Eradication of large tumors expressing human papillomavirus E7 protein by therapeutic vaccination with E7 fused to the extra domain a from fibronectin.

Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8(+) T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 5-7 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma.

Mansilla C ，Berraondo P ，Durantez M ，Martínez M ，Casares N ，Arribillaga L ，Rudilla F ，Fioravanti J ，Lozano T ，Villanueva L ，Sarobe P ，Borrás F ，Leclerc C ，Prieto J ，Lasarte JJ ... -  《-》

Bivalent therapeutic vaccine against HPV16/18 genotypes consisting of a fusion protein between the extra domain A from human fibronectin and HPV16/18 E7 viral antigens.

In vivo targeting of human papillomavirus (HPV) derived antigens to dendritic cells might constitute an efficient immunotherapeutic strategy against cervical cancer. In previous works, we have shown that the extra domain A from murine fibronectin (mEDA) can be used to target antigens to toll-like receptor 4 (TLR4) expressing dendritic cells and induce strong antigen-specific immune responses. In the present study, we have produced a bivalent therapeutic vaccine candidate consisting of the human EDA (hEDA) fused to E7 proteins from HPV16 and HPV18 (hEDA-HPVE7-16/18) and evaluate its potential as a therapeutic vaccine against cervical cancer. Recombinant fusion proteins containing HPV E7 proteins from HPV16 and HPV18 virus subtypes fused to hEDA were produced and tested in vitro on their capacity to bind TLR4 and induce the production of tumor necrosis factor-α or interleukin (IL)-12 by human monocytes and dendritic cells. The immunogenicity and potential therapeutic activity of the vaccine in combination with cisplatin or with the TLR3 agonist molecules polyinosinic-polycytidylic acid (Poly IC) or Poly ICLC was evaluated in mice bearing subcutaneous or genital orthotopic HPV16 TC-1 tumors. hEDA-HPVE7-16/18 prototype vaccine binds human TLR4 and stimulate TLR4-dependent signaling pathways and IL-12 production by human monocyte-derived dendritic cell. Vaccination with hEDA-HPVE7-16/18 induced strong HPVE7-specific Cytotoxic T lymphocyte (CTL) responses and eliminated established tumors in the TC-1-based tumor model. The antitumor efficacy was significantly improved by combining the fusion protein with cisplatin or with the TLR-3 ligand Poly IC and especially with the stabilized analog Poly ICLC. Moreover, hEDA-HPVE7-16/18+Poly ICLC induced full tumor regression in 100% of mice bearing orthotopic genital HPV tumors. Our results suggest that this therapeutic vaccine formulation may be an effective treatment for cervical tumors that do not respond to current therapies.

Arribillaga L ，Echeverria I ，Belsue V ，Gomez T ，Lozano T ，Casares N ，Villanueva L ，Domingos-Pereira S ，Romero PJ ，Nardelli-Haefliger D ，Hervás-Stubbs S ，Sarobe P ，Rodriguez MJ ，Carrascosa JL ，Zürcher T ，Lasarte JJ ... -  《Journal for ImmunoTherapy of Cancer》

A novel, broad spectrum therapeutic HPV vaccine targeting the E7 proteins of HPV16, 18, 31, 45 and 52 that elicits potent E7-specific CD8T cell immunity and regression of large, established, E7-expressing TC-1 tumors.

Persistent infection by high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, which remains one of the most common cancers among women worldwide. In addition, there is a growing appreciation that high risk HPVs are associated with a number of other cancers including anogenital cancers as well as a subset of head and neck cancers. Recently, prophylactic HPV vaccines targeting the two most prevalent high risk HPVs (HPV16 and HPV18) have been deployed in large-scale vaccination campaigns. However, the extent to which these prophylactic vaccines confer protection against other high risk HPV genotypes is largely unknown and prophylactic vaccines have been shown to be ineffective against pre-existing infection. Thus there continues to be an urgent need for effective therapeutic vaccines against HPV. The E7 protein of HPV16 has been widely studied as a target for therapeutic vaccines in HPV-associated cancer settings because HPV16 is the most prevalent of the high risk HPV genotypes. However, HPV16 accounts for only about 50% of cervical cancers and there are at least 15 other high risk HPVs that are known to be oncogenic. We have developed a novel, broad-spectrum, therapeutic vaccine (Pentarix) directed at the E7 proteins from five of the most prevalent high-risk genotypes of HPV worldwide (HPV16, 18, 31, 45 and 52) that together account for more than 80% of all HPV-associated cancers. Pentarix is a recombinant protein-based vaccine that elicits strong, multi-genotype specific CD8 T cell immunity when administered to mice in combination with adjuvants comprised of agonists of the TLR3 or TLR9 family of innate immune receptors. Furthermore, large, established E7-expressing TC-1 tumors undergo rapid and complete regression after therapeutic vaccination of mice with Pentarix. Together, these data suggest that Pentarix may be of clinical value for patients with E7-positive, HPV-associated precancerous lesions or malignant disease.

Wick DA ，Webb JR 《-》

A novel therapeutic vaccine composed of a rearranged human papillomavirus type 16 E6/E7 fusion protein and Fms-like tyrosine kinase-3 ligand induces CD8(+) T cell responses and antitumor effect.

The development of cervical cancer is mainly caused by infection with high risk genotypes of human papillomavirus, particularly type 16 (HPV16), which accounts for more than 50% of cervical cancer. The two early viral oncogenes, E6 and E7, are continuously expressed in cervical cancer cells and are necessary to maintain the malignant cellular phenotype, thus providing ideal targets for immunotherapy of cervical cancer. In this study, a novel vaccine strategy was developed based on a rationally shuffled HPV16 E6/E7 fusion protein, the addition of Fms-like tyrosine kinase-3 ligand (Flt3L) or the N domain of calreticulin (NCRT), and the usage of a CpG adjuvant. Four recombinant proteins were constructed: m16E6E7 (mutant E6/E7 fusion protein), rm16E6E7 (rearranged mutant HPV16 E6/E7 fusion protein), Flt3L-RM16 (Flt3L fused to rm16E6E7), and NCRT-RM16 (NCRT fused to rm16E6E7). Our results suggest that Flt3L-RM16 was the most potent of these proteins in terms of inducing E6- and E7-specific CD8+ T cell responses. Additionally, Flt3L-RM16 significantly induced regression of established E6/E7-expressing TC-1 tumors. Higher doses of Flt3L-RM16 trended toward higher levels of antitumor activity, but these differences did not reach statistical significance. In summary, this study found that Flt3L-RM16 fusion protein is a promising therapeutic vaccine for immunotherapy of HPV16-associated cervical cancer.

Li J ，Chen S ，Ge J ，Lu F ，Ren S ，Zhao Z ，Pu X ，Chen X ，Sun J ，Gu Y ... -  《-》

Successful therapeutic vaccination with integrase defective lentiviral vector expressing nononcogenic human papillomavirus E7 protein.

Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.

Grasso F ，Negri DR ，Mochi S ，Rossi A ，Cesolini A ，Giovannelli A ，Chiantore MV ，Leone P ，Giorgi C ，Cara A ... -  《-》