GTL001, a bivalent therapeutic vaccine against human papillomavirus 16 and 18, induces antigen-specific CD8+ T cell responses leading to tumor regression.

BACKGROUND: Prophylactic vaccines are available for women and girls not yet infected with HPV, but women already infected with HPV need a treatment to prevent progression to high-grade cervical lesions and cancer. GTL001 is a bivalent therapeutic vaccine for eradicating HPV-infected cells that contains HPV16 E7 and HPV18 E7 both fused to detoxified adenylate cyclase from Bordetella pertussis, which binds specifically to CD11b+ antigen-presenting cells. This study examined the ability of therapeutic vaccination with GTL001 adjuvanted with topical imiquimod cream to induce functional HPV16 E7- and HPV18 E7-specific CD8+ T cell responses. METHODS: Binding of GTL001 to human CD11b was assessed by a cell-based competition binding assay. Cellular immunogenicity of intradermal vaccination with GTL001 was assessed in C57BL/6 mice by enzyme-linked immunospot assay and in vivo killing assays. In vivo efficacy of GTL001 vaccination was investigated in the TC-1 murine HPV16 E7-expressing tumor model. RESULTS: GTL001 bound specifically to the human CD11b/CD18 receptor. GTL001 adjuvanted with topical 5% imiquimod cream induced HPV16 E7 and HPV18 E7-specific CD8+ T cell responses. This CD8+ T-cell response mediated in vivo killing of HPV E7-expressing cells. In the HPV16 E7-expressing tumor model, GTL001 adjuvanted with imiquimod but not imiquimod alone or a combination of unconjugated HPV16 E7 and HPV18 E7 caused complete tumor regression. CONCLUSIONS: GTL001 adjuvanted with topical 5% imiquimod is immunogenic and induces HPV16 E7 and HPV18 E7-specific CD8+ T cell responses that can kill HPV E7-expressing cells and eliminate HPV E7-expressing tumors.

GTL001 and bivalent CyaA-based therapeutic vaccine strategies against human papillomavirus and other tumor-associated antigens induce effector and memory T-cell responses that inhibit tumor growth.

GTL001 is a bivalent therapeutic vaccine containing human papillomavirus (HPV) 16 and HPV18 E7 proteins inserted in the Bordetella pertussis adenylate cyclase (CyaA) vector intended to prevent cervical cancer in HPV-infected women with normal cervical cytology or mild abnormalities. To be effective, therapeutic cervical cancer vaccines should induce both a T cell-mediated effector response against HPV-infected cells and a robust CD8+ T-cell memory response to prevent potential later infection. We examined the ability of GTL001 and related bivalent CyaA-based vaccines to induce, in parallel, effector and memory CD8+ T-cell responses to both vaccine antigens. Intradermal vaccination of C57BL/6 mice with GTL001 adjuvanted with a TLR3 agonist (polyinosinic-polycytidylic acid) or a TLR7 agonist (topical 5% imiquimod cream) induced strong HPV16 E7-specific T-cell responses capable of eradicating HPV16 E7-expressing tumors. Tumor-free mice also had antigen-specific memory T-cell responses that protected them against a subsequent challenge with HPV18 E7-expressing tumor cells. In addition, vaccination with bivalent vaccines containing CyaA-HPV16 E7 and CyaA fused to a tumor-associated antigen (melanoma-specific antigen A3, MAGEA3) or to a non-viral, non-tumor antigen (ovalbumin) eradicated HPV16 E7-expressing tumors and protected against a later challenge with MAGEA3- and ovalbumin-expressing tumor cells, respectively. These results show that CyaA-based bivalent vaccines such as GTL001 can induce both therapeutic and prophylactic anti-tumor T-cell responses. The CyaA platform can be adapted to different antigens and adjuvants, and therefore may be useful for developing other therapeutic vaccines.

GTL001, A Therapeutic Vaccine for Women Infected with Human Papillomavirus 16 or 18 and Normal Cervical Cytology: Results of a Phase I Clinical Trial.

PURPOSE: Women infected with human papillomavirus (HPV) with normal cytology to mild abnormalities currently have no treatment options other than watchful waiting or surgery if high-grade cervical lesions or cancer develop. A therapeutic vaccine would offer the possibility of preventing high-grade lesions in HPV-infected women. GTL001 is a therapeutic vaccine composed of recombinant HPV16 and HPV18 E7 proteins fused to catalytically inactive Bordetella pertussis CyaA. This study examined the tolerability and immunogenicity of GTL001 in women infected with HPV16 or HPV18 with normal cytology. EXPERIMENTAL DESIGN: This was a phase I trial (EudraCT No. 2010-018629-21). In an open-label part, subjects received two intradermal vaccinations 6 weeks apart of 100 or 600 µg GTL001 + topical 5% imiquimod cream at the injection site. In a double-blind part, subjects were randomized 2:1:1 to two vaccinations 6 weeks apart of 600 µg GTL001 + imiquimod, 600 µg GTL001 + placebo cream, or placebo + imiquimod. RESULTS: Forty-seven women were included. No dropouts, treatment-related serious adverse events, or dose-limiting toxicities occurred. Local reactions were transient and mostly mild or moderate. HPV16/18 viral load decreased the most in the 600 µg GTL001 + imiquimod group. In post hoc analyses, the 600 µg GTL001 + imiquimod group had the highest rates of initial and sustained HPV16/18 clearance. Imiquimod increased antigen-specific T-cell response rates but not rates of solicited reactions. All subjects seroconverted to CyaA. CONCLUSIONS: For women infected with HPV16 or HPV18 with normal cervical cytology, GTL001 was immunogenic and had acceptable safety profile. Clin Cancer Res; 22(13); 3238-48. ©2016 AACR.

Preclinical antitumor activity of cabazitaxel, a semisynthetic taxane active in taxane-resistant tumors.

PURPOSE: Taxanes are important chemotherapeutic agents with proven efficacy in human cancers, but their use is limited by resistance development. We report here the preclinical characteristics of cabazitaxel (XRP6258), a semisynthetic taxane developed to overcome taxane resistance. EXPERIMENTAL DESIGN: Cabazitaxel effects on purified tubulin and on taxane-sensitive or chemotherapy-resistant tumor cells were evaluated in vitro. Antitumor activity and pharmacokinetics of intravenously administered cabazitaxel were assessed in tumor-bearing mice. RESULTS: In vitro, cabazitaxel stabilized microtubules as effectively as docetaxel but was 10-fold more potent than docetaxel in chemotherapy-resistant tumor cells (IC50 ranges: cabazitaxel, 0.013-0.414 µmol/L; docetaxel, 0.17-4.01 µmol/L). The active concentrations of cabazitaxel in these cell lines were achieved easily and maintained for up to 96 hours in the tumors of mice bearing MA16/C tumors treated with cabazitaxel at 40 mg/kg. Cabazitaxel exhibited antitumor efficacy in a broad spectrum of murine and human tumors (melanoma B16, colon C51, C38, HCT 116, and HT-29, mammary MA17/A and MA16/C, pancreas P03 and MIA PaCa-2, prostate DU 145, lung A549 and NCI-H460, gastric N87, head and neck SR475, and kidney Caki-1). Of particular note, cabazitaxel was active in tumors poorly sensitive or innately resistant to docetaxel (Lewis lung, pancreas P02, colon HCT-8, gastric GXF-209, mammary UISO BCA-1) or with acquired docetaxel resistance (melanoma B16/TXT). CONCLUSIONS: Cabazitaxel is as active as docetaxel in docetaxel-sensitive tumor models but is more potent than docetaxel in tumor models with innate or acquired resistance to taxanes and other chemotherapies. These studies were the basis for subsequent clinical evaluation.

Superior antitumor activity of SAR3419 to rituximab in xenograft models for non-Hodgkin's lymphoma.

PURPOSE: To investigate the activity of SAR3419, a novel humanized anti-CD19 antibody (huB4), conjugated to a cytotoxic maytansine derivative N(2)'-deacetyl-N(2)'-(4-mercapto-4-methyl-1-oxopentyl) maytansine, in preclinical xenograft models for non-Hodgkin's lymphoma. EXPERIMENTAL DESIGN: Antitumor activity of SAR3419 was assessed as a single agent and in comparison with conventional therapies using a subcutaneous model for diffuse large B-cell lymphoma (WSU-DLCL2) and a systemic model for follicular small cleaved cell lymphoma (WSU-FSCCL) in mice with severe combined immune deficiency. RESULTS: Our results showed that in these chemotherapy-resistant models, SAR3419 was more effective than CHOP (cyclophosphamide-Adriamycin-vincristine-prednisone) regimen or rituximab. Only treatment with SAR3419 led to survival of the whole group of animals to the end of the experiment (150-155 days) in both models. Higher doses of SAR3419 (15 and 30 mg/kg) were more effective than lower dose of 7.5 mg/kg. The immunoconjugation was necessary because neither huB4 nor DM4 alone had significant activity. Treatment with rituximab resulted in antitumor activity in both models comparable with the low dose of SAR3419. Cyclophosphamide-Adriamycin-vincristine-prednisone alone showed modest activity in both models. Necropsy and tissue staining in the WSU-FSCCL systemic model revealed that all deaths featured leptomeningeal lymphoma in the control and treated groups. Interestingly, some of the animals that survived to the end of the experiment and seemed healthy at time of euthanasia did show microscopic evidence of lymphoma. CONCLUSIONS: Overall, SAR3419 is a very active immunotoxin in preclinical models for human B-cell lymphoma and holds promise as a novel and well-tolerated therapy in B-cell non-Hodgkin's lymphoma.

Class III beta-tubulin isotype predicts response in advanced breast cancer patients randomly treated either with single-agent doxorubicin or docetaxel.

PURPOSE: To evaluate the role of microtubule-associated variables as potential predictors of response and clinical outcome in patients with advanced breast cancer receiving single-agent docetaxel or doxorubicin chemotherapy. EXPERIMENTAL DESIGN: The analysis was done on 173 tumor samples from patients with locally advanced or metastatic breast cancer who have participated in the TAX-303 phase III trial in which patients were randomly assigned to receive docetaxel or doxorubicin. Expression of total alpha- and beta-tubulin, classes II to IV beta-tubulin isotypes, and tau protein was evaluated by immunohistochemistry on formalin-fixed, paraffin-embedded tumors from the primary breast cancer. RESULTS: We observed that patients with "high" expression of class III beta-tubulin isotype had a higher probability of response to docetaxel than to doxorubicin treatment (odds ratio, 1.9; 95% confidence interval, 1.01-3.7; P = 0.05). No difference was observed in terms of time to progression or in terms of overall survival. CONCLUSIONS: This study suggests that the superiority of docetaxel over doxorubicin seems to be confined to the subgroup of patients with "high" expression of class III beta-tubulin isotype.

Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography.

OBJECTIVES: To evaluate the early tumor vasculature disrupting effects of the AVE8062 molecule and the feasibility of dynamic contrast-enhanced ultrasonography (DCE-US) in the quantitative assessment of these effects. MATERIAL AND METHODS: AVE8062 was administered at a single dose (41 mg/kg) to 40 melanoma-bearing nude mice, which were all imaged before and after drug administration (5 + 15 minutes, 1, 6, and 24 hours). Using an ultrasound scanner (Aplio, Toshiba), intratumor vessels were counted in power Doppler mode and tumor microvasculature was assessed in a specific harmonic mode associated with a perfusion and quantification software for contrast-uptake quantification (Sonovue, Bracco). The peak intensity (PI), time-to-PI (T PI), and full-width at half maximum (FWHM) were extracted from the time-intensity curves expressed as linear raw data. Histologic analysis evaluated microvessel density (MVD) and necrosis at each time point studied. Statistical significance was estimated (paired sum rank and Mann-Whitney tests) to evaluate drug activity and to compare its efficacy at the different time points. RESULTS: In power Doppler mode, intratumoral vessels depletion started 15 minutes postinjection (32%, P = 0.004) and the decrease was maximal at 6 hours (51%, P = 0.002). PI decreased by 3.5- and 45.7-fold at 1 and 6 hours, respectively, compared with preinjection values (P = 0.016 and P = 0.008). The decrease at 6 hours was significantly different from the variation at 1 hour (P = 0.0012) and at 24 hours (P = 0.0008). T PI and FWHM showed a significant increase exclusively at 6 hours (P = 0.0034, P = 0.0039). Histology revealed significantly decreased MVD and increased necrosis at 24 hours (P < 0.01). CONCLUSION: DCE-US allowed quantitative in vivo evaluation of the functional effects of AVE8062, which was found most effective on tumoral microvasculature 6 hours after its administration. A clinical phase-1 study of AVE8062 is ongoing using the same ultrasonography methodology before and 6 and 24 hours postadministration.

[Antitumor effect of docetaxel against human endometrial tumor cell lines].

The antitumor effect of docetaxel against human endometrial tumor cell lines was investigated in vitro and in vivo. In the in vitro study,docetaxel showed concentration-dependent inhibition of the growth of 4 tumor cell lines having different degrees of differentiation (AN3 CA, KLE, HEC-1-A and HEC-1-B), with IC(50) values ranging from 2.48 to 82.40 ng/ml. These values represent ca. 1/900-1/30 of the mean maximum plasma concentration of 2.27 microg/ml attained when the recommended dose of 70 mg/m(2) for patients with endometrial cancer was administered to patients with various types of cancer in phase I trial. In addition, the activity was nearly equal to paclitaxel, and much more potent than fluorouracil, cisplatin and doxorubicin. Docetaxel also showed strong antitumor activity against xenografts of the AN3 CA human endometrial adenocarcinoma cell line in nude mice. In the docetaxel treated group at its MTD (33 mg/kg/dose, q 6 d x 3, iv), all of the animals were tumor-free survivors on Day 62 after xenografting. The antitumor effect in the MTD-administered group was the strongest of all of the tested anticancer drug groups (cyclophosphamide, mitomycin C, fluorouracil, cisplatin, doxorubicin). Even at two docetaxel dosages below its MTD (20.5 and 12.5 mg/kg/day), the drug showed a marked cytotoxic activity. These results demonstrated that docetaxel shows potent antitumor efficacy against human endometrial tumor cell lines, leading to the expectation that it will be useful as a therapeutic agent for endometrial cancer.

The mechanism of action of docetaxel (Taxotere) in xenograft models is not limited to bcl-2 phosphorylation.

Docetaxel is a new taxoid compound with a broad spectrum of antitumor activity. Previous studies have shown that in vitro treatment of specific human tumor lines with docetaxel is associated with the phosphorylation and inactivation of the bcl-2 protein and the occurrence of apoptosis. The goal of this study was to examine whether bcl-2 expression is truly required for in vivo responsiveness to docetaxel. The expression and state of phosphorylation of bcl-2 was examined in human MX-1 breast or DU-145 prostate tumors explanted from nu/nu mice treated with docetaxel. The MX-1 cells accumulated in the G2/M phase of the cell cycle and exhibited phosphorylation of bcl-2 after treatment with docetaxel. By Western blot analysis DU-145 prostate tumor cells did not express bcl-2 protein before or following in vivo treatment with docetaxel. However, docetaxel was highly active against the DU-145 tumor xenograft model. Thus, docetaxel induces apoptosis and cell death through a different, bcl-2-independent mechanism in the DU-145 human prostate tumor, indicating that bcl-2 may not have prognostic value for treatment with docetaxel.

Combined effects of docetaxel and angiostatin gene therapy in prostate tumor model.

The anti-tumor effects of adenovirus-delivered angiostatin (AdK3) in association with docetaxel (Taxotere) have been evaluated in a human-origin prostate tumor model. In vitro, human endothelial cells were 50- to 100-fold more sensitive to docetaxel than prostate cell lines (PC3, LNCaP, and DU145), and the combination regimen of docetaxel and AdK3 was significantly more cytotoxic for endothelial cells than either treatment alone. PC3 cells, which display the highest sensitivity to docetaxel, were then grafted onto athymic mice for an evaluation of the combined regimen as a therapy. The combination of a single intratumoral injection of AdK3 (2 x 10(9) pfu) and a single intravenous injection of docetaxel (15 mg/kg) was compared with the injection of AdK3 alone on preestablished mice bearing PC3-derived tumors with a mean tumor volume of 60 +/- 11 or 205 +/- 46 mm3. Significant antitumoral effects were observed only in mice receiving the combined treatment. We showed that all PC3 tumors regressed in the AdK3-docetaxel combination group and that 40 to 83% totally regressed. In all cases, this regimen was tightly correlated with a marked decrease in intratumoral vascularization. Our experimental data show that attacking both endothelial and tumoral compartments is an efficient and logical strategy, making this bitherapy approach clinically promising.

Microtubule-associated parameters as predictive markers of docetaxel activity in advanced breast cancer patients: results of a pilot study.

Docetaxel is currently one of the most active agents for breast cancer. Predictive markers of docetaxel efficacy are clearly needed in order to avoid unnecessary toxicity in nonresponding or resistant patients and to improve the cost-effectiveness ratio of docetaxel. This pilot study correlates the clinical efficacy of docetaxel in 54 metastatic or locally advanced breast cancer patients with the expression of microtubule-associated parameters evaluated by immunohistochemistry in archival tumor samples. Among the 41 eligible patients (evaluable response to docetaxel and available predocetaxel treatment paraffin-embedded tumor tissue), response to docetaxel was: partial response 54%, stable disease 29%, and progressive disease 17%. Alfa- and b-tubulin and Tau protein were expressed in the majority of tumor samples. Class II, III, and IV b-tubulin isotypes were expressed in 56%, 65%, and 82% of samples, respectively. No clear association was found between response to docetaxel and the level of expression of Tau protein, a- and b-tubulin, and class III and IV b-tubulin isotypes. In patients with class II b-tubulin-positive tumors, the response rate was 39%, while in class II b-tubulin-negative tumors the response rate was 79% (P = 0.04). Therefore, we conclude that the class II b-tubulin isotype seems to be a promising predictive marker of docetaxel activity. Nevertheless, further investigations are needed due to the limited number of patients evaluated in this pilot study.