Evis Havari

Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro

Alemtuzumab is a humanized monoclonal antibody specific for the CD52 protein present at high levels on the surface of B and T lymphocytes. In clinical trials, alemtuzumab has shown a clinical benefit superior to that of interferon‐β in relapsing–remitting multiple sclerosis patients. Treatment with alemtuzumab leads to the depletion of circulating lymphocytes followed by a repopulation process characterized by alterations in the number, proportions and properties of lymphocyte subsets. Of particular interest, an increase in the percentage of T cells with a regulatory phenotype (Treg cells) has been observed in multiple sclerosis patients after alemtuzumab. Since Treg cells play an important role in the control of autoimmune responses, the effect of alemtuzumab on Treg cells was further studied in vitro. Alemtuzumab effectively mediated complement‐dependent cytolysis of human T lymphocytes and the remaining population was enriched in T cells with a regulatory phenotype. The alemtuzumab‐exposed T cells displayed functional regulatory characteristics including anergy to stimulation with allogeneic dendritic cells and ability to suppress the allogeneic response of autologous T cells. Consistent with the observed increase in Treg cell frequency, the CD25hi T‐cell population was necessary for the suppressive activity of alemtuzumab‐exposed T cells. The mechanism of this suppression was found to be dependent on both cell–cell contact and interleukin‐2 consumption. These findings suggest that an alemtuzumab‐mediated increase in the proportion of Treg cells may play a role in promoting the long‐term efficacy of alemtuzumab in patients with multiple sclerosis.

Immune status following alemtuzumab treatment in human CD52 transgenic mice

Alemtuzumab is a monoclonal antibody against the CD52 antigen present at high levels on the surface of lymphocytes. While treatment of multiple sclerosis patients with alemtuzumab results in marked depletion of lymphocytes from the circulation, it has not been associated with a high incidence of serious infections. In a human CD52 transgenic mouse, alemtuzumab treatment showed minimal impact on the number and function of innate immune cells. A transient decrease in primary adaptive immune responses was observed post-alemtuzumab but there was little effect on memory responses. These results potentially help explain the level of immunocompetence observed in alemtuzumab-treated MS patients.

Reduction of inflammation and preservation of neurological function by anti-CD52 therapy in murine experimental autoimmune encephalomyelitis

Alemtuzumab, a monoclonal antibody directed against human CD52, is used in the treatment of MS. To characterize the impact of anti-CD52 administration, a monoclonal antibody to mouse CD52 (anti-muCD52) was generated and evaluated in EAE mouse models of MS. A single course of anti-muCD52 provided a therapeutic benefit accompanied by a reduction in the frequency of autoreactive T lymphocytes and production of pro-inflammatory cytokines. Examination of the CNS revealed a decrease in infiltrating lymphocytes, demyelination and axonal loss. Electrophysiological assessment showed preservation of axonal conductance in the spinal cord. These findings suggest that anti-CD52 therapy may help preserve CNS integrity.

Expression of TMPRSS4 in non-small cell lung cancer and its modulation by hypoxia

Overexpression of TMPRSS4, a cell surface-associated transmembrane serine protease, has been reported in pancreatic, colorectal and thyroid cancers, and has been implicated in tumor cell migration and metastasis. Few reports have investigated both TMPRSS4 gene expression levels and the protein products. In this study, quantitative RT-PCR and protein staining were used to assess TMPRSS4 expression in primary non-small cell lung carcinoma (NSCLC) tissues and in lung tumor cell lines. At the transcriptional level, TMPRSS4 message was significantly elevated in the majority of human squamous cell and adenocarcinomas compared with normal lung tissues. Staining of over 100 NSCLC primary tumor and normal specimens with rabbit polyclonal anti-TMPRSS4 antibodies confirmed expression at the protein level in both squamous cell and adenocarcinomas with little or no staining in normal lung tissues. Human lung tumor cell lines expressed varying levels of TMPRSS4 mRNA in vitro. Interestingly, tumor cell lines with high levels of TMPRSS4 mRNA failed to show detectable TMPRSS4 protein by either immunoblotting or flow cytometry. However, protein levels were increased under hypoxic culture conditions suggesting that hypoxia within the tumor microenvironment may upregulate TMPRSS4 protein expression in vivo. This was supported by the observation of TMPRSS4 protein in xenograft tumors derived from the cell lines. In addition, staining of human squamous cell carcinoma samples for carbonic anhydrase IX (CAIX), a hypoxia marker, showed TMPRSS4 positive cells adjacent to CAIX positive cells. Overall, these results indicate that the cancer-associated TMPRSS4 protein is overexpressed in NSCLC and may represent a potential therapeutic target.

Alemtuzumab induction of intracellular signaling and apoptosis in malignant B lymphocytes

Abstract The molecular changes induced by alemtuzumab following binding of CD52 on B tumor cells were investigated. Alemtuzumab alone had no detectable impact on cell signaling but cross-linking of alemtuzumab on the surface of B tumor lines with anti-human Fc antibodies induced a transient Ca2 + flux followed by phosphorylation of several kinases involved in stress and survival pathways, and expression of associated proteins including TNF-α. Cross-linking of alemtuzumab also induced capping and caspase-dependent apoptosis of the tumor lines. When using primary cells from B-CLL patients, alemtuzumab alone was capable of inducing protein phosphorylation and apoptosis through the cross-linking of alemtuzumab by FcγRIIb receptors on B-CLL cells. Apoptosis was prevented by blocking of FcγRIIb receptors with anti-CD32 antibody. Overall, our results indicate that cross-linking of alemtuzumab on B tumor cells can occur naturally through Fc receptor interaction and leads to the activation of specific cellular pathways and induction of apoptosis.

Anti-murine CD52 antibody treatment does not adversely affect the migratory ability of immune cells

OBJECTIVE: To evaluate the migratory properties of various immune cell populations following anti-muCD52 treatment in murine models of inflammation. BACKGROUND: Alemtuzumab is an anti-CD52 humanized monoclonal antibody that causes depletion of circulating lymphocytes followed by a distinctive pattern of repopulation. Alemtuzumab showed superior efficacy vs. subcutaneous interferon beta-1a in phase 3 trials in active relapsing-remitting multiple sclerosis patients who were treatment-naive (CARE-MS I) or had relapsed on prior therapy (CARE-MS II). An anti-muCD52 antibody was utilized to expand our understanding of anti-CD52 therapy in mouse models of immune cell migration. DESIGN/METHODS: Immune cell migration was evaluated using both in vitro and in vivo analysis. Lymphocyte migration in vitro was evaluated in transwell assays using CD4 T cells purified from anti-muCD52-treated mice. In vivo migration was assessed using an acute CNS inflammation model in which mice were injected intracerebroventricularly with lipopolysaccharide (LPS) following anti-muCD52 treatment. Similarly, migration of innate immune cell subsets was evaluated in peritonitis model of inflammation, with animals being injected intraperitoneally with thioglycollate 3 days following anti-muCD52 treatment. The numbers of CNS-infiltrating lymphocytes or peritoneum-infiltrating cells were measured by polychromatic flow cytometry. RESULTS: Immune cells from anti-muCD52-treated mice retained their ability to migrate. In vitro, CD4 T cells were able to migrate in response to SDF-1alpha as efficiently as cells from vehicle-treated animals. In vivo, lymphocytes in anti-muCD52-treated animals were able to migrate into the CNS following local induction of inflammation by LPS. Similarly, innate immune cell migration was not affected by anti-muCD52 treatment, as similar numbers and types of cells were observed in inflamed peritoneum of anti-muCD52- and vehicle-treated animals. CONCLUSIONS: These results indicate that following anti-muCD52 treatment, immune cells retain their ability to migrate. These findings suggest that anti-muCD52 treatment may not compromise immune surveillance, but further studies are required. Study Supported by: Genzyme, a Sanofi company Disclosure: Dr. Havari has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Turner has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Dodge has received personal compensation for activities with Genzyme Corp. as an employee. Mr. Treleaven has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Shihabuddin has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Roberts has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Kaplan has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Siders has received personal compensation for activities with Genzyme, Corp. as an employee.

Abstract C166: Cancer cells expressing TMPRSS4 colocalized with carbonic anhydrase IX (CAIX)‐positive cells in lung and pancreatic carcinomas

TMPRSS4, a cell surface transmembrane serine protease, is over expressed at the transcriptional level in pancreatic, colorectal and thyroid cancers compared with normal tissues. Recent studies have indicated a role for TMPRSS4 in tumor cell migration and tumor metastasis. We examined TMPRSS4 expression in dissected non‐small cell lung cancer (NSCLC) tissues by quantitative RT‐PCR and immunohistochemisty. At the transcriptional level, TMPRSS4 expression was elevated in adeno and squamous cell (SC) carcinomas when compared to the matching normal tissues (p=0.0035). At the protein level, over 100 tumor and normal specimens were examined with rabbit polyclonal anti‐TMPRSS4 antibodies via immunohistochemistry. Adeno and SC carcinomas were positive for TMPRSS4 (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C166.