AE36 HER2/neu-derived peptide linked to positively charged liposomes with CpG-ODN as an effective therapeutic and prophylactic vaccine for breast cancer

In the present day and age, cancer is still a life-limiting factor whose one of the therapeutic strategies is immunotherapy with vaccines. This research aims to prepare and characterize nanoliposomal vaccine formulation attached to HER2/neu-derived peptide (AE36) with or without CpG-ODN, and to evaluate its immunological responses to the therapy using BALB/c mice with HER2 overexpressing breast cancer. Methods: AE36 was conjugated to the liposomes containing DOTAP, DOPE and Cholesterol. Such formulations are able to produce CD8+ and CD4+ responses and induce synthesis of cytokines detectable via Enzyme-linked immunosorbent assay kits, cytotoxicity testing and intracellular cytokine staining combined with flow cytometry. Therapeutic and prophylactic effectiveness were evaluated through the formulation in samples. The highest effectiveness was found in DDC-peptide + CpG-ODN in both prophylactic and therapeutic studies, which decreased the size of tumors significantly and increased time of survival. These nanoliposomes linked to AE36 could be regarded as an appropriate candidate for the treatment and also for prophylaxis of HER2+ breast cancer; however further studies are needed.


Introduction
During 20 past years, cancer immunotherapy has drawn a great deal of scientific attention to itself due to ineffectiveness of other ways of treatment, such as chemotherapy, radiotherapy and surgery [[1]]. In cancer immunotherapy, cellular immunity could be induced when cytotoxic T lymphocytes (CTLs), particularly CD8+ T cells (Cytotoxic T cells with CD8 surface protein), are activated [ [2,3]]. The immune cells can recognize the cancer cells following active production of immune responses. Epidermal growth factor receptor proteins in human (HER2 or HER2/neu), are overexpressed in metastatic and non-metastatic cancer cells. This overexpression causes unrestricted cell division, which ultimately results in tumors. The immune system is stimulated via sequences of this protein and the sequences (AE36) are possibly applied in prophylaxis and treatment of HER2+ cancers. AE36 is taken into account as one of those sequences [ [4][5][6]]. Immunity against HER2/neu sequences could reduce tumor progression and improve prognosis [ [7,8]]. Liposomes are carriers for proteins or peptide antigens. Materials may attach to their outer surface, be encapsulated in the internal aqueous spaces, or be reconstituted with the liposomes lipid bilayers. These carriers prompt immune reactions by interactions with antigen presenting cells. Moreover, it has been demonstrated that antigens could be presented by liposomes to helper T lymphocytes. [ [9,10]]. Contents of liposomes can be reserved from environmental hazards before releasing at the target area [ [11]]. In the present research, AE36 (Ac-GVGSPYVSRLLGICL-NH2), as a peptide derived from HER2 intracellular domain containing 15 amino acids attached to the liposomes, was employed as a vaccine [ [12][13][14]]. AE36 is attached to the MHC II molecules producing T cell responses to peptides in mice and also synthesizes T lymphocytes in patients in vitro [ [15,16]]. Additionally, plasmacytoid dendritic cells and B cells are activated by CpG motifs in order to express Toll-like receptor 9 for generating an innate immune response. Generation of this response is attainable with production of T helper 1 (Th1) together with pro inflammatory cytokines synthesis, which is currently applied as an effective adjuvant in cancer immunotherapy studies [ [17][18][19]]. In the previous study, the peptide was encapsulated in liposome and here, the peptide was linked to liposome in order to get better effects [20]. In the present study, the effectiveness of AE36 peptide conjugated to liposomal formulation composed of DOTAP: DOPE: CHOLESTROL for the commencement of CTL response was evaluated in BALB/c mice model with TUBO breast cancer tumor, which shows the overexpression of HER2/neu oncogene.

Synthesis of AE36 peptide-PEG2000-DSPE
Thin layer chromatography was done to determine binding of Maleimide-PEG2000-DSPE to AE36 peptide. Disappearing the PEG2000-DSPE spot in mixture, revealed the end of the reaction (Fig.1.B).  Table 1 demonstrates the physical characteristics of liposomal formulations. Based on table 1, particle volume was in the range of 185.6± 3 nm and 200± 0.6nm prior to adding CpG-ODN and there were no significant differences in the particle volume in the prepared formulations (P.value>0.05).

Liposomes characteristics
Liposomes volume enhanced by adding CpG; however, zeta potential reduced. Moreover, particles revealed the polydispersity index (PdI) of approximately 0.2. .

Intracellular cytokine assay by flowcytometry on splenocytes
Flow cytometry was utilized for intracellular cytokine assay for IL-4 and IFN-γ on CD4+ and CD8+ T cell splenocytes. As shown in Figure 3, which is rather similar to result of ELISA assay, Peptide + CpG group was produced the highest amount of IFN-γ from CD8+ T-cells, yet it was not significant.

Fig 3 demonstrates Intracellular cytokine production in splenocytes of mice immunized with three booster doses of 10 µg AE36 in different liposomal vaccines. Fourteen days after the last booster, spleens of mice (three per group) were isolated and stained with PE labeled CD8 or CD4 surface markers, and then stained with anti-IFN-γ-FITC or anti-IL-4-PE antibodies, respectively. The frequency of IFN-γ producing cells within the CD8+ or CD4+ population (c & d) and IL-4 producing cells within the CD4+ population (a) were subsequently determined by FACSCalibur Cell analyzer. The data represent mean ± SEM (n = 3). (P.value<0.05)
compared to the buffer group (P.value < 0.0001) ( fig.4).

Prophylactic model of mice
The model was initiated by vaccination of the studied formulations for three times and a two-week interval. Two weeks after the last vaccination, the groups were exposed to TUBO cells. The animals were monitored in order to make sure whether there are mice with a touchable tumor at the injection (right flank) area. Weights and sizes were calculated until 3 mentioned situations occurred. As it is shown in fig.5 and table 2, DDC-P + CpG group decreased the size of tumor significantly. (P.value < 0.01). One of the mice in this group was cured and completely became tumor free. However, without any remarkable statistical significance, the survival time increased in the mentioned group compared to other groups.   (n = 5).

Discussion
In the previous study of ours, the peptide was encapsulated in liposome, whereas here, the peptide was linked to liposome to get better effects [ [20]]. AE36, as a sequence of HER2 protein, was employed for development of a liposomal base vaccine for prophylaxis as well as HER2+ breast cancer therapy.
In fact, the hybrid peptide AE37 includes Ii-Key (LRMK) plus AE36 (GVGSPYVSRLLGICL). Ii-Key, as a section of (MHC) class II invariant chain (Ii), facilitates the epitopes to be charged to MHC class II molecules [ [24]]. The vaccines have demonstrated immunologic responses to AE37 in vitro and in vivo, and also AE36 with or without GM-CSF (Granulocyte-macrophage colony-stimulating factor) in a dose-dependent manner [ [19]]. Not only does GM-CSF impose costs, but it is also associated with many adverse effects when applied in clinical settings; for instance, fever, myalgia, malaise, rash and reactions at the site of injection [ [25]].
In the present study, AE36 immunogenicity without Ii-Key peptide increased when linked to a positively charged liposome (DOTAP/DOPE/Cholesterol). As reported previously, positively charged liposomes can greatly induce cytotoxic T lymphocyte compared to the neutral or negatively charged liposomes [ [26]]. CD4+ T helper activation has implied remarkable effects on cancer immunotherapy done by AE37. CD4+and CD8+ immune responses were observed for AE37 and AE36 peptide in most patients [ [27]].
DOTAP phospholipid in the formulations produced positively charged liposomes leading to promotion of antigen transportation to macrophages and dendritic cells, which is due to quaternary ammonium components exhibits native adjuvanticity. Moreover, dendritic cells and chemokine induction could be stimulated with DOTAP, which stimulates migration of dendritic cells to lymph node and results in appropriate responses to antigen-specific CD8+T lymphocyte [ [28]].
Cholesterol in liposomal formulations enhance the in vivo liposomes stability. The more inflexible the structure is, the more CD8+T-cell responses would be stimulated. On top of that, secretion of antigens from cytoplasm increases with cholesterol. It also prevents vesicle lysosomal degradation.
In the liposome formulation, DOPE as a pH sensitive lipid is effective for presenting antigens to MHC I pathway as well as inducing CTL response [ [29][30][31][32]].
The immune system of vertebrates seems to have protective immune responses to CpG motifs that are shown to be Th1-based. Toll-like receptor 9 is expressed via dendritic and B cells for generation of an innate immune response to CpG motifs detectable through Th1 and pro inflammatory cytokines generation. CpG-ODN is a vaccine adjuvant in tumor immunization. CpG DNA is of lower toxicity than other adjuvants and stimulates stronger immune responses [ [33][34][35]].
Liposomes defend peptides from being degraded to the target area, like lymphocytes and cells which have antigen presentation. AE36 is able to attach to MHC II molecules of APC resulting in generation of T cell responses against peptides in cancer mouse models [ [36,37]].
Results of the present paper indicated that the stable particles and the liposomes sizes were between 186 -200 nm before adding CpG and 290 -303nm after adding it, CpG was found to be suitable to be appropriately delivered to the lymphatic system. Liposomes over 100 nm can generate a Th1 type of immune response versus those below 100 nm [ [38,39]]. Furthermore, particles net charge reduced Buffer 30 29±6 0 after addition of CpG to liposomes in view of the negatively charged CpG, which can electrostatistically bind to the liposome surface followed by neutralization of the DOTAP charge.

Materials
Peptide AE36 with the purity of more than 95% was produced by

Liposome preparation
Solution of lipids were mixed, dried to a thin film by rotary evaporation, and freeze-dried as mentioned before [ [20]]. Afterwards, hydration in HEPES/ dextrose buffer (10 mM HEPES with 5% dextrose, pH=7) was done. Lipid dispersed in buffer properly by vortexing and sonicating. Large vesicles were extruded with a mini extruder (Avestin, Canada) and polycarbonate membranes (200 and 100 nm) in order to form nanoparticles. Ultimately, 10µg peptide in micelle with 100µl of liposome were co-incubated at 50 oC for 4 hours under stirring. Liposomes were then put at 4° C under argon [ [21]].

Liposome characterization
Physical properties such as particle size, zeta potential and polydispersity index of liposomes were determined by dynamic light scattering (Malvern Instruments, Malvern, UK). Phosphate assay method was used to determine the amount of phospholipid in formulations [ [22]].

Immunization
BALB/c mice (Five mice per group) were immunized with different formulations (with or without CpG), three times with two-week intervals. Each dose of injection was 10 µg peptide for per mice. Some of the formulations were mixed with CpG ODN, 30 minutes before injection. The amount of CpG was 15µg for per animal. Two weeks after the last vaccination, three mice per group were sacrificed and their spleens were aseptically collected for evaluation of immune responses. Liposomes hydration buffer and liposome without any peptide (Empty liposomes) were used as control groups.
Where ITest, and I100% indicate Calcein AM intensity in splenocytes, and thoroughly lysed with 2% TX100, respectively. Iblank also represents the untreated cells intensity.

Prophylactic Model of TUBO Challenge
Five mice from each group were vaccinated three times with a two-week interval with different formulations. 14 days after the third vaccination, each mouse was subcutaneously injected with 5 × 105 TUBO cells in 50 μl PBS buffer. After five days, using a caliper, 3 orthogonal diameters a, b, c was calculated. Tumor volume was calculated with the following formula: [length × width × height] × 0.52) [ [23]]. Calculating the tumor size and studying survivals of mice continued until one of these conditions for euthanizing was observed: the tumor volume found to be over 1000 mm3; the body weight showed less than 15% of primary mass or lethargic or sick mice which were impossible to be nourished.

Therapeutic Model of TUBO Challenge
Primarily, five mice per group were challenged with subcutaneous injection of 5 × 105 TUBO cells in 50 μl PBS buffer in the right flank. When the dimension of the tumor reached the approximate size of 3 mm, the immunization schedule was subcutaneously done three times with a two-week interval. The tumor volume was measured employing this formula: ([length × width × height] × 0.52). The mice without any tumor were considered as tumor-free at the end of the experiment. The mice were euthanized provided that the tumor volume was greater than 1000 mm3, or the body weight reached below 15% of initial mass or the mice became lethargic, sick or unable to feed.

Statistical analysis
Two-way analysis ANOVA followed by Tukey's post-test were done in order to evaluate the significance of the differences among various formulations. The survival data was analyzed by logrank test between the groups. P <0.05 was considered to be significant. Graph Pad Prism 6 Software was applied for data analysis. The significant differences are shown as P. values of less than 0.05, 0.01, 0.001 and 0.0001 flagged with one to four stars, respectively.

Conclusions
In conclusion, the present findings implied that the anti-tumor immune activity of AE36 significantly increased by liposomal formulations rather than the control groups. Moreover, the mentioned formulations were found beneficial in the treatment as well as prophylaxis of breast cancer. Accordingly, they could be regarded as the appropriate and promising candidates for vaccine delivery which could delay the tumor growth rate of HER2+ breast cancer. Further studies are needed to confirm the clinical aspects of AE36 peptide linked liposomal formulations.