Peter Öhlschläger

Human Papillomavirus Type 16 L1 Capsomeres Induce L1-Specific Cytotoxic T Lymphocytes and Tumor Regression in C57BL/6 Mice

ABSTRACT We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1165-173 peptide for ex vivo restimulation of splenocytes prior to analysis (51Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a Db-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and 51Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1ΔN10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and 51Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins.

Enhancement of immunogenicity of a therapeutic cervical cancer DNA‐based vaccine by co‐application of sequence‐optimized genetic adjuvants

Treatment of patients with cervical cancer by conventional methods (mainly surgery, but also radiotherapy and chemotherapy) results in a significant loss in quality of life. A therapeutic DNA vaccine directed to tumor‐specific antigens of the human papilloma virus (HPV) could be an attractive treatment option. We have developed a nontransforming HPV‐16 E7‐based DNA vaccine containing all putative T cell epitopes (HPV‐16 E7SH). DNA vaccines, however, are less immunogenic than protein‐ or peptide‐based vaccines in larger animals and humans. In this study, we have investigated an adjuvant gene support of the HPV‐16 E7SH therapeutic cervical cancer vaccine. DNA encoded cytokines (IL‐2, IL‐12, GM‐CSF, IFN‐γ) and the chemokine MIP1‐α were co‐applied either simultaneously or at different time points pre‐ or post‐E7SH vaccination. In addition, sequence‐optimized adjuvant genes were compared to wild type genes. Three combinations investigated lead to an enhanced IFN‐γ response of the induced T cells in mice. Interestingly, IFN‐γ secretion of splenocytes did not strictly correlate with tumor response in tumor regression experiments. Gene‐encoded MIP‐1α applied 5 days prior to E7SH‐immunization combined with IFN‐γ or IL‐12 (3 days) or IL‐2 (5 days) postimmunization lead to a significantly enhanced tumor response that was clearly associated with granzyme B secretion and target cells lysis. Our results suggest that a conditioning application and combination with adjuvant genes may be a promising strategy to enhance synergistically immune responses by DNA immunization for the treatment of cervical cancer.

Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7

To allow vaccination irrespective of HLA type, DNA vaccines encoding full‐length antigens are required. However, here, we demonstrate that the immunogenicity of DNA vaccines encoding the full‐length human papillomavirus (HPV) type 16 E7 and E6 proteins is highly reduced compared to vaccines encoding only the immunodominant epitope. Furthermore, the low remaining immunogenicity is essentially lost for both E7 and E6 when a nononcogenic “gene‐shuffled” variant is utilized. To address these issues, we tested whether alterations in transgene design can restore the immunogenicity of full‐length and gene‐shuffled DNA vaccines. Remarkably, genetic fusion of E7 with tetanus toxin fragment C (TTFC) resulted in a dramatic increase in immunogenicity both for the full‐length and the gene‐shuffled version of E7. Moreover, the TTFC fusion vaccines were more immunogenic than a vaccine encoding a fusion of E7 and mycobacterial heat shock protein‐70, which has recently been tested in a clinical trial. Interestingly, vaccination with these TTFC fusion vaccines also resulted in extremely persistent T‐cell responses. The E7‐specific CD8+ T cells induced by TTFC fusion vaccines were functional in terms of IFN‐γ production, formation of immunological memory, in vivo cytolytic activity and tumor eradication. Finally, we show that genetic fusion with TTFC also improves the immunogenicity of a gene‐shuffled E6 DNA vaccine. These data demonstrate that genetic fusion with tetanus toxin fragment C can dramatically improve the immunogenicity of full‐length and gene‐shuffled DNA vaccines. The DNA fusion vaccines developed here will be evaluated for the treatment of HPV‐positive carcinomas in future studies.

Titanium Salan Complexes Displays Strong Antitumor Properties In Vitro and In Vivo in Mice

The anticancer activity of titanium complexes has been known since the groundbreaking studies of Köpf and Köpf-Maier on titanocen dichloride. Unfortunately, possibly due to their fast hydrolysis, derivatives of titanocen dichloride failed in clinical studies. Recently, the new family of titanium salan complexes containing tetradentate ONNO ligands with anti-cancer properties has been discovered. These salan complexes are much more stabile in aqueous media. In this study we describe the biological activity of two titanium salan complexes in a mouse model of cervical cancer. High efficiency of this promising complex family was demonstrated for the first time in vivo. From these data we conclude that titanium salan complexes display very strong antitumor properties exhibiting only minor side effects. Our results may influence the chemotherapy with metallo therapeutics in the future.

T cell response to human papillomavirus 16 E7 in mice: Comparison of Cr release assay, intracellular IFN-γ production, ELISPOT and tetramer staining

Successful vaccination against infections by high-risk papillomaviruses aiming at the prevention of cervical cancer most likely requires the induction of neutralizing antibodies and human papillomavirus (HPV)-specific T cells directed against early viral proteins such as E7. Whereas the technology for detection of antibodies is well established, measurement of T cells is more cumbersome and standardization of assays is difficult. By using chromium release assay, ELISPOT, tetramer staining and intracellular IFN-γ assay, we compared the levels of HPV 16 E7-specific T cells obtained after immunization of C57BL/6 mice with different DNA expression vectors. We found that all four assays gave highly comparable results. ELISPOT can be recommended for future studies as it indicates the presence of activated (i.e. IFN-γ-secreting) T cells in a quantitative manner and combines high sensitivity with relatively low T cell demand.

Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice

BackgroundHuman papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer.MethodsIn this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 – the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize γ-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems.ResultsHigh-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression.ConclusionsThe fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs.

Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4.

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

Design of a highly effective therapeutic HPV16 E6/E7-specific DNA vaccine: optimization by different ways of sequence rearrangements (shuffling).

Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism.

Design, synthesis, in vitro stability and cytostatic effect of multifunctional anticancer drug‐bioconjugates containing GnRH‐III as a targeting moiety

Bioconjugates containing the GnRH-III hormone decapeptide as a targeting moiety are able to deliver chemotherapeutic agents specifically to cancer cells expressing GnRH receptors, thereby increasing their local efficacy while limiting the peripheral toxicity. However, the number of GnRH receptors on cancer cells is limited and they desensitize under continuous hormone treatment. A possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of the bioconjugates would be the attachment of more than one chemotherapeutic agent to one GnRH-III molecule. Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing GnRH-III as a targeting moiety and daunorubicin as a chemotherapeutic agent. Two different drug design approaches were pursued. The first one was based on the bifunctional [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) containing two lysine residues in positions 4 and 8, whose ε-amino groups were used for the coupling of daunorubicin. In the second drug design, the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was used as a scaffold; an additional lysine residue was coupled to the ϵ-amino group of (8) Lys in order to generate two free amino groups available for conjugation of daunorubicin. The in vitro stability/degradation of all synthesized compounds was investigated in human serum, as well as in the presence of rat liver lysosomal homogenate. Their cellular uptake was determined on human breast cancer cells and the cytostatic effect was evaluated on human breast, colon and prostate cancer cell lines. Compared with a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect.

An artificial PAP gene breaks self-tolerance and promotes tumor regression in the TRAMP model for prostate carcinoma

Prostate cancer (PCa) is the most commonly diagnosed type of cancer in men in western industrialized countries. As a public health burden, the need for the invention of new cost-saving PCa immunotherapies is apparent. In this study, we present a DNA vaccine encoding for the prostate-specific antigen prostatic acid phosphatase (PAP) linked to the J-domain and the SV40 enhancer sequence. The PAP DNA vaccine induced a strong PAP-specific cellular immune response after electroporation (EP)-based delivery in C57BL/6 mice. Splenocytes from mice immunized with PAP recognized the naturally processed PAP epitopes, indicating that vaccination with the PAP-J gene broke its self-tolerance against PAP. Remarkably, DNA vaccination with PAP-J inhibited tumor growth in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model that closely resembled human PCa. Therefore, this study highlights a novel cancer immunotherapy approach with the potential to control PCa in clinical settings.Prostate cancer (PCa) is the most commonly diagnosed type of cancer in men in western industrialized countries. As a public health burden, the need for the invention of new cost-saving PCa immunotherapies is apparent. In this study, we present a DNA vaccine encoding for the prostate-specific antigen prostatic acid phosphatase (PAP) linked to the J-domain and the SV40 enhancer sequence. The PAP DNA vaccine induced a strong PAP-specific cellular immune response after electroporation (EP)-based delivery in C57BL/6 mice. Splenocytes from mice immunized with PAP recognized the naturally processed PAP epitopes, indicating that vaccination with the PAP-J gene broke its self-tolerance against PAP. Remarkably, DNA vaccination with PAP-J inhibited tumor growth in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model that closely resembled human PCa. Therefore, this study highlights a novel cancer immunotherapy approach with the potential to control PCa in clinical settings.