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PDF file, 54KB, Stability of CEACAM1 expression on injected T cells and its persistence in in vivo model.

PDF file, 32KB, Validation of the immune-protective effect of CEACAM1 in CEACAM1-silenced melanoma cells.

PDF file, 39KB, Quantification of CEACAM1 molecules in normal cell strains and melanoma cell lines.

CEACAM1 (biliary glycoprotein-1, CD66a) was reported as a strong clinical predictor of poor prognosis in melanoma. We have previously identified CEACAM1 as a tumor escape mechanism from cytotoxic lymphocytes. Here, we present substantial evidence in vitro and in vivo that blocking of CEACAM1 function with a novel monoclonal antibody (MRG1) is a promising strategy for cancer immunotherapy. MRG1, a murine IgG1 monoclonal antibody, was raised against human CEACAM1. It recognizes the CEACAM1-specific N-domain with high affinity (KD ∼ 2 nmol/L). Furthermore, MRG1 is a potent inhibitor of CEACAM1 homophilic binding and does not induce any agonistic effect. We show using cytotoxicity assays that MRG1 renders multiple melanoma cell lines more vulnerable to T cells in a dose-dependent manner, only following antigen-restricted recognition. Accordingly, MRG1 significantly enhances the antitumor effect of adoptively transferred, melanoma-reactive human lymphocytes using human melanoma xenograft models in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice. A significant antibody-dependent cell cytotoxicity response was excluded. It is shown that MRG1 reaches the tumor and is cleared within a week. Importantly, approximately 90% of melanoma specimens are CEACAM1+, implying that the majority of patients with melanoma could be amenable to MRG1-based therapy. Normal human tissue microarray displays limited binding to luminal epithelial cells on some secretory ducts, which was weaker than the broad normal cell binding of other anticancer antibodies in clinical use. Importantly, MRG1 does not directly affect CEACAM1+ cells. CEACAM1 blockade is different from other immunomodulatory approaches, as MRG1 targets inhibitory interactions between tumor cells and late effector lymphocytes, which is thus a more specific and compartmentalized immune stimulation with potentially superior safety profile. Mol Cancer Ther; 11(6); 1300–10. ©2012 AACR.

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PDF file, 57KB, The effect of MRG1 was not mediated by ADCC.

PDF file, 277KB, MRG1 specifically recognizes the N-domain of the CEACAM1 protein and has a high binding affinity.

PDF file, 42KB, MRG1 synergizes with adoptively transferred human TIL in xenograft model.

Adoptive cell transfer therapy with reactive T cells is one of the most promising immunotherapeutic modalities for metastatic melanoma patients. Homing of the transferred T cells to all tumor sites in sufficient numbers is of great importance. Here, we seek to exploit endogenous chemotactic signals in order to manipulate and enhance the directional trafficking of transferred T cells toward melanoma. Chemokine profiling of 15 melanoma cultures shows that CXCL1 and CXCL8 are abundantly expressed and secreted from melanoma cultures. However, the complimentary analysis on 40 melanoma patient-derived tumor-infiltrating lymphocytes (TIL) proves that the corresponding chemokine receptors are either not expressed (CXCR2) or expressed at low levels (CXCR1). Using the in vitro transwell system, we demonstrate that TIL cells preferentially migrate toward melanoma and that endogenously expressing CXCR1 TIL cells are significantly enriched among the migrating lymphocytes. The role of the chemokines CXCL1 and CXCL8 is demonstrated by partial abrogation of this enrichment with anti-CXCL1 and anti-CXCL8 neutralizing antibodies. The role of the chemokine receptor CXCR1 is validated by the enhanced migration of CXCR1-engineered TIL cells toward melanoma or recombinant CXCL8. Cytotoxicity and IFNγ secretion activity are unaltered by CXCR1 expression profile. Taken together, these results mark CXCR1 as a candidate for genetic manipulations to enhance trafficking of adoptively transferred T cells. This approach is complimentary and potentially synergistic with other genetic strategies designed to enhance anti-tumor potency.

CEACAM1 adhesion molecule is broadly expressed, participates in pivotal cellular and immunological processes and is involved in cancer. Originally identified as a tumor suppressor, it is now known that in several cancers, including malignant melanoma, CEACAM1 expression correlates with tumor progression and poor survival. Here we review the findings connecting CEACAM1 to malignant melanoma, encompassing in-vitro, in-vivo and patients-derived data. A CEACAM1-mediated mechanism used by melanoma cells to evade immune attack is described in detail. Finally, the potential value of CEACAM1 as a melanoma biomarker and therapeutic target is being discussed.

It was previously shown that CEACAM1 on melanoma cells strongly predicts poor outcome. Here, we show a statistically significant increase of serum CEACAM1 in 64 active melanoma patients, as compared to 48 patients with no evidence of disease and 37 healthy donors. Among active patients, higher serum CEACAM1 correlated with LDH values and with decreased survival. Multivariate analysis with neutralization of LDH showed that increased serum CEACAM1 carries a hazard ratio of 2.40. In vitro, soluble CEACAM1 was derived from CEACAM1(+), but neither from CEACAM1(-) melanoma cells nor from CEACAM1(+) lymphocytes, and directly correlated with the number of CEACAM1(+) melanoma cells. Production of soluble CEACAM1 depended on intact de novo protein synthesis and secretion machineries, but not on metalloproteinase function. An unusually high percentage of CEACAM1(+) circulating NK and T lymphocytes was demonstrated in melanoma patients. CEACAM1 inhibited killing activity in functional assays. CEACAM1 expression could not be induced on lymphocytes by serum from patients with high CEACAM1 expression. Further, expression of other NK receptors was impaired, which collectively indicate on a general abnormality. In conclusion, the systemic dysregulation of CEACAM1 in melanoma patients further denotes the role of CEACAM1 in melanoma and may provide a basis for new tumor monitoring and prognostic platforms.

Malignant melanoma is a devastating disease whose incidences are continuously rising. The recently approved antimelanoma therapies carry new hope for metastatic patients for the first time in decades. However, the clinical management of melanoma is severely hampered by the absence of effective screening tools. The expression of the CEACAM1 adhesion molecule on melanoma cells is a strong predictor of poor prognosis. Interestingly, a melanoma-secreted form of CEACAM1 (sCEACAM1) has recently emerged as a potential tumor biomarker. Here we add novel evidences supporting the prognostic role of serum CEACAM1 by using a mice xenograft model of human melanoma and showing a correlation between serum CEACAM1 and tumor burden. Moreover, we demonstrate that serum CEACAM1 is elevated over time in progressive melanoma patients who fail to respond to immunotherapy as opposed to responders and stable disease patients, thus proving a correlation between sCEACAM1, response to treatment, and clinical deterioration.

Lissencephaly 1 (LIS1) was the first gene implicated in the pathogenesis of type-1 lissencephaly. More than a decade of research by multiple laboratories has revealed that LIS1 is a key node protein, which participates in several pathways, including association with the molecular motor cytoplasmic dynein, the reelin signaling pathway, and the platelet-activating factor pathway. Mutations in LIS1-interacting proteins, either in human, or in mouse models has suggested that LIS1 might play a role in the pathogenesis of numerous diseases such as male sterility, schizophrenia, neuronal degeneration, and viral infections.

CEACAM1 (biliary glycoprotein-1, CD66a) was reported as a strong clinical predictor of poor prognosis in melanoma. We have previously identified CEACAM1 as a tumor escape mechanism from cytotoxic lymphocytes. Here, we present substantial evidence in vitro and in vivo that blocking of CEACAM1 function with a novel monoclonal antibody (MRG1) is a promising strategy for cancer immunotherapy. MRG1, a murine IgG1 monoclonal antibody, was raised against human CEACAM1. It recognizes the CEACAM1-specific N-domain with high affinity (KD ∼ 2 nmol/L). Furthermore, MRG1 is a potent inhibitor of CEACAM1 homophilic binding and does not induce any agonistic effect. We show using cytotoxicity assays that MRG1 renders multiple melanoma cell lines more vulnerable to T cells in a dose-dependent manner, only following antigen-restricted recognition. Accordingly, MRG1 significantly enhances the antitumor effect of adoptively transferred, melanoma-reactive human lymphocytes using human melanoma xenograft models in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice. A significant antibody-dependent cell cytotoxicity response was excluded. It is shown that MRG1 reaches the tumor and is cleared within a week. Importantly, approximately 90% of melanoma specimens are CEACAM1+, implying that the majority of patients with melanoma could be amenable to MRG1-based therapy. Normal human tissue microarray displays limited binding to luminal epithelial cells on some secretory ducts, which was weaker than the broad normal cell binding of other anticancer antibodies in clinical use. Importantly, MRG1 does not directly affect CEACAM1+ cells. CEACAM1 blockade is different from other immunomodulatory approaches, as MRG1 targets inhibitory interactions between tumor cells and late effector lymphocytes, which is thus a more specific and compartmentalized immune stimulation with potentially superior safety profile. Mol Cancer Ther; 11(6); 1300–10. ©2012 AACR.

The immune system fights cancer and sometimes temporarily eliminates it or reaches an equilibrium stage of tumor growth. However, continuous immunological pressure also selects poorly immunogenic tumor variants that eventually escape the immune control system. Here, we focus on metastatic melanoma, a highly immunogenic tumor, and on anti-melanoma immunotherapies, which recently, especially following the FDA approval of Ipilimumab, gained interest from drug development companies. We describe new immunomodulatory approaches currently in the development pipeline, focus on the novel CEACAM1 immune checkpoint, and compare its potential to the extensively described targets, CTLA4 and PD1. This paper combines multi-disciplinary approaches and describes anti-melanoma immunotherapies from molecular, medical, and business angles.

Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1–Ndel1–Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine-tuning the activity of the retrograde motor cytoplasmic dynein.

Radial neuronal migration is key in structuring the layered cortex. Here we studied the role of MARK2/Par-1 in this process. The dual name stands for the MAP/microtubule affinity-regulating kinase 2 (MARK2) and the known polarity kinase 1 (Par-1). Reduced MARK2 levels usingin uteroelectroporation resulted in multipolar neurons stalled at the intermediate zone border. Reintroduction of the wild-type kinase postmitotically improved neuronal migration. Our results indicated that reduction in MARK2 affected centrosomal dynamics in migrating neurons of the cerebral cortex. Increased MARK2 has been shown to destabilize microtubules, and here we show for the first time that reduced MARK2 stabilized microtubules in primary cultured neurons. Kinase-independent activity permitted multipolar-to-bipolar transition but did not restore proper migration. Increased MARK2 levels resulted in a different phenotype, which is loss of neuronal polarity. MARK2 kinase activity reduction hindered migration in the developing brain, which was rescued by increasing kinase activity. Our results stress the necessity of maintaining dynamic microtubules for proper neuronal migration. Furthermore, the exact requirements for MARK2 and its kinase activity vary during the course of neuronal migration. Collectively, our results stress the requirements for the different roles of MARK2 during neuronal migration.

Mutations in doublecortin (DCX) cause X-linked lissencephaly ("smooth brain") and double cortex syndrome in humans. DCX is highly phosphorylated in migrating neurons. Here, we demonstrate that dephosphorylation of specific sites phosphorylated by JNK is mediated by Neurabin II, which recruits the phosphatase PP1. During cortical development, the expression pattern of PP1 is widespread, while the expression of DCX and Neurabin II is dynamic, and they are coexpressed in migrating neurons. In vitro, DCX is site-specific dephosphorylated by PP1 without the presence of Neurabin II, this dephosphorylation requires an intact RVXF motif in DCX. Overexpression of the coiled-coil domain of Neurabin II, which is sufficient for interacting with DCX and recruiting the endogenous Neurabin II with PP1, induced dephosphorylation of DCX on one of the JNK-phosphorylated sites. We hypothesize that the transient recruitment of DCX to different scaffold proteins, JIP-1/2, which will regulate its phosphorylation by JNK, and Neurabin II, which will regulate its dephosphorylation by PP1, plays an important role in normal neuronal migration.

The mammalian cortex is generally subdivided into six organized layers, which are formed during development in an organized fashion. This organized cortical layering is disrupted in case of mutations in the doublecortin (DCX) gene. DCX is a Microtubule Associated Protein (MAP). However, besides stabilization of microtubules, it may be involved in additional functions. The participation of this molecule in signal transduction is beginning to emerge via discovery of interacting molecules and its regulation by phosphorylation using several different kinases. We raise the hypothesis, that the combinatorial phosphorylation of DCX by different kinases and at different sites may be a molecular regulatory switch in the transition of a migrating neuron through multiple phases of migration. Our recent research has suggested the involvement of DCX in the JNK (Jun-N-terminal Kinase) pathway. The JNK pathway is linked to the reelin pathway, known to regulate cortical layering. Positioning of DCX in this signaling pathway opens up additional possibilities of understanding how migrating neurons are controlled.

Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).