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We describe the synthesis of a series of 2-arylbenzimidazole derivatives bearing sulfonamide functionality (4a-d, 7a-c and 10) as well as hydroxamic acid (15a-b), carboxylic acid (16a-b), carboxamide (17a-b) and boronic acid (22a-b and 26) functionalities, which act as human carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors. The newly synthesized benzimidazole derivatives were evaluated against 4 physiologically relevant CA isoforms (hCA I, II, IX, and XII), and especially the sulfonamide-containing benzimidazoles demonstrated intriguing inhibitory activity against tumor associated CA IX and XII with K I values in the range of 5.2-29.3 nM and 9.9-41.7 nM, respectively. Notably, compound 4c was the most potent and selective CA IX (K I  = 6.6 nM) and XII (K I  = 9.9 nM) inhibitor with a significant selectivity ratio over cytosolic CA I and II isoforms in the range of 3.4-25.2. In addition, compounds having hydroxamic acid (15a-b) or carboxylic acid (16a-b) functionalities resulted in greater selectivity ratios for CA IX/XII over CAI/II in the range of 4.1-121.5 although with K I values in lower micromolar potency (K I s = 0.36-0.85 μM for CA IX/XII)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Uterine serous carcinoma is an aggressive form of endometrial cancer that carries an extremely poor prognosis. Solitomab is a novel bispecific single-chain antibody construct that targets epithelial cell adhesion molecule on tumor cells and also contains a CD3 binding region. We evaluated the expression levels of epithelial cell adhesion molecule and the in vitro activity of solitomab against primary uterine serous carcinoma cell lines in vitro and ex-vivo in the ascites of patients with uterine serous carcinoma., Objective: The purpose of this study was to determine the frequency of expression of epithelial cell adhesion molecule on uterine serous carcinoma cell lines and the ability of solitomab to modulate immune responses (T-cell proliferation, activation, cytokine production, and tumor killing) to tumor cells when it is combined with lymphocytes and epithelial cell adhesion molecule-positive cell lines or epithelial cell adhesion molecule-positive ascitic fluid in vitro., Study Design: Epithelial cell adhesion molecule expression was evaluated by flow cytometry in a total of 14 primary uterine serous carcinoma cell lines. Sensitivity to solitomab-dependent cellular-cytotoxicity was tested against a panel of primary uterine serous carcinoma cell lines that express different levels of epithelial cell adhesion molecule in standard 4-hour chromium release assays. The proliferative activity, activation, cytokine secretion (ie, type I vs type II), and cytotoxicity of solitomab in autologous tumor-associated T cells in the ascitic fluid of patients with uterine serous carcinoma was also evaluated by carboxyfluorescein succinimidyl ester and flow-cytometry assays. Differences in epithelial cell adhesion molecule expression, solitomab-dependent cellular-cytotoxicity levels were analyzed with the use of an unpaired t test. T-cell activation marker increase and cytokine release were analyzed by a paired t test., Results: Surface expression of epithelial cell adhesion molecule was found in 85.7% (12 of 14) of the uterine serous carcinoma cell lines that were tested by flow cytometry. Epithelial cell adhesion molecule-positive cell lines were found resistant to natural killer cells or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean killing ± standard of the mean, 2.7% ± 3.1% after incubation of epithelial cell adhesion molecule-positive cell lines with control bispecific antibody construct). In contrast, after incubation with solitomab, epithelial cell adhesion molecule-positive uterine serous carcinoma cells became highly sensitive to T-cell cytotoxicity (mean killing, 25.7% ± 4.5%; P < .0001) by peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with epithelial cell adhesion molecule that expressed malignant cells in ascites with solitomab resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (ie, CD25 and HLA-DR), and a reduction in number of viable uterine serous carcinoma cells in ascites (P < .001)., Conclusion: Solitomab induces robust immunologic responses in vitro that result in increased T-cell activation, proliferation, production of cytokines, and direct killing of tumor cells. These findings suggest that solitomab may represent a novel, potentially effective agent for the treatment of recurrent/metastatic and/or chemo-resistant uterine serous carcinoma-overexpressing epithelial cell adhesion molecule., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate poor survival and significant treatment morbidity with standard therapy. The immune profile in HNSCC, whether caused by carcinogen exposure or human papillomavirus (HPV), is notably immunosuppressive. Early clinical trials of immunotherapy in HNSCC were troubled by systemic toxicity or difficulties in local administration. Now, interest in immunotherapy has been revitalized by mechanistic insights into immune evasion by HNSCC, coupled to ongoing development of novel immunotherapies. This review will summarize immune escape mechanisms in HNSCC, namely downregulation of tumor antigen (TA) presentation, aberrant regulation of the signal transducer and activator of transcription (STAT) family, the immunosuppressive cytokine milieu, and dysregulation of immune effector cells. Therapeutic strategies hypothesized to specifically counter HNSCC immunosuppression will then be discussed. We will survey TA- targeted monoclonal antibodies (mAb), including the prototype cetuximab, as well as adjunctive strategies to enhance antibody-dependent cell-mediated cytotoxicity. We will review immunomodulation to restore STAT1/STAT3 activation balance. Examples of mAb therapy to block immunosuppressive cytokines, such as interleukin-6 or VEGF, will be provided. mAbs which release co-inhibitory T cell receptors such as CTLA-4 and PD-1, overexpressed in HNSCC, also hold therapeutic promise. Finally, we will describe principles for therapeutic vaccination in HPV-associated HNSCC, where non-host TAs such as viral oncoproteins represent ideal targets, and HPV-negative HNSCC, where p53 is a promising target. Insights into immunosuppression in HNSCC have elucidated mechanistic targets for immunotherapy. Rational clinical investigation may lead to effective stand alone or combinatorial treatment approaches., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Previously, we reported that (-)-xanthatin, a naturally occurring xanthanolide present in the Cocklebur plant, exhibits potent anti-proliferative effects on human breast cancer cells, accompanied by an induction of the growth arrest and DNA damage-inducible gene 45γ (GADD45γ), recognized recently as a novel tumor suppressor gene. However, the mechanisms mediating this activation were unknown. Topoisomerase IIα (Topo IIα) inhibition has been reported to produce a cell death response accompanied by an atypical DNA laddering fragmentation profile, similar to that noted previously for (-)-xanthatin. Therefore we hypothesized that (-)-xanthatin's GADD45γ activation was mediated through the Topo IIα pathway. Here, we identify that (-)-xanthatin does function as a catalytic inhibitor of Topo IIα, promoting DNA damage. In addition, reactive oxygen species (ROS) were elevated in cells treated with this agent. Mechanistically, it was determined that the induced levels of GADD45γ mRNA resulting from (-)-xanthatin exposures were stabilized by coordinately produced ROS, and that the consequent induction of GADD45γ mRNA, GADD45γ protein and ROS generation were abrogated by co-treatment with N-acetyl-l-cysteine. Taken together, the data support the concept that Topo IIα inhibition by (-)-xanthatin is a trigger that stimulates expression of DNA damage-inducible GADD45γ mRNA and that concomitantly produced ROS act downstream to further enhance the GADD45γ mRNA/GADD45γ protein induction process, resulting in breast cancer cell death., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Melanoma-associated antigen-encoding (MAGE) genes are expressed in melanoma and other cancers but not in normal somatic cells. MAGE expression is associated with aggressive tumor growth, poor clinical outcome, and resistance to chemotherapy, but the mechanisms have not been completely elucidated. In this study, we show that downregulation of MAGE-C2 in A375 melanoma cells and low-passage cultures from human metastatic melanomas (MRA cells) results in increased apoptosis and decreased growth of tumor xenografts in athymic nude mice. Previously, we showed that MAGE-C2 binds KAP1, a scaffolding protein that regulates DNA repair. Phosphorylation of KAP1-Serine 824 (Ser824) by ataxia-telangiectasia-mutated (ATM) kinase is necessary for repair of DNA double-strand breaks (DSBs); now we show that MAGE-C2 knockdown reduces, whereas MAGE-C2 overexpression increases, ATM kinase-dependent phosphorylation of KAP1-Ser824. We demonstrate that MAGE-C2 increases co-precipitation of KAP1 with ATM and that binding of MAGE-C2 to KAP1 is necessary for increased KAP1-Ser824 phosphorylation. Furthermore, ectopic expression of MAGE-C2 enhances repair of I-SceI endonuclease-induced DSBs in U-2OS cells. As phosphorylation of KAP1-Ser824 facilitates relaxation of heterochromatin, which is necessary for DNA repair and cellular proliferation, our results suggest that MAGE-C2 can promote tumor growth by phosphorylation of KAP1-Ser824 and by enhancement of DNA damage repair.

Background: After surgery and anthracycline adjuvant treatment, about 60% of early advanced breast cancer patients develop recurrence. These differences in treatment outcome indicate the need to identify markers for risk of recurrence. The aim of this study was the retrospective analysis of relationship between tumour features (topoisomerase IIα (TOPOIIα), human epidermal growth factor receptor 2 (HER2), hormone receptors, cytokeratin (CK)5/6 expression and proliferation rate) and disease-free survival (DFS) of breast cancer patients treated with anthracyclines in adjuvant setting., Material and Methods: The study was performed in the group of 172 patients (mean age: 52.8 years, T1-T2, N1-N2, M0). HER2, TOPOIIα, estrogen receptor (ER) and progesterone receptor (PgR) expression and proliferation rate were studied immunohistochemically. HER2 overexpression was confirmed by fluorescence in situ hybridisation (FISH). These data were correlated with 5-year DFS., Results: In univariate analysis, lower TOPOIIα expression (median value ≤ 11.9%) and tumour grade G1 + G2 were favourable prognostic factors. All tumours were classified into four subtypes: (1) lower TOPOIIα expression and G1 + G2, (2) lower TOPOIIα expression and G3, (3) higher TOPOIIα expression and G3, and (4) higher TOPOIIα expression and G1 + G2. In Cox multivariate regression analysis, tumour subtype distinguished by TOPOIIα expression and grade was independent prognostic factor for DFS. All patients (n = 52) with TOPOIIα lower expression and G1 + G2 tumours, survived 5 years without any evidence of disease., Conclusion: The results suggest that lower TOPOIIα expression and lower tumour grade are favourable prognostic factors for early advanced breast cancer patients after adjuvant anthracycline chemotherapy., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

One avenue towards the development of more selective anti-cancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. In this context, the targeted delivery of therapeutic agents to newly-formed blood vessels ("vascular targeting") is particularly attractive, because of the dependence of tumors on new blood vessels to sustain growth and invasion, and because of the accessibility of neo-vascular structures for therapeutic agents injected intravenously. Ligand-based vascular targeting strategies crucially rely on good-quality vascular tumor markers. Here we describe a number of established technologies for the enrichment of accessible vascular proteins based on the isolation of glycoproteins, the in vivo coating of accessible cell surfaces with colloidal silica and the in vivo perfusion with reactive ester derivatives of biotin. Label-free as well as isotopic labeling based strategies for the subsequent MS-based protein quantification are outlined. Finally, bioinformatic workflows for protein quantification are depicted aiming at assisting in the evaluation of appropriate strategies for individual projects. This review gives an overview of current chemical proteomic strategies for the enrichment and quantification of the accessible vascular proteome and helps in selecting bioinformatic strategies for data analysis and validation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: In order to compensate for the paucity of defined tumor antigens (Ag) in non-Hodgkin's lymphomas, a promising approach might be the use of whole tumor cells as a source of tumor Ag to pulse antigen-presenting cells (APC). However, it is not presently known how the tumor cells should be delivered to APC to optimize the cross-presentation of tumor Ag to anti-tumor CD8 T cells. We aimed to compare CD20-opsonized, apoptotic and necrotic human tumor cells for their capacity to induce endocytosis and cross-presentation of tumor-associated Ag by dendritic cells (DC) or macrophages., Methods: Endocytosis of human tumor-derived material by macrophages or DC was monitored by flow cytometry. We used a previously described influenza model and studied cross-presentation of viral Ag as cellular surrogate tumor-associated Ag by APC after endocytosis of lymphoma B cells treated by inactivated influenza virus., Results: Optimal endocytosis was obtained when tumor cells were opsonized by an anti-CD20 antibody and, as expected, macrophages were more phagocytic than DC. However, Ag from opsonized, apoptotic and live cells, but not from necrotic lymphoma cells, were efficiently cross-presented by DC but not by macrophages., Discussion: We have developed a new model with human primary lymphoma cells to study the cross-presentation of tumor-associated Ag by APC. The results we have obtained support the use of whole lymphoma cells from patients to pulse DC to induce an anti-tumor immune response.

Cytokines interleukin (IL)-12 and IL-23 are implicated in the pathogenesis of psoriasis. IL-12 causes differentiation of CD4+ T cells to interferon-gamma (IFN-gamma)-producing T helper 1 (Th1) cells, while IL-23 induces differentiation to IL-17-producing pathogenic Th17 cells. The effects of the monoclonal antibody to IL-12/23 p40 subunit (CNTO 1275) on IL-12 receptor (IL-12R) expression, markers associated with skin homing, activation, and cytokine secretion were investigated in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated in the presence or absence of recombinant human (rh) IL-12 or rhIL-23, with or without CNTO 1275. CNTO 1275 inhibited upregulation of CLA, IL-12R, IL-2Ralpha and CD40L expression and also inhibited IL-12- and IL-23-induced IFN-gamma, IL-17A, tumor necrosis factor (TNF)-alpha, IL-2, and IL-10 secretion. Thus, the therapeutic effect of CNTO 1275 may be attributed to the IL-12/23 neutralization, resulting in decreased expression of skin homing and activation markers, and IL-12- and IL-23-induced cytokine secretion.

Cytotoxic T lymphocyte antigen-4 has become recognized as one of the key negative regulators of adaptive immune responses, having a central role in the maintenance of peripheral tolerance and in shaping the repertoire of emergent T cell responses. Concurrent recognition of the potential importance of inhibitory immune regulators in limiting antitumor responses, either as a result of chronic antigenic stimulation or the self-nature of many tumor-selective target antigens, has led to the development of cytotoxic T lymphocyte antigen-4-blocking antibodies as therapeutic anticancer agents. Following extensive preclinical modeling, these agents have entered clinical trials, where they are showing encouraging activity in heavily pretreated patients with advanced-stage disease, particularly with melanoma or renal carcinoma. Finding ways to dissociate antitumor activity from adverse immune events should enable actualization of their therapeutic potential in the coming years.

Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to the antibody fragment scFvC54 specific for the cell surface target antigen EGP2. In the present study, we report that the selective binding of scFvC54:sTRAIL to EGP2-positive target cells conveys an exceptionally potent pro-apoptotic effect toward neighboring tumor cells that are devoid of EGP2 expression (bystander cells). The anti-tumor bystander activity of scFvC54:sTRAIL was detectable at target-to-bystander cell ratios as low as 1:100. Treatment in the presence of EGP2-blocking or TRAIL-neutralizing antibody strongly inhibited apoptosis in both target and bystander tumor cells. In the absence of target cells, bystander cell apoptosis induction was abrogated. The bystander apoptosis activity of scFvC54:sTRAIL did not require internalization, enzymatic conversion, diffusion, or communication (gap junctional intracellular communication) between target and bystander cells. Furthermore, scFvC54:sTRAIL showed no detectable signs of innocent bystander activity toward freshly isolated blood cells. Further development of this new principle is warranted for approaches where cancer cells can escape from antibody-based therapy due to partial loss of target antigen expression.

Background: The autoimmune cytopenias encompass the disorders of immune thrombocytopenia purpura (ITP), pure red-cell aplasia (PRCA), autoimmune hemolytic anemia (AIHA), autoimmune neutropenia and various combinations of these conditions. T lymphocytes are thought to play an important role in the pathogenesis of autoimmune cytopenias, and the presence of autoantibody may represent an epiphenomenon, rather than the primary pathogenetic mechanism. The majority of patients usually respond to standard immunosuppressive therapy and can mostly be treated as out-patients. A small proportion, however, have severe, resistant and life-threatening disease, or may experience major morbidity from side effects of drugs given to treat their disease., Methods: We have treated 21 patients with autoimmune cytopenias with the MAb Campath-1H, and for later patients in this series, in combination with low dose CYA., Results: Responses were seen in 14 of 20 evaluable patients, although relapse occurred in seven patients. In many patients corticosteroid therapy could be discontinued or greatly reduced., Discussion: We conclude that Campath-1H can induce remissions in autoimmune cytopenias and we critically review its role in the treatment of these disorders.

Generating an antitumor immune response in tumor-bearing host has been impaired by several characteristics of both patient and tumor cells. Amongst those requirements is the necessity of generating immune effectors that are specific to tumor cells. The last two decades have seen the description of many so called tumor "specific" antigens. Indeed, strictly specific tumor antigens are scarce. Most antigens are tumor-associated antigens, also shared by normal tissues. Telomerase and its activity have recently been recognized as a major marker of tumoral growth in more than 80% of cancers. Some telomerase subunits might be ideal, if not universal, targets to an antitumor immune response in patients with cancer. Many other major parameters remain to be understood and to be mastered. Nevertheless, experiments reported in this overview give hope to the possibility of generating an immune response aimed at elements of telomerase, both in healthy subjects and in patients with advanced disease.

Major surgery impairs cellular and humoral immunity, in particular natural cytotoxicity, and this may facilitate the dissemination of tumour cells in the peri-operative period. Recombinant interleukin-2 (rIL-2) has been used to modulate peri-operative immune function. Eighteen patients were randomized to receive either rIL-2 or placebo for three days, as a subcutaneous injection, prior to surgical resection for colorectal cancer. Natural cytotoxicity (natural killer (NK) and lymphokine-activated killer (LAK) cell activity), monocyte phagocytosis and immune cell surface activation marker (CD14+HLA-DR) expression were assessed during therapy and for up to 21 days after surgery. rIL-2 therapy enhanced both NK and LAK cell-mediated cytotoxicity and augmented circulating lymphocyte CD16+ and CD56+ cell subset populations. Circulating monocyte phagocytosis was also increased. Hence, rIL-2 may be used to enhance immune function in the peri-operative period in patients undergoing curative cancer surgery.

Proliferative activity is a potential prognostic indicator of neoplastic cell growth. Usually the assessment of the tumor growth fraction requires specially processed or frozen tissue. We have raised a monoclonal antibody, Ki-S1, suitable for the detection of proliferating cells in routinely processed and paraffin-embedded tissue specimens. A retrospective study was conducted on 83 mammary carcinoma patients with a median follow-up of 45.6 months. Ki-S1 positivity was significantly (P < 0.0001) correlated with the S-phase fraction. A high proliferative activity of more than 30% of tumor cells being Ki-S1-positive was significantly correlated with recurrence (P < 0.001). Cumulative survival was significantly reduced in the high Ki-S1 expressing subgroup. In a multivariate model including Ki-S1, lymph node metastases, tumor size, and progesterone receptor positivity, independent significant information was provided by Ki-S1. We conclude that in mammary carcinomas, Ki-S1-determined growth fraction represents a significant and independent prognostic predictor.