VACCINE CONJUGATES

Abstract

The present invention relates to conjugates comprising B- and T-cell epitopes, vaccine compositions comprising said conjugates, their use in the prevention and treatment of cancer, such as prostate cancer, as well as kits comprising the conjugates and/or vaccine compositions. Also claimed are particular T-cell epitope-containing antigenic peptides, and nucleic acids encoding them and constructs and vectors comprising such nucleic acids.

Claims

1. A conjugate comprising at least one B-cell epitope-containing peptide conjugated to a T-cell epitope-containing antigen, wherein: (i) said at least one B-cell epitope-containing peptide comprises a minimal tetanus toxoid epitope (MTTE), said MTTE comprising: (a) an amino acid sequence of at least 10 amino acids which are contiguous in SEQ ID NO: 22 and comprise the amino acid sequence GITELKKL set forth in SEQ ID NO: 23; or (b) an amino acid sequence with at least 70% sequence identity to an amino acid sequence of (a); wherein said B-cell epitope-containing peptide is not the complete tetanus toxin beta chain; (ii) said T-cell epitope-containing antigen is a polypeptide comprising from N-terminus to C-terminus: (a) a translocation peptide; (b) a CD8+ T-cell cancer epitope; and (c) a CD4+ T-cell cancer epitope; (iii) the N-terminus of said T-cell epitope-containing antigen is conjugated to said B-cell epitope-containing peptide; and wherein (iv) the conjugation of the at least one B-cell epitope-containing peptide and the T-cell epitope-containing antigen may be direct or indirect.

2. The conjugate of claim 1, further comprising a proteasome cleavage site positioned between the CD8+ T-cell epitope and the CD4+ T-cell epitope.

3. The conjugate of claim 2, wherein the proteasome cleavage site is provided by a spacer.

4. The conjugate of any one of claims 1 to 3, comprising a spacer sequence between the B-cell epitope-containing peptide and the T-cell epitope-containing antigen, said spacer being C-terminal to the MTTE.

5. The conjugate of any one of claims 1 to 4, wherein the translocation peptide comprises the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence with at least 75% sequence identity thereto.

6. The conjugate of any one of claims 1 to 5, wherein the CD8+ T-cell cancer epitope is a prostate cancer epitope.

7. The conjugate of any one of claims 1 to 6, wherein the CD4+ T-cell cancer epitope is a prostate cancer epitope.

8. The conjugate of any one of claims 1 to 7, wherein the CD8+ T-cell cancer epitope comprises an 8-15 amino acid fragment of SEQ ID NO: 24 or of SEQ ID NO: 25, or an amino acid sequence with at least 65% sequence identity to any such fragment.

9. The conjugate of claim 8, wherein the CD8+ T-cell cancer epitope comprises an 8-9 amino acid fragment of SEQ ID NO: 24 or of SEQ ID NO: 25, or an amino acid sequence with at least 65% sequence identity to any such fragment.

10. The conjugate of claim 8 or 9, wherein the CD8+ T-cell cancer epitope comprises an amino acid sequence selected from any one of SEQ ID NOs: 2, 3, 4, 5 and 6, or an amino acid sequence with at least 65% sequence identity thereto.

11. The conjugate of any one of claims 1 to 10, wherein the CD4+ T-cell cancer epitope comprises an 11-30 amino acid fragment of SEQ ID NO: 24 or of SEQ ID NO: 25, or an amino acid sequence with at least 75% sequence identity to any such fragment.

12. The conjugate of claim 11, wherein said CD4+ T-cell cancer epitope comprises a 12-18 amino acid fragment of SEQ ID NO: 24 or of SEQ ID NO: 25, or an amino acid sequence with at least 75% sequence identity to any such fragment.

13. The conjugate of claim 11 or 12, wherein the CD4+ T-cell cancer epitope comprises an amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10 and 11, or an amino acid sequence with at least 75% sequence identity thereto.

14. The conjugate of claim 10 or 13, being Conjugate I, wherein the CD8+ T-cell cancer epitope comprises the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence with at least 65% sequence identity thereto, and the CD4+ T-cell cancer epitope comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence with at least 75% sequence identity thereto.

15. The conjugate of claim 14, wherein the T-cell epitope-containing antigen comprises the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence with at least 70% sequence identity thereto.

16. The conjugate of claim 10 or 13, being Conjugate II, wherein the CD8+ T-cell cancer epitope comprises the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence with at least 65% sequence identity thereto, and the CD4+ T-cell cancer epitope comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence with at least 75% sequence identity thereto.

17. The conjugate of claim 16, wherein the T-cell epitope-containing antigen comprises the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence with at least 70% sequence identity thereto.

18. The conjugate of claim 10 or 13, being Conjugate III, wherein the CD8+ T-cell cancer epitope comprises the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence with at least 65% sequence identity thereto, and the CD4+ T-cell cancer epitope comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence with at least 75% sequence identity thereto.

19. The conjugate of claim 18, wherein the T-cell epitope-containing antigen comprises the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence with at least 70% sequence identity thereto.

20. The conjugate of claim 10 or 13, being Conjugate IV, wherein the CD8+ T-cell cancer epitope comprises the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence with at least 65% sequence identity thereto, and the CD4+ T-cell cancer epitope comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence with at least 75% sequence identity thereto.

21. The conjugate of claim 20, wherein the T-cell epitope-containing antigen comprises the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence with at least 70% sequence identity thereto.

22. The conjugate of claim 10 or 13, being Conjugate V, wherein the CD8+ T-cell cancer epitope comprises the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence with at least 65% sequence identity thereto, and the CD4+ T-cell cancer epitope comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence with at least 75% sequence identity thereto.

23. The conjugate of claim 22, wherein the T-cell epitope-containing antigen comprises the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence with at least 70% sequence identity thereto.

24. The conjugate of any one of claims 1 to 23, wherein the MTTE comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence with at least 70% sequence identity thereto.

25. The conjugate of any one of claims 1 to 23, wherein the MTTE comprises the amino acid sequence set forth in any one of SEQ ID NOs: 30-86 or an amino acid sequence with at least 70% sequence identity thereto.

26. The conjugate of any one of claims 1 to 25, wherein the N-terminus of the T-cell epitope-containing antigen is conjugated to the C-terminal amino acid of said at least one B-cell epitope-containing peptide.

27. The conjugate of any one of claims 1 to 26, wherein the B-cell epitope-containing peptide further comprises a cysteine residue.

28. The conjugate of any one of claim 21 or 23-25, wherein the at least one B-cell epitope-containing peptide comprises the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 100, or an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 100.

29. The conjugate of any one of claims 1 to 28, wherein said conjugate comprises at least three B-cell epitope-containing peptides.

30. The conjugate of any one of claims 27-29, wherein said conjugate has a chemical structure selected from: ##STR00007## or an isomer or enantiomer of Formula VI; wherein BCECP indicates a B-cell epitope-containing peptide and TCECA indicates a T-cell epitope containing antigen, and in each of said structures each sulphur atom linking a B-cell epitope-containing peptide to an open or closed succinimide ring is from the thiol group of a cysteine residue of the attached B-cell epitope-containing peptide, and the linkage of the T-cell epitope-containing antigen to the chemical structure is a peptide bond to the N-terminus of the T-cell epitope-containing antigen.

31. The conjugate of claim 30, wherein the B-cell epitope-containing peptide comprises the amino acid sequence set forth in SEQ ID NO: 21, and the T-cell epitope-containing antigen comprises the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 20.

32. A conjugate, being Conjugate VI, comprising at least one B-cell epitope-containing peptide conjugated to a T-cell epitope-containing antigen, wherein: said B-cell epitope-containing peptide is as defined in any one of claims 1, 24-25 or 27-28; said T-cell epitope-containing antigen is a peptide comprising a 20-35 amino acid fragment of SEQ ID NO: 18, or an amino acid sequence with at least 70% sequence identity to such a fragment; and the N-terminus of said antigen is conjugated to said B-cell epitope-containing peptide.

33. The conjugate of claim 32, wherein said T-cell epitope-containing antigen comprises the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence with at least 65% sequence identity thereto.

34. The conjugate of claim 33, wherein said T-cell epitope-containing antigen comprises the amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence with at least 70% sequence identity thereto.

35. The conjugate of any one of claims 32 to 34, wherein: (a) said conjugate comprises at least three B-cell epitope-containing peptides; (b) said conjugate has a chemical structure as defined in claim 30; and/or (c) in said conjugate the N-terminus of the T-cell epitope-containing antigen is conjugated to the C-terminus of the B-cell epitope-containing peptide.

36. A vaccine composition comprising at least one conjugate as defined in any one of claims 1 to 35.

37. The vaccine composition of claim 36, said vaccine composition comprising one or more of Conjugate I, II, III, IV or V of claims 13 to 30.

38. The vaccine composition of claim 37, said vaccine composition comprising Conjugates I, II, III, IV and V of claims 13 to 30.

39. The vaccine composition of any one of claims 36 to 38, further comprising Conjugate VI of claims 32 to 35.

40. The vaccine composition of any one of claims 36 to 39, wherein each B-cell epitope-containing peptide of each conjugate in the vaccine composition is identical.

41. The vaccine composition of claim 40, said vaccine composition comprising Conjugate I, Conjugate II, Conjugate IV and Conjugate V of claims 14 to 17 and 20 to 23.

42. The vaccine composition of claim 40, said vaccine composition comprising Conjugate I, Conjugate III and Conjugate V of claims 14 to 15, 18 to 19 and 22 to 23.

43. The vaccine composition of any one of claims 36 to 42, further comprising one or more pharmaceutically-acceptable diluents, carriers or excipients.

44. A conjugate as defined in any one of claims 1 to 35, or a vaccine composition as defined in any one of claims 36 to 43, for use in therapy.

45. A conjugate as defined in any one of claims 1 to 35, or a vaccine composition as defined in any one of claims 36 to 43, for use in prevention or treatment of cancer.

46. The conjugate or vaccine composition for use according to claim 45, wherein said cancer is prostate cancer.

47. A method for the prevention or treatment of cancer in a subject in need of such prevention or treatment, comprising administering to said subject a therapeutically effective amount of a conjugate as defined in any one of claims 1 to 35 or a vaccine composition as defined in any one of claims 36 to 43.

48. Use of a conjugate as defined in any one of claims 1 to 35, or a vaccine composition as defined in any one of claims 36 to 42, in the manufacture of a medicament for use in the prevention or treatment of cancer.

49. A polypeptide comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 13-17, or an amino acid sequence with at least 70% sequence identity thereto, wherein said polypeptide comprises from N-terminus to C-terminus: (a) a translocation peptide; (b) a CD8+ T-cell cancer epitope; and (c) a CD4+ T-cell cancer epitope; wherein a proteasome cleavage site is optionally present between said CD8+ T-cell cancer epitope and said CD4+ T-cell cancer epitope, optionally wherein said cleavage site is provided by a spacer; wherein said translocation peptide is able to mediate TAP-driven transport of said polypeptide or said CD8+ T-cell cancer epitope into the endoplasmic reticulum of a host cell.

50. A nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a polypeptide as defined in claim 49.

51. A construct or vector comprising a nucleic acid molecule as defined in claim 50.

52. A kit comprising a vaccine composition as defined in any one of claims 36 to 43, and a second therapeutically active agent.

Description

[0261] The present invention may be more fully understood from the non-limiting Examples below and in reference to the drawings, in which:

[0262] FIG. 1 shows exemplary reaction schemes for synthesising conjugates of the invention, following the protocols described in Example 2. As shown in the reaction scheme, compound 12 may be either conjugated directly to an SLP, yielding a closed-ringed conjugate of the invention (Conjugate 14) or may first undergo ring-opening and then be conjugated to an SLP, to yield an open-ringed conjugate exemplified by Conjugate 16. Thus the two reaction pathways shown from compound 12 are alternative pathways, one of which yields open-ringed conjugates and the other closed-ringed conjugates.

[0263] FIG. 2 shows cytokine (TNFα and IFNγ) production by T-cells in donor blood, as analysed by flow cytometry. Peptides and conjugates were incubated in human whole blood from prostate cancer patients and healthy donors, pre- and post DTP vaccination (A) or with and without a mouse anti-MTTE IgG2a antibody (B). The change for each individual donor is shown, the result pre-vaccination (or without anti-MTTE antibody, left) being linked to the result post-vaccination (or with anti-MTTE antibody, right). The cells were gated as CD45RO+CD3+CD4+CD8− or CD45RO+CD3+CD4−CD8+, and the % of IFNγ+ and TNFα+ cells are displayed. The blood was either untreated (0 time point) or treated with saline solution (NaCl), Conjugates I-VI of the invention (LUR1-6) or the T-cell epitope-containing antigens of Conjugates I-VI (SLP1-6). The [MTTE].sub.3-NLV conjugate containing the HLA-A*0201-restricted epitope pp65(NLV) from CMV and CMV lysate were used as positive controls, and the MTTE3-irrelevant (MTTE3-irrel) conjugate was used as a further control. This conjugate contained a scrambled SLP sequence (DGLQGLLLGLRQRIETLEGK, SEQ ID NO: 88) without any known human T cell epitopes. The dot plots of the three responding donors from A and B are displayed in C and D.

[0264] FIG. 3 shows the titres of anti-MTTE antibodies in cancer patients' plasma before and after receipt of a DTP booster. FIG. 3A shows the titre of total IgG antibodies, FIG. 3B the titre of IgM antibodies, FIG. 3C the titre of IgG1 antibodies and FIG. 3D the titre of IgG4 antibodies. **equals p-value <0.01 and ns=non-significant, assessed by paired t-test.

[0265] FIG. 4 shows the results of in vitro antigen presentation experiments, using antigen provided in constructs synthesised according to Examples 2 and 3. FIG. 4A shows T-cell activation levels using various concentrations of a conjugate with intact succinimide rings; FIG. 4B shows T-cell activation levels using various concentrations of an equivalent conjugate in which the succinimide rings have been opened.

[0266] FIG. 5 shows cytokine (IFNγ) production by memory (CD45RO+) or non-memory (CD45RO−) CD8+ T-cells in donor blood from a patient that responded in FIG. 2. The blood is either untreated (0 time point) or treated with saline solution (NaCl). Blood from the donor was subjected to either a mix of conjugates I-VI of the invention (LUR1-6) or each individual conjugate was assessed alone. Results are shown as the fold increase of IFNγ production compared to vehicle-exposed blood.

[0267] FIG. 6 is a schematic diagram showing the general structure of SLP1-6, the T-cell epitope-containing antigens of Conjugates I-VI respectively. Each SLP contains the same TAP sequence, but the CD8 and CD4 epitopes, and the proteasome cleavage site, differ between the SLPs. The N- and C-termini of the SLPs are indicated.

[0268] FIG. 7 shows the binding of GMP LUG1-6 constructs to human anti-MTTE antibodies. A shows binding of conjugates to monoclonal human IgG1 anti-MTTE antibodies. Conjugates were coated onto an ELISA plate at a range of concentrations (from 0.000457-1 nmol/ml). Human recombinant anti-MTTE IgG1 antibody was used as primary antibody and detection was performed using an anti-human kappa light chain secondary antibody. B shows binding of conjugates to polyclonal human anti-MTTE antibodies from a donor. Conjugates were coated onto an ELISA plate at a range of concentrations (from 0.004-1 nmol/ml) and incubated with diluted donor plasma. Detection was performed using an anti-human kappa light chain secondary antibody.

[0269] FIG. 8 shows the effects on anti-MTTE titre of vaccination of animals with LUG2 conjugates (A), and (B) ELISPOT analysis of T cell responses after HLA-DR4 mice had been vaccinated with LUG2. HLA-DR4 transgenic mice were subcutaneously vaccinated with LUG2 (20 μg) using a prime/boost schedule. A week later the mice were sacrificed, heart bleed was performed, serum was analysed by anti-MTTE ELISE and the splenocytes were analysed using IFNγ ELISPOT. ELISPOT was performed by incubation of the splenocytes with the SLPs UV02 (SEQ ID NO: 14) and UV08 (SEQ ID NO: 107) for 48 h. SEB was used as positive control and untreated splenocytes as negative control.

[0270] FIG. 9 shows an analysis of TENDU toxicity in a human blood loop assay and in male rabbits (the TENDU vaccine comprises the LUG1-6 conjugates, which as described below correspond to Conjugates I-VI described herein manufactured to GMP standard). Fresh blood from five Boostrix vaccinated prostate cancer patients and five healthy individuals was transferred to the loops. The LUG1-6 constructs were added at the respective concentrations or NaCl was added as a vehicle. Alemtuzumab (3 μg/ml) was added to the respective loops as positive control. Plasma samples were collected at 0 min and 15 min for measurement of C3a and C5a by ELISA (A-B). For analysis of IL-8 (C), IFNγ (F), IL-6 (G), IL-1β (H) and TNFα (I) plasma samples were collected at 0 min and 4 h. Plasma samples were analysed using the MSD array and MSD software. The LLOD and ULOD were defined as described in the methods. Male rabbits were subcutaneously vaccinated four times with Equip-T followed by four subcutaneous TENDU vaccination at either low, intermediate or high dose (See Table VI). The vaccinations were given every two weeks and plasma samples were collected on week 8 and week 15 before the first and last administration of TENDU and 4 h and 24 h after TENDU administration. Plasma was analysed using rabbit ELISA Kits. Concentrations of IFNγ (D) and IL-8 (E) were calculated.

EXAMPLES

Example 1—B-Cell Epitope-Containing Peptide Design

[0271] A variety of designs of B-cell epitope-containing peptides comprising the amino acid sequence set forth in SEQ ID NO: 1 were synthesised, and analysed to determine a design to allow for antibody binding to the MTTE sequence. As seen previously in the published patent application WO 2011/115483, N-terminal modification hampered antibody binding to the MTTE.

[0272] The synthesised peptides were conjugated to biotin (at either their C- or N-terminus). Certain of the peptides included an additional amino acid sequence which formed a spacer between the biotin and the MTTE of SEQ ID NO: 1. The peptides designed are set forth in Table 1, below. A control peptide was also synthesised, comprising a scrambled MTTE sequence with a C-terminal spacer sequence (SEQ ID NO: 103) conjugated to biotin.

TABLE-US-00002 TABLE 1 SEQ ID NO of Amino Biotin Acid Location Peptide Sequence Sequence C-terminal F-I-G-I-T-E-L-K-K-L-E-S-K-I-N-K-V-F-S-S-A- 104 F-A-D-V-E-A-A C-terminal F-I-G-I-T-E-L-K-K-L-E-S-K-I-N-K-V-F   1 C-terminal F-I-G-I-T-E-L-K-K-L-E-S-K-I-N-K-V-F-A-A-K- 105 Y-A-R-V-R-A C-terminal E-K-L-l-N-K-L-S-K-l-F-K-G-T-I-E-V-F-S-S-A- 103 F-A-D-V-E-A-A N-terminal F-I-G-I-T-E-L-K-K-L-E-S-K-I-N-K-V-F-A-A-K- 105 Y-A-R-V-R-A

[0273] Antibody binding to each peptide was analysed by ELISA. The biotinylated peptides were incubated on a streptavidin-coated Nunc-Immuno MaxiSorp plate. A polyclonal rabbit anti-MTTE antibody batch was used to titrate the titre to each individual peptide coated on the plate. S-shaped curves were calculated using the Bolzmann formula. The titre-value, herein the dilution of 50% of max-absorbance, was extracted from the Bolzmann data of the curve. A goat anti-rabbit IgG conjugated to alkaline phosphatase was used as secondary antibody, and 4-nitrophenyl phosphate disodium salt hexahydrate was used to develop the assay. Absorbance was then read at 405 nm to determine the titre. The results are presented in Table 2, below:

TABLE-US-00003 TABLE 2 Biotin Location Peptide Sequence Titre (EC50) C-terminal SEQ ID NO: 104 500 C-terminal SEQ ID NO: 1 400 C-terminal SEQ ID NO: 105 800 C-terminal SEQ ID NO: 103 0 N-terminal SEQ ID NO: 105 200

[0274] The biotinylated peptide without a spacer displays a titre of 400, whereas the two peptides comprising spacers C-terminal to SEQ ID NO: 1 display similar or enhanced titres, indicating that a C-terminal spacer does not negatively influence antibody binding. Conjugation of biotin to the C-terminus of the peptide was found to be important for optimal antibody binding. As shown above, conjugation of biotin to the N-terminus of the MTTE resulted in antibody binding to the MTTE being reduced by half. The scrambled MTTE sequence with a C-terminal spacer does not display any titre, indicating that no antibody bound the peptide, regardless of the inclusion of a spacer.

Example 2—Conjugate Synthesis

[0275] In this Example the synthesis of a construct of the invention is described. This Example relates to the synthesis of a construct containing 3 B-cell epitope-containing peptides, which comprise the MTTE sequence FIGITELKKLESKINKVF (SEQ ID NO: 1) and a C-terminal spacer with the sequence AAKYARVRAKC (SEQ ID NO: 102) (i.e. they have the sequence set forth in SEQ ID NO: 21); and an example T-cell epitope-containing antigen with the sequence LEQLESIINFEKLAAAAAK (SEQ ID NO: 87) derived from ovalbumin (UniProt accession number P01012). The synthesis was performed as described on pp. 40-45 of EP 2547364 B1 (WO 2011/115483). For completeness, the reaction scheme is shown in FIG. 1 (all compound numbers (bold) in this example refer to the compounds of FIG. 1).

[0276] Core Synthesis

[0277] The core of the conjugate (10) was synthesised as described in [113]-[121] of EP 2547364 B1.

[0278] Peptide Synthesis

[0279] The two peptides used in this Example were synthesised as described in [122] of EP 2547364 B1. The peptides synthesised were:

TABLE-US-00004 (i) F-I-G-I-T-E-L-K-K-L-E-S-K-I-N-K-V-F-A-A-K-Y- A-R-V-R-A-K-C (MTTE-spacer-SH, peptide 11); and (ii) Azidohexanoyl-L-E-Q-L-E-S-I-I-N-F-E-K-L-A- A-A-A-A-K (azido-antigen, peptide 13):

Construct Synthesis

[0280] Construct 14 was synthesised as described in [123] of EP 2 547 364 E1.

Ring Opening

[0281] Succinimide rings can cause molecular instability, meaning that compounds containing succinimide rings can show limited stability under particular conditions, especially under basic conditions and at elevated temperatures. An assessment of stability of the constructs with succinimide rings (e.g. construct 14) was performed, in which the constructs were incubated at pH 8.7 and 30° C. for 46 hours. Under these conditions, after 46 hours virtually all succinimide rings were hydrolysed, and some of the molecules had lost an MTTE group (data not shown). Thus, to avoid instability issues, an extra incubation step for succinimide ring opening was introduced to the conjugate synthesis pathway, yielding stable constructs.

[0282] Open ring constructs were obtained as follows: 10 mg MTTE-spacer-SH (peptide 11) was dissolved in 300 μl Milli-Q water. A solution of core structure 10 in 100 μl acetonitrile was added and the pH adjusted to 6 with 4.2% NaHCO.sub.3. The reaction was allowed to proceed at room temperature for about 1 hour, yielding compound 12. The succinimide rings of compound 12 were then opened as follows: 425 μl tBuOH/water (9:1, v/v) and 100 μl 4.2% NaHCO.sub.3 were added to the reaction mixture containing the newly-synthesised compound 12. The ring opening reaction was allowed to proceed for 72 hours at 30° C. The reaction mixture was brought to pH 6 with 0.5 M acetic acid. Ring opening yields a mixture of 8 open-ringed isomers, as each succinimide ring may be opened such that the sulphide group is adjacent to either the amide bond or the carboxyl group. An example of an open-ringed isomer obtained from ring opening is presented as Compound 15. To this mixture was added azidohexanyl SLP (13) in DMSO, and an open-ringed conjugate comprising an MTTE and T-cell epitope-containing SLP generated (Compound 16, shown, is the conjugate obtained from attachment of an SLP to Compound 15).

Example 3—Synthesis of Alternative Open-Ringed Conjugates

[0283] All construct numbers are the same as in Example 2/FIG. 1.

[0284] Constructs 10 and 11 were synthesised as described above. An azido-peptide comprising an antigen with the amino acid sequence:

TABLE-US-00005 (SEQ ID NO: 15) A-R-W-W-S-L-S-L-G-F-L-F-L-A-A-A-G-K-V-F-R-G-N-K- V-K-N-A-Q-L-A
was synthesised using the same protocol as for the synthesis of compound 13, described above, with the exception that azidopropanoic acid was used instead of azidohexanoic acid.

[0285] Compound 12 was synthesised, and its rings opened, as above. To this mixture was added azidopropionyl SLP in DMSO, and open-ringed conjugates comprising an MTTE and T-cell epitope-containing SLP generated. The resultant compounds were analysed by mass spectrometry as described above. The constructs have a calculated mass of 14698.4 Da, and a measured deconvoluted average mass of 14699.0 Da.

Example 4—Selection of T-Cell Epitope Combinations and TTES

[0286] 11 known prostate cancer CD8+ T-cell epitopes (C1-11) and 6 known prostate cancer CD4+ T-cell epitopes (H1-H6) were selected from the literature:

TABLE-US-00006 CTL-epitope SEQ ID Code sequence NO: HLA Allele Bound C1 ILLWQPIPV 90 A2 C2 YLPFRNCPR 91 A3, A11, A31, A33 C3 LYCESVHNF 92 A24 C4 GMPEGDLVY  5 A1 C5 LLHETDSAV  3 A2 C6 MMNDQLMFL 93 A2 C7 VLAGGFFLL 94 A2 C8 KVFRGNKVK  6 A3, A11, A31, A33 C9 NYARTEDFF  2 A24 C10 LLAVTSIPSV 95 A2 C11 SLSLGFLFL  4 A2 Helper-epitope SEQ ID Code sequence NO: HLA Allele Bound H1 GQDLFGIWSKVYDPL  7 Promiscuous H2 TEDTMTKLRELSELS 96 Promiscuous H3 GKVFRGNKVKNAQLA  9 Promiscuous H4 TGNFSTQKVKMHIHS 10 DR4 H5 NYTLRVDCTPLMYSL 11 DR1/DR9 H6 RQIYVAAFTVQAAAE  8 DR4/DR9

[0287] It was decided that the conjugates of the prostate cancer vaccine would contain a single long peptide (SLP) comprising one each of C1-11 and H1-6, and that the vaccine would comprise 5 such conjugates, each with a different CD8+ and CD4+ epitope. Epitope combinations were selected based on the requirement that a candidate long peptide should contain a CTL-epitope that is properly TAP translocated and of which the C-terminus is generated in the context of the longer peptide also containing the helper T-cell epitope.

[0288] SLPs containing all 66 possible combinations of the listed CTL-epitopes and Helper-epitopes were synthesised. These peptides were treated with commercially-available immuno-proteasome according to the protocol of the supplier. Each peptide (1 μl of the DMSO stock solution) was added to 300 μl aqueous buffer containing 0.5 μg immunoproteasome 20S (human, purified, BML-PW9645-0050, Enzo Life Science), 30 mM Tris-HCl (pH 7.2), 10 mM KCl, 5 mM MgCl.sub.2 and 1 mM DTT. The mixture was vortexed and incubated at 37° C. for various time periods. At each time point an aliquot (50 μl) was taken from the digestion mixture, added to 4 μl formic acid and the solution obtained was homogenised by vortexing and stored at −20° C. until analysis. For analysis 1 μl of this solution was mixed with 1 μl matrix solution (10 mg/ml α-cyano-4-hydroxy cinnamic acid (ACH) in acetonitrile/water 1/1 containing 0.2% TFA) and spotted on a MALDI-TOF target plate. Samples at all time points were analysed with MALDI-TOF mass spectrometry (Bruker Microflex) revealing the proteasome-induced peptide fragments (>800 Da).

[0289] Proteasomal degradation was monitored after 24 hr digestion using MALDI-TOF mass spectrometry with a Bruker Microflex or a Bruker Ultraflex instrument. Epitope combinations which were incorrectly cleaved (i.e. were not cleaved between the two epitopes) were resynthesised with spacer sequences between the CD8+ and CD4+ T-cell epitopes, and their cleavage retested. Appropriate spacer sequences were predicted using the online programme NetChop 3.1 (prediction method: C-term 3.0; threshold: 0.5). Optimal cleavage was identified for the following epitope combinations: C9-H1 (with a spacer of SEQ ID NO: 29); C5-H6 (with a spacer with the sequence A-A-A); C11-H3 (with a spacer with the sequence A-A-A); C4-H4 (with no spacer); and C8-H5 (with no spacer). Following cleavage of these peptides, N-terminal fragments comprising the CTL epitope and C-terminal fragments comprising the Helper epitope were identifiable.

[0290] To enhance translocation of the selected CTL epitopes into the endoplasmic reticulum (ER), the algorithm TAPREG (http://imed.med.ucm.es/Tools/tapreg; Diez-Rivero et al. (2010), Proteins 78: 63-72) was used to identify a TTES (Tap Translocation Enhancing Sequence). Based on the TAPREG analysis, the amino acid sequence ARWW (SEQ ID NO: 12) was selected. The SLPs developed as described above were synthesised with the identified TTES at the N-terminus and incubated in vitro and tested by a TAP translocation assay. The TAP translocation assay was performed as described in Neefjes et al., Science 261: 769-771 (1993). The general structure of the designed SLPs is shown in FIG. 6.

Example 5—Proof of Concept Studies with Specific Conjugates of Invention

[0291] Methods

[0292] Blood Loop Assay

[0293] Blood from donors was taken in an open system and immediately mixed with the anti-coagulant heparin (Leo Pharma AB, Sweden) to a final concentration of 1 IU/ml. All materials in direct contact with the blood were surface-heparinised using the heparin coating kit from Corline (Sweden). Blood and conjugates were applied to heparinised PVC tubings from Corline, which were then sealed using specialised metal connectors, forming loops. The blood loops were rotated on a wheel within a 37° C. incubator. At the end-point sampled blood was mixed with EDTA to a final concentration of 10 mM immediately to stop any ongoing reaction and to prevent clotting of blood. The platelets were counted at 0 and at the end time-point using either a Coulter® Ac-T Diff™ Analyser (Beckman Coulter, Miami, Fla.) or XP-300 (Sysmex, Japan) to ensure that coagulation had not occurred during the experimental procedure and as a response to the reagents added. Plasma was collected and stored at −80° C.

[0294] Intracellular Staining & Flow Cytometry Analysis

[0295] The intracellular staining of IFNγ and TNFα was performed by adding brefeldin A (Sigma-Aldrich) after 2 hours of circulation of conjugates in the blood loop system. The experiment was terminated after another 4 hours as described for the blood loop system above.

[0296] Antibodies for flow cytometry analysis were purchased from Biolegend: anti-CD3 (Clone UCHT1), anti-CD4 (Clone OKT-4), anti-CD8 (clone SK1), anti-CD45RO (Clone UCHL1), anti-IFNγ (Clone 4S.B3) and anti-TNFα (Clone MAb11). Whole blood was stained with cell surface-specific antibodies before red blood cell lysis using FACS lysing solution (BD Biosciences) according to the manufacturer's instructions. The remaining cells were washed and fixed with BD Cytofix/Cytoperm buffer at 4° C. in the dark for 20 minutes. To permeabilize the cells they were first washed and then incubated with Perm/Wash Buffer (BD Biosciences) at RT for 10 minutes. The cells were stained for IFNγ and TNFα for 30 minutes at 4° C. in the dark and subsequently washed in PBS with 1% BSA and 3 mM EDTA (Sigma-Aldrich).

[0297] Following staining, the cells were analysed using a Canto II flow cytometer (BD Biosciences) or Cytoflex (Beckman coulter). The cell populations were gated and analysed using FlowJo (Tree Star).

[0298] Ethical Considerations

[0299] Blood sampling and DTP vaccination of healthy volunteers were approved by the local ethical committee. In short, an 18G gauge needle attached to heparinized tubing was used to draw blood. The blood was collected in a 50 ml surface-heparinized tube and subsequently transferred to the loop tubing and then set to rotate as described above. The DTP vaccination was performed by routine personnel at the hospital using a standard vaccine cocktail.

[0300] Results

[0301] Blood was taken from donors (prostate cancer patients and healthy volunteers) and the loop assay was performed.

[0302] The blood was set to rotate in plastic tubings. Three blood samples from each donor were used: to one of these LUR1-6 conjugates were administered, to another the corresponding naked T-cell epitope-containing antigens (SLP1-6) and to the third saline solution. The LUR1-6 conjugates correspond to Conjugates I-VI as described herein. They were synthesised as described in Example 2; they comprise B-cell epitope-containing peptides of SEQ ID NO: 100 and T-cell epitope containing antigens of SEQ ID NOs: 13-17 and 20, respectively. SLP1-6 correspond to peptides of SEQ ID NOs: 13-17 and 20, respectively.

[0303] 2 hours after administration of LUR1-6 or SLP1-6, brefeldin was added and after yet another 4 hours the blood was sampled and intracellular staining was performed to analyse cytokine production. Low levels of cytokine production by memory CD8+ T-cells were observed. The donors were then given a booster vaccine comprising TTd (a DTP vaccine) to boost their levels of anti-TTx antibodies. Within approximately 1-2 weeks from this booster, the loop experiment and cytokine production analysis were repeated. Cytokine production in the memory CD8+ T cells was now found in the LUR1-6-treated blood of the two individuals with the highest anti-MTTE-IgG1 levels pre-vaccination (one patient and one healthy volunteer), as shown in FIG. 2A, C. Other cell populations including CD4+CD45RO+ memory T cells (FIG. 2A, C), CD8CD45RO− and CD4CD45RO− (not shown) were unresponsive to treatment with LUR1-6.

[0304] These results show that Conjugates I-VI can induce an immune response. The healthy individual in whose blood cytokine production was found was also a male, as such the cytokine production in his blood may be due to previous or ongoing prostatitis that has triggered activation and expansion of auto-reactive T cells.

[0305] Blood from a non-DTP-vaccinated patient (donor PMO30) was treated with mouse anti-MTTE IgG2a together with the LUR1-6 mixture, inducing TNFα release by CD8+CD45RO+T memory cells (FIG. 2B, D).

Example 6—DTP Booster Increases Anti-TTx Antibody Titre in Cancer Patients

[0306] The results of Example 5 suggested that administration of a DTP booster vaccine to cancer patients causes an increase in the titres of anti-TTx antibodies, including antibodies which recognise the MTTE of SEQ ID NO: 1 (as is the case in healthy volunteers, Fletcher et al., Journal of Immunology 201(1): 87-97 2018). This was tested experimentally.

[0307] Methods

[0308] Plasma was obtained from patients as described above in Example 5. Plasma was taken both before a patient received a DTP vaccination and 7-10 days afterwards.

[0309] Anti-MTTE antibody titres in plasma from patients (pre- and post-DTP vaccination) were determined using an in-house ELISA. Streptavidin plates (Thermo Scientific) were coated with the peptide of SEQ ID NO: 104, biotinylated at its C-terminus and a scrambled peptide (ETTM) of SEQ ID NO: 103 (also biotinylated at its C-terminus) overnight at 4° C. The plates were washed with PBS (0.05% Tween) and blocked with PBS (10% BSA and 0.05% Tween) for 1 hour at RT. The plasma was serially diluted in PBS (1% BSA and 0.05% Tween), applied to the plates and incubated for 2 hours at RT. MTTE-specific IgM and IgG antibodies were detected with secondary HRP-conjugated antibodies: rabbit anti-human IgG (polyclonal antibody from Dako; diluted 1:4000), anti-IgG1 (Clone HP6070 from Thermo Fisher; diluted 1:500), anti-IgG4 (Clone HP6023 from Thermo Fisher; diluted 1:500) and anti-IgM (polyclonal from Dako; diluted 1:1000). The secondary HRP-conjugated antibodies were diluted in PBS (1% BSA) and incubated on the plates for 1 hour at RT. The reaction was developed with the substrate TMB (Dako) and stopped with 1 M H.sub.2SO.sub.4. The absorbance was read at 450-570 nm using an iMark microplate reader (Bio-Rad).

[0310] Results

[0311] The results of the analysis are shown in FIG. 3. FIG. 3A shows the titres of IgG antibodies obtained from patients' plasma before and after DTP vaccination; FIG. 3B shows the titres of IgG1 antibodies, FIG. 3C the titres of IgG4 antibodies and FIG. 3D the titres of IgM antibodies.

[0312] As shown, a statistically significant increase in the titre of IgG1 antibodies which recognise the MTTE of SEQ ID NO: 1 was seen following administration of a DTP booster to the patients, relative to beforehand. No increase in the titres of IgG4 or IgM antibodies was seen post DTP boost. The data was analysed using paired t-test.

Example 7—In Vitro Antigen Presentation

[0313] Methods

[0314] Cells

[0315] D1 cells are growth factor-dependent immature dendritic cells (DCs) initially derived from a C57BL/B6 mice. Immature D1 cells were cultured with GM-CSF (20 ng/ml). B3Z is a murine T-cell hybridoma specific for the OVA-derived CD8+ epitope SIINFEKL (SEQ ID NO: 89) in the context of the murine Class I MHC H-2Kb, and which expresses β-galactosidase under the control of the IL-2 promoter (Karttunen et al., PNAS 89(13): 6020-6024, 1992). B3Z cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) with 10% heat-inactivated FBS, 1% penicillin/streptavidin, 50 μM β-mercaptoethanol and supplemented with Hygromycin B (Invitrogen, Life technologies, Rockville, USA). The generation and culturing of hybridoma cell lines producing mouse anti-MTTE IgG1 and IgG2a antibodies (i.e. antibodies which recognise SEQ ID NO: 1) was performed as described in Fletcher et al. (supra).

[0316] In Vitro Antigen Presentation Assay

[0317] The antigen presentation assay was performed as previously described (Mangsbo et al, Molecular Immunology 93: 115-124 (2018)). Briefly, immune complexes were pre-formed by incubating the conjugates synthesised in Examples 2 and 3 (which contain the SIINFEKL T-cell epitope recognised by B3Z cells) with an antigen-specific antibody (anti-MTTE IgG1 or IgG2a) at 37° C. for 30 minutes. The immune complexes were incubated with D1 cells (2.5×10.sup.4/well) for 24 hours, supernatant was removed and subsequently B3Z cells were added and incubated for another 24 hours (5×10.sup.4/well) at a DC:T-cell ratio of 1:2. The immune complexes were pre-formed at concentrations 3-fold higher than their working concentrations. Addition of the complexes to the D1 cells resulted in their dilution to their working concentrations. The cells were then lysed with a lysing solution (100 mM β-mercaptoethanol, 0.125% IGEPAL CA-630, 9 mM MgCl.sub.2) containing the β-galactosidase substrate chlorophenol red-β D-galactopyranoside (CPRG; 1.8 μg/ml) at 37° C. for 6 hours before the absorbance was measure at 595 nm using an iMark microplate reader (Bio-Rad).

[0318] Binding of GMP LUG1-6 Constructs to Human Monoclonal Anti-MTTE IgG1 Antibody

[0319] An in-house ELISA was used to confirm binding of GMP-produced LUG1-6 constructs to a recombinant human monoclonal anti-MTTE IgG1 antibody. ELISA plates were coated with 100 μl/well conjugates diluted in Milli-Q water at a range of concentrations (0.000457-1 nmol/ml, a single conjugate per well). The plates were covered and incubated at 4° C. overnight. The plates were subsequently washed four times and blocked with 200 μl/well PBS containing 10% BSA and 0.05% Tween20 and incubated at room temperature (RT) for 1 hour. After washing, the human chimeric anti-MTTE IgG1 antibody (custom made by Evitria AG, Switzerland, >99% monomeric content and <0.1 EU/mg endotoxin), at 0.1 μg/ml in PBS supplemented with 1% BSA and 0.05% Tween20 was added. The plates were washed four times with 250 μl/well PBS containing 0.05% Tween20 and the secondary antibody diluted 1:8000 in PBS supplemented with 1% BSA (anti-human kappa light chain secondary antibody, Thermo Fisher Scientific #A18853) was added to all wells (100 μl/well). After incubation for 1 hour at RT in the dark the plates were washed and 100 μl TMB was added to the wells. The reaction was stopped with 100 μl/well 1 M H.sub.2SO.sub.4 and the absorbance was measured at 450-570 nm wavelength.

[0320] Binding of GMP LUG1-6 Constructs to Human Polyclonal Anti-MTTE Antibody

[0321] The same in-house ELISA as above was used to confirm binding of GMP-produced LUG1-6 constructs to human polyclonal anti-MTTE antibody from plasma from a human donor previously confirmed to have anti-MTTE antibodies.

[0322] ELISA plates were coated with 100 μl/well conjugate diluted in Milli-Q water at a range of concentrations (0.004, 0.03, 0.4 and 1 nmol/ml, a single conjugate per well). The plates were covered and incubated at RT for 2 hours. The plates were then washed 4 times with 250 μl/well PBS containing 0.05% Tween20. The plates were then blocked 3 times with 200 μl/well Superblock T20 (Thermo Scientific) for 5 mins at RT. Plates were washed 4 times with 250 μl/well PBS containing 0.05% Tween20. Donor human plasma was diluted 1:200 in PBS supplemented with 1% BSA and 0.05% Tween20, and 100 μl/well applied to the plates, which were then incubated for 2 hours at RT. Plates were again washed 4 times with 250 μl/well PBS containing 0.05% Tween20, and the secondary antibody diluted 1:8000 in PBS supplemented with 1% BSA (anti-human kappa light chain secondary antibody, Thermo Fisher Scientific #A18853) was added to all wells (100 μl/well). After incubation for 1 hour at RT in the dark the plates were washed and 100 μl TMB was added to the wells. The reaction was stopped with 100 μl/well 1 M H.sub.2SO.sub.4 and the absorbance was measured at 450-570 nm wavelength.

[0323] Results

[0324] DC1 dendritic cells were incubated with immune complexes formed from conjugates synthesised according to Examples 2 and 3. These conjugates are essentially identical, except that the conjugates synthesised according to Example 2 contain intact succinimide rings, whereas the rings of the conjugates synthesised according to Example 3 are opened. The results of these experiments are shown in FIG. 4, in which a higher absorbance at 595 nm indicates a higher level of T-cell activation.

[0325] The results obtained with presentation of antigen from conjugates with intact rings are shown in FIG. 4A; the results obtained with presentation of antigen from conjugates with opened rings are shown in FIG. 4B. As can be seen, both conjugates were able to activate B3Z T-cells in the antigen presentation assay, though the conjugate with opened succinimide rings drove a greater level of T-cell activation. Conjugates with open rings were also confirmed to bind anti-MTTE antibodies as analysed by ELISA (both recombinant monoclonal human IgG1 antibody, FIG. 7A; and polyclonal human donor antibody, FIG. 7B).

Example 8—HLA Profile of Responders and Memory CD8 T-Cell Responses to an Individual Construct in One Patient and One Healthy Individual

[0326] The two individuals whose blood showed an increased response to the mix of LUR1-6 conjugates following DTP booster vaccination in Example 5 were analysed to determine their HLA profiles, and thus which conjugate(s) they may have been responding to. In addition one patient that did not receive a DTP booster but that displayed a response when rabbit anti-MTTE antibodies were given in conjunction with the constructs was assessed. However this donor that was also HLA profiled, displayed blood clotting during sampling and as such the experimental plan was not fully executed and all loops were not run. Thus this patient was removed from the data analysis and is not displayed below:

[0327] Based on the peptide's CD8 epitope and donor's HLA-type class I the analysed patient can respond to LUG2, 3 and 6 and the healthy individual can respond to LUG2, 3, 5 and 6.

[0328] LUG1=SEQ ID NO: 13; LUG2=SEQ ID NO: 14; LUG3=SEQ ID NO: 15; LUG4=SEQ ID NO: 16; LUG5=SEQ ID NO: 17; LUG6=SEQ ID NO: 20.

TABLE-US-00007 HLA-TYPE HLA-TYPE DONOR CLASS I CLASS II PATIENT HLA*A2:01 HLA-DRB1*04 HLA-DRB1*13 HEALTHY HLA*A2:01 HLA-DRB1*15 INDIVIDUAL HLA*A03:01:01

[0329] Methods

[0330] The loop assay was performed as in Example 5 but with the following loops assessed per individual: [0331] 1. Vehicle (NaCl 0.9%) [0332] 2. anti-MTTE IgG2a (40 μg/ml) [0333] 3. LUG1-6 (6×125 nM) [0334] 4. LUG1-6 (6×125 nM)+anti-MTTE IgG2a (40 μg/ml) [0335] 5. LUG1 (125 nM) [0336] 6. LUG1 (125 nM)+anti-MTTE IgG2a (40 μg/ml) [0337] 7. LUG2 (125 nM) [0338] 8. LUG2 (125 nM)+anti-MTTE IgG2a (40 μg/ml) [0339] 9. LUG3 (125 nM) [0340] 10. LUG3 (125 nM)+anti-MTTE IgG2a (40 μg/ml) [0341] 11. LUG4 (125 nM) [0342] 12. LUG4 (125 nM)+anti-MTTE IgG2a (40 μg/ml) [0343] 13. LUG5 (125 nM) [0344] 14. LUG5 (125 nM)+anti-MTTE IgG2a (40 μg/ml) [0345] 15. LUG6 (125 nM) [0346] 16. LUG6 (125 nM)+anti-MTTE IgG2a (40 μg/ml)

[0347] An average value was calculated from each duplicate loop, regardless of whether the loop was spiked with rabbit anti-MTTE antibodies or not. Both were used in the analysis as values with or without anti-MTTE spiked loops did not differ and as endogenous antibodies are present to the MTTE in these individuals from the previous DTP vaccination. The mean value of loop 1 and 2 was used as the background vehicle value. The fold change was calculated by the mean value of the compound exposed loop divided by the mean value of the background vehicle sample.

[0348] The results of the fold increase in recall response of IFNγ-producing CD8+ memory (CD45RO+) T-cells is shown in FIG. 5. CD4 responses were not detectable at the tested time point (not shown).

[0349] Results

[0350] The results show that the patient responded with IFNγ production to both the mix of the conjugates and to individual conjugates and that the response to the individual conjugates matches the expected response based on the HLA type. The healthy individual did not display a response above background during this analysis, possibly reflecting that any inflammatory cause that led to a spike in auto-immune T-cells in circulation at the first analysis (FIG. 2) was not present and as such those auto-reactive T-cells had vanished.

[0351] The patient also displayed a response to LUG5 which cannot be predicted based on the HLA profile of the selected CD8 epitope. However it cannot be excluded that the CD4 epitope harbours an HLA class I epitope that the patient responds to. Via the IEBD analysis resource Consensus tool (Kim et al., Protein Sci 12: 1007-1017 (2003)) the sequence YTLRVDCTPL (SEQ ID NO: 97) in the CD4 epitope in LUG5 was predicted to bind to HLA-A*02:01 with a low percentile rank.

[0352] LUG5 also displayed elevated IFNγ responses in the CD4+CD45RO negative population (not shown).

[0353] Whenever the abbreviation LUG is used, it means that a construct is of GMP quality, and whenever the abbreviation LUR is used, it means that a construct is made for research purposes. Structurally each construct LUG1, LUG2, LUG3, LUG4, LUG5 and LUG6 corresponds to the construct LUR 1, LUR2, LUR3, LUR4 LUR5 5 and LUR6.

Example 9—Testing of Conjugates in Mice

[0354] Methods

[0355] Evaluation of Epitope-Specific T Cell Responses in Humanised HLA-DR4 Mice

[0356] Female HLA-DR4 transgenic mice on a C57/B16 background (12 weeks old at the start of the study) were acquired from Taconic (Germantown, Md., USA). HLA-DR4 animals were administered a LUG2 construct (20 μg or 5 μg) subcutaneously at the tail base followed by a boost two weeks later. A week later the mice were sacrificed, and the spleens were collected for generation of single cell suspensions for analysis by ELISPOT as described below. Heart bleed was performed to analyze anti-MTTE titers after LUG2 exposure. Tail vein-sampled HLA-DR4 animals that had not been exposed to LUG2 were used as controls for baseline titre assessment (unexposed animals).

[0357] Evaluation of Immune Responses

[0358] Antibody titres against the MTTE were determined using an in-house ELISA. Streptavidin plates (Thermo Scientific) were coated with the peptide of SEQ ID NO: 104, biotinylated at its C-terminus, overnight at 4° C. The plates were washed with PBS (0.05% Tween) and blocked with PBS (10% BSA and 0.05% Tween) for 1 hour at RT. The mouse serum was serially diluted in PBS (1% BSA and 0.05% Tween), applied to the plates and incubated for 2 hours at RT. Mouse MTTE-specific IgG antibodies were detected with secondary HRP-conjugated antibody: goat anti-mouse IgG (polyclonal antibody from Dako; diluted 1:4000). The secondary HRP-conjugated antibody was diluted in PBS (1% BSA) and incubated on the plates for 1 hour at RT. The reaction was developed with the substrate TMB (Dako) and stopped with 1 M H.sub.2SO.sub.4. The absorbance was read at 450-570 nm using an iMark microplate reader (Bio-Rad).

[0359] The immunogenicity of the HLA-DR4 epitope was assessed by stimulating splenocytes with SLPs containing the embedded HLA-DR4 sequence. This was performed using an ex vivo IFNγ ELISpot assay (ELISpot kit for mouse IFNγ/3321-2A, Mabtech, Stockholm, Sweden). The LUG2 SLP with the TAP sequence has the amino acid sequence set forth in SEQ ID NO: 14, and the LUG2 SLP without the TAP sequence is set forth in SEQ ID NO: 107; both contain the embedded HLA-DR4 sequence. One day before spleens were harvested, 96-well ELISpot plates (Millipore) for the IFN-γ ELISpot assay were pre-coated with capture antibody according to the manufacturer's protocol. After 5 washes with PBS/Tween and blocking for a minimum of 30 min with T cell medium including RPMI 1640 (Life Technologies/Thermo Fisher Scientific), containing 1% w/v L-Glutamine (SLS/Lonza), 10% v/v FBS (Fisher/GE Healthcare), 2% HEPES (SLS/Lonza), 0.1% v/v Fungizone (Promega), 0.5×10.sup.6/well freshly isolated splenocytes were seeded in triplicate into the plate along with 100 μl of the respective SLPs at a final concentration of 10 μg/ml. The cells were then incubated at 37° C. in a 5% CO.sub.2 incubator for 48 hours, and the plates then washed 5 times with DPBST. 50 μl/well biotinylated detection antibody (1/1000 dilution) against mouse IFNγ was then added, and the plates incubated for 2 hours at room temperature. Plates were then washed 5 times with DPBST, followed by the addition of 50 μl/well streptavidin alkaline phosphatase (1/1000 dilution). Plates were then incubated for 1 h 30 min at room temperature. After incubation, plates were washed again 6 times with DPBST and then 50 μl/well development solution (BCIP/NBT, BioRad) was added. The plates were left in the dark at room temperature until spots could be seen. Once spots developed, the reaction was stopped by rinsing the plates with tap water. Plates were then left to dry and the spots were quantified using an ELISpot plate reader (Cellular Technology Limited, Shaker Heights, Ohio, USA). SEB, the staphylococcal enterotoxin-B (at 2.5 μg/ml) was used as positive control, and unstimulated splenocytes (cells alone) were used as a negative control for every ELISpot assay. All experiments were performed in triplicate. Animals were scored as having a positive reaction when the number of spots in the cells-alone wells did not reach more than 20 and when the response in the peptide-containing wells was at least twice that of the standard deviation of the mean of the control wells.

[0360] Results

[0361] Evaluation of cytokine-expressing T cells in the human whole blood loop assay identified a small fraction of both healthy individuals and prostate cancer patients that responded with IFNγ and/or TNFα expression upon formation of ICs with the LUR/LUG 1-6 constructs. However, this assay was limited by the low frequency of epitope-specific T cells in the human blood and the lack of tetramers/multimers that could increase the sensitivity of the method. Therefore, to address in vivo priming and expansion of epitope-specific T cells commercial HLA-DR4 mice were used. As LUG2 includes an HLA-DR4 restricted PSMA epitope it was possible to expose animals to LUG2 conjugates and evaluate CD4+ T cell priming. HLA-DR4 mice received a prime/boost vaccination schedule with the LUG2 constructs. From serum collected from the LUG2 exposed animal and un-exposed animals as controls we identified that mice exposed to LUG2 increased their anti-MTTE antibody titers. Upon treatment of splenocytes from the LUG2 vaccinated animals with the SLP contained in the LUG2 construct (UV02, SEQ ID NO: 14) or the SLP without the TAP ARWW sequence (UV08, SEQ ID NO: 107), an increased number of IFN-γ producing T cells was noted (FIG. 8 shows the results obtained from mice vaccinated with 20 μg LUG2; a similar pattern of results was obtained from mice vaccinated with 5 μg LUG2—data not shown).

Example 10—Conjugate Safety

[0362] Methods

[0363] Cytokine and Complement Analysis in Plasma from Boostrix-Vaccinated Patients

[0364] Approximately two weeks after vaccination with the TDP vaccine Boostrix (GSK, Brentford, UK), blood was collected from five healthy individuals and five prostate cancer patients.

[0365] For evaluation of infusion reactions with regards to cytokine release and complement activation, the blood was treated with 3 different concentrations of the TENDU vaccine mixed constructs LUG1-6 using 0.05 μg/ml, 0.5 μg/ml and 2.5 μg/ml of each individual construct. Plasma harvested after 0 and 4 hours in the blood loop assay was used for concentration determination of IFN-γ, IL-1β, IL-2, IL-6, IL-8, IL-10 and TNF-α using Mesoscale V-plex kit (MSD Discovery®, Kenilworth, N.J., USA) according to the manufacturer's instructions. Lower limit of detection (LLOD) was calculated using MSD software and defined as 2.5×SD above the zero calibrator. Upper limit of detection (ULOD) was calculated using MSD software from the signal value of the Standard-1. Lower and upper limit of quantifications (LLOQ and ULOQ) are verified using MSD and calculated from the standard curve and percentage recovery of diluent standards with precision of 20% and accuracy 80-120%.

[0366] Plasma harvested after 0 and 15 minutes in the blood loop assay was analysed for complement activation (C3a and C5a) with ELISA kits from Hycult Biotech (Uden, Netherlands) according to the manufacturer's instructions.

[0367] Rabbit Toxicity

[0368] The TENDU vaccine was tested for toxicity in male rabbits by Meditox (Konerovice, Czech Republic). The rabbits were subcutaneously vaccinated four times (with two-week intervals) with tetanus toxoid vaccine (Equip® T vet.≥30 IU/ml, Orion Pharma Animal Health, Danderyd, Sweden) to generate TTd seropositive animals. After another two weeks the rabbits were subcutaneously vaccinated four times (with two-week intervals) with TENDU at low (10 μg/construct, n=5), intermediate (100 μg/construct, n=5) or high (240 μg/construct, n=8) dose. The two control groups were rabbits only receiving the tetanus vaccination (n=5) and rabbits that only received the high dose of TENDU (n=5). Clinical observations were made such as body weight, body temperature, food consumption, ophthalmoscopy, blood analysis, serum chemistry, urine analysis and a pathological examination.

[0369] Blood samples were collected in K3 EDTA tubes on week 15 before TENDU administration and post-TENDU administration at 4 h and 24 h. Blood samples were centrifuged (3500 rpm for 10 min, at 4° C.). The plasma was collected and stored at −20° C. until analysis with ELISA.

[0370] ELISA for Cytokine Detection in Rabbit Plasma

[0371] The following ELISA kits were used for analysis of cytokines in rabbit plasma: RayBio Rabbit IL-8 (cat. no ELL-IL-8-1), RayBio IL-1β (cat. no ELL-IL1b-1) and RayBio Rabbit IFNγ (cat. no ELL-IFNg-1) (Norcross, Ga., USA). Cytokine analysis was performed according to manufacturer's instructions.

[0372] Results

[0373] The safety of the vaccine constructs was evaluated using a blood loop system with blood samples from both healthy individuals and prostate cancer patients vaccinated with Boostrix. We assessed cytokine release and complement activation at three doses. The highest dose of each conjugate administrated was 240 μg. In humans this dose would lead to a Cmax of approximately 0.024 μg/ml per conjugate with an estimation of that a body contains 10 L blood/extracellular liquid. Complement activation in response to immune complex formation can lead to release of C3a and C5a components which act as anaphylatoxins and increase inflammatory response. We analysed the concentration and production of the cleaved complement components C5a and C3a (FIG. 9A-B). C3a concentration increased slightly in response to both 0.5 μg/ml and 2.5 μg/ml LUG1-6 constructs in healthy individuals and prostate cancer patients, concentrations that are above any expected Cmax concentration from subcutaneous administration of the conjugates. C5a concentration was similarly increased only upon treatment with 2.5 μg/ml of each LUG1-6 construct. For the lowest dose, in the range of any expected systemic exposure range, no complement activation was detected. As expected alemtuzumab, an antibody that is known to lead to complement fixation, led to increased concentrations of both C3a and C5a in both prostate cancer patients and healthy individuals. Infusion of biotherapeutics in blood can through different mechanisms induce cytokine release, and therefore we analysed the production of a set of cytokines after stimulation with the LUG1-6 constructs. Alemtuzumab, an antibody known to induce cytokine release, led to a significant increase in production of cytokines IL-8 (FIG. 9C), IFNγ, IL-6 IL-1bTNFα (FIG. 9F-I) and IL-10 (data not shown). LUG1-6 constructs only led to a notable elevation of IL-8 at the highest concentration of 2.5 μg/ml of each vaccine construct, while the remaining cytokines IFNγ, IL-6 and TNFα were not affected by treatment with the TENDU constructs at any of the concentrations analysed. Additionally, IL-2, IL-10 and IL-1β production was not affected by any of the TENDU concentrations used (data not shown).

[0374] Safety of TENDU was also assessed in vivo, in either tetanus toxoid seronegative or seropositive male rabbits. Rabbits were vaccinated four times with low, intermediate or high dose of TENDU and no clinical signs of toxicity were observed in any of the groups. The subcutaneous injections did not induce any local adverse reactions and there was no effect on body weight, food consumption, or body temperature of the rabbits in the study. To evaluate possible risks due to cytokine release after subcutaneous administration of TENDU and since IL-8 was released upon direct exposure of blood to TENDU at 2.5 μg/ml of each LUG construct, plasma collected from rabbits was analysed for IFN-γ, IL-8 and IL-1b. IFN-γ was undetectable in the majority of the samples without any increase in the concentration noticed after TENDU administration (FIG. 9E) however IL-8 concentrations in plasma from DTP-vaccinated rabbits were slightly increased over time (up to 24 h) in some of the animals in the different dose groups without a clear association with administration of high TENDU dose (FIG. 9D). IL-1β was undetectable at all time points and TENDU doses analysed (data not shown).