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

Abstract

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 1 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.There is consistent evidence that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types cause cervical cancer. 1 In contrast to the prophylactic HPV vaccines that exhibit great promise in reducing the burden of cervical cancer, there is limited progress towards the development of immune therapeutic strategies for those women already infected with HPV who do not benefit from the current vaccines. The efficacy of these HPV vaccines correlates with the presence of serum neutralizing antibodies which may prevent virus infection (reviewed in ref. 2). However, it is believed that T cell immune responses, in particular against E7 protein, may play an important role in viral clearance and resolution of HPV-induced lesions. 3 Thus, a vaccination strategy able to activate a strong T cell immune response with the capacity to recognize and kill cancer cells expressing HPV proteins might be considered as therapeutic alternative for cervical carcinoma.Dendritic cells (DCs) are considered to be at the centre of acquired T cell responses due to their outstanding ability to capture antigens (Ag) and process them for their presentation as short peptides in the context of MHC molecules to naïve T cells. The unique capacity of DC to elicit immune responses has prompted many laboratories to use DC in clinical trials. However, ex vivo manipulation of DC for the production of DC-based vaccines is expensive, time consuming and difficult to standardize. Indeed, several variables, such as DC lineage, antigen loading to...

Vaccine-induced but not tumor-derived Interleukin-10 dictates the efficacy of Interleukin-10 blockade in therapeutic vaccination

“…This immunogen, which we had previously demonstrated to be useful in the treatment of TC-1 tumors, 32 contains HPVE7 protein bound to the 91-amino acid extra domain A (EDA) from fibronectin, a TLR4 ligand which targets antigens to DC and induces its maturation, promoting the induction of T cell responses. As in B16-OVA-bearing mice, no clear differences were observed in tumor homogenates (data not shown), whereas serum IL-10 levels increased after vaccination (Fig.…”

“…Proteins containing the EDA from fibronectin EDA-OVA and EDA-HPV were produced as described. 32,42 Endotoxin levels in these proteins were below 0.2 EU/mg protein. OVA protein (low endotoxin) was purchased from Hyglos (321001).…”

Inhibition of FOXP3/NFAT Interaction Enhances T Cell Function after TCR Stimulation

“…In the second model, TC-1 P3 (A15) cells (5 3 10 5 cells/mouse) with a downregulated MHC class I expression (27) were injected s.c. in C57BL/6 mice (n = 6). Five days later, mice were vaccinated with 3 nmol recombinant fusion protein EDA-HPVE7 in saline (vaccine) (28). A group of vaccinated mice received also peritumoral administration of the peptide FOXP3 393-403 (a daily administration of 100 mg peptide per mouse from day 5 to day 15).…”

The extradomain A of fibronectin (EDA) combined with poly(I:C) enhances the immune response to HIV-1 p24 protein and the protection against recombinant Listeria monocytogenes-Gag infection in the mouse model

“…hepatitis C virus peptide [13] or Human Papilomavirus E7 protein [14]. We have also observed that EDA exerts its adjuvant effect more efficiently when delivered not far 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 apart but in close contact with the antigen (San Román et al, submitted for publication).…”

“…Altogether, these observations suggest that the expression of EDA in response to tissue injury, or other cellular warning signals, might induce recruitment of dendritic cells to the site of injury and trigger their subsequent maturation. Indeed, previous work demonstrates that EDA, as fusion protein, may constitute a strategy to target viral or tumoral antigens to dendritic cells in vivo [11,13,14]. Therefore, the capacity of EDA to induce dendritic cell maturation through TLR4 activation may also favor antigen uptake, expression of co-stimulatory signals, antigen presentation and the induction of anti-viral or antitumoral T cell responses [11,13].…”