. Evidence is emerging that nutriment-induced ß-cell dysfunction could be related to indirect induction of a state of low grade inflammation. Our aim was to study whether hyperphagia associated obesity could promote an inflammatory response in pancreatic islets leading to ß-cell dysfunction. In the hyperphagic obese insulin resistant male Zucker rat, we measured the level of circulating pro-inflammatory cytokines and estimated their production as well as the expression of their receptors in pancreatic tissue and ß-cells. Our main findings concern intra-islet pro-inflammatory cytokines from fa/fa rats: IL-1ß, IL-6 and TNFα expressions were increased; IL-1R1 was also over-expressed with a cellular redistribution also observed for IL-6R. To get insight into the mechanisms involved in phenotypic alterations, abArrays were used to determine the expression profile of proteins implicated in different membrane receptors signaling, apoptosis and cell cycle pathways. Despite JNK overexpression, cell viability was unaffected probably because of decreases in cleaved caspase3 as well as in SMAC/DIABLO and APP, involved in the induction and amplification of apoptosis. Concerning ß-cell proliferation, decreases in important cell cycle regulators (Cyclin D1, p35) and increased expression of SMAD4 probably contribute to counteract and restrain hyperplasia in fa/fa rat islets. Finally and probably as a result of IL-1ß and IL-1R1 increased expressions with sub-cellular redistribution of the receptor, islets from fa/fa rats were found more sensitive to both stimulating and inhibitory concentrations of the cytokine; this confers some physiopathological relevance to a possible autocrine regulation of ß-cell function by IL-1ß. These results support the hypothesis that pancreatic islets from prediabetic fa/fa rats undergo an inflammatory process. That the latter could contribute to ß-cell hyperactivity/proliferation and possibly lead to progressive ß-cell failure in these animals, deserves further investigations.

CD4 ligation excludes the Carma1-Bcl10-MALT1 complex from GM1-positive membrane rafts in CD3/CD28 activated T cells.

The antibody 13B8.2, which is directed against the CDR3-like loop on the D1 domain of CD4, induces CD4/ZAP-70 reorganization and ceramide release in membrane rafts. Here, we investigated whether CD4/ZAP-70 compartmentalization could be mediated by an effect of 13B8.2 on the Carma1-Bcl10-MALT1 complex in membrane rafts. We report that treatment of CD3/CD28-activated Jurkat T cells with 13B8.2, but not rituximab, excluded Carma1-Bcl10-MALT1 proteins from GM1(+) membrane rafts and concomitantly decreased NF-κB activation. Fluorescence confocal imaging confirmed that Carma1-Bcl10 and Carma1-MALT1 co-patching, observed in GM1(+) membrane rafts following CD3/CD28 activation, were abrogated after a 24h-treatment with 13B8.2. The CD4/ZAP-70 compartmentalization in membrane rafts induced by 13B8.2 is thus related to Carma1-Bcl10-MALT1 raft exclusion.

The lipid-modulating effects of a CD4-specific recombinant antibody correlate with ZAP-70 segregation outside membrane rafts.

We previously reported that the anti-tumoral effects of the recombinant IgG(1) antibody 13B8.2, which is directed against the CDR3-like loop on the D1 domain of CD4, are linked to accumulation/retention of CD4 inside membrane rafts, recruitment of signaling molecules of the TCR/CD3 pathway and raft exclusion of the ZAP-70 kinase and its downstream targets Vav-1, PLCγ1 and SLP-76. We thus wanted to assess whether this compartmentalization could be related to a possible effect of 13B8.2 on the lipid composition of rafts. Here we show that 13B8.2 treatment of Jurkat T cells did not affect neutral lipids and particularly cholesterol content in GM1-positive membrane rafts, but decreased phosphatidylserine synthesis. C18:0 saturated fatty acid level in GM1-positive membrane rafts and ceramide release were concomitantly increased following treatment with 13B8.2. Antibody-induced ceramide release in membrane rafts occurred through enhanced acid sphingomyelinase activity and was blocked by the acid sphingomyelinase inhibitor imipramine, but was not affected by inhibitors of de novo ceramide synthesis, myriocin and fumonisin B1. Similarly to 13B8.2, addition of bacterial sphingomyelinase increased ceramide release and segregated ZAP-70 outside GM1-positive membrane rafts from Jurkat T cells. Besides CD4/ZAP-70 modulation in membrane rafts, the 13B8.2-induced activation of the acid sphingomyelinase/ceramide pathway is an important event for structuring raft platforms and transducing CD4-related intracellular signals, which can further fine-tune antibody-triggered tumoral effects.

Recombinant anti-CD4 antibody 13B8.2 blocks membrane-proximal events by excluding the Zap70 molecule and downstream targets SLP-76, PLC gamma 1, and Vav-1 from the CD4-segregated Brij 98 detergent-resistant raft domains.

The biological effects of rIgG(1) 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-kappaB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG(1) 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG(1) 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37 degrees C, together with recruitment of TCR, CD3zeta, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Ctheta, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cgamma1, and p95(vav). Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr(292) and Tyr(319) phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10-30 s following rIgG(1) 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-kappaB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG(1) 13B8.2 acts to induce its biological effects.

In vitro antitumoral activity of baculovirus-expressed chimeric recombinant anti-CD4 antibody 13B8.2 on T-cell lymphomas.

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule. The recombinant IgG1 antibody 13B8.2 was previously shown to inhibit HIV-1 replication and to abrogate the one-way mixed-lymphocyte reaction and block proliferation of CD3-stimulated peripheral blood CD4 lymphocytes from healthy donors. Before testing this recombinant anti-CD4 antibody in in vivo preclinical trials, in vitro mechanisms of action of rIgG1 13B8.2 were assessed using various CD4 T-cell lymphomas. The baculovirus-expressed rIgG1 13B8.2 antibody led to 14% to 40% proliferation inhibition of the lymphoblastic leukaemia-derived SUP-T1, the acute T lymphoma-derived CCRF-CEM and Jurkat, and the cutaneous T-Cell lymphoma-derived HUT-78 cell lines, but it did not affect the cell cycle nor induce cell apoptosis. rIgG1 antibody 13B8.2 bound the C1q fraction, leading to 9% to 17% complement-mediated lysis of the HUT-78, H9, Sup-T1, and the CCRF-CEM cell lines. No correlation was observed between cell sensitivity to rIgG1 13B8.2-triggered complement-dependent lysis and CD35-, CD46-, CD55-, and CD59-surface expression on T lymphoma cells. Using fluorescence-activated cell sorter analysis, the antibody was shown to bind to FcgammaRI/CD64-transfected IIA1.6, FcgammaRII/CD32-transfected CDw32L, and FcgammaRIII/CD16-transfected Jurkat CD16 cell lines. In correlation with these findings, rIgG1 13B8.2 induced 11% to 31% antibody-dependent cell-mediated cytotoxicity of the CCRF-CEM, SUP-T1, A2.01 CD4, and Jurkat cell lines. These convincing results on the activity of the recombinant chimeric anti-CD4 antibody 13B8.2 have led us to perform in vivo preclinical study in a murine xenograft model of CD4 lymphomas.

PIN-bodies: a new class of antibody-like proteins with CD4 specificity derived from the protein inhibitor of neuronal nitric oxide synthase.

By inserting the CB1 paratope-derived peptide (PDP) from the anti-CD4 13B8.2 antibody binding pocket into each of the three exposed loops of the protein inhibitor of neuronal nitric oxide synthase (PIN), we have combined the anti-CD4 specificity of the selected PDP with the stability, ease of expression/purification, and the known molecular architecture of the phylogenetically well-conserved PIN scaffold protein. Such "PIN-bodies" were able to bind CD4 with a better affinity and specificity than the soluble PDP; additionally, in competitive ELISA experiments, CD4-specific PIN-bodies were more potent inhibitors of the binding of the parental recombinant antibody 13B8.2 to CD4 than the soluble PDP. The efficiency of CD4-specific CB1-inserted PIN-bodies was confirmed in biological assays where these constructs showed higher potencies to block antigen presentation by inhibition of IL-2 secretion and to inhibit the one-way and two-way mixed lymphocyte reactions, compared with soluble anti-CD4 PDP CB1. Insertion of the PDP into the first exposed loop (position 33/34) of PIN appeared to be the most promising scaffold. Taken together, our findings demonstrate that the PIN molecule is a suitable scaffold to expose new peptide loops and generate small artificial ligand-binding products with defined specificities.

Biological activities on T lymphocytes of a baculovirus-expressed chimeric recombinant IgG1 antibody with specificity for the CDR3-like loop on the D1 domain of the CD4 molecule.

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody (mAb) 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule and which inhibits HIV-1 replication. Chimeric rIgG1 antibody 13B8.2 blocked, in a dose-dependent manner, antigen presentation through inhibition of subsequent IL-2 secretion by stimulated T cells. The one-way mixed lymphocyte reaction was abrogated by previous addition of baculovirus-produced rIgG1 13B8.2 in the T-cell culture. Anti-proliferative activity of rIgG1 was demonstrated on CD3-activated CD4+ T lymphocytes from healthy donors, such effect being associated with reduced IL-2 secretion of activated T cells. On the other hand, no proliferation inhibition was observed on CD4+ T lymphocytes activated with phorbol ester plus ionomycin, suggesting that rIgG1 13B8.2 preferentially acts on a proximal TCR-induced signaling pathway. Treatment of DBA1/J human CD4-transgenic mice with 100 microg of recombinant antibody for three consecutive days led to in vivo recovery of rIgG1 antibody 13B8.2 both coated on murine T lymphocytes and free in mouse serum, without CD4 depletion or down-modulation. These findings predict that the baculovirus-expressed chimeric rIgG1 anti-CD4 antibody 13B8.2 is a promising candidate for immunotherapy.