Homophilic Interaction of CD147 Promotes IL-6-Mediated Cholangiocarcinoma Invasion via the NF-κB-Dependent Pathway

Cholangiocarcinoma (CCA), an aggressive cancer of bile ducts, is a well-known chronic inflammation-related disease. The major impediment in CCA treatment is limited treatment options for advanced disease; hence, an alternative is urgently required. The role of CD147 on cytokine production has been observed in inflammation-related diseases, but not in CCA. Therefore, this study was focused on CD147-promoting proinflammatory cytokine production and functions. Proinflammatory cytokine profiles were compared between CD147 expressing CCA cells and CD147 knockout cells (CD147 KO). Three cytokines, namely interleukin (IL)-6, IL-8, and granulocyte–monocyte colony-stimulating factor (GM-CSF), were dramatically diminished in CD147 KO clones. The involvement of the CD147-related cytokines in CCA invasion was established. CD147-promoted IL-6, IL-8, and GM-CSF secretions were regulated by NF-κB nuclear translocation, Akt activation, and p38 phosphorylation. CD147-fostering IL-6 production was dependent on soluble CD147, CD147 homophilic interaction, and NF-κB function. The overexpression of specific genes in CCA tissues compared to normal counterparts emphasized the clinical importance of these molecules. Altogether, CD147-potentiated proinflammatory cytokine production leading to CCA cell invasion is shown for the first time in the current study. This suggests that modulation of CD147-related inflammation might be a promising choice for advanced CCA treatment.

203 Preclinical characterization and development of MG1124, a Novel Immune Checkpoint inhibitor targeting CEACAM1 for NSCLC patients

BackgroundCEACAM1 is the only member of CEACAM family which is expressed on lymphocytes such as T cells and NK cells that mediate suppression of inflammatory T cell response. It is known that CEACAM1-CEACAM1 homophilic interaction induces downregulation of ZAP70 phosphorylation in response to T cell receptor (TCR) stimulation. There is a wealth of research demonstrating the correlation between CEACAM1 expression and cancer progression, in a wide range of indications. We developed a fully human monoclonal antibody (mAb) MG1124 that specifically binds to CEACAM1 but not to other CEA family members, thereby exerting anti-tumor effect via triggering immune response.MethodsT cell activation of MG1124 was determined by an NFAT-luciferase reporter assay with CEACAM1 overexpressing Jurkat stable cells. In vitro efficacy of MG1124 was examined using an NK cell- or cytotoxic T cell-mediated tumor cell killing assay. The anti-tumor efficacy of MG1124 alone or in combination was studied in a humanized mouse model. As MG1124 binds to monkey CEACAM1 with high affinity, pharmacokinetics assessment of MG1124 was performed in cynomolgus monkeys.ResultsAn anti-CEACAM1 antibody MG1124 bound to CEACAM1 but not to other CEA family members. MG1124 blocked CEACAM1 homophilic interaction by binding to the N domain of CEACAM1. Especially the homophilic interaction induced downregulation of ZAP70 phosphorylation in response to TCR stimulation in a CEACAM1 overexpressing Jurkat stable cell line, which was rescued by MG1124 resulting in augmentation of NFAT activity and IL-2 expression. NK cell or cytotoxic T cell-mediated tumor lysis was increased by MG1124 in a CEACAM1 expression-dependent manner. MG1124 inhibited tumor growth in CEACAM1 expressing NSCLC CDX humanized mouse models. In an NSCLC PDX humanized mouse model, MG1124 dose-dependently inhibited tumor growth as monotherapy. Moreover, MG1124 showed synergistic anti-cancer activity with pembrolizumab in NSCLC huPDX models. Pharmacokinetic (PK) analysis in cynomolgus monkeys showed that the half-life (T1/2) of MG1124 was estimated to range from 14 to 17 days, and the peak plasma concentration (Cmax) and overall exposure (AUC) were found to be generally dose proportional. Following this PK study, a toxicity study in cynomolgus monkeys is ongoing.ConclusionsMG1124, a novel anti-CEACAM1 mAb, blocked CEACAM1-mediated negative regulation and restored NK or cytotoxic T cell activities. MG1124 showed effective anti-tumor activity in in vivo mouse models and its combination with PD-1 blockade further enhanced treatment efficacy. The data presented herein support further advancement of MG1124 towards clinical development. MG1124 is a potential therapeutic candidate for immune checkpoint blockade in cancer therapy.ReferencesScott D Gray-Owen, Richard S Blumberg. CEACAM1: contact-dependent control of immunity. Nat Rev Immunol 2006; 6(6): 433–46.Anka Thies. et al., CEACAM1 expression in cutaneous malignant melanoma predicts the development of metastatic disease. J Clin Oncol 2002; 20(10): 2530–6.Sebastian Dango, et al., Elevated expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) is associated with increased angiogenic potential in non-small-cell lung cancer. Lung Cancer 2008; 60(3): 426–33.Matthew Dankner, et al., CEACAM1 as a multi-purpose target for cancer immunotherapy. OncoImmunology 2017;6(7):e1328336.

Preclinical characterization and efficacy of 4R9, a novel immune checkpoint blockade targeting CEACAM1 for cancer therapy.

e14155 Background: CEACAM1 is one of the several immune checkpoint receptors expressed on T cells that mediate suppression of inflammatory T cell response. It is known that CEACAM1-CEACAM1 homophilic interaction induces downregulation of ZAP70 phosphorylation in response to T cell receptor (TCR) stimulation. Also, CEACAM1 is highly expressed on non-small cell lung cancer (NSCLC) and its expression correlated with cancer progression and poor prognosis. We developed a fully human monoclonal antibody 4R9, human CEACAM1-targeting antibody. Methods: T cell activation of 4R9 was determined by NFAT-luciferase reporter assay with CEACAM1 overexpressing Jurkat stable cells. In vitro efficacy of 4R9 was examined using NK-mediated tumor cell killing assay. The anti-tumor efficacy of 4R9 alone or in combination was studied in vivo in a humanized mouse model engrafted with NSCLC patient-derived tumor xenografts. Results: Anti-CEACAM1 antibody 4R9 binds to CEACAM1 overexpressed in HEK293 or Jurkat cells but not to other CEA family members. 4R9 blocks CEACAM1-CEACAM1 homophilic interaction by binding to N domain of CEACAM1. CEACAM1-CEACAM1 homophilic interaction induced downregulation of ZAP70 phosphorylation in response to TCR stimulation in CEACAM1 overexpressing Jurkat stable cell line, which was rescued by 4R9 resulting in augmentation of NFAT activity and IL-2 expression. NK cell-mediated tumor lysis was increased by 4R9 in a CEACAM1 expression-dependent manner. Out of 49 NSCLC tumor tissues, 20 cases exhibited dominant expression of CEACAM1 over PD-L1 with 6 cases showing > 50% of CEACAM1 positivity. In a single mouse trial using NSCLC PDX-huNSG mouse model, 4R9 (20 mpk, 2qW) suppressed tumor progression more than 30% as monotherapy (10/19) as well as in combination (16/22) with pembrolizumab (5 mpk, 2qW). Moreover, PDX of adenocarcinoma origin with more than 50% of CEACAM1 expression were more efficiently prohibited for progression with 4R9, suggesting potential therapeutic use of 4R9 in patients with NSCLC. Conclusions: Anti-CEACAM1 antibody blocked CEACAM1-mediated negative regulation and restored T/NK cell activities. Different expression patterns of CEACAM1 compared with PD-L1 and efficacies in a single mouse trial support the rationale for developing anti-CEAEAM1 antibody as cancer therapeutics.

Protein production, crystallization and preliminary X-ray analysis of two isoforms of the Dscam1 Ig7 domain

DrosophilaDown syndrome cell adhesion molecule 1 (Dscam1) plays a critical role in neural development. It can potentially form 38 016 isoforms through alternative RNA splicing, and exhibits isoform-specific homophilic interaction through three variable Ig domains (Ig2, Ig3 and Ig7). The diversity and homophilic interaction are essential for its functions. Ig7 has 33 isoforms and is the most variable among the three variable Ig domains. However, only one isoform of Ig7 (isoform 30) has been structurally determined to date. Here, two isoforms of Dscam1 Ig7 (isoforms 5 and 9; Ig75and Ig79) were produced and crystallized. Diffraction data from Ig75and Ig79crystals were processed to resolutions of 1.95 and 2.37 Å, respectively. Comparison of different Dscam1 Ig7 isoforms will provide insight into the mechanism of its binding specificity.