Patent ID: 12220387

EXAMPLES

In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: In Vitro Human Dendritic Cell Maturation

The goal of this study was to investigate the capacity of a complex for use according to the present invention to induce maturation of dendritic cells. In the present study, the complex for use according to the present invention is a fusion protein, comprising the cell-penetrating peptide “Z13”, a protein “MAD5”, which consists of different CD8+and CD4+epitopes from various antigens, and the TLR4 peptide agonist “EDA”. Accordingly, a fused protein with the EDA peptide at the N-terminal position and different control conjugated proteins without Z13 or EDA or both were designed.

Namely, the following constructs were designed, whereby in the amino acid sequence the cell-penetrating peptide “Z13” is shown underlined and the TLR peptide agonist “EDA” is shown in italics:

EDAZ13Mad5

Sequence:

[SEQ ID NO: 26]MHHHHHHNIDRPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGEDDTAELQGLR PGSEYTVSVVALHDDMESQPLIGIQSTKRY KNRVASRKSR AKFKQLLQHYREVAAAKSSE NDRLRLLLKE SLKISQAVHA AHAEINEAGREVVGVGALKV PRNOQDWLGVP RFAKFASFEA QGALANIAVDKANILDVEQLE SIINFEKLTE WTGS

Molecular weight: 25′057 Da

Characteristics:

Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopesContains EDA TLR agonist (Lasarte, J. J., et al., The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo. J Immunol, 2007. 178(2): p. 748-56)Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM DTT, 1 M L-Arginine, pH 8Endotoxin level: <0.01EU/ug
Z13Mad5

[SEQ ID NO: 29]MHHHHHHKRY KNRVASRKSR AKFKQLLQHY REVAAAKSSENDRLRLLLKE SLKISQAVHA AHAEINEAGR EVVGVGALKVPRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLESIINFEKLTE WTGS

Molecular weight: 15′196 Da

Characteristics:

Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopesStorage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM DTT, 1 M L-Arginine, pH 9Endotoxin level:Batch 1: 0.32EU/mgBatch 2: 0.44EU/mg
Mad5

Sequence:

[SEQ ID NO: 30]MHHHHHHE SLKISQAVHA AHAEINEAGR EVVGVGALKVPRNQDWLGVP RFAKEASFEA QGALANIAVD KANLDVEQLESIINFEKLTE WTGS

Molecular weight: 10′154.6 Da

Characteristics:

Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopesStorage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM DTT, 0.5 M L-Arginine, pH 8Endotoxin level: 0.069EU/mg

The EDAZ13Mad5, Z13Mad5 and Mad5 proteins were investigated for their capacity to induce human dendritic cell (DC) maturation. After incubation during 48 h with 300 nM of protein, activation markers expression (CD86, CD40, CD83 and HLA-DR) was assessed on the human DCs by FACS (FIGS.1-4). Specific buffers of each protein were used as negative controls.

Results are shown for CD40 inFIG.1, for CD86 inFIG.2, for HLADR inFIG.3, and for CD83 inFIG.4. Whereas EDAZ13Mad5 induced maturation of human DCs, shown by the up-regulation of CD86, HLADR and CD83, Z13Mad5 and Mad5 proteins were not able to activate human DCs. These results indicate that the EDA portion of the protein is responsible for the up-regulation of the activation markers on the human DCs.

Example 2: In Vitro Epitope Presentation (MHC 1)

The goal of this study was to assess functional M HC class I-restricted cross-presentation in a murine in an vitro system using bone marrow derived dendritic cells (BMDCs) and splenocytes from different TCR transgenic mice. To this end, the constructs EDAZ13Mad5 and Mad5 (described above in Example 1) and the construct EDAMad5 were used:

EDAMad5

Sequence

[SEQ ID NO: 31]MHHHHHHNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGI RELFPAPDGEDDTAELQGLR PGSEYTVSVVALHDDMESQPLIGIQSTE SLKISQAVHA AHAEINEAGREVVGVGALKV PRNQDWLGVP RFAKFASFEA QGALANIAVDKANLDVEQLE SIINFEKLTE WTGS

Molecular weight: 20′017 Da

Characteristics:

Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopesContains EDA TLR agonistStorage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM DTT, 0.5 M L-Arginine, pH 8Endotoxin level: 1.8EU/mg

BMDCs were loaded overnight with 300 nM of with the EDAMad5, EDAZ13Mad5 and Mad5 proteins containing OVACD8, OVACD4 and gp100 epitopes. Processing and presentation of these MHC I-restricted OVACD8 and gp100 epitopes were monitored by measuring the in vitro proliferation of naïve OVA257-264-specific CD8+T cells from OT-1 T cell receptor (TCR) transgenic mice and gp100-specific CD8+T cells from P-mel T cell TCR transgenic mice respectively. Accordingly, efficient MHC class I-restricted presentation of OVACD8 epitope and gp100 epitope was monitored after 4 days with CFSE-labeled OT1 cells and P-Mel cells respectively. Processing and presentation of MHC II-restricted OVACD4 epitope was monitored by measuring the in vitro proliferation of naïve OVA323-339-specific CD4+T cells from OT-2 T cell receptor (TCR) transgenic mice. Accordingly, efficient M HC class II-restricted presentation of OVACD4 epitope was monitored after 4 days with CFSE-labeled OT2 cells. As control, BMDCs were pulsed for 1 h with 5 uM peptide (one experiment representative of 2 individual experiments).

Results are shown inFIG.5. Similar cross-presentation and processing capacity of all assessed Mad5-based proteins were observed.

Example 3: CD8 T Cell Immune Response

To investigate the efficacy of EDA-conjugated proteins in inducing polyclonal CD8+T cell response, C57BL/6 mice were vaccinated twice (Wk0 and Wk2), by subcutaneous injection of either 2 nmol or 10 nmol of the constructs EDAZ13Mad5 or EDAMad5 (described in Examples 1 and 2). Positive control group was vaccinated with Mad5 and the TLR4 agonist MPLA (equimolar to EDA). Two doses were assessed 2 nmol of the construct (FIG.6) and 10 nmol of the construct (FIG.7). 3-4 mice were used per group.

Seven days after the last vaccination, mice were bled and pentamer staining was performed to monitor the OVA-specific immune response in the blood. InFIG.8, the percentage of pentamer positive CD8+ T cells is shown for all groups and both doses tested.

These data show that interestingly the immune response is lower at 10 nmol compared to 2 nmol. At both doses, 2 nmol and 10 nmol, the vaccine mediated immune response was observed more consistently in the EDAZ13Mad5 group in contrast to the EDAMad5 group. Moreover, there is an increased immune response when the TLR4 agonist is conjugated with the vaccine.

Example 4: Vaccine Efficacy on Tumor Growth in a Benchmark EG.7-OVA Tumor Model

To evaluate the effect of EDA construct proteins on tumor growth control, the s.c. model of EG.7-OVA thymoma cells was chosen. C57BL/6 mice were implanted s.c. with 3×105EG7-OVA tumor cells in the left flank. After tumor implantation, mice were vaccinated at day 5 and 13 with 10 nmol of one of the following constructs (cf. Examples 1 and 2 for construct description): EDAZ13Mad5, EDAMad5, Mad5, or Mad5 and MPLA (equimolar to EDA) s.c. in the right flank. Tumor size was measured with a caliper.

FIG.9shows the tumor growth of 7 mice per group (mean±SEM); *, p<0.05 EDAZ13Mad5 versus control group (2-way Anova test).FIG.10shows individual tumor growth curves (7 individual mice per group).FIG.11Ashows the survival curve of 7 mice per group; *, p<0.05 EDAZ13Mad5 versus control group (Log-rank test).FIG.11Bshows the tumor-free progression curve of 7 mice per group; *, p<0.05 EDAZ13Mad5 versus control group (Log-rank test).

The results show that in a therapeutic setting, EDAZ13Mad5 was the only protein vaccine to significantly control the tumor growth compared to the control group with a significant better tumor free progression curve and survival curve.

The results therefore suggest that the construct protein EDAZ13Mad5 is a highly potent vaccine for controlling the tumor growth in a therapeutic setting.

Example 5: Vaccine Efficacy on Tumor Growth in a Melanoma Metastasis Model

To assess the efficacy in a lung metastasis model using B16-OVA tumor cells in a semi-therapeutic setting, different construct proteins were used: EDAMad5, EDAZ13Mad5, Z13Mad5+MPLA (cf. Examples 1 and 2 for design of the constructs), and MPLA alone. C57BL/6 mice were implanted i.v. with 1×105B16-OVA melanoma tumor cells and at the same time (d0) 2 nmol of the vaccine (EDAMad5, EDAZ13Mad5, Z13Mad5+MPLA, MPLA alone) was administered by subcutaneous injection in the right flank. Nine days later, mice were vaccinated a second time with the same dose. Further control groups were vaccinated with 2 nmol of Z13Mad5 and the TLR4 agonist MPLA (equimolar to EDA) or MPLA alone. Mice were euthanized at day 13 and lung recovered. Number of metastasis foci was counted for each lung. The results are shown inFIG.12.

The results show that the conjugate EDAZ13Mad5 is as potent as Z13Mad5+MPLA to inhibit tumor metastasis in the lung. Furthermore, EDA-Mad5 is less potent than EDAZ13Mad5, indicating a crucial role of Z13 in vaccine efficacy.

Example 6: Vaccine Efficacy on Tumor Growth in a Melanoma Metastasis Model—Prophylactic Setting

Furthermore, the efficacy of the different construct proteins EDAMad5, EDAZ13Mad5, and Z13Mad5+MPLA (cf. Examples 1 and 2 for design of the constructs) was assessed in a lung metastasis model in a prophylactic setting. C57BL/6 mice were vaccinated 21 and 7 days before implantation of tumor cells (d-21 and d-7) by subcutaneous injection of 2 nmol of EDAZ13Mad5, EDAMad5 or Z13Mad5+MPLA (equimolar to EDA) s.c. in the right flank. At day 0, mice were implanted i.v. with 1×105B16-OVA melanoma tumor cells. Mice were euthanized at day 14 and lung recovered. Results are shown inFIG.13.

Example 7: Design of Further Constructs Comprising a TLR2 Peptide Agonist

Herein, the complex for use according to the present invention is again a fusion protein, comprising the cell-penetrating peptide “Z13”, the protein “MAD5”, which consists of different CD8+and CD4+epitopes from various antigens, and the TLR2 peptide agonist “Anaxa”. Accordingly, fused proteins with the Anaxa peptide at the C-terminal or N-terminal position were designed.

Namely, the following constructs were designed, whereby in the amino acid sequence the cell-penetrating peptide “Z13” is shown underlined and the TLR peptide agonist “Anaxa” is shown in italics:

AnaxaZ13Mad5

Sequence:

MHHHHHHSTV HEILCKLSLE GDHSTPPSAY GSVKPYTNFDAEKRYKNRVA SRKSRAKFKQ LLQHYREVAA AKSSENDRLRLLLKESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQDWLGVPRFAKF ASFEAQGALA NIAVDKANLD VEQLESIINFEKLTEWIGS

Molecular weight: 18973 Da

Characteristics:

Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopesContains the 35-mer peptide of AnnexinStorage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM DTT, 0.5 M L-Arginine, pH 8Endotoxin level: 5.17 EU/mg
Z13Mad5Anaxa

Sequence:

[SEQ ID NO: 28]MHHHHHHKRYKNRVA SRKSRAKFKQ LLQHYREVAA AKSSENDRLRLLLKESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQDWLGVPRFAKF ASFEAQGALA NIAVDKANLD VEQLESIINFEKLTEWTGSSTVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAE

Molecular weight: 18973 Da

Characteristics:

Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopesContains the 35-mer peptide of AnnexinStorage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM DTT, 0.5 M L-Arginine, pH 8

Endotoxin level: 3.1 EU/mg

Example 8: TLR2 Binding (HEK-hTLR2 Cell Lines

The goal of this study was to assess whether the Z13Mad5Anaxa and AnaxaZ13Mad5 construct proteins (cf. Example 7 for design of these construct proteins) were able to bind TLR2 as an agonist. HEK-Blue™ hTLR2 were seeded in flat 96-well plate in culture medium, stimulated with 0.3 μM, 1 μM or 3 μM of AnaxaZ13Mad5 or Z13Mad5Anaxa and incubated at 37° C. for 24 h. Positive control was performed with 500 ng/ml of Pam3CSK4, a TLR2 agonist.

To monitor the activation of NF-κB/AP1, twenty microliters of the supernatant were added to QuantiBlue® detection medium and incubated at 37° C. for 1 h before OD reading (620 nm). Results are shown inFIG.14A.

The secretion of IL-8 in the supernatant was quantified by ELISA. Results are shown inFIG.14B.

Results (FIG.14A, B) showed that Z13Mad5Anaxa and AnaxaZ13Mad5 are similarly able to bind to TLR2 in a dose dependent manner.

Example 9: In Vivo Induction of Specific CD8+T Cells

To investigate the efficacy of the Anaxa-conjugated proteins of Example 7 in the induction of CD8+T cell responses, C57BL/6 mice were vaccinated twice (Wk0 and Wk2), by subcutaneous injection of 2 nmol of AnaxaZ13Mad5 or 2 nmol of Z13Mad5Anaxa. Seven days after the last vaccination, mice were bled and to monitor the OVA-specific immune response in the blood, pentamer staining was performed (one experiment with 4 mice per group). Results are shown inFIGS.15and16.

These data indicate that both, the Z13Mad5Anaxa vaccine and the AnaxaZ13Mad5 construct, elicit a strong immune response.

Example 10: Therapeutic Effect on Tumor Growth

To evaluate the effect of the Anaxa-conjugated construct proteins designed in Example 7 on tumor growth control, a benchmark tumor model was used, namely the s.c. implantation of EG.7-OVA thymoma cells.

C57BL/6 mice were implanted s.c. with 3×105EG7-OVA tumor cells in the left flank. After tumor implantation, the three groups of 7 mice each were vaccinated s.c. in the right flank at day 5 and 13 by subcutaneous injection of 10 nmol of either AnaxZ13Mad5 (group 1), Z13Mad5Anaxa (group 2) or Z13Mad5 and Pam3CSK4 (equimolar to Anaxa; group 3). In order to compare the effect to a protein mixed with an external adjuvant, a control group was vaccinated with Z13Mad5 and Pam3CSK4 (equimolar to Anaxa). Tumor size was measured with a caliper. Results are shown inFIG.17-19.

In a therapeutic schedule, Z13Mad5Anaxa and AnaxaZ13Mad5 are better protein vaccines for controlling tumor growth compared to the control group, i.e. co-injection of Z13Mad5 and Pam3CSK showing a significant better survival curve. In particular, Z13Mad5Anaxa and AnaxaZ13Mad5 demonstrate significantly higher efficacy than Z13Mad5 administrated separately with Pam3CSK4. The results therefore suggest that the construct proteins Z13Mad5Anaxa and AnaxaZ13Mad5 are promising conjugate-vaccines for controlling the tumor growth in a therapeutic setting.

Example 11: Therapeutic Effect on Tumor Growth—Comparison of Constructs with Different TLR Agonists

The goal of this study was to compare the efficacy of the different construct protein vaccines conjugated to different TLR agonist, namely EDAZ13Mad5 and Z13Mad5Anaxa of Example 1 and 7, on tumor growth control. To this end, C57BL/6 mice were implanted s.c. with 3×105EG.7-OVA thymoma cells in the left flank as described previously in Example 10. Mice (7 individual mice per group) were vaccinated s.c. in the right flank at day 5 and 13 with 2 nmol of either EDAZ13Mad5, Z13Mad5Anaxa or co-injection of Z13Mad5+MPLA (equimolar to EDA).

Results are shown inFIGS.20,21and22. In this experimental setting, Z13Mad5Anaxa, EDAZ13Mad5, and Z13Mad5+MPLA were similarly able to significantly control tumor growth. Moreover, these data indicate that Z13Mad5Anaxa is the best construct to significantly control tumor growth and EDAZ13Mad5 was slightly better than Z13Mad5+MPLA in this experimental setting.

Example 12: Dose Effect of Z13Mad5Anaxa on Tumor Growth Control

To identify the optimal dose of the conjugate vaccine, three different doses (0.5 nmol, 2 nmol and 10 nmol) of Z13Mad5Anaxa (cf. Example 7) were assessed for their ability to control tumor growth. The dose effect of Z13Mad5Anaxa construct was evaluated in the s.c. model of EG.7-OVA thymoma cells as described previously in Example 10. After tumor implantation, mice were vaccinated twice (at day 5 and at day 13 after tumor implantation) in a therapeutic setting at 0.5, 2 or 10 nmol of Z13Mad5Anaxa.

C57BL/6 mice were implanted s.c. with 3×105EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13) by subcutaneous injection of either 0.5 nmol, 2 nmol or 10 nmol of Z13Mad5Anaxa in the right flank. Tumor size was measured with a caliper.

The tumor growth of 7 mice per group is depicted inFIG.23. Those data show that the doses of 0.5 and 2 nmol are at least as efficacious as 10 nmol for controlling tumor growth.

Example 13: Effect of Different Routes of Administration of Z13Mad5Anaxa

This study was based on the previous Examples demonstrating the efficacy of Z13Mad5Anaxa conjugate vaccine (cf. Example 7), which is able to elicit specific immune responses and is efficacious for controlling tumor growth in the subcutaneous tumor model EG7 as shown above. To investigate the effect of subcutaneous, intramuscular and intradermal routes of administration, immune responses elicited by subcutaneous, intramuscular and intradermal injection were compared. Intradermal injections were performed using the PLEASE® device from Pantec Biosolutions.

Mice were vaccinated three times every two weeks (Wk0, Wk2 and Wk4) with 0.5 or 2 nmol of Z13Mad5Anaxa (cf. Example 7). In order to target several lymph nodes, the 1st and the 3rd vaccinations were performed in the right flank whereas the 2nd was done in the left flank. SIINFEKL-specific CD8+ T cell response was analyzed 1 week after the 2nd and the 3rd vaccination in the blood.FIG.24shows the SIINFEKL-specific CD8 T cell responses after each vaccination detected in the blood of C57BL/6 mice vaccinated three times (Wk0, Wk2 and Wk4) s.c., i.d. or i.m. with 0.5 nmol (FIG.24A) or 2 nmol (FIG.24B) of Z13Mad5Anaxa. Blood was obtained from mice 7 days after the 2nd and the 3rd vaccination and multimer staining was performed (one experiment with 4 mice per group).

The results indicate that at the two doses assessed (0.5 and 2 nmol), (i) all routes of administration tested elicited a SIINFEKL-specific CD8 immune response and (ii) the subcutaneous vaccination elicited the strongest SIINFEKL-specific CD8 immune response. For subcutaneous administration, the maximum response was reached after the 3nd vaccination and still maintained after the 3rd vaccination. The SIINFEKL-specific CD8 immune response after the 2nd vaccination elicited by intradermal and intramuscular vaccinations is lower compared to subcutaneous vaccination and is not enhanced after the 3rd vaccination.

Next, the effector function and the exhaustion status of SIINFEKL-specific CD8 T cells was evaluated by analyzing KLRG 1 (Killer cell lectin-like receptor subfamily G member 1) and PD-1 respectively.

To this end, C57BL/6 mice were vaccinated three times (Wk0, Wk2 and Wk4) s.c., i.d. or i.m. with 2 nmol of Z13Mad5Anaxa (cf. Example 7). Blood was obtained from mice 7 days after the 2nd and the 3rd vaccination and FACS staining was performed. KLRG1 and PD-1 expression were analyzed on multimer-positive CD8 T cells (one experiment with 4 mice per group). Results are shown inFIG.25.

These data indicate that the expression of KLRG 1 is strongly increasing on SIINFEKL-specific CD8 T cells after subcutaneous vaccination. After i.d. or i.m. vaccination, the observed effects were lower. The percentage of KLRG 1-positive cells among SIINFEKL-specific CD8 T cells is also enhanced after s.c. vaccination (data not shown).

In contrast to KLRG 1, PD-1 expression is decreasing with the time and the vaccinations, for subcutaneous and intramuscular vaccination routes. This suggests that SIINFEKL-specific CD8 T cells are not exhausted. The percentage of PD1-positive cells among SIINFEKL-specific CD8 T cells is also reduced after s.c. and i.m. vaccination (data not shown). It is important to note that PD-1 expression is higher after the 2nd vaccination when mice were vaccinated subcutaneously, reflecting the early activation status of specific T cells (Keir, M. E., et al., PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol, 2008. 26: p. 677-704).

The expression of the late exhaustion marker Tim-3 was also analyzed. A very low expression as observed for all groups.

Taken together, results indicate that subcutaneous vaccination elicits the best specific CD8 immune response compared to intramuscular or intradermal injections.

Example 14: Intranodal Route of Administration

Based on the previous experiments (Example 13), the intranodal route of administration was additionally investigated. To this end, the immune response elicited by intranodal injection of Z13Mad5Anaxa (cf. Example 7) was investigated.

For this purpose, mice were first injected with Evans Blue subcutaneously in order to allow easily visualizing the lymph nodes for injection and inject intranodally without invasive surgery, for example as described in Jewell, C. M., S. C. Lopez, and D. J. Irvine, In situ engineering of the lymph node microenvironment via intranodal injection of adjuvant-releasing polymer particles. Proc Natl Acad Sci US A, 2011. 108(38): p. 15745-50.

C57BL/6 mice were vaccinated two times every two weeks (Wk0 and Wk2) intranodally with 0.5 nmol of Z13Mad5Anaxa (cf. Example 7). The 1st vaccination was performed in the right inguinal lymph node, whereas the second vaccination was done in the left inguinal lymph node. Blood was obtained from mice 7 days after the 2nd vaccination and multimer staining was performed (3 mice per group). In other words, SIINFEKL-specific CD8+ T cell response was analyzed one week after the 2nd vaccination in the blood.FIG.26shows the SIINFEKL-specific CD8 T cell responses. Those data indicate that also intranodal injection was able to elicit SIINFEKL-specific CD8 T cells.

Example 15: Vaccination Schedule

The vaccination schedule evaluation work was initiated with the objective to identify the impact of the third vaccination using the same Z13Mad5Anaxa construct as described above (cf. Example 7). The subcutaneous route was chosen given the previous results.

In the experiment first two vaccinations were performed at wk0 and wk2 with a 3rd vaccination either at wk4 (FIG.27A) or at wk8 (FIG.27B). Thus, C57BL/6 mice were vaccinated three times (FIGS.27Aand C: Wk0, Wk2 and Wk4 andFIGS.27Band D: Wk0, Wk2 and Wk8) s.c. with 2 nmol of Z13Mad5Anaxa. Blood was obtained from mice 7 days after last vaccination and pentamer staining was performed (one experiment with 4 mice per group). Accordingly, SIINFEKL-specific CD8+ T cell response was analyzed 1 week after the 2nd and the 3rd vaccination (FIGS.27Aand B). Additionally, the effector function of SIINFEKL-specific T cells was evaluated by analyzing the expression of KLRG 1 on specific CD8 T cells (FIGS.27Cand D).

The data indicate that compared to control the percentage of SIINFEKL-specific CD8 T cells was significantly increased at all time points tested (Vac2 and Vac3) as well as in both vaccination schedules (FIGS.27Aand B).

Interestingly, the third vaccination at Wk4 allowed to most prominently increasing the percentage of SIINFEKL-specific CD8 T cells (FIG.27A). The same cells also demonstrate an improved effector function through higher KLRG 1 expression (FIG.27C). In contrast, with a third vaccination performed at Wk8 no improvement from the second to the third vaccination could be observed in the SIINFEKL-specific immune response and in the KLRG 1 expression.

Taken together, these results indicate that the CD8 immune response could be increased by shorten the delay between the second and the third vaccination.

Given that an earlier third vaccination seems to increase immune response, in the next study two short schedules of vaccination were investigated:i) three vaccinations at day 0, day 3 and day 7 andii) three vaccinations at day 0, day 7 and day 14.

Again, C57BL/6 mice were used and vaccination was performed s.c. with 0.5 nmol of Z13Mad5Anaxa (cf. Example 7). Multimer staining was performed on blood samples obtained one week after the 2nd and the 3rd vaccination (one experiment with 4 mice per group).

Thus, SIINFEKL-specific CD8+ T cell response was analyzed one week after the 2nd and the 3rd vaccination (FIGS.28Aand D). Additionally, the effector function of SIINFEKL-specific T cells was evaluated by analyzing the expression of KLRG 1 on specific CD8 T cells (FIGS.28B and28E) and the exhaustion status by analyzing the PD-1 expression of specific T cells (FIGS.28C and28F).

The data indicate that—similarly to the first study regarding the vaccination schedule described above—compared to control the percentage of SIINFEKL-specific CD8 T cells was increased at all time points tested (Vac2 and Vac3) as well as in both vaccination schedules (FIGS.28Aand B).

However, compared to the schedule wk0-wk2-wk4, a schedule with vaccinations at Day0, Day3 and Day7 did not elicit such a high SIINFEKL-specific CD8 T cell immune response. Concerning the schedule with vaccinations at Day0, Day7 and Day14, the SIINFEKL-specific CD8 T cell immune response elicited is better compared to the previous schedule (d0-d3-d7) but is not maintained after the 3rd vaccination.

Taken together, vaccination schedule data set indicates that the Wk0-Wk2-Wk4 vaccination schedule is the best vaccination schedule for inducing potent OVA-specific CD8 immune response with high effector function.

Example 16: Capacity of TLR Agonist-CPP Conjugate Constructs to Activate Murine Antigen-Presenting Cells (APCs

To investigate the effect of both, the CPP component and the TLR agonist component in a complex for use according to the present invention, again the fusion proteins as described above (cf. Examples 1, 2 and 7) were used.

In addition, a further “control peptide” was designed, which is also a fusion protein and which comprises the protein “MAD5”, which consists of different CD8+and CD4+epitopes from various antigens, and the TLR2 peptide agonist “Anaxa” (i.e. without cell penetrating peptide). Accordingly, the following control construct was additionally designed:

Mad5Anaxa

Sequence:

[SEQ ID NO: 32]MHHHHHHESL KISQAVHAAH AEINEAGREV VGVGALKVPRNQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESIINFEKLTEWT GSSTVHEILC KLSLEGDHST PPSAYGSVKPYTNFDAE

Molecular weight: 13933 Da

Characteristics:

Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopesContains the 35-mer peptide of Annexin in C-terminal positionStorage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM DTT, 0.5 M L-Arginine, pH 8Endotoxin level: Batch 1-12.15 EU/mg

The aim of this study was to evaluate the capacity of two exemplary complexes according to the present invention, namely EDAZ13Mad5 (cf. Example 1) and Z13Mad5Anaxa (cf. Example 7), to promote antigen-presenting cells activation in comparison to reference complexes lacking either the cell penetrating peptide component Z13 (Mad5Anaxa, cf. above; EDAMad5, cf. Example 2) or the TLR agonist (Z13Mad5, cf. Example 1).

To this end, the capacity of the above mentioned constructs to promote antigen-presenting cells (APC) activation was assessed in bone marrow-derived dendritic cells (BMDCs), which express all TLRs except TLR7.

BMDCs were seeded in flat 96-well plate in culture medium, stimulated with 1 μM of either Z13Mad5Anaxa (cf. Example 7), Mad5Anaxa (cf. above), Z13Mad5 (cf. Example 1), EDAZ13Mad5 (cf. Example 1) or EDAMad5 (cf. Example 2) and incubated for 24 h at 37° C.

The APC activation was investigated by monitoring the secretion of IL-6 in the culture supernatant of BMDCs. IL-6 secretion was quantified by ELISA in the supernatant.

The results are shown inFIG.29. These data clearly show that Z13Mad5Anaxa was able to activate BMDCs, whereas no such activation was observed when the cells were cultured in presence of Z13Mad5 or Mad5Anaxa. This suggests that not only the TLR agonist (Anaxa or EDA) is critical for the activation of macrophages and dendritic cells, but that the CPP is also needed. Also the presence of the CPP without the TLR agonist is not sufficient, but indeed both, CPP and TLR agonist are critical for the activation of macrophages and dendritic cells.

Those results were confirmed by using another cell line, namely in the Raw 264.7 mouse macrophage cell line, which expresses all TLRs except TLR5 (Applequist, S. E., R. P. Wallin, and H. G. Ljunggren, Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines. Int Immunol, 2002. 14(9): p. 1065-74).

Raw 264.7 cells were seeded in flat 96-well plate in culture medium, stimulated with 1 μM of either Z13Mad5Anaxa (cf. Example 7), Mad5Anaxa (cf. above) or Z13Mad5 (cf. Example 1) and incubated for 24 h at 37° C.

In Raw 264.7 cells the APC activation was investigated by monitoring the secretion of TNF-α in the culture supernatant of Raw 264.7. TNF-α secretion was quantified by ELISA in the supernatant. The results are shown inFIG.30.

It is thought that the CPP may facilitate the entry of the molecule into the cells, allowing a better targeting of intracellular TLR.

Taken together, the data reveal the critical role of both, CPP and TLR agonist, within the conjugate constructs to activate APC. This effect may be due to helping the entry of the construct into the cells, therefore resulting in an optimal targeting of the intracellular TLR.

Example 17: Ability of the Conjugate Constructs to Bind to Human TLR4

It was recently shown that the Anaxa peptide owns an adjuvant activity by signaling through TLR2 (WO 2012/048190 A1), whereas the EDA peptide is a natural ligand for TLR4 (Okamura, Y., et al., The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem, 2001. 276(13): p. 10229-33).

Moreover, as shown above in Example 8 andFIG.14, a complex for use according to the present invention comprising the Anaxa peptide as TLR agonist, for example Z13Mad5Anaxa, is able to bind to human TLR2 and to promote the secretion of IL-8 by HEK-hTLR2 cells (cf. Example 8,FIG.14).

In the present study, the ability of complexes according to the present invention comprising either the Anaxa peptide as TLR agonist or the EDA peptide as TLR agonist to bind to human TLR4 was evaluated. To this end, HEK cells transfected with human TLR4 (HEK-hTLR4) were seeded in flat 96-well plate in culture medium, stimulated with 1 μM of either Z13Mad5Anaxa (cf. Example 7), Mad5Anaxa (cf. above), Z13Mad5 (cf. Example 1), EDAZ13Mad5 (cf. Example 1) or EDAMad5 (cf. Example 2) and incubated for 24 h at 37° C. IL-8 secretion was quantified by ELISA in the supernatant.

Results are shown inFIG.31. As expected, incubation of HEK-hTLR4 with EDAZ13Mad5 resulted in remarkable IL-8 secretion, indicating binding of EDAZ13Mad5 to TLR4. In line with the results obtained in Example 16, the IL-8 secretion of EDAMad5 (without the CPP) was remarkably lower as compared to EDAZ13Mad5, showing the effect of the presence of a CPP. The Z13Mad5 construct, which does not comprise a TLR agonist, showed no IL-8 secretion, indicating—as expected—a lack of binding to TLR4.

Interestingly, incubation of HEK-hTLR4 with the construct Z13Mad5Anaxa resulted in the most pronounced IL-8 secretion, indicating binding of Z13Mad5Anaxa to TLR4. This is astonishing, since Anaxa was previously hypothesized to be a TLR2 agonist. Again, the same construct but without the CPP (Mad5Anaxa) resulted in remarkably lower IL-8 secretion, confirming the results obtained in Example 16.

Taken together, these data (i) confirm the results obtained in Example 16, (ii) confirm that EDA is indeed a TLR4 agonist, and (iii) show surprisingly that the Anaxa peptide is also a TLR4 agonist (in addition to being a TLR2 agonist, cf. Example 8 andFIG.14).

Example 18: Vaccine Efficacy on Tumor Growth in a Lung Metastasis Model—Semi-Therapeutic Setting: TLR Agonist EDA

This study is based on Example 6, showing the efficacy of a complex for use according to the present invention, namely EDAZ13Mad5, in a melanoma lung metastasis model in a prophylactic setting (cf.FIG.13).

In the present study the same lung metastasis model was used as well as the construct proteins EDAZ13Mad5 and Z13Mad5+MPLA (cf. Examples 1 and 2 for design of the constructs). However, in the semi-therapeutic setting, C57BL/6 mice were vaccinated at the same time as tumor cells were implanted (d0) and, for a second time, at nine days after implantation (d9). Vaccination was performed by subcutaneous injection of 2 nmol of EDAZ13Mad5, Z13Mad5+MPLA (equimolar to EDA) or MPLA s.c. in the right flank. At day 0, mice were implanted i.v. with 1×105B16-OVA melanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneous injection of 2 nmol of EDAZ13Mad5, Z13Mad5+MPLA (equimolar to EDA) or MPLA alone s.c. in the right flank. Mice were euthanized at day 13 and lung recovered. Results are shown inFIG.32.

The results show that EDAZ13Mad5 is slightly more potent than Z13Mad5+MPLA to inhibit the growth of melanoma metastasis. In addition, no adjuvant effect was observed in mice injected with MPLA only.

Both, EDAZ13Mad5 and Z13Mad5+MPLA, significantly inhibit the growth of melanoma metastasis in the lung in prophylactic and semitherapeutic settings.

Example 19: Vaccine Efficacy on Tumor Growth in a Lung Metastasis Model—Semi-Therapeutic Setting: TLR Agonist Anaxa

This study is based on Example 18 with the same model (semitherapeutic settings) and experimental schedule. However, the effect of complexes according to the present invention comprising the “Anaxa” peptide as TLR agonist were investigated—instead of the EDA TLR agonist as in Example 18.

To this end, C57BL/6 mice were implanted i.v. with 1×105B16-OVA melanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneous injection of 0.5 nmol of Z13Mad5Anaxa, Mad5Anaxa or Z13Mad5+Pam3CSK4 (equimolar to Anaxa) s.c. in the right flank. Mice were euthanized at day 21 and the lung was recovered. Number of metastasis foci was counted for each lung. The results are shown inFIG.33.

The results show that Z13Mad5Anaxa is sensibly more potent than Z13Mad5+Pam3CSK4 to inhibit the growth of melanoma metastasis. In contrast, Mad5Anaxa was not able to control metastasis growth in the lung, underlining again the importance of CPP.

Altogether, the B16-OVA lung metastasis experiment showed that Z13Mad5Anaxa was highly efficacious in inhibiting the growth of melanoma metastasis in the lung.

Example 20: Vaccine Efficacy in a Glioblastoma Model

In this study, another cancer model was used, namely a glioblastoma model. Glioma is the most frequent form of primary brain tumors in adults, with glioblastoma multiforme (GBM) being the most lethal. This tumor is notorious for its highly invasive and aggressive behavior. Currently, the best treatment against GBM is a regimen involving a combination of surgery, chemotherapy and radiotherapy, which has a median survival period of only 14.6 months. There is an urgent, unmet medical need for new treatment modalities that improve the prognosis of glioma patients. T-cell mediated immunotherapy is a conceptually attractive treatment option to use in conjunction with existing modalities for glioma, in particular highly invasive GBM.

The Gl261 glioma is a carcinogen-induced mouse glioma model. This model represents one of the very few brain tumor models developed in immunocompetent animals, that has growth characteristics similar to human GBM (Newcomb, E. and D. Zagzag, The murine GL261 glioma experimental model to assess novel brain tumor treatments, in CNS Cancer Models, Markers, Prognostic, Factors, Targets, and Therapeutic Approaches, E. G. Van Meir, Editor. 2009, Humana Press: Atlanta. p. 227-241; Jacobs, V. L., et al., Current review of in vivo GBM rodent models: emphasis on the CNS-1 tumour model. ASN Neuro, 2011. 3(3): p. e00063). Low numbers of intracranially transplanted Gl261 cells formed intracranial tumors in C57BL/6 mice (Zhu, X., et al., Poly-/CLC promotes the infiltration of effector T cells into intracranial gliomas via induction of CXCL10 in IFN-alpha and IFN-gamma dependent manners.Cancer Immunol Immunother,2010. 59(9): p. 1401-9; Zhu, X., et al., Toll like receptor-3 ligand poly-/CLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models. J Transl Med, 2007. 5: p. 10). The cells are moderately immunogenic: they are able to elicit tumor-specific immune response at the tumor site. However, the tumor-specific immune cells are not capable of complete tumor clearance.

Recently, M. Ollin generated a new Gl261 model (Ohlfest, J. R., et al., Vaccine injection site matters: qualitative and quantitative defects in CDB T cells primed as a function of proximity to the tumor in a murine glioma model. J Immunol, 2013. 190(2): p. 613-20) by transfecting Gl261 cell line with the “Quad Cassette” expressing four peptides presented by H-2b class I or II molecules: human gp10025-33, chicken OVA257-264, chicken OVA323-339, and mouse I-Eα52-68. The Quad-Gl261 cell line also stably expresses luciferase, which allows the follow-up of tumor growth by bioluminescence.

The goal of this study was to assess the efficacy of a complex for use according to the present invention in the Quad-Gl261 glioblastoma model.

The effect of a complex for use according to the present invention, namely Z13Mad5Anaxa (cf. Example 7) was evaluated in the above described glioblastoma model. T cell homing at the tumor site was therefore analyzed in Gl261-Quad tumor-bearing mice vaccinated twice (Wk1 and Wk3) with Z13Mad5Anaxa vaccine. A group vaccinated with Z13Mad5 and Anaxa (equimolar to Z13Mad5Anaxa) administrated separately was used as control. Briefly, C57BL/6 mice were implanted i.e. (intracranially) with 5×105Gl261-Quad tumor cells and vaccinated twice (at d7 and d21 following implantation) by s.c. injection of 2 nmol of Z13Mad5Anaxa (group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa (group 2). At Wk4, the blood and the brain infiltrating leukocytes (BILs) were analyzed, whereby SIINFEKL-specific CD8 T cells were quantified in blood and in BILs at d28 by multimer staining (5-8 mice per group). Results are shown inFIG.34.

In general, low frequency of SIINFEKL-specific CD8 T cells was quantified in the blood. However, a higher percentage of SIINFEKL-specific CD8 T cells was observed in the blood of Z13Mad5Anaxa-vaccinated mice. In all groups, there was a sensibly stronger accumulation of SIINFEKL-specific CD8 T cells in the BILs.

After two vaccinations with Z13Mad5Anaxa, the frequency of SIINFEKL-specific cells CD8+ T cells in the BILs was 2-fold higher (24%) than with Z13Mad5+Anaxa (12%).

Next, cytokine secretion was assessed. To this end, C57BL/6 mice were implanted i.e. with 5×105Gl261-Quad tumor cells and vaccinated twice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmol of Z13Mad5 and 2 nmol of Anaxa. BILs were isolated and cultured during 6 h with matured BMDCs loaded or not with SIINFEKL peptide in presence of BrefeldinA before intracellular staining for cytokines. Results are shown inFIG.35.

Despite heterogeneity, a high level of cytokine secretion was observed for brain-infiltrating CD8 T cells from mice vaccinated with Z13Mad5Anaxa. These results demonstrate that Z13Mad5Anaxa vaccine was able to elicit a stronger SIINFEKL specific CD8 T cell immune response in the brain of tumor-bearing mice with potent effector function.

The results obtained are indicating that Z13Mad5Anaxa is efficacious for eliciting high brain infiltrating SIINFEKL-specific CD8 immune response. Z13Mad5Anaxa is able to promote the secretion of cytokine by antigen-specific CD8 T cells in the brain.

Example 21: Vaccine Efficacy on Survival in the Gl261-Quad Glioblastoma Model

In an independent experiment, the survival of control and Z13Mad5Anaxa-vaccinated mice was monitored. The therapeutic settings were three consecutive vaccinations with 2 nmol of Z13Mad5Anaxa at day 7, 21 and 35, post i.e. tumor implantation.

C57BL/6 mice were implanted i.e. with 5×105Gl261-Quad tumor cells and vaccinated three times (d7, d21 and d35) by s.c. injection of 2 nmol of Z13Mad5Anaxa. Mice were weight daily and euthanized when weight loss reached more than 15%. Results are shown inFIG.36.

The results show that Z13Mad5Anaxa therapeutic vaccination is more efficacious than the control group with a median survival prolonged by 10 days.

Example 22: Vaccine Efficacy in a Subcutaneous Tumor Model—Prophylactic Setting

This study is based on the results obtained in Example 10 as shown inFIGS.17-19.

To evaluate the effect of the Anaxa-conjugated construct proteins designed in Example 7 on tumor growth control, a benchmark tumor model was used, namely the s.c. implantation of EG.7-OVA thymoma cells. In contrast to Example 10, wherein vaccination was performed on days 5 and 13, in the present study a prophylactic setting was evaluated, wherein mice were vaccinated 21 and 7 days before tumor implantation.

C57BL/6 mice were vaccinated twice (d-21 and d-7) by s.c. injection of 0.5 nmol of Z13Mad5Anaxa in the right flank and then implanted at day 0 s.c. with 3×105EG7-OVA tumor cells in the left flank and. Tumor size was measured with a caliper.

The results are shown inFIG.37with tumor volume (FIG.37A) and survival rate (FIG.37B). The data is showing that prophylactic vaccination with Z13Mad5Anaxa is highly efficacious for controlling tumor growth and survival rate. The volume of the tumor is highly significantly decreased in mice treated with Z13Mad5Anaxa as compared to control mice. The survival rate is highly significantly increased in mice treated with Z13Mad5Anaxa as compared to control mice.

Example 23: Vaccine Efficacy in a Subcutaneous Tumor Model—Therapeutic Setting with Established Tumor

This study is based on the results obtained in Example 10 as shown inFIGS.17-19and on the results obtained in Example 22 shown inFIG.37. It was the goal of this study to evaluate the effect of Z13Mad5Anaxa (cf. Example 7) on an established tumor.

For this purpose, the s.c. model of B16-OVA melanoma cells was used. In this model tumor cells are spreading slowly, therefore allowing a bigger vaccination time window.

The first vaccination with the low dose of 0.5 nmol of Z13Mad5Anaxa was performed once the tumor was established and visible i.e. at day 14 after tumor cell implantation. A second vaccination was done at day 21.

Thus, C57BL/6 mice were implanted s.c. with 1×105B16-OVA tumor cells in the left flank and vaccinated twice (d14 and d21) by s.c. injection of 0.5 nmol of Z13Mad5Anaxa in the right flank. Tumor growth and survival curves were monitored. Results are shown inFIG.38.

The results indicate that Z13Mad5Anaxa efficaciously controls the growth of an established and visible tumor. Moreover, despite an established and visible tumor survival rates increased in mice treated with Z13Mad5Anaxa as compared to controls.

Example 24: Vaccine Efficacy in a Subcutaneous Tumor Model—Therapeutic Setting: Effect of the CPP

The protocol of this study corresponds to the study described in Example 10, with the difference that an additional group “Mad5Anaxa” (cf. Example 16) was evaluated.

Briefly, a benchmark tumor model was used, namely the s.c. implantation of EG.7-OVA thymoma cells. C57BL/6 mice were implanted s.c. with 3×105EG7-OVA tumor cells in the left flank. After tumor implantation, groups of 7 mice each were vaccinated s.c. in the right flank at day 5 and 13 by subcutaneous injection of 0.5 nmol of either Z13Mad5Anaxa (group 1) or Mad5Anaxa (group 2) and compared to a control group. Tumor size was measured with a caliper. Results are shown inFIG.39.

The results show that the mice treated with Z13Mad5Anaxa show a significantly decreased tumor volume and a significantly increased survival rate compared to both, control mice and mice treated with Mad5Anaxa, i.e. a construct without CPP. These results indicate that the presence of a CPP results in significantly decreased tumor volume and a significantly increased survival rate, i.e. in increased efficiency of vaccination. Therefore, the results indicate—together with the results obtained in Example 10—that the presence of a CPP and the TLR agonist exert a synergic effect on tumor growth and survival rate.

Example 25: Comparison of the Kinetic of Immune Responses with Complexes Having Different Cell Penetrating Peptides

To investigate the effect of different CPPs in the complex for use according to the present invention the fusion protein Z13Mad5Anaxa as described above (cf. Example 7) was used.

In addition, further fusion proteins were designed, which comprise CPPs other than Z13—namely Z14 (SEQ ID NO: 7) or Z18 (SEQ ID NO: 11). Those fusion proteins also comprise the protein “MAD5”, which consists of different CD8+and CD4+epitopes from various antigens, and the TLR2 peptide agonist “Anaxa”. Accordingly, the following constructs were additionally designed:

Z14Mad5Anaxa

Sequence:

(SEQ ID NO: 33)MHHHHHHKRY KNRVASRKSR AKFKQLLQHY REVAAAKESLKISQAVHAAH AEINEAGREV VGVGALKVPR NQDWLGVPRFAKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWTGSSTVHFILC KLSLEGDHST PPSAYGSVKP YTNFDAE
Z18Mad5Anaxa

Sequence:

(SEQ ID NO: 34)MHHHHHHREV AAAKSSENDR LRLLLKESLK ISQAVHAAHAEINEAGREVV GVGALKVPRN QDWLGVPRFA KFASFEAQGALANIAVDKAN LDVEQLESII NFEKLTEWTG SSTVHEILCKLSLEGDHSTP PSAYGSVKPY TNFDAE

C57BL/6 mice were assigned to eight different groups (4 mice per group): three groups receiving 2 nmol of either Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa and a respective control and three groups receiving 0.5 nmol of Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa and a respective control. The mice were vaccinated five times (Week0, Week2, Week4, Week6 and Week8) s.c. Mice were bled 7 days after the 2nd, 3rd, 4thand 5thvaccination and multimer staining was performed (one experiment with 4 mice per group).

The results are shown inFIG.40. All groups vaccinated with Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa showed an increased percentage of multimer-positive cells compared to the control group (except for the second vaccination of Z18Mad5Anaxa). These results indicate that complexes according to the present invention having different cell penetrating peptides are able to elicit an immune response at different doses.

Example 26: Comparison of T Cell Immune Responses with Complexes Having Different Cell Penetrating Peptides

To investigate the CD8 T cell immune responses in more detail, C57BL/6 mice were assigned to three different groups (3-4 mice per group): naïve, Z13Mad5Anaxa or Z14Mad5Anaxa.

C57BL/6 mice of the Z13Mad5Anaxa group and of the Z14Mad5Anaxa group were vaccinated five times (Week0, Week2, Week4, Week6 and Week8) s.c. with 2 nmol of either Z13Mad5Anaxa (cf. Example 7) or Z14Mad5Anaxa (cf. Example 25). Nine days after the 5thvaccination, mice were euthanized, organs recovered and multimer staining was performed to identify the percentage of SIINFEKL-specific CD8 T cells in the spleen, bone marrow and draining lymph nodes (inguinal and axillary).

The results are shown inFIG.41. Mice vaccinated with Z13Mad5Anaxa or with Z14Mad5Anaxa showed a similar increase in multimer-positive cells, in particular in the spleen and bone marrow as well as a slight increase in draining lymph nodes.

To further investigate the CD8 T cell effector function after vaccination with complexes with different CPPs, in the same groups of mice as described above Elispot assay was performed on spleen cells stimulated with SIINFEKL OVACD8 peptide (SEQ ID NO: 35) nine days after the 5thvaccination in order to quantify IFN-γ producing cells.

The results are shown inFIG.42A. Mice vaccinated with Z13Mad5Anaxa showed a significant increase in IFN-γ producing cells compared to naïve mice. Mice vaccinated with Z14Mad5Anaxa showed also an increase in IFN-γ producing cells compared to naïve mice, however, the increase was not significant, which may be due to the low number of mice (3 mice in Z14Mad5Anaxa group).

To investigate the CD4 T cell responses after vaccination with complexes with different CPPs, in the same groups of mice as described above Elispot assay was performed on spleen cells stimulated with OVACD4 peptide (SEQ ID NO: 36) nine days after the 5th vaccination in order to quantify IFN-γ producing cells.

The results are shown inFIG.42B. Mice vaccinated with Z13Mad5Anaxa showed a highly significant increase in IFN-γ producing cells compared to naïve mice. Mice vaccinated with Z14Mad5Anaxa showed also an increase in IFN-γ producing cells compared to naïve mice, however, the increase was not significant, which may be due to the low number of mice (3 mice in Z14Mad5Anaxa group).

In addition, in the above described groups of mice, intracellular staining was performed on spleen cells stimulated with SIINFEKL OVACD8 peptide (SEQ ID NO: 35) to identify CD107a+IFN-γ+TNF-α+cells. Results are shown inFIG.43. Mice vaccinated with Z13Mad5Anaxa or with Z14Mad5Anaxa showed a similar increase in CD107a+IFN-γ+TNF-α+cells.

Example 27: Comparison of the Effect of Complexes Having Different Cell Penetrating Peptides on Tumor Growth and Survival in the EG.7-OVA s.c. Model

To investigate the effects of complexes having different cell penetrating peptides on tumor growth and survival the EG.7-OVA s.c. model was used. On d0 C57BL/6 mice were implanted s.c. with 3×105EG7-OVA tumor cells in the left flank and assigned to three different groups (naïve, Z13Mad5Anaxa and Z14Mad5Anaxa). Mice were vaccinated twice at d5 and d13 after tumor implantation by s.c. injection of either 0.5 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa in the right flank.

Results are shown inFIG.44. Vaccination with Z13Mad5Anaxa or with Z14Mad5Anaxa resulted in significantly decreased tumor volumes compared to control mice (FIG.44A) as well as to significantly increased survival rates compared to control mice (FIG.44B). Those results indicate that both complexes, Z13Mad5Anaxa and Z14Mad5Anaxa, are able to significantly decrease tumor growth and to significantly prolong survival.

Example 28: Comparison of the Immune Responses after Vaccination with Complexes Having Different Cell Penetrating Peptides

In this experiment the effect of different CPPs in the complex for use according to the present invention was investigated by using a complex with the TLR agonist “EDA”. Therefore, the fusion protein EDAZ13Mad5 as described above (cf. Example 1) was used.

In addition, further fusion proteins were designed, which comprise CPPs other than Z13—namely Z14 (SEQ ID NO: 7) or Z18 (SEQ ID NO: 11). Those fusion proteins also comprise the protein “MAD5”, which consists of different CD8+and CD4+epitopes from various antigens, and the TLR4 peptide agonist “EDA”. Accordingly, the following constructs were additionally designed:

EDAZ14Mad5

Sequence:

(SEQ ID NO: 37)MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGE DDTAELQGLR PGSEYTVSVVALHDDMESQP LIGIQSTKRY KNRVASRKSR AKFKQLLQHYREVAAAKESL KISQAVHAAH AEINEAGREV VGVGALKVPRNQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESIINFEKLTEWT GS
EDAZ18Mad5

Sequence:

(SEQ ID NO: 38)MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYRVTYSSPEDGI RELFPAPDGE DDTAELQCLR PGSEYTVSVVALHDDMESQP LIGIQSTREV AAAKSSENDR LRLLLKESLKISQAVHAAHA EINEAGREVV GVGALKVPRN QDWLGVPRFAKFASFEAQGA LANIAVDKAN LDVEQLESII NFEKLTEWTG S

C57BL/6 mice were assigned to eight different groups (4 mice per group): three groups receiving 2 nmol of either EDAZ13Mad5, EDAZ14Mad5 or EDAZ18Mad5 and a respective control and three groups receiving 0.5 nmol of either EDAZ13Mad5, EDAZ14Mad5 or EDAZ18Mad5 and a respective control group. The mice were vaccinated three times (Week0, Week2 and Week4) s.c. Mice were bled 7 days after the 2ndand 3rdvaccination and multimer staining was performed (one experiment with 4 mice per group).

The results are shown inFIG.45. All groups vaccinated with EDAZ13Mad5, EDAZ14Mad5 or EDAZ18Mad5 showed an increased percentage of multimer-positive cells compared to the control group. These results indicate that complexes according to the present invention having different cell penetrating peptides are able to elicit an immune response at different doses.

Example 29: Effect of EDAZ14Mad5 on Tumor Growth and Survival in the EG.7-OVA s.c. Model

To investigate the effect of EDAZ14Mad5 on tumor growth and survival the EG.7-OVA s.c. model was used (cf. Example 4 andFIGS.9-11for the effect of EDAZ13Mad5 in the same model).

On d0 C57BL/6 mice were implanted s.c. with 3×105EG7-OVA tumor cells in the left flank and assigned to two different groups (naïve and EDAZ14Mad5). Mice were vaccinated twice at d5 and d13 after tumor implantation by s.c. injection of 0.5 nmol of EDAZ14Mad5 in the right flank.

Results are shown inFIG.46. Similarly to EDAZ13Mad5 (cf. Example 4,FIGS.9-11) vaccination with EDAZ14Mad5 resulted in significantly decreased tumor volumes compared to control mice (FIG.46A) as well as to significantly increased survival rates compared to control mice (FIG.46B). Those results indicate that EDAZ14Mad5 is able to significantly decrease tumor growth and to significantly prolong survival—similarly to EDAZ13Mad5 (cf. Example 4,FIGS.9-11).

Example 30: Superior Efficacy of Z13Mad5Anaxa Fusion Construct Compared to Z13Mad5 and Anaxa in a Glioblastoma Model

To investigate the efficacy of a complex according to the present invention the glioblastoma model was chosen (cf. Example 20). Namely, Z13Mad5Anaxa (cf. Example 7; SEQ ID NO: 28) was administered to one group of mice, whereas Z13Mad5 (SEQ ID NO: 29) and Anaxa (SEQ ID NO: 15) were administered (both together) to another group of mice.

T cell homing at the tumor site was analyzed in Gl261-Quad tumor-bearing mice (7-16 mice per group) vaccinated twice, namely at day 7 and at day 21 after tumor implantation (day 0), with 2 nmol Z13Mad5Anaxa vaccine. A group vaccinated with both, Z13Mad5 and Anaxa (equimolar to Z13Mad5Anaxa), was used as control. Briefly, C57BL/6 mice were implanted i.e. (intracranially) with 5×105Gl261-Quad tumor cells and vaccinated twice (at d7 and d21 following implantation) by s.c. injection of 2 nmol of Z13Mad5Anaxa (group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa (group 2). At day 28, the blood and the brain infiltrating leukocytes (BILs) were analyzed, whereby SIINFEKL-specific CD8 T cells were quantified in blood and in BILs at d28 by multimer staining (7-16 mice per group).

Results are shown inFIG.47. A significantly higher percentage of SIINFEKL-specific CD8 T cells was observed in the blood of Z13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with both, Z13Mad5 and Anaxa (FIG.47A). Similarly, a stronger accumulation of SIINFEKL-specific CD8 T cells was observed in the BILs of Z13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with Z13Mad5 and Anaxa separately (FIG.47B, p=0.0539).

Next, cytokine secretion was assessed. To this end, C57BL/6 mice were implanted i.e. with 5×105Gl261-Quad tumor cells and vaccinated twice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmol of Z13Mad5 and 2 nmol of Anaxa. BILs were isolated and cultured during 6 h with matured BMDCs loaded or not with SIINFEKL peptide (SEQ ID NO: 35) in presence of BrefeldinA before intracellular staining for cytokines.

Results are shown inFIG.48. In general, a high level of cytokine secretion was observed for brain-infiltrating CD8 T cells from mice vaccinated with Z13Mad5Anaxa. In particular, a significantly higher secretion of total IFN-γ and of IFN-γ and TNF-α together was observed for brain-infiltrating CD8 T cells from mice vaccinated with Z13Mad5Anaxa as compared to mice vaccinated with Z13Mad5 and Anaxa separately.

Taken together, these results demonstrate that Z13Mad5Anaxa vaccine (as compared to Z13Mad5 and Anaxa administered separately) was able to elicit a stronger SIINFEKL specific CD8 T cell immune response in the brain of tumor-bearing mice with potent effector function.

The results obtained are indicating that Z13Mad5Anaxa is efficacious for eliciting high brain infiltrating SIINFEKL-specific CD8 immune response. Z13Mad5Anaxa is able to promote the secretion of cytokine by antigen-specific CD8 T cells in the brain.

Example 31: Effect of Another Antigenic Cargo in the Complex According to the Present Invention

To investigate the effect of a different antigenic cargo (“Mad8”), another complex comprising a cell penetrating peptide, different antigens and a TLR peptide agonist was designed (“Z13Mad8Anaxa”). Z13Mad8Anaxa differs from Z13Mad5Anaxa (described in Example 7) in the antigenic cargoes. In particular, “Z13Mad8Anaxa” is a fusion protein comprising the cell-penetrating peptide “Z13”, the antigenic cargo “MAD8” comprising CD8 and CD4 epitopes of glycoprotein 70, and the TLR peptide agonist “Anaxa”. In the following, the amino acid sequence of Z13Mad8Anaxa is shown with the cell-penetrating peptide “Z13” shown underlined and the TLR peptide agonist “Anaxa” shown in italics:

(SEQ ID NO: 39)KRYKNRVASR KSRAKFKQLL QHYREVAAAK SSENDRLRLLLKVTYHSPSYAY HQFERRAILN RLVQFIKDRI SVVQALVLTSTVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAE

Naïve Balb/c mice (4 mice per group) were vaccinated four times s.c. (week0, week2, week4 and week6 with 2 nmol of Z13Mad8Anaxa.

To investigate the CD4 T cell responses after vaccination, one week after the 4th vaccination, mice were euthanized; organs recovered and ex vivo Elispot assay was performed on spleen cells stimulated with gp70CD4 peptide (SEQ ID NO: 64) or gp70CD8 peptide (SEQ ID NO: 65) in order to quantify IFN-γ-producing epitope-specific CD4 and CD8 T cells.

The results are shown inFIG.49. Mice vaccinated with Z13Mad8Anaxa showed a significant increase in IFN-γ-producing cells compared to naïve mice. These data show that Z13Mad8Anaxa vaccine was able to elicit potent epitope-specific CD8 and CD4 T cell immune response and thus that the complex according to the present invention is able to elicit self-antigen immune response.

Example 32: Effect of Another Antigenic Cargo in the Complex According to the Present Invention

To investigate the effect of a further different antigenic cargo (“Mad11”), another complex comprising a cell penetrating peptide, different antigens and a TLR peptide agonist was designed (“Z13Mad11Anaxa”). Z13Mad11Anaxa differs from Z13Mad5Anaxa (described in Example 7) in the antigenic cargoes. In particular, “Z13Mad11Anaxa” is a fusion protein comprising the cell-penetrating peptide “Z13”, the antigenic cargo “MAD11” comprising two CD8 epitopes of survivin as described in Derouazi M, Wang Y, Marlu R, et al. Optimal epitope composition after antigen screening using a live bacterial delivery vector: Application to TRP-2. Bioengineered Bugs. 2010; 1(1):51-60. doi:10.4161/bbug.1.1.9482, and the TLR peptide agonist “Anaxa”. In the following, the amino acid sequence of Z13Mad11Anaxa is shown:

(SEQ ID NO: 40)KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNYRIATFKNWPFLEDCAMEELTVSEFLKLDRQRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Naïve C57BL/6 mice (5 mice per group) were implanted i.v. with 1×105B16 melanoma tumor cells and vaccinated twice (d0 and d10) by subcutaneous injection of 1 nmol of Z13Mad11Anaxa.

On day 18 mice were euthanized, organs recovered and ex vivo Elispot assay was performed on spleen cells stimulated with survivin peptides survivin20-28 (SEQ ID NO: 67) and survivin97-104: (SEQ ID NO: 68) in order to quantify IFN-γ producing survivin-specific T cells.

The results are shown inFIG.50. Mice vaccinated with Z13Mad11Anaxa showed less metastasis compared to naïve mice (FIG.50A). Moreover, in the spleen of mice vaccinated with Z13Mad11Anaxa significantly higher numbers of IFN-γ producing survivin-specific T cells were observed (FIG.49B).

The results obtained show that Z13Mad11Anaxa is efficacious for reducing the number of metastasis and Z13Mad11Anaxa is able to promote the secretion of cytokines by antigen-specific CD8 T cells in the spleen.

Example 33: Effect of Another Antigenic Cargo in the Complex According to the Present Invention

To investigate the effect of a further different antigenic cargo (“Mad9”), another complex comprising a cell penetrating peptide, a different antigen and a TLR peptide agonist was designed (“Z13Mad9Anaxa”). Z13Mad9Anaxa differs from Z13Mad5Anaxa (described in Example 7) in the antigenic cargo. In particular, “Z13Mad9Anaxa” is a fusion protein comprising the cell-penetrating peptide “Z13”, the antigenic cargo “Mad9” comprising the neoantigen as identified by Yadav et al. Nature. 2014 Nov. 27; 515(7528):572-6 from MC-38 tumor cell line, and the TLR peptide agonist “Anaxa”. In the following, the amino acid sequence of Z13Mad9Anaxa is shown with the cell-penetrating peptide “Z13” shown underlined and the TLR peptide agonist “Anaxa” shown in italics:

(SEQ ID NO: 41)KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKHLELASMTNMELMSSIVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Naïve C57BL/6 mice (4 mice per group) were vaccinated four times s.c. (week0, week2, week4 and week6 with 2 nmol of Z13Mad9Anaxa. To investigate the CD8 T cell responses after vaccination, one week after the 4th vaccination, mice were euthanized, organs recovered and Elispot assay was performed on spleen cells after a 7-day in vitro restimulation with stimulated with adpgk peptide (SEQ ID NO: 66) in order to quantify to quantify IFN-γ-producing epitope-specific CD8 T cells.

The results are shown inFIG.51. Mice vaccinated with Z13Mad9Anaxa showed a significant increase in effector neoantigen-specific CD8 T cells compared to naïve mice.

Example 34: Comparison of the Immune Responses after Vaccination with Complexes Having Different Cell Penetrating Peptides

In this experiment the effect of a further different CPP in the complex according to the present invention was investigated by using a complex with the TLR agonist “Anaxa”. Therefore, the fusion protein Z13Mad5Anaxa as described above (cf. Example 7, SEQ ID NO: 28) was used.

In addition, a further fusion protein was designed, which comprise the TAT CPP combined to furin linkers as described in Lu et al., Multiepitope trojan antigen peptide vaccines for the induction of antitumor CTL and Th immune responses J. Immunol., 172 (2004), pp. 4575-4582. That fusion protein also comprises the protein “MAD5”, which consists of different CD8+and CD4+epitopes from various antigens, and the TLR4 peptide agonist “Anaxa”. Accordingly, the following construct was additionally designed:

TatFMad5Anaxa

Sequence:

(SEQ ID NO: 46)RKKRRQRRRRVKRISQAVHAAHAEINEAGRRVKRKVPRNQDWLRVKRASFEAQGALANIAVDKARVKRSIINFEKLRVKRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

C57BL/6 mice were assigned to three different groups (8 mice per group): one group receiving 2 nmol of Z13Mad5Anaxa, one group receiving 2 nmol of TatFMad5Anaxa and a respective control. The mice were vaccinated two times (Week0 and Week2) s.c. with either 2 nmol of Z13Mad5Anaxa or 2 nmol of TatFMad5Anaxa. Mice were bled 7 days after the 2ndvaccination and multimer staining was performed (8 mice per group).

The results are shown inFIG.52. Mice vaccinated with Z13Mad5Anaxa or TatFMad5Anaxa showed an increased percentage of multimer-positive cells compared to the control group. These results indicate that complexes according to the present invention having different cell penetrating peptides are able to elicit an immune response at different doses. However, the CPP derived from ZEBRA (Z13) was better than the TAT CPP.

Example 35: Superior Efficacy of Z13Mad5Anaxa Fusion Construct Compared to Z13Mad5 and Anaxa in Naïve Mice

Next, the efficacy of a complex according to the present invention was investigated in naïve mice. Namely, Z13Mad5Anaxa (cf. Example 7; SEQ ID NO: 28) was administered to one group of mice, whereas Z13Mad5 (SEQ ID NO: 29) and Anaxa (SEQ ID NO: 15) were administered (both together) to another group of mice.

C57BL/6 mice of the Z13Mad5Anaxa group and of the Z13Mad5+Anaxa group were vaccinated once (Week0) by s.c. injection of 2 nmol of Z13Mad5Anaxa (group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa (group 2). At day 14, the blood was analyzed, whereby SIINFEKL-specific CD8 T cells were quantified in blood by multimer staining (4-8 mice per group).

Results are shown inFIG.53. A significantly higher percentage of SIINFEKL-specific CD8 T cells was observed in the blood of Z13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with Z13Mad5 and Anaxa separately (FIG.53).

Taken together, these results demonstrate that Z13Mad5Anaxa vaccine (as compared to Z13Mad5 and Anaxa administered separately) was able to elicit a stronger SIINFEKL specific CD8 T cell immune response in the periphery.

Example 36: Effect of Another Antigenic Cargo in the Complex According to the Present Invention

To investigate the effect of a further different antigenic cargo (“Mad12”), another complex comprising a cell penetrating peptide, a different antigen and a TLR peptide agonist was designed (“Z13Mad12Anaxa”). Z13Mad12Anaxa differs from Z13Mad5Anaxa (described in Example 7) in the antigenic cargo. In particular, “Z13Mad12Anaxa” is a fusion protein comprising the cell-penetrating peptide “Z13”, the antigenic cargo “MAD12” comprising three neoantigens as identified by Yadav et al. Nature. 2014 Nov. 27; 515(7528):572-6 from MC-38 tumor cell line, and the TLR peptide agonist “Anaxa”. In the following, the amino acid sequence of Z13Mad12Anaxa is shown:

(SEQ ID NO: 69)KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEGSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Naïve C57BL/6 mice (4 mice per group) were vaccinated twice s.c. (week0, week2) with 2 nmol of Z13Mad12Anaxa. To investigate the CD8 T cell responses after vaccination, one week after the 2ndvaccination, the blood was analyzed, whereby neoantigen reps1-specific CD8 T cells were quantified in blood by multimer staining (4 mice per group).

The results are shown inFIG.54. Mice vaccinated with Z13Mad12Anaxa showed a significant increase in effector neoantigen-specific CD8 T cells compared to naïve mice.

Example 37: In Vitro Human Dendritic Cell Maturation

The goal of this study was to investigate the capacity of a complex for use according to the present invention (“Z13Mad5Anaxa”, SEQ ID NO: 28, cf. Example 7) to induce maturation of dendritic cells in comparison to a complex lacking a TLR peptide agonist (“Z13Mad5”, SEQ ID NO: 29, cf. Example 1).

The Z13Mad5Anaxa polypeptide and the Z13Mad5 polypeptide were investigated for their capacity to induce human dendritic cell (DC) maturation. After incubation over night with 300 nM of protein, activation markers expression (CD86, CD80, CD83 and HLA-DR) was assessed on the human DCs by FACS (FIG.55). Same buffer volumes of each protein were used as negative controls.

Results are shown inFIG.55. Whereas Z13Mad5Anaxa induced maturation of human DCs, shown by the up-regulation of CD86, HLADR and CD83, Z13Mad5 was not able to activate human DCs. These results indicate that the Anaxa portion of the protein is responsible for the up-regulation of the activation markers on the human DCs.

Example 38: CD8 T Cell Immune Response Over the Course of Repeated Vaccination

To investigate whether a pool of effector T cells could be maintained over several months, repeated vaccination was performed. Namely, C57BL/6 mice were vaccinated subcutaneously six times (weeks 0, 2, 4, 8, 12, 16) with 2 nmol of the construct Z13Mad5Anaxa (SEQ ID NO: 28). Seven days after the each vaccination (and before some vaccinations), mice were bled and pentamer staining was performed to monitor the OVA-specific immune response in the blood (two experiments with 4 mice per group). InFIG.56, the percentage of pentamer positive CD8+ T cells is shown for Z13Mad5Anaxa vaccinated mice and for control mice.

These data show that a pool of effector T cells could be maintained over four months (17 weeks) with six vaccinations.

Example 39: Evaluation of the Complex According to the Present Invention in the Murine MC38 Colorectal Cancer Model

To evaluate the effects of the complex according to the present invention in a murine colorectal cancer model, the murine MC38 colorectal cancer model was chosen. MC38 is a colon carcinoma cell line.

Naïve C57BL/6 mice were implanted s.c. with 2×105MC38 tumor cells in the left flank and vaccinated twice (-day 21 & -day 7 before tumor implantation) by subcutaneous injection of 2 nmol of the construct Z13Mad11Anaxa (cf. Example 32, SEQ ID NO: 40) in the right flank. The “Mad11” antigenic cargo contains two survivin epitopes expressed by the murine MC38 colorectal model.

The results are shown inFIG.57. Mice vaccinated with Z13Mad11Anaxa showed significantly less tumor volume compared to naïve mice (FIG.57A). Moreover, mice vaccinated with Z13Mad11Anaxa showed a significantly higher survival rate (FIG.57B).

The results obtained show that Z13Mad11Anaxa was able to significantly reduce and delay the tumor growth as compared to the control. Moreover, the survival rate of mice vaccinated with Z13Mad11Anaxa was increased.

Example 40: Preliminary Toxicity Study in Mice

To assess the toxicity of the complex according to the present invention in mice, Z13Mad5Anaxa (SEQ ID NO: 28) was injected at 2 nmol s.c. and i.v. in naïve C57BL/6 mice. Blood sampling was performed at 0.5, 1.5 and 3 h after administration. A commercial multiplex kit (Luminex) was used for the detection and quantification of cytokines in blood and four mice were sampled at each time point. However, no expression of any pro-inflammatory cytokines could be detected. It should be noted that the multiplex kit was compared to a classical ELISA with similar results (data not shown).

Next, the dose of Z13Mad5Anaxa (SEQ ID NO: 28) was increased to 10 nmol. However, again no cytokines were detected following s.c. administration. In contrast to s.c. administration, a transient increase of IL-6 and TNFα was observed at 1.5 h after i.v. administration. However, this slight increase disappeared after 3 h (FIG.58). Subcutaneous injection of 10 nmol of Z13Mad5Anaxa in mice did not induce any cytokines release up to 6 h after treatment (data not shown).

Example 41: Effects of a Complex Comprising a Cell Penetrating Peptide, Different Antigens and a TLR Peptide Agonist on Homing of T Cells to the MC-38 Tumor Site

In order to assess the effects of a complex comprising a cell penetrating peptide, different antigens and a TLR peptide agonist on homing of T cells to the tumor site, mice were vaccinated with cell penetrating peptide, different antigens and a TLR peptide agonist in the MC-38 tumor model. On day 27, mice were euthanized and FACS staining was performed to monitor the neoantigen-specific immune response in the TILs (tumor-infiltrating lymphocytes).

C57BL/6 mice (four mice per group, female, 7 week old) were implanted s.c. with 2×105MC-38 tumor cells in the left flank (day 0). After tumor implantation, mice of the groups “Z13Mad12Anaxa” were vaccinated at days 3, day 10 and 17 subcutaneously with 2 nmol of Z13Mad12Anaxa (cf. Example 36; SEQ ID NO: 69) at the tail base.

As shown inFIGS.59,60and61, neoantigen-specific T cells accumulate at the tumor site in vaccinated mice. The percentage of multimer-positive cells was significantly increased in mice vaccinated with Z13Mad12Anaxa. The lowest percentage of multimer-positive cells was found in control mice.

Example 42: Human Dendritic Cell Activation by Human Constructs

The goal of this study was to investigate the capacity of different complexes according to the present invention comprising different human antigenic cargoes to induce maturation of human dendritic cells. To this end, each of the constructs ATP110 (SEQ ID NO: 72), ATP112 (SEQ ID NO: 74), ATP115 (SEQ ID NO: 77), ATP117 (SEQ ID NO: 79), ATP118 (SEQ ID NO: 80), ATP119 (SEQ ID NO: 81), ATP120 (SEQ ID NO: 82), ATP122 (SEQ ID NO: 84), ATP123 (SEQ ID NO: 85), and ATP125 (SEQ ID NO: 87) was tested. The biological indicator used for the evaluation of DC activation was the Activation Index which indicates the percentage of activation based on the expression intensity of four membrane antigens: HLA-DR, CD80, CD83 and CD86.

After incubation over night with 300 nM or 600 nM of each of the above-mentioned constructs (ATP110 (SEQ ID NO: 72), ATP112 (SEQ ID NO: 74), ATP115 (SEQ ID NO: 77), ATP117 (SEQ ID NO: 79), ATP118 (SEQ ID NO: 80), ATP119 (SEQ ID NO: 81), ATP120 (SEQ ID NO: 82), ATP122 (SEQ ID NO: 84), ATP123 (SEQ ID NO: 85), and ATP125 (SEQ ID NO: 87)), activation markers expression (CD86, CD80, CD83 and HLA-DR) was assessed on the human dendritic cells by FACS (FIGS.62-71). Same buffer volumes of each construct were used as negative controls.

Results are shown inFIG.62-71with all constructs tested showing dendritic cell maturation shown by the up-regulation of CD86, HLADR and CD83.

Example 43: Effects of Combination of a PD1 Inhibitor and a Complex Comprising a Cell Penetrating Peptide, Different Antigens and a TLR Peptide Agonist on Tumor Growth and Survival Rate in a Colon Carcinoma Model

In order to assess the effects of combination of a PD1 inhibitor and a complex comprising a cell penetrating peptide, different antigens and a TLR peptide agonist in treating colorectal cancer, the MC-38 tumor model was used. MC-38 is a colon carcinoma cell line.

To this end, C57BL/6 mice (thirteen to fourteen mice per group, female, 7 week old) were implanted s.c. with 2×105MC-38 tumor cells in the left flank (day 0). After tumor implantation, mice of the groups “Z13Mad12Anaxa” and “Z13Mad12Anaxa+anti-PD1” were vaccinated at days 3, 10 and 17 subcutaneously with 2 nmol of Z13Mad12Anaxa (cf. Example 36; SEQ ID NO: 69) at the tail base. 200 μg of anti-PD1 antibody RMP1-14 (BioXcell, West Lebanon, NH, USA) were administered i.p. on each of days 6, 10, 13, 17, 20, 24 and 27 to mice of groups “anti-PD1” and “Z13Mad12Anaxa+anti-PD1”. At days 10 and 17, when both, Z13Mad12Anaxa and antibody, were administered, the antibody was administered i.p. just after s.c. administration of Z13Mad12Anaxa. Tumor size was measured with a caliper.

As shown inFIGS.72and73, treatment with the PD1 inhibitor alone or with Z13Mad12Anaxa alone resulted in significantly reduced tumor volume (FIG.72A) and increased survival (FIG.72B), as compared to the control group. However, the combination of both, the PD1 inhibitor and Z13Mad12Anaxa, resulted in the most pronounced improvement, namely in strongly decreased tumor volume and strongly increased survival rates. Of note, in the “Z13Mad12Anaxa+aPD1” group only three mice developed tumors (whereas 10 mice remained tumor-free), whereas in the “aPD1” group and in the “Z13Mad12Anaxa” group eight and ten mice, respectively, developed tumors. In the control group all mice developed tumors. These data show that a combination of both, anti-PD1 therapy and Z13Mad12Anaxa vaccination, is more efficient than anti-PD1 therapy alone or Z13Mad12Anaxa vaccination alone.

As shown inFIG.73, the number of tumor-free mice in the “Z13Mad12Anaxa+aPD1” group (10 mice out of 13 mice) is larger than the sum of the numbers of tumor-free mice in the “aPD1” group (6 mice out of 14 mice) and in the “Z13Mad12Anaxa” group (3 mice out of 13 mice) together. These results thus indicate a synergistic effect of anti-PD1 therapy and Z13Mad12Anaxa vaccination.

Example 44: Immune Response Elicited in Mice after Vaccination with ATP128

Another human construct, ATP128 (SEQ ID NO: 89), comprising a cell penetrating peptide, epitopes of three antigens (Survivin, CEA and ASCL2) and a TLR peptide agonist was designed. In particular, “ATP128” (SEQ ID NO: 89) is a fusion protein comprising the cell-penetrating peptide “Z13” (SEQ ID NO: 6), epitopes of three the antigens survivin, CEA and ASCL2, and a sequence variant of the TLR peptide agonist “Anaxa” (namely, a TLR peptide agonist according to SEQ ID NO: 71). In the following, the amino acid sequence of ATP128 is shown:

(SEQ ID NO: 89)KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPSEGALSPAERELLDFSSWLGGYSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAE

Naïve C57BL/6 mice (5 mice per group, female, 7-week-old) were vaccinated by subcutaneous injection of 4 nmol of ATP128 (SEQ ID NO: 89). Control mice received vehicle (vaccine buffer). Seven days after treatment mice were bled, and ex vivo Elispot assay was performed on blood cells stimulated with mouse dendritic cells loaded with ATP128 in order to quantify IFN-γ producing vaccine-specific T cells.

The results are shown inFIG.74. Mice vaccinated with ATP128 showed a higher ATP128-specific immune response compared to naïve mice.

The results obtained show that ATP128 is efficacious for eliciting immune response in mice.

Example 45: Human Dendritic Cell Activation by ATP128 Construct

The goal of this study was to investigate the capacity of ATP128 (SEQ ID NO: 89) comprising human antigenic cargo to induce maturation of human dendritic cells (human DCs). To this end, ATP128 (SEQ ID NO: 89) was tested. The biological indicator used for the evaluation of DC activation was the Activation Index (FIG.75) which indicates the percentage of activation based on the expression intensity of four membrane antigens: HLA-DR, CD80, CD83 and CD86.

After incubation over night with 300 nM of ATP128 (SEQ ID NO: 89), activation markers expression (CD86, CD80, CD83 and HLA-DR) was assessed on the human dendritic cells by FACS (FIG.75). Same buffer volume was used as negative control whereas MPLA was used as positive control.

Results are shown inFIG.75. The results indicate that ATP128 can potently activate human DCs.

Example 46: Human Dendritic Cell Activation by ATP128 Construct

In order to confirm that human cells are able to process and present the epitopic peptides included in ATP128 (SEQ ID NO: 89), human dendritic cells (human DCs) from 2 different donors (named donor “9” and “10”) were over-night loaded with ATP128 and processed for HLA Class I and Class II/presented peptide purification, peptide elution and characterization by mass spectroscopy. Briefly, peptide pools from shock-frozen human DC samples were obtained by immune precipitation using HLA-specific antibodies, acid treatment and ultrafiltration. The HLA peptide pools were separated according to their hydrophobicity by reversed-phase chromatography and the eluting peptides were analysed in a mass spectrometer (Thermo Fisher Scientific). The data were then collected and automatically processed by analysing the mass signals of unfragmented peptides as well as fragment spectra containing peptide sequence information. False discovery rates (FDR) were determined by the Percolator algorithm (Käll L, Canterbury J D, Weston J, Noble W S, MacCoss M J (2007) Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4(11):923-925)) based on processing against a decoy database consisting of the shuffled target database. For HLA class I, peptide lengths were limited to 8-12 aa of length. For HLA class 11, peptides were limited to 12-25 aa of length. HLA annotation was performed using SYFPEITHI (www.syfpeithi.de).

Data have shown that human DCs loaded with ATP128 are able to process and present both class I and class II epitopic peptides derived from the multiantigenic domain (FIG.76). Peptides derived from all antigens were identified in the class I pool. A higher number of class II bound peptides were identified from all the antigen portions of the vaccine.

TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING):SEQ ID NOSequenceRemarksSEQ ID NO: 1RQIKIYEQNRRMKWKKCPP: PenetratinSEQ ID NO: 2YGRKKRRQRRRCPP: TAT minimalSEQ ID NO: 3MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVZEBRA amino acidYQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAYsequence (naturalHVSTAPTGSWFSAPQPAPENAYQAYAAPQLFPsequence fromVSDITQNQQINQAGGEAPQPGDNSTVQTAAEpstein-Barr virusAVVFACPGANQGQQLADIGVPQPAPVAAPAR(EBV)) (YP_401673)RTRKPQQPESLEECDSELEIKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNFSEQ ID NO: 4KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSECPPI (Z11)NDRLRLLLKQMCSEQ ID NO: 5KRYKNRVASRKCRAKEKQLLQHVREVAAAKSSECPP2 (Z12)NDRLRLLLKSEQ ID NO: 6KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSECPP3 (Z13)NDRLRLLLKSEQ ID NO: 7KRYKNRVASRKSRAKFKQLLQHYREVAAAKCPP4 (Z14)SEQ ID NO: 8KRYKNRVASRKSRAKFKCPP5 (Z15)SEQ ID NO: 9QHYREVAAAKSSENDCPP6 (Z16)SEQ ID NO: 10QLLQHYREVAAAKCPP7 (Z17)SEQ ID NO: 11REVAAAKSSENDRLRLLLKCPP8 (Z18)SEQ ID NO: 12KRYKNRVACPPP (Z19)SEQ ID NO: 13VASRKSRAKFKCPP10 (Z20)SEQ ID NO: 14ESLKISQAVHAAHAEINEAGREVVGVGALMAD5 cargoKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSEQ ID NO: 15STVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAETLR2 peptide agonistAnaxaSEQ ID NO: 16DDDKenterokinase targetsiteSEQ ID NO: 17IEDGRfactor Xa target siteSEQ ID NO: 18LVPRGSthrombin target siteSEQ ID NO: 19ENLYFQGprotease TEV targetsiteSEQ ID NO: 20LEVLFQGPPreScissionprotease targetSEQ ID NO: 21RX(R/K)Rfurin target siteSEQ ID NO: 22GGGGGpeptidic linkerSEQ ID NO: 23GGGGpeptidic linkerSEQ ID NO: 24EQLEpeptidic linkerSEQ ID NO: 25TEWTpeptidic linkerSEQ ID NO: 26MHHHHHHNIDRPKGLAFTDVDVDSIKIAEDAZ13Mad5WESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSEQ ID NO: 27MHHHHHHSTVHEILCKLSLEGDHSTPPSAAnaxaZ13Mad5YGSVKPYTNFDAEKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSEQ ID NO: 28MHHHHHHKRYKNRVASRKSRAKFKQLLZ13Mad5AnaxaQHYREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 29MHHHHHHKRYKNRVASRKSRAKFKQLLZ13Mad5QHYREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSEQ ID NO: 30MHHHHHHESLIKISQAVHAAHAEINEAGREVMad5VGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSEQ ID NO: 31MHHHHHHNIDRPKGLAFTDVDVDSIKIAEdaMad5WESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSEQ ID NO: 32MHHHHHHESLKISQAVHAAHAEINEAGMad5AnaxaREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLSLEGDHSTPPSAYGSVKPYINFDAESEQ ID NO: 33MHHHHHHKRYKNRVASRKSRAKFKQLLZ14 Mad5AnaxaQHYREVAAAKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 34MHHHHHHREVAAAKSSENDRLRLLLKESZ18 Mad5AnaxaLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLSLEGDHSIPPSAYGSVKPYTNFDAESEQ ID NO: 35SIINFEKLSIINFEKL OVACD8SEQ ID NO: 36ISQAVHAAHAEINEAGROVACD4 peptideSEQ ID NO: 37MHHHHHHNIDRPKGLAFTDVDVDSIKIAEDAZ14Mad5WESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTKRYKNRVASRKSRAKFKQLLQHYREVAAAKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSEQ ID NO: 38MHHHHHHNIDRPKGLAFTDVDVDSIKIAEDAZ18Mad5WESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSFYIVSVVALHDDMESQPLIGIQSTREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSEQ ID NO: 39KRYKNRVASRKSRAKFKQLLQHYREVAAAZ13Mad8AnaxaKSSENDRLRLLLKVTYHSPSYAYHQFERRAILNRLVQFIKDRISVVQALVLTSTVHEREILCKLSLEGDHSTPPSAYGSVKPYTN FDAESEQ ID NO: 40KRYKNRVASRKSRAKFKQLLQHYREVAAAZ13Mad11AnaxaKSSENDRLRLLLKNYRIATFKNWPFLEDCAMEELTVSEFLKLDRQRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 41KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENZ13Mad9AnaxaDRLRLLLKHLELASMTNMELMSSIVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 42HLELASMTNMELMSSIVMad9SEQ ID NO: 43VTYHSPSYAYHQFERRAILNMad8SEQ ID NO: 44NYRIATFKNWPFLEDCAMEELTVSEFLKLDMad11SEQ ID NO: 45NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYREDAVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTSEQ ID NO: 46RKKRRQRRRRVKRISQAVHAAHAEINEAGRRVKTatFMad5AnaxaRKVPRNQDWLRVKRASFEAQGALANIAVDKARVKRSIINFEKLRVKRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 47MAPPQVLAFGLLLAAATATFAAAQEECVCENYKEpCAMLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAVVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNASEQ ID NO: 48GLKAGVIAVEpCAM epitopeSEQ ID NO: 49MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEMUC-1KETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPCSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAATSANLSEQ ID NO: 50GSTAPPVHNMUC-1 epitopeSEQ ID NO: 51TAPPAHGVTSMUC-1 epitopeSEQ ID NO: 52MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACsurvivinTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAKKVRRAIEQLAAMDSEQ ID NO: 53RISTFKNWPFsurvivin epitopeSEQ ID NO: 54MESPSAPPHRWCIPWQRLLLTASLLTWNPPTTACEAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLILFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALSEQ ID NO: 55YLSGANLNLSCEA epitopeSEQ ID NO: 56SWRINGIPQQCEA epitopeSEQ ID NO: 57MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDKirsten RasPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIMSEQ ID NO: 58VVVGAGGVGKirsten Ras epitopeSEQ ID NO: 59MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATMAGE-A3EEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKAFSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEESEQ ID NO: 60KVAELVHFLMAGE-A3 epitopeSEQ ID NO: 61MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQIL13Ralpha2DFEIVDPGYLGYLYLQWQPPLSLDHFKECTVEYELKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEVQSSWAETTYWISPQGIPETKVQDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDHALQCVDYIKADGQNIGCRFPYLEASDYKDFYICVNGSSENKPIRSSYFTFQLQNIVKPLPPVYLTFTRESSCEIKLKWSIPLGPIPARCFDYEIEIREDDTTLVTATVENETYTLKTTNETRQLCFVVRSKVNIYCSDDGIWSEWSDKQCWEGEDLSKKTLLRFWLPFGFILIIVIFVTGLLLRKPNTYPKMIPEFFCDTSEQ ID NO: 62LPFGFILIL13Ralpha2 epitopeSEQ ID NO: 63LFRAAQANDVVLQIMEHLELASMTNMELMSSIMad12VVISASIIVFNLLELEGSEQ ID NO: 64LVQFIKDRISVVQAgp70CD4 peptideSEQ ID NO: 65SPSYvYHQFgp70CD8 peptideSEQ ID NO: 66ASMTNMELMadpgk peptideSEQ ID NO: 67ATKNWPFLsurvivin20-28SEQ ID NO: 68TVSEFLKLsurvivin97-104SEQ ID NO: 69KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENZ13Mad12AnaxaDRLRLLLKLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEGSTVHEILCKLSLEGDHSTPPSAYGSVKPYINFDAESEQ ID NO: 70MELAALCRWGLLLALLPPGAASTQVCTGTDMKLHer2/neuRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGPYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMGVGSPYVSRLLGICLTSTVQLTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHKHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDCYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAFNPEYLGLDVPVSEQ ID NO: 71STVHEILSKLSLEGDHSTPPSAYGSVKPYTNEDAETLR peptide agonist“Anaxa” sequencevariantSEQ ID NO: 72KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP110DRLRLLLKAPPQVLAFGLLLAAATAYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILCKLSLEGDHSTPPSAYGSVKPYNFDAESEQ ID NO: 73KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP111DRLRLLLKRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHIQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALILGDPKKLLTQHFVQENYLEYRQVPGSDPASYEFLWGPRALVETSYVKVALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVAPPQVLAFGLLLAAATAYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILCKLSLEGDHSTPRSAYGSVKPYTNFDAESEQ ID NO: 74KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP112DRLRLLLKRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSLGDPKKLLTQHFVQENYLEYRQVPGSDPASYEFLWGPRALVETSYVKVALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVAPPQVLAFGLLLAAATAYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 75KRYKNRVASRKSRAKFKOLLQHYREVAAAKSSENATP113DRLRLLLKRTLTFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSLGDPKKLLTQHFVQENYLEYRQVPGSDPASYLELWGPRALVETSYVKVALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVAPPQVLAFGLLLAAATAYVDEKAPEFSMQGLKAGVIAVIVVAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHNVTSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 76KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP114DRLRLLLKRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVVSNLATGRNNSIVKSITVSASGTSPGLSAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAPPQVLAFGLLLAAATAYVDEKAPEFSMQGLKAGVIAVIVVAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPIDRPALGSTAPPVHNVTSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 77KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP115DRLRLLLKRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSEYKLVVVGAVGVGKSALTAPPQVLAFGLLLAAATAYVDEKAPEFSMQGLKAGVIAVIVVAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHINVTSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 78KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP116DRLRLLLKRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAPPQVLAFGLLLAAATAYVDEKAPEFSMQGLKAGVIAVIVVLGDPKKLLTQHFVQENYLEYRQVPGSDPASYEFLWGPRALVETSYVKVALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 79KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP117DRLRLLLKRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAPPQVLAFGLLLAAATAYVDEKAPEFSMGLKAGVIAVIVVAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHNVTSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 80KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP118DRLRLLLKRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQOHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAPPQVLAFGLLLAAATAYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 81KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP119DRLRLLLKLGDPKKLLTQHFVQENYLEYRQVPGSDPASYEFLWGPRALVETSYVKVALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHNVTSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 82KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP120DRLRLLLKRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 83KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP121DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALIAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHNVTSAPPQVLAFGLLLAAATALIYYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 84KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP122DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSSHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHNVTSAPPQVLAFGLLLAAATALIYYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILCKLSLEGDHSTPPSAYGSVKYPYTNFDAESEQ ID NO: 85KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP123DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPCSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHNVTSAPPQVLAFGLLLAAATALIYYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 86KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP124DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHNVTSAPPQVLAFGLLLAAATALIYYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ D NO: 87KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP125DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDRPALGSTAPPVHNVTSAPPQVLAFGLLLAAATALIYYVDEKAPEFSMQGLKAGVIAVIVVSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 88KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP127DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAPPQVLAFGLLLAAATALIYYVDEKAPEFSMQGLKAGVIAVIVVAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPSEGALSPAERELLDFSSWLGGYSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 89KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP128DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPSEGALSPAERELLDFSSWLGGYSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 90KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENATP129DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAKNKIAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPSEGALSPAERELLDFSSWLGGYSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 91KRYKNRVASRKSRAKFQLLQHYREVAAAKSSENATP130DRLRLLLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAKNKIAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPPGTTPVAASPSRASSSPGRGGSSEPGSPRSAYSSDDSGSEGALSPAERELLDFSSWLGGYSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAESEQ ID NO: 92MDGGTLPRSAPPAPPVPVGCAARRRPASPELLREASCL2SRRRRRATAETGGGAAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPPGTTPVAASPSRASSSPGRGGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSSWLGGYSEQ ID NO: 93SAVEYIRALQASCL2 epitopeSEQ ID NO: 94ERELLDFSSWASCL2 epitopeSEQ ID NO: 95APTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKSurvivin fragmentQFEELTLGEFLKLDRERSEQ ID NO: 96NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVCEA fragmentTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSASEQ ID NO: 97AAVARRNERERNRVKLVNLGFQALRQHVPHGGASCL2 fragmentASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPSEGALSPAERELLDFSSWLGGYSEQ ID NO: 98NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVantigenic cargo ofTLDVLPDSSYLSGANLNLSCHSASPQYSWRINGIATP128PQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELTLGEFLKLDRERAAVARRNERERNRVKLVNLCFQALRQHVPIHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPSEGALSPAERELLDFSSWLGGY