A Novel B7-H6-Targeted IgG-Like T Cell-Engaging Antibody for the Treatment of Gastrointestinal Tumors.

PURPOSE: Advanced-stage gastrointestinal cancers represent a high unmet need requiring new effective therapies. We investigated the antitumor activity of a novel T cell-engaging antibody (B7-H6/CD3 ITE) targeting B7-H6, a tumor-associated antigen that is expressed in gastrointestinal tumors. EXPERIMENTAL DESIGN: Membrane proteomics and IHC analysis identified B7-H6 as a tumor-associated antigen in gastrointestinal tumor tissues with no to very little expression in normal tissues. The antitumor activity and mode of action of B7-H6/CD3 ITE was evaluated in in vitro coculture assays, in humanized mouse tumor models, and in colorectal cancer precision cut tumor slice cultures. RESULTS: B7-H6 expression was detected in 98% of colorectal cancer, 77% of gastric cancer, and 63% of pancreatic cancer tissue samples. B7-H6/CD3 ITE-mediated redirection of T cells toward B7-H6-positive tumor cells resulted in B7-H6-dependent lysis of tumor cells, activation and proliferation of T cells, and cytokine secretion in in vitro coculture assays, and infiltration of T cells into tumor tissues associated with tumor regression in in vivo colorectal cancer models. In primary patient-derived colorectal cancer precision-cut tumor slice cultures, treatment with B7-H6/CD3 ITE elicited cytokine secretion by endogenous tumor-infiltrating immune cells. Combination with anti-PD-1 further enhanced the activity of the B7-H6/CD3 ITE. CONCLUSION: These data highlight the potential of the B7-H6/CD3 ITE to induce T cell-redirected lysis of tumor cells and recruitment of T cells into noninflamed tumor tissues, leading to antitumor activity in in vitro, in vivo, and human tumor slice cultures, which supports further evaluation in a clinical study.

LY6G6D is a selectively expressed colorectal cancer antigen that can be used for targeting a therapeutic T-cell response by a T-cell engager.

Colorectal cancer (CRC) is one of the most common cancers worldwide and demands more effective treatments. We sought to identify tumor selective CRC antigens and their therapeutic potential for cytotoxic T-cell targeting by transcriptomic and immunohistochemical analysis. LY6G6D was identified as a tumor selectively expressed CRC antigen, mainly in the microsatellite stable (MSS) subtype. A specific anti LY6G6D/CD3 T cell engager (TcE) was generated and demonstrated potent tumor cell killing and T cell activation in vitro. Ex vivo treatment of primary patient-derived CRC tumor slice cultures with the LY6G6D/CD3 TcE led to IFNγ secretion in LY6G6D positive tumor samples. In vivo, LY6G6D/CD3 TcE monotherapy demonstrated tumor regressions in pre-clinical mouse models of engrafted human CRC tumor cells and PBMCs. Lastly, 2D and 3D cocultures of LY6G6D positive and negative cells were used to explore the bystander killing of LY6G6D negative cells after specific activation of T cells by LY6G6D positive cells. LY6G6D/CD3 TcE treatment was shown to lyse target negative cells in the vicinity of target positive cells through a combined effect of IFNγ, TNFα and Fas/FasL. In summary, LY6G6D was identified as a selectively expressed CRC antigen that can be utilized to potently re-direct and activate cytotoxic T-cells to lyse LY6G6D expressing CRC using a TcE. This effect can be spread to target negative neighboring tumor cells, potentially leading to improved therapeutic efficacy.

A Bispecific DLL3/CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer.

PURPOSE: Small cell lung cancer (SCLC) is the most lethal and aggressive subtype of lung carcinoma characterized by highly chemotherapy-resistant recurrence in the majority of patients. To effectively treat SCLC, we have developed a unique and novel IgG-like T-cell engaging bispecific antibody (ITE) that potently redirects T-cells to specifically lyse SCLC cells expressing Delta-like ligand 3 (DLL3), an antigen that is frequently expressed on the cell surface of SCLC cells, with no to very little detectable expression in normal tissues. EXPERIMENTAL DESIGN: The antitumor activity and mode of action of DLL3/CD3 ITE was evaluated in vitro using SCLC cell lines and primary human effector cells and in vivo in an SCLC xenograft model reconstituted with human CD3+ T-cells. RESULTS: Selective binding of DLL3/CD3 ITE to DLL3-positive tumor cells and T-cells induces formation of an immunological synapse resulting in tumor cell lysis and activation of T-cells. In a human T-cell engrafted xenograft model, the DLL3/CD3 ITE leads to an increase in infiltration of T-cells into the tumor tissue resulting in apoptosis of the tumor cells and tumor regression. Consistent with the mode of action, the DLL3/CD3 ITE treatment led to upregulation of PD-1, PD-L1, and LAG-3. CONCLUSIONS: This study highlights the ability of the DLL3/CD3 ITE to induce strictly DLL3-dependent T-cell redirected lysis of tumor cells and recruitment of T-cells into noninflamed tumor tissues leading to tumor regression in a preclinical in vivo model. These data support clinical testing of the DLL3/CD3 ITE in patients with SCLC.

Interaction of Snail and p38 mitogen-activated protein kinase results in shorter overall survival of ovarian cancer patients.

Epithelial ovarian cancer is a highly metastatic disease and the leading cause of death among cancer of the female genital tract. Abnormal epidermal growth factor receptor (EGFR) signalling has been shown to be involved in epithelial-mesenchymal transition (EMT), an early step during metastasis. Additionally, over-expression of the E-cadherin repressor Snail, a key regulator of EMT, has previously been found to be associated with unfavourable prognostic features. Thus, the aim of our study was to elucidate the role of EGFR-dependent signalling pathways for Snail expression in ovarian cancer. For this purpose, we analysed 25 formalin-fixed and paraffin-embedded (FFPE) primary tumours and their corresponding metastases for the expression of 25 signalling pathway molecules by reverse phase protein arrays. We found a significant correlation of Snail with EGFR((Tyr1086)) and p38 MAPK((Thr180/Tyr182)) in primary ovarian carcinoma and with EGFR((Tyr1086)) in their corresponding metastasis. Additionally, we showed that high expression levels of Snail in primary tumours combined with high expression levels of the phosphorylated p38 MAPK((Thr180/Tyr182)) in metastasis lead to an increased risk for death in ovarian carcinoma patients. Thus, for future combinatorial cancer therapy, drug combinations that best target the deregulated protein network in each individual patient should be selected.

Molecular profiling of signalling pathways in formalin-fixed and paraffin-embedded cancer tissues.

In most hospitals word-wide, histopathological cancer diagnosis is currently based on formalin-fixed and paraffin-embedded (FFPE) tissues. In the last few years new approaches and developments in patient-tailored cancer therapy have raised the need to select more precisely those patients, who will respond to personalised treatments. The most efficient way for optimal therapy and patient selection is probably to provide a tumour-specific protein network portrait prior to treatment. The discovery and characterisation of deregulated signalling molecules (e.g. human epidermal growth factor receptor 2, mitogen-activated protein kinases) are very promising candidates for the identification of new suitable therapy targets and for the selection of those patients who will receive the greatest benefit from individualised treatments. The reverse phase protein array (RPPA) is a promising new technology that allows quick, precise and simultaneous analysis of many components of a network. Importantly it requires only limited amounts of routine clinical material (e.g. FFPE biopsies) and can be used for absolute protein measurements. We and other research groups have described successful protein extraction from routine FFPE tissues. In this manuscript we show how these recent developments might facilitate the implementation of RPPA in clinical trials and routine settings.

Activation of epidermal growth factor receptor results in snail protein but not mRNA overexpression in endometrial cancer.

Reduced E-cadherin expression is associated with tumour progression of many carcinomas, including endometrial cancers. The transcription factor Snail is known as one of the most prominent transcriptional E-cadherin repressors; its regulation in cancer tissues, however, still remains unclear. Here, we report that activation of epidermal growth factor receptor (EGFR) resulted in overexpression of Snail and also identified critical downstream signalling molecules. Stimulation of two endometrial carcinoma cell lines with epidermal growth factor (EGF) lead to an increase of Snail protein expression. In primary human endometrioid endometrial carcinomas Snail protein expression correlated with the activated, phosphorylated form of EGFR (Tyr1086) as revealed by profiling 24 different signalling proteins using protein lysate microarrays. In addition, we observed an inverse correlation between Snail and E-cadherin protein levels in these tumours. Most likely, p38 MAPK, PAK1, AKT, ERK1/2 and GSK-3beta are involved in the up-regulation of Snail downstream of EGFR. Snail mRNA expression did not show a correlation with activated EGFR in these tumours. Taken together, profiling of signalling proteins in primary human tissues provided strong evidence that EGFR signalling is involved in Snail protein overexpression.

Precise measurement of the E-cadherin repressor Snail in formalin-fixed endometrial carcinoma using protein lysate microarrays.

Over-expression of the zinc finger transcription factor Snail leads to down-regulation of the epithelial proteins E-cadherin and Cytokeratin 18 and to upregulation of the mesenchymal protein Vimentin. The aim of our study was to characterize for the first time Snail protein expression in formalin-fixed primary tumour tissues using protein lysate microarrays and correlate Snail with E-cadherin, Cytokeratin 18, and Vimentin protein abundances. In a first feasibility study, we examined 17 formalin-fixed endometrioid adenocarcinomas by protein lysate microarrays. Snail expression showed a statistical significant inverse correlation with the expression of E-cadherin (P<0.001). A trend for correlation between Snail and Cytokeratin 18 (P=0.043) and the tumour grade (P=0.074) was seen. For Snail and Vimentin no correlation was found (P=0.384). In conclusion, our results fit to the proposed function of Snail as a transcriptional repressor of E-cadherin and Cytokeratin 18 in primary human carcinomas and demonstrate the usefulness of protein lysate microarrays for the precise determination of proteins involved in epithelial-mesenchymal-transition.

Mammalian target of rapamycin inhibition induces cell cycle arrest in diffuse large B cell lymphoma (DLBCL) cells and sensitises DLBCL cells to rituximab.

Diffuse large B-cell lymphoma (DLBCL) is a common lymphoma entity. Although a significant amount of DLBCL patients can be cured with modern chemotherapeutic regimens, a substantial proportion of patients die because of progressive disease. Therefore, new therapeutic strategies are clearly needed. Inhibitors of mTOR [mammalian target of rapamycin (Rap)] represent a new class of antiproliferative drugs with applications as immunosuppressive and anticancer agents. Extensive safety data exist on the mTOR inhibitor RAD001, which is already approved as an immunosuppressant in organ transplant recipients. Rap and RAD001 inhibited cell cycle progression in DLBCL cells by inducing a G1 arrest without inducing apoptosis. Phosphorylation of the main targets of mTOR, p70 s6 kinase and 4-EBP-1 was reduced in cells cultured in the presence of RAD001. Cell cycle arrest was accompanied by reduced phosphorylation of the retinoblastoma protein (RB) as well as reduced expression of cyclin D3 and A in all cell lines. Although the effect of the chemotherapeutic agent vincristine (vin) was not enhanced by RAD001, rituximab-induced cytotoxicity was augmented in the rituximab-sensitive cell lines. mTOR inhibition is a promising therapeutic strategy in DLBCL by inducing a G1 arrest and augments rituximab-induced cytotoxicity. Therefore, combination of these drugs might be an interesting new therapeutic approach in DLBCL patients.

Inhibition of the mammalian target of rapamycin and the induction of cell cycle arrest in mantle cell lymphoma cells.

Mammalian target of rapamycin (mTOR) inhibitors represent a new class of potential anticancer agents. The mTOR inhibitor, rapamycin, inhibited proliferation in three mantle cell lymphoma (MCL) cell lines and reduced cyclin D3 expression while cyclin D1 levels remained unchanged. This finding was confirmed in cells from a MCL patient.

Expression of cyclin E in resting and activated B-chronic lymphocytic leukaemia cells: cyclin E/cdk2 as a potential therapeutic target.

Disease progression in B-cell chronic lymphocytic leukaemia (B-CLL) is determined by the interplay between proliferation kinetics in the proliferating compartment and cell death in the accumulating compartment. Improving our knowledge of cell cycle regulation in B-CLL cells might therefore be important for identifying therapeutic targets. Cyclin E was detected by Western blotting in purified B-CLL cells from peripheral blood samples of all 12 patient tested but not in normal peripheral blood B cells. While cyclin-dependent kinase 2 (cdk2) expression was similar in different samples, p27 and cyclin E expression was highly variable. We further investigated the regulation of p27, cyclin E and cdk2 in an in vitro model of cycling B-CLL cells. Cyclin E and cdk2 expression was increased in B-CLL cells stimulated with a CpG-oligodeoxynucleotide and interleukin-2, while p27 expression rapidly declined. This was accompanied by the increased formation of cyclin E-cdk2 complexes, which were able to phosphorylate Histone H1 in vitro. Pharmacological inhibition of cdk2 activity with Roscovitine-inhibited thymidine incorporation and Histone H1 phosphorylation. We conclude that further evaluation of cyclin E and p27 in peripheral blood cells might help to identify prognostic subgroups. In addition, inhibition of Cyclin E-cdk2 activity by Roscovitine might be a new therapeutic strategy in B-CLL.

Cycling B-CLL cells are highly susceptible to inhibition of the proteasome: involvement of p27, early D-type cyclins, Bax, and caspase-dependent and -independent pathways.

OBJECTIVE: Although peripheral blood B-CLL cells are arrested in G0 phase of the cell cycle, a proliferating pool of cells in proliferation centers might be involved in disease progression. We have previously described an in vitro model of this proliferating pool of cells using B-CLL cells stimulated with immunostimulatory oligonucleotides (CpG-ODN) and interleukin-2. Lactacystin is a specific inhibitor of the proteasome and is a potent apoptosis inductor in resting peripheral B-CLL cells. In the present study, we investigated the effect of proteasome inhibition in proliferating B-CLL cells. METHODS: The effect of proteasome inhibition was analyzed using thymidine incorporation, annexin V assays, and TUNEL staining. Immunoblots were performed to evaluate expression of proteins involved in cell cycle and apoptosis regulation. RESULTS: Lactacystin blocked cell cycle progression in activated B-CLL cells and inhibited degradation of p27. Upregulation of cyclin D2 and D3 in activated B-CLL cells was inhibited while the expression of cdk2, cdk4, and cyclin E remained unchanged. Activated B-CLL cells were more susceptible to apoptosis induction as compared to resting B-CLL cells. Apoptosis induction was accompanied by cleavage of Bax, procaspase 8, procaspase 9, and procaspase 3. However, a broad-spectrum caspase inhibitor (z-VAD.fmk) only partially inhibited cell death although DNA degradation was completely inhibited. CONCLUSION: Proteasome inhibition is highly effective in proliferating B-CLL cells and induces apoptosis using a caspase-dependent and -independent pathway.

Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A, and survivin.

In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G(0)/early G(1) phase of the cell cycle, and defective apoptosis might be involved in disease progression. However, increasing evidence exists that B-CLL is more than a disease consisting of slowly accumulating resting B cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells, and has shown activity against a broad range of human tumor cell lines. Therefore, we investigated the ability of rapamycin to block cell cycle progression in proliferating B-CLL cells. We have recently demonstrated that stimulation with CpG-oligonucleotides and interleukin-2 provides a valuable model for studying cell cycle regulation in malignant B cells. In our present study, we demonstrated that rapamycin induced cell cycle arrest in proliferating B-CLL cells and inhibited phosphorylation of p70s6 kinase (p70(s6k)). In contrast to previous reports on nonmalignant B cells, the expression of the cell cycle inhibitor p27 was not changed in rapamycin-treated leukemic cells. Treatment with rapamycin prevented retinoblastoma protein (RB) phosphorylation in B-CLL cells without affecting the expression of cyclin D2, but cyclin D3 was no longer detectable in rapamycin-treated B-CLL cells. In addition, rapamycin treatment inhibited cyclin-dependent kinase 2 activity by preventing up-regulation of cyclin E and cyclin A. Interestingly, survivin, which is expressed in the proliferation centers of B-CLL patients in vivo, is not up-regulated in rapamycin-treated cells. Therefore, rapamycin interferes with the expression of many critical molecules for cell cycle regulation in cycling B-CLL cells. We conclude from our study that rapamycin might be an attractive substance for therapy for B-CLL patients by inducing a G(1) arrest in proliferating tumor cells.

Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase Cdelta.

In the present study we analyzed the role of phophatidylinositol-3 kinase (PI-3K) in B chronic lymphocytic leukemia (B-CLL) cells. PI-3K is activated by many stimuli and is linked to several different signaling pathways. We demonstrated that inhibition of PI-3K by a specific inhibitor, LY294002, induced apoptosis in B-CLL cells in vitro. This effect was specific for the inhibition of PI-3K because inhibition of other signaling pathways such as extracellular signaling-regulated kinase (ERK), p38, or p70S6 kinase did not affect spontaneous apoptosis. Furthermore, PI-3K was constitutively activated in freshly isolated B-CLL cells. Corresponding to enhanced apoptosis, LY294002 down-regulated expression of the antiapoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and Mcl-1. Next, we investigated which factors downstream of PI-3K were activated in B-CLL cells. We demonstrated that protein kinase B/Akt is expressed in all tested CLL samples but no activation of Akt was detected. In contrast, we observed a constitutive activation of protein kinase Cdelta (PKCdelta) in freshly isolated B-CLL cells. PKCdelta is linked to PI-3K and is phosphorylated at Thr505 in response to PI-3K activation. We further demonstrated that tyrosine phosphorylation and activity of PKCdelta were dependent on PI-3K activity in B-CLL cells. Inhibition of PKCdelta by the specific inhibitor Rottlerin strikingly enhanced apoptosis. In contrast, peripheral blood B cells of healthy donors were resistant to inhibition of PI-3K or PKCdelta. We conclude that activated PI-3K might be important in the pathogenesis of B-CLL, and survival signals might be mediated via PKCdelta. Therefore, inhibition of PI-3K or PKCdelta may be an innovative approach to treat B-CLL.

Sensitization of B-cell chronic lymphocytic leukemia cells to recombinant immunotoxin by immunostimulatory phosphorothioate oligodeoxynucleotides.

A recombinant anti-CD25 immunotoxin, LMB-2, has shown clinical efficacy in hairy cell leukemia and T-cell neoplasms. Its activity in B-cell chronic lymphocytic leukemia (B-CLL) is inferior but might be improved if B-CLL cells expressed higher numbers of CD25 binding sites. It was recently reported that DSP30, a phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN) induces immunogenicity of B-CLL cells by up-regulation of CD25 and other antigens. The present study investigated the antitumor activity of LMB-2 in the presence of DSP30. To this end, B-CLL cells from peripheral blood of patients were isolated immunomagnetically to more than 98% purity. Incubation with DSP30 for 48 hours augmented CD25 expression in 14 of 15 B-CLL samples, as assessed by flow cytometry. DSP30 increased LMB-2 cytotoxicity dose dependently whereas a control ODN with no CpG motif did not. LMB-2 displayed no antitumor cell activity in the absence of CpG-ODN as determined colorimetrically with an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. In contrast, B-CLL growth was inhibited in 12 of 13 samples with 50% inhibition concentrations (IC(50)) in the range of LMB-2 plasma levels achieved in clinical studies. Two samples were not evaluable because of spontaneous B-CLL cell death in the presence of DSP30. Control experiments with an immunotoxin that does not recognize hematopoietic cells, and an anti-CD22 immunotoxin, confirmed that sensitization to LMB-2 was specifically due to up-regulation of CD25. LMB-2 was much less toxic to normal B and T lymphocytes compared with B-CLL cells. In summary, immunostimulatory CpG-ODNs efficiently sensitize B-CLL cells to a recombinant immunotoxin by modulation of its target. This new treatment strategy deserves further attention.