TNFα-targeting therapy with the use of the drugs Etanercept, Infliximab, and Adalimumab is used in the clinical treatment of various inflammatory and immune diseases. Although all of these reagents function to disrupt the interaction between TNFα and its receptors, clinical investigations showed the advantages of Adalimumab treatment compared with Etanercept and Infliximab. However, the underlying molecular mechanism of action of Adalimumab remains unclear. In our previous work, we presented structural data on how Infliximab binds with the E-F loop of TNFα and functions as a TNFα receptor-binding blocker. To further elucidate the variations between TNFα inhibitors, we solved the crystal structure of TNFα in complex with Adalimumab Fab. The structural observation and the mutagenesis analysis provided direct evidence for identifying the Adalimumab epitope on TNFα and revealed the mechanism of Adalimumab inhibition of TNFα by occupying the TNFα receptor-binding site. The larger antigen-antibody interface in TNFα Adalimumab also provided information at a molecular level for further understanding the clinical advantages of Adalimumab therapy compared with Infliximab.
<div>Abstract<p>Modest clinical outcomes of dendritic cell (DC) vaccine trials call for novel strategies. In this study, we have created a chimeric CD40 molecule that incorporates a single chain Fv (scFv) molecule specific for human ErbB2 antigen and fusing to the membrane spanning and cytosolic domains of murine CD40. After adenoviral transfer to bone marrow–derived DC, this chimeric receptor (CR) induced nuclear factor-κB (NF-κB)–dependent DC activation and effector function when cultured with immobilized ErbB2 protein or ErbB2-positive tumor cells <i>in vitro. In vivo</i> migration assays showed that ∼40% injected CR-modified DC (scFv-CD40-DC) effectively migrated to ErbB2-positive tumors, where they were activated after ErbB2 antigen stimulation, and sequentially homed into the draining lymph nodes. In murine ErbB2-positive D2F2/E2 breast tumor (BALB/c) and EL4/E2 thymoma (C57BL/6) models, i.v. injection of 1 × 10<sup>6</sup> scFv-CD40-DC significantly inhibited tumor growth and cured established tumors. Importantly, the cured mice treated by injection of scFv-CD40-DC were effective in preventing both ErbB2-positive and parental ErbB2-negative tumor rechallenge. Analysis of the underlying mechanism revealed that i.v. infusion of scFv-CD40-DC elicited tumor-specific CTL responses, and the transfer of CTLs from scFv-CD40-DC–treated mice protected naive mice against a subsequent tumor challenge. These results support the concept that genetic modification of DC with tumor-associated antigen-specific CD40 chimeric receptor might be a useful strategy for treatment of human cancers. [Cancer Res 2008;68(10):3854–62]</p></div>
Although monoclonal antibodies, including CD20 antibody rituximab, are standard therapeutics for several cancers, their efficacy remains variable and often modest. There is an urgent need to enhance the efficacy of the current generation of anticancer antibodies. Flt3 ligand, a soluble protein, has the ability to induce substantial expansion of dendritic cells (DCs). In this study, we constructed a bispecific immunoglobulin G-like bispecific fusion protein (BiFP) targeting both CD20 and Flt3 (CD20-Flex) by using CrossMab technology. We found that the BiFP exhibited stabilities that were comparable with the parental antibody rituximab and were able to bind to both targets with unaltered binding affinity. Notably, our data indicated that CD20-Flex BiFP could not only eliminate lymphoma temporarily but also potentiate tumor-specific T-cell immunity, which affords a long-lasting protection from tumor recurrence. The results showed that the expansion and infiltration of DCs into tumor tissues by CD20-Flex BiFP could be an effective way to generate protective immune responses against cancer, suggesting that the CD20-Flex BiFP could be a promising therapeutic agent against B-cell lymphomas.
<div>Abstract<p>Modest clinical outcomes of dendritic cell (DC) vaccine trials call for novel strategies. In this study, we have created a chimeric CD40 molecule that incorporates a single chain Fv (scFv) molecule specific for human ErbB2 antigen and fusing to the membrane spanning and cytosolic domains of murine CD40. After adenoviral transfer to bone marrow–derived DC, this chimeric receptor (CR) induced nuclear factor-κB (NF-κB)–dependent DC activation and effector function when cultured with immobilized ErbB2 protein or ErbB2-positive tumor cells <i>in vitro. In vivo</i> migration assays showed that ∼40% injected CR-modified DC (scFv-CD40-DC) effectively migrated to ErbB2-positive tumors, where they were activated after ErbB2 antigen stimulation, and sequentially homed into the draining lymph nodes. In murine ErbB2-positive D2F2/E2 breast tumor (BALB/c) and EL4/E2 thymoma (C57BL/6) models, i.v. injection of 1 × 10<sup>6</sup> scFv-CD40-DC significantly inhibited tumor growth and cured established tumors. Importantly, the cured mice treated by injection of scFv-CD40-DC were effective in preventing both ErbB2-positive and parental ErbB2-negative tumor rechallenge. Analysis of the underlying mechanism revealed that i.v. infusion of scFv-CD40-DC elicited tumor-specific CTL responses, and the transfer of CTLs from scFv-CD40-DC–treated mice protected naive mice against a subsequent tumor challenge. These results support the concept that genetic modification of DC with tumor-associated antigen-specific CD40 chimeric receptor might be a useful strategy for treatment of human cancers. [Cancer Res 2008;68(10):3854–62]</p></div>
Escape from immune recognition has been hypothesized to be a factor in carcinogenesis. It may be mediated for many cancers through down-regulation in the MHC class 1 antigen processing and presentation pathway. TAP-1, TAP-2, tightly linked to LMP-2 and LMP-7 are multiple components of the endogenous, antigen presentation pathway machinery. We addressed the question of alterations in this pathway in human Glioblastoma (HGB) and of its relationship to modulation in expression of IGF-1 that is highly expressed in this cancer. Deficiencies in expression of TAP-1 were demonstrated by RT-PCR and/or by immuno-flow cytometry in the HGB cell line T98G obtained from ATCC, and in 3 of 4 human cell lines established from patients with Glioblastoma Multiforme. Deficiencies in expression of TAP-2 were observed in 3 of 4, deficiencies in expression of LMP-2 in 4 of 4 and deficiencies in LMP-7 in 3 of 4 HGB cell lines examined by RT-PCR and Western blot. Following down-regulation of IGF-1 by transfection with the pAnti IGF-1 vector that expresses IGF-1 RNA in antisense orientation, or by the exogenous addition of IGF-1 receptor monoclonal antibody to cell culture media, the deficiencies in components of the MHC-1 antigen presentation pathway were up-regulated and/or rescued in all HGB cell lines tested. Moreover, this up-regulation in expression was aborted by addition of 100 ng/ml of IGF-1 to the culture media. Unlike in the case of IFN-γ, the restoration of TAP-1 and LMP-2 by down-regulation of IGF-1 in Glioblastoma cells was not correlated to the tyrosine phosphorylation of STAT 1. In summary, the simultaneous reversion in expression of the multiple constituents of MHC-1 antigen processing path and up-regulation in expression of MHC-1 occurring with down-regulation in IGF-1 may have a role in reinforcement of immunity against tumor antigen(s) in some animal cancers and in humans with Glioblastoma Multiforme.
IL-17 and IL-22 are linked to the development of intestinal inflammation and colorectal cancer (CRC). However, the maintenance of IL-17 and IL-22 production, as well as the cell type (Th17) that mediates these cytokines in CRC patients, remains unknown. To examine this, untreated CRC patients and healthy controls were recruited in this study. We first observed that CRC patients contained significantly elevated levels of IL-17- and IL-22-producing CD4+ T cells. The vast majority of IL-22-expressing CD4+ T cells also expressed IL-17. We then found that the production of both IL-17 and IL-22 required support from autologous monocytes, since the depletion of monocytes significantly downregulated IL-17 and IL-22 secretion. Naive T cells from CRC patients did not secrete IL-17 or IL-22 initially, but long-term coculture with autologous monocytes significantly upregulated IL-17 and IL-22 production in an IL-6-dependent manner. Blockade of IL-6 significantly reduced the levels of both IL-17 and IL-22. We then observed that CD163+ M2 macrophages were the main contributor of IL-6. Interestingly, incubation of monocytes with CCR4+CCR6+ Th17 cells resulted in significantly higher levels of CD163+ macrophages as well as higher IL-6 secretion, than incubation with non-Th17 CD4+ T cells. Together, our study discovered a positive feedback mechanism between Th17 and M2 macrophages in CRC patients.
Cell adhesion molecule 2 (CADM2) is an immunoglobulin (Ig)-like cell adhesion molecule, which belongs to the CADMs family. The four members of CADMs family including three Ig-like domains and a short cytoplasmic tail share high degree of identity with each other, making it difficult to specifically identify each members using western blotting or immunohistochemistry. And most of anti-CADM2 antibodies available commercially or used in published papers are rabbit polyclonal antibodies, exhibiting nonspecific recognition in studies. In this study, we developed a monoclonal antibody (mAb) specific to an epitope on the extracellular domain of CADM2 using conventional hybridoma technology. Western blot assays indicated that the established mAb, named as 2B11, was specific for CADM2 recognition without interference of other members of CADMs family. Furthermore, 2B11 was competent to detect CADM2 expression specifically on the surface membranes of several types of tumor tissues, avoiding the false results with the polyclonal antibodies due to nonspecific staining. In conclusion, the mAb 2B11 could be suitable for specific detection of CADM2 expression in tumor cells or tissues.
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