Robert Hofmeister

Nuclear factor κB is activated in macrophages and epithelial cells of inflamed intestinal mucosa

BACKGROUND & AIMSnTranscription factors of the nuclear factor kappaB (NF-kappaB) family play an important role in the regulation of genes involved in inflammation. In inflammatory bowel diseases, proinflammatory cytokines known to be regulated by NF-kappaB are involved. The aim of this study was to investigate the role of NF-kappaB activation during mucosal inflammation in situ.nnnMETHODSnA monoclonal antibody, alpha-p65mAb, was applied for immunofluorescence and immunohistochemical analysis that recognizes activated NF-kappaB. Electrophoretic mobility shift assay was used to directly demonstrate the presence of active DNA-binding NF-kappaB.nnnRESULTSnUsing the alpha-p65mAb antibody, activated NF-kappaB could be found in biopsy specimens from inflamed mucosa but was almost absent in uninflamed mucosa. The number of cells showing NF-kappaB activation correlated with the degree of mucosal inflammation but was not significantly different between inflamed mucosa from patients with Crohns disease, ulcerative colitis, and nonspecific colitis or diverticulitis. NF-kappaB activation was localized in macrophages and in epithelial cells as identified by double-labeling techniques. Electrophoretic mobility shift assay with isolated lamina propria mononuclear cells and epithelial cells confirmed these results.nnnCONCLUSIONSnThis study shows for the first time the activation of NF-kappaB during human mucosal inflammation in situ. In addition to macrophages, epithelial cells contained activated NF-kappaB, indicating an involvement in the inflammatory process.

Serial killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3-bispecific single-chain antibody construct.

Certain bispecific antibodies exhibit an extraordinary potency and efficacy for target cell lysis by eliciting a polyclonal T‐cell response. One example is a CD19‐/CD3‐bispecific single‐chain antibody construct (bscCD19xCD3), which at femtomolar concentrations can redirect cytotoxic T cells to eliminate human B lymphocytes, B lymphoma cell lines and patient‐derived malignant B cells. Here we have further explored the basis for this high potency. Using video‐assisted microscopy, bscCD19xCD3 was found to alter the motility and activity of T cells from a scanning to a killing mode. Individual T cells could eliminate multiple target cells within a 9 hr time period, resulting in nuclear fragmentation and membrane blebbing of target cells. Complete target cell elimination was observed within 24 hr at effector‐to‐target cell ratios as low as 1:5. Under optimal conditions, cell killing started within minutes after addition of bscCD19xCD3, suggesting that the rate of serial killing was mostly determined by T‐cell movement and target cell scanning and lysis. At all times, T cells remained highly motile, and no clusters of T and target cells were induced by the bispecific antibody. Bystanding target‐negative cells were not detectably affected. Repeated target cell lysis by bscCD19xCD3‐activated T cells increased the proportion of CD19/CD3 double‐positive T cells, which was most likely a consequence of transfer of CD19 from B to T cells during cytolytic synapse formation. To our knowledge, this is the first study showing that a bispecific antibody can sustain multiple rounds of target cell lysis by T cells.

BiTEs: bispecific antibody constructs with unique anti-tumor activity

Bispecific T-cell engager molecules (BiTEs) constitute a class of bispecific single-chain antibodies for the polyclonal activation and redirection of cytotoxic T cells against pathogenic target cells. BiTEs combine a unique set of properties that have not yet been reported for any other kind of bispecific antibody construct, namely extraordinary potency and efficacy against target cells at low T-cell numbers without the need for T-cell co-stimulation. Here we review novel insights into the mechanism of BiTE action, which help to explain the unique features of BiTEs, as well as data from various animal models demonstrating the outstanding therapeutic potential of BiTEs for the treatment of malignant diseases.

The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct

BiTE molecules comprise a new class of bispecific single-chain antibodies redirecting previously unstimulated CD8+ and CD4+ T cells for the elimination of target cells. One example is MT103 (MEDI-538; bscCD19xCD3), a CD19-specific BiTE that can induce lysis of normal and malignant B cells at low picomolar concentrations, which is accompanied by T cell activation. Here, we explored in cell culture the impact of the glucocorticoid derivative dexamethasone on various activation parameters of human T cells in response to MT103. In case cytokine-related side effects should occur with BiTE molecules and other T cell-based approaches during cancer therapy it is important to understand whether glucocorticoids do interfere with the cytotoxic potential of T cells. We found that MT103 induced in the presence of target cells secretion by peripheral T cells of interleukin (IL)-2, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6, IL-10 and IL-4 into the cell culture medium. Production of all studied cytokines was effectively reduced by dexamethasone at a concentration between 1 and 3xa0×xa010−7xa0M. In contrast, upregulation of activation markers CD69, CD25, CD2 and LFA-1 on both CD4+ and CD8+ T cells, and T cell proliferation were barely affected by the steroid hormone analogue. Most importantly, dexamethasone did not detectably inhibit the cytotoxic activity of MT103-activated T cells against a human B lymphoma line as investigated with lymphocytes from 12 human donors. Glucocorticoids thus qualify as a potential co-medication for therapeutic BiTE molecules and other cytotoxic T cell therapies for treatment of cancer.

Combination of rituximab with blinatumomab (MT103/MEDI-538), a T cell-engaging CD19-/CD3-bispecific antibody, for highly efficient lysis of human B lymphoma cells

We have compared the cytotoxic activity of rituximab with that of blinatumomab (MT103/MEDI-538), a single-chain CD19-/CD3-bispecific antibody engaging human T cells. Blinatumomab consistently led to a higher degree of lysis of human lymphoma lines than rituximab, and was active at much lower concentration. The cytotoxicity mediated by blinatumomab and rituximab both caused a potent activation of pro-caspases 3 and 7 in target cells, a key event in induction of granzyme-mediated apoptotic cell death. Combination of rituximab with blinatumomab was found to greatly enhance the activity of rituximab, in particular at low effector-to-target cell ratios and at low antibody concentration.

Exchanging human Fcγ1 with murine Fcγ2a highly potentiates anti-tumor activity of anti-EpCAM antibody adecatumumab in a syngeneic mouse lung metastasis model

An important mode of action shared by human IgG1 antibody therapies is antibody-dependent cellular cytotoxicity (ADCC). ADCC relies on the interaction of the antibody’s Fc portion with Fc-gama receptors (FcγR) on immune effector cells. The anti-tumor activity of human IgG1 antibodies is frequently assessed in mouse models. Binding of human IgG1 to murine FcγRs is however of reduced affinity. We here show that ADCC of adecatumumab (MT201), a fully human IgG1 antibody specific for epithelial cell adhesion molecule (EpCAM/CD326), is drastically lower if human peripheral blood mononuclear cells are replaced by murine splenocytes as effector cells. When the variable domains of adecatumumab were genetically fused to a murine IgG2a backbone (yielding mu-adecatumumab), ADCC with murine effector cells was much improved, but at the same time significantly reduced with human effector cells. The serum half-lives of adecatumumab and mu-adecatumumab were determined in mice and dosing schedules established that gave similar serum trough levels during a 4-week antibody treatment. The anti-tumor activities of adecatumumab and mu-adecatumumab were then compared side-by-side in a lung metastasis mouse model established with a syngeneic B16 melanoma line expressing human EpCAM at physiologically relevant levels. Treatment of mice with mu-adecatumumab led to an almost complete prevention of lung metastases, while the human version of the antibody was much less active. This shows that adecatumumab has high anti-tumor activity when tested in a form that is better compatible with the species’ immune system. Moreover, our data suggest to routinely compare in mouse models human IgG1 and murine IgG2a versions of antibodies to properly assess the contribution of ADCC to overall anti-tumor activity.

In vivo treatment with the fungal metabolite gliotoxin suppresses intestinal inflammation in dextran sulphate sodium [DSS]-induced colitis

Back~,round: NF-kB is a transcription factor, which plays a central role in immune and inflammatory responses. Gliotoxin, a toxic metabolite produced by pathogenic fungi, inhibits the activation of NF-kB in vitro (Pahl, HL et aL; J.Exp. Med. 183: 1829-40; 1996), which indicates antiinfiammatory properties. We therefore investigated whether gliotoxin was able to downregulate inflammatory cytokine production in vitro and intestinal inflammation in the mouse model of acute DSS-induced colitis. Methods: In vitro experiments: Gel mobility shift assays were performed using nuclear extracts of EL-4 cells, stimulated with PMA plus IL-1 in the presence or absence of gliotoxin. RAW-264.7 cells were stimulated with 10pg LPS and different amounts of gliotoxin (0.1-10pg/ml) for 8 hours. Supernatants were collected and secreted TNF-a and IL-6 were measured by ELISA. In vivo experiments: Colitis was induced by oral administration of 5% DSS dissolved in the drinking water given for 7 days to Balb/c mice. Treatment with intraperitoneal administration of gliotoxin (20 or 2x20 pg/mouse daily for 5 days) or saline was performed in 2 independent experiments (n=5/group, 2x20 ~g Gliotoxin only 1 Exp.) and was begun on day 3 of DSS application Mice were killed and evaluated on day 8. Extent of colonic inflammation was estimated histologically [max.grade 4] and tissue expression of IL 1 and TNF was analyzed by RT-PCR. Colonic IL-6 protein production was measured by ELISA. Results: Gliotoxin inhibited PMA plus IL-1 stimulated activation of NF-kB nuclear translocation. Furthermore, Gliotoxin downregulated IL-6 and TNF protein production in a dose dependent manner. In vivo a significant amelioration of the histological score was achieved with gliotoxin treatment compared to placebo treated animals [ Mean + SEM: 2.8 -+ 0.3 DSS/Placebo; 1.4 + 0.3 DSS/Gliotoxin 20pg*; 0.4 + 0.1 DSS/Gliotoxin 2x20pg*; 0.6-+ 0A H20/placebo; *p<0.01; n=10 except n=4 2x20 DSS/gliotoxin]. Giving 2x20 ~g gliotoxin resulted in death of one mouse. Gliotoxin treatment suppressed in vivo mRNA expression of IL-1 and TNF-a and downregulated IL-6 protein production in colonic specimens. Conclusion: The fungal metabolite gliotoxin is a powerful immunsuppressive agent, although when administered systemically at higher doses a toxic effect can not be excluded. Further studies of this agent as a potential therapeutic alternative in inflammatory bowel disease are warranted.