The evidence that virus-induced tumors generally elicit T-cell responses prompts the notion that HPV-related cervical carcinoma would be amenable to treatment by T-cell-mediated adoptive therapy. Therefore, we cultured and cloned tumor-infiltrating lymphocytes (TIL) from a patient with cervical carcinoma and studied the in vitro characteristics of these TIL by using the established autologous tumor-cell line. After stimulation of bulk TIL cultures with 1,000 Units/ml recombinant interleukin 2 (rIL-2), followed by limiting dilution, T-cell clones were generated in the presence of 20 U/ml rIL-2 and irradiated autologous tumor cells, PBLs and EBV-transformed B-cell lines. Phenotypically, all clones were CD3/CD8-positive with a heterogeneous CD56 expression. All expressed preferential cytolytic activity against autologous tumor cells, did not lyse autologous lymphoblasts, and were cytotoxic against the NK-sensitive cell line K562. A minor lytic capacity was detectable on allogeneic cervical tumor-cell lines or tumor-cell lines of other histologic types. Cytotoxicity against the autologous tumor could be inhibited by anti-CD3, anti-CD8 and anti-ICAM1 but not by anti-HLA class-1 (W6/32, B9.12.1), anti-allele-specific HLA determinants and anti-LFA-3 antibodies. We demonstrate a highly specific autologous lytic activity of cervical carcinoma TIL, in which a CD3-associated surface antigen recognition is involved. These results may prove useful in further studies on adoptive immunotherapy of cervical cancer patients.

Bi-specific monoclonal antibodies (MAbs) were developed by somatic hybridization of 2 mouse hybridomas, one producing MAb against the G250 renal-cell carcinoma (RCC)-associated antigen and the other against the T-cell antigen CD3 (OKT3). The dual specificity of the hybrid MAb produced by these so-called quadromas was analyzed by immunohistochemistry on tissue sections and by cytotoxicity assays with relevant target and effector cells. The bi-specific MAb could induce TCR alpha beta/CD3+ and TCR gamma delta/CD3+ cloned lymphocytes to kill RCC cells. A noteworthy finding was that the TCR alpha beta and gamma delta lymphocyte clones showed different triggering abilities. The specificity of target-cell lysis by the cytotoxic T cells (CTL) was dictated by the specificity of the G250 MAb. Control bi-specific MAb, recognizing a cell-surface structure not involved in T-cell activation, did not induce lysis. Several IgG subclass switch variants of the G250 hybridoma, i.e., IgG1, 2a, 2b and IgE, were used for somatic hybridization with the OKT3 hybridoma (IgG2a). Except for IgE, all IgG subclass combinations could equally induce cytolysis. Induction of cytolysis was inhibited only by excess OKT3 MAb. Comparison of 2 bi-specific MAb preparations of the same combination (IgG2a/1), produced by 2 quadromas derived from the same parental hybridomas after identical purification procedures, produced different amounts of bispecific MAb.

BackgroundEvaluation of resection margins during cancer surgery can be challenging, often resulting in incomplete tumour removal. Fluorescence-guided surgery (FGS) aims to aid the surgeon to visualize tumours and resection margins during surgery. FGS relies on a clinically applicable imaging system in combination with a specific tumour-targeting contrast agent. In this study EpCAM (epithelial cell adhesion molecule) is evaluated as target for FGS in combination with the novel Artemis imaging system.MethodsThe NIR fluorophore IRDye800CW was conjugated to the well-established EpCAM specific monoclonal antibody 323/A3 and an isotype IgG1 as control. The anti-EpCAM/800CW conjugate was stable in serum and showed preserved binding capacity as evaluated on EpCAM positive and negative cell lines, using flow cytometry and cell-based plate assays. Four clinically relevant orthotopic tumour models, i.e. colorectal cancer, breast cancer, head and neck cancer, and peritonitis carcinomatosa, were used to evaluate the performance of the anti-EpCAM agent with the clinically validated Artemis imaging system. The Pearl Impulse small animal imaging system was used as reference. The specificity of the NIRF signal was confirmed using bioluminescence imaging and green-fluorescent protein.ResultsAll tumour types could clearly be delineated and resected 72 h after injection of the imaging agent. Using NIRF imaging millimetre sized tumour nodules were detected that were invisible for the naked eye. Fluorescence microscopy demonstrated the distribution and tumour specificity of the anti-EpCAM agent.ConclusionsThis study shows the potential of an EpCAM specific NIR-fluorescent agent in combination with a clinically validated intraoperative imaging system to visualize various tumours during surgery.

BackgroundOvarian cancer remains still the leading cause of death of gynecological malignancy, in spite of first-line chemotherapy with cisplatin and paclitaxel. Although initial response is favorably, relapses are common and prognosis for women with advanced disease stays poor. Therefore efficacious approaches are needed.MethodsPreviously, an anti-cancer agent, EPD exhibited potent cytotoxic effects towards ovarian cancer and not towards normal cells. Cell viability and cell cycle analysis studies were performed with EPD, in combination with cisplatin and/or paclitaxel, using the ovarian carcinoma cell lines: SK-OV-3, OVCAR-3, JC, JC-pl and normal fibroblasts. Cell viability was measured using Presto Blue and cell cycle analysis using a flow cytometer. Apoptosis was measured in JC and JC-pl , using the caspase 3 assay kit.ResultsIn JC-pl, SK-OV-3 and JC, synergistic interactions between either EPD and cisplatin or EPD and paclitaxel were observed. For the first time the effects of EPD on the cell cycle of ovarian cancer cells and normal cells was studied. EPD and combinations of EPD with cisplatin and/ or paclitaxel showed cell cycle arrest in the G2/M phase. The combination of EPD and cisplatin showed a significant synergistic effect in cell line JC-pl, while EPD with paclitaxel showed synergistic interaction in JC. Additionally, synergistic drug combinations showed increased apoptosis.ConclusionsOur results showed a synergistic effect of EPD and cisplatin in an ovarian drug resistant cell line as well as a synergistic effect of EPD and paclitaxel in two other ovarian cell lines. These results might enhance clinical efficacy, compared to the existing regimen of paclitaxel and cisplatin.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0157-2) contains supplementary material, which is available to authorized users.

Down-regulation of HLA in tumor cells, low numbers and dysfunctionality of NK cells are commonly observed in patients with end-stage cervical cancer. Adoptive transfer of high numbers of cytotoxic NK cells might be a promising treatment approach in this setting. Here, we explored the cytotoxic efficacy on ten cervical cancer cell lines of activated allogeneic NK cells from two sources, i.e., peripheral blood (PBNK) with and without cetuximab (CET), a tumor-specific monoclonal antibody directed against EGFR, or derived from umbilical cord blood (UCB-NK). Whereas CET monotherapy was ineffective against the panel of cervical cancer cell lines, irrespective of their EGFR expression levels and despite their RAS wt status, it significantly enhanced the in vitro cytotoxic efficacy of activated PBNK (P = 0.002). Equally superior cytotoxicity over activated PBNK alone was achieved by UCB-NK (P < 0.001). Both PBNK- and UCB-NK-mediated cytotoxic activity was dependent on the NK-activating receptors natural killer group 2, member D receptor (NKG2D) and DNAX accessory molecule-1 (DNAM-1) (P < 0.05) and unrelated to expression levels of the inhibitory receptors HLA-E and/or HLA-G. Most strikingly, whereas the PBNK’s cytotoxic activity was inversely correlated with HLA-ABC levels (P = 0.036), PBNK + CET and UCB-NK cytotoxicity were entirely independent of HLA-ABC expression. In conclusion, this study provides a rationale to initiate a clinical trial for cervical cancer with adoptively transferred allogeneic NK cells, employing either UCB-NK or PBNK + CET for EGFR-expressing tumors. Adoptive transfer of UCB-NK might serve as a generally applicable treatment for cervical cancer, enabled by HLA-, histology- and HPV-independent killing mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-016-1919-1) contains supplementary material, which is available to authorized users.