Characterization of zolbetuximab in pancreatic cancer models.

In healthy tissue, the tight junction protein Claudin 18.2 (CLDN18.2) is present only in the gastric mucosa. Upon malignant transformation of gastric epithelial tissue, perturbations in cell polarity lead to cell surface exposure of CLDN18.2 epitopes. Moreover, CLDN18.2 is aberrantly expressed in malignancies of several other organs, such as pancreatic cancer (PC). A monoclonal antibody, zolbetuximab (formerly known as IMAB362), has been generated against CLDN18.2. In a phase 2 clinical trial (FAST: NCT01630083), zolbetuximab in conjunction with chemotherapy prolonged overall and progression-free survival over chemotherapy alone and improved quality of life. In this study, the mechanism of action and antitumor activity of zolbetuximab were investigated using nonclinical PC models. Zolbetuximab bound specifically and with strong affinity to human PC cells that expressed CLDN18.2 on the cell surface. In ex vivo systems using immune effector cells and serum from healthy donors, zolbetuximab induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), resulting in the lysis of cultured human PC cells. The amplitude of ADCC and CDC directly correlated with cell surface CLDN18.2 levels. The chemotherapeutic agent gemcitabine upregulated CLDN18.2 expression in cultured human PC cells and enhanced zolbetuximab-induced ADCC. In mouse xenograft tumors derived from human PC cell lines, including gemcitabine-refractory ones, zolbetuximab slowed tumor growth, benefited survival, and attenuated metastases development. The results presented here validate CLDN18.2 as a targetable biomarker in PC and support extension of the clinical development of zolbetuximab to patients with CLDN18.2-expressing PC.

A monoclonal rat anti-mouse EMAP II antibody that functionally neutralizes pro- and mature-EMAP II in vitro.

EMAP II is an endothelial cell and monocyte activating proinflammatory cytokine, which has been demonstrated to induce endothelial cell apoptosis. In order to analyze its role in disease models linked to inflammation and endothelial cell death, we aimed to develop a neutralizing antibody against mouse EMAP II. Therefore, we generated rat monoclonal anti-mouse EMAP II antibodies by immunization with recombinant full length, mouse pro-EMAP II protein. We could identify by ELISA, hybridoma clones from fusion with mouse myeloma SP2/0 cells which produced antibodies recognizing both full length and mature EMAP II. We further characterized one antibody, M7/1 and demonstrated its ability to detect both EMAP II forms in Western blotting and to neutralize EMAP II directed migration of human peripheral blood monocytes as well as EMAP II induced apoptosis of tumor and endothelial cells. We conclude that this antibody can be useful to both target and analyze murine disease models, in which EMAP II may be involved.

The noncanonical WNT pathway is operative in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease that comprises sustained vasoconstriction, enhanced proliferation of pulmonary vascular cells, and in situ thrombosis. The discovery of several contributing signaling pathways in recent years has resulted in an expanding array of novel therapies; however, IPAH remains a progressive disease with poor outcome in most instances. To identify new regulatory pathways of vascular remodeling in IPAH, we performed transcriptome-wide expression profiling of laser-microdissected pulmonary arterial resistance vessels derived from explanted IPAH and nontransplanted donor lung tissues. Statistical analysis of the data derived from six individuals in each group showed significant regulation of several mediators of the canonical and noncanonical WNT pathway. As to the noncanonical WNT pathway, the planar cell polarity (PCP) pathway, the ras homolog gene family member A (RHOA), and ras-related C3 botulinum toxin substrate-1 (RAC1) were strongly up-regulated. Real-time PCR of laser-microdissected pulmonary arteries confirmed these array results and showed in addition significant up-regulation of further PCP mediators wingless member 11 (WNT11), disheveled associated activator of morphogenesis-1 (DAAM1), disheveled (DSV), and RHO-kinase (ROCK). Immunohistochemical staining and semiquantitative expression analysis confirmed the markedly enhanced expression of the PCP mediators in the pulmonary resistance vessels, in particular in the endothelial layer in IPAH. Therefore we propose the PCP pathway to be critically involved in the regulation of vascular remodeling in IPAH.

A placenta-specific gene ectopically activated in many human cancers is essentially involved in malignant cell processes.

The identification and functional characterization of tumor-specific genes is a prerequisite for the development of targeted cancer therapies. Using an integrated data mining and experimental validation approach for the discovery of new targets for antibody therapy of cancer, we identified PLAC1. PLAC1 is a placenta-specific gene with no detectable expression in any other normal human tissue. However, it is frequently aberrantly activated and highly expressed in a variety of tumor types, in particular breast cancer. RNAi-mediated silencing of PLAC1 in MCF-7 and BT-549 breast cancer cells profoundly impairs motility, migration, and invasion and induces a G1-S cell cycle block with nearly complete abrogation of proliferation. Knockdown of PLAC1 is associated with decreased expression of cyclin D1 and reduced phosphorylation of AKT kinase. Moreover, PLAC1 is localized on the surface of cancer cells and is accessible for antibodies which antagonize biological functions of this molecule. These features, in summary, make PLAC1 an attractive candidate for targeted immunotherapeutic approaches.

Multivalent recombinant proteins for probing functions of leucocyte surface proteins such as the CD200 receptor.

CD200 (OX2) is a membrane glycoprotein that interacts with a structurally related receptor (CD200R) involved in the regulation of macrophage function. The interaction is of low affinity (K(D) approximately 1 microm) but can be detected using CD200 displayed in a multivalent form on beads or with dimeric fusion proteins consisting of the extracellular region of CD200 and immunoglobulin Fc regions. We prepared putative pentamers and trimers of mouse CD200 with sequences from cartilage oligomeric matrix protein (COMP) and surfactant protein D (SP-D), respectively. The COMP protein gave high-avidity binding and was a valuable tool for showing the interaction whilst the SP-D protein gave weak binding. In vivo experiments showed that an agonistic CD200R monoclonal antibody caused some amelioration in a model of experimental autoimmune encephalomyelitis but the COMP protein was cleared rapidly and had minimal effect. Pentameric constructs also allowed detection of the rat CD48/CD2 interaction, which is of much lower affinity (K(D) approximately 70 microm). These reagents may have an advantage over Fc-bearing hybrid molecules for probing cell surface proteins without side-effects due to the Fc regions. The CD200-COMP gave strong signals in protein microarrays, suggesting that such reagents may be valuable in high throughput detection of weak interactions.

Negative regulatory role of PI3-kinase in TNF-induced tumor necrosis.

Tissue factor is the prime initiator of blood coagulation. Expression of tissue factor in tumor endothelial cells leads to thrombus formation, occlusion of vessels and development of hemorrhagic infarctions in the tumor tissue, often followed by regression of the tumor. Tumor cells produce endogenous vascular endothelial growth factor (VEGF), which sensitizes endothelial cells for systemically administered tumor necrosis factor alpha (TNF alpha) and synergistically enhances the TNF-induced expression of tissue factor. We have analyzed the pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells (HUVECs) after combined stimulation with TNF and VEGF. By using specific low molecular weight inhibitors, we demonstrated that protein kinase C (PKC), p44/42 and p38 mitogen-activated protein (MAP) kinases, and stress-activated protein kinase (JNK) are essentially involved in the induction of tissue factor. In contrast, the application of wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, led to strongly enhanced expression of tissue factor in TNF- and VEGF-treated cells, implicating a negative regulatory role for PI3-kinase. In vivo, the application of wortmannin promoted the formation of TNF-induced hemorrhages and intratumoral necroses in murine meth A tumors. The co-injection of wortmannin lowered the effective dose of applied TNF. Therefore, it is conceivable that the treatment of TNF-sensitive tumors with a combination of TNF and wortmannin will ensure the selective damage of the tumor endothelium and minimize the risk of systemic toxicity of TNF. TNF-treatment in combination with specific inhibition of PI3-kinase is a novel concept in anti-cancer therapy.

Expression of the human CD4 receptor is sufficient for the transduction of murine T-cells with MLV/HIV pseudotyped vectors.

Murine leukemia virus (MLV) can be pseudotyped with a variant of the HIV envelope gene encoding the surface glycoprotein gp120-SU and a carboxyl-terminally truncated transmembrane (TM) protein, with only seven cytoplasmic amino acids. MLV/HIV pseudotyped retroviral vectors selectively target human CD4+ cells and can be used as tools to study entry of HIV into cells. Mouse T-cells are immune to HIV infection, which is primarily caused by the weak binding affinity of HIV gp120 to the murine CD4 receptor. Here we show that expression of the human CD4 receptor in murine T-cells is sufficient for syncytia formation with HIV-1 envelope expressing cells and entry of MLV/HIV pseudotyped retroviral vectors. This implies that the murine CXCR4 receptor is a functional coreceptor for MLV/HIV pseudotyped vectors and confirms previous data that the inability of HIV to replicate in murine T-cells is due to a post entry block.

Specific transduction of HIV-1 envelope expressing cells by retroviral vectors pseudotyped with hybrid CD4/CXCR4 receptors.

Infection of a target cell by HIV is initiated by the interaction of the envelope glycoprotein with the CD4 receptor molecule on the surface of the target cell. This is followed by binding of a coreceptor of the chemokine receptor family and subsequently fusion of viral and cellular membranes. Membrane fusion is independent of whether the viral envelope protein is on the viral or on the cellular membrane. Accordingly, targeting of HIV infected cells by retroviral vectors has been previously achieved both by coincorporation of CD4 and coreceptors into murine leukemia virus (MLV) and lentivirus based vector particles. It was, therefore, tested whether hybrid genes of CD4 and CXCR4 are also able to yield 'receptor' vectors. A construct containing the four extracellular loops of CD4 fused to CXCR4 (CD4-D4-X4) allowed gene transfer into HIV-1 envelope expressing cells by vectors based on either MLV or lentiviruses. The CD4-D2-X4 hybrid receptor, containing the first two extracellular CD4 domains, allowed gene transfer only by lentiviral vectors. Attempts to increase vector titres by deletion of the intracellular part of CXCR4 failed. Vector titres obtained by hybrid receptors were slightly lower than published titres obtained by separate expression of CD4 and CXCR4. Thus, CD4-D4-CXCR4 hybrids are useful for the generation of retroviral and lentiviral vectors with specificity for HIV-1 envelope expressing cells.