Anti-HER2 vaccine based upon AAV derived multimeric structures

Abstract

The present invention relates to parvovirus mutated structural proteins comprising insertions of mimotopes of a HER2, compositions, multimeric structures, medicaments and vaccines comprising the same, nucleic acids, expression cassettes, constructs, vectors and cells comprising the nucleic acids, methods of preparing the structural proteins and methods of inducing a B-cell response or of treating a HER2-related disease.

Claims

1. Parvovirus mutated structural protein for inducing a B-cell response against human epidermal growth factor receptor (HER2), which comprises one or more mimotopes of HER2 capable of specifically binding to an antibody directed against HER2, wherein at least one of the mimotopes comprises an amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98 or SEQ ID NO: 99.

2. Parvovirus mutated structural protein according to claim 1, wherein the antibody is Trastuzumab or Pertuzumab.

3. Parvovirus mutated structural protein according to claim 1, wherein a plurality of structural proteins is capable of forming a capsomeric structure, capsid or virus-like particle.

4. Parvovirus mutated structural protein according to claim 3, wherein the one or more mimotopes of HER2 are arranged in the parvovirus mutated structural protein to be located on the surface of the capsomeric structure, capsid or virus-like particle.

5. Parvovirus mutated structural protein according to claim 1, wherein the parvovirus is selected from the group consisting of adeno-associated virus (AAV), bovine AAV (b-AAV), canine AAV (CAAV), canine parvovirus (CPV), mouse parvovirus, minute virus of mice (MVM), B19, H1, avian AAV (AAAV), feline panleukopenia virus (FPV), and goose parvovirus (GPV).

6. Parvovirus mutated structural protein according to claim 5, wherein the AAV is AAV-1, AAV-2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, or AAV-12.

7. Parvovirus mutated structural protein according to claim 1, wherein the parvovirus mutated structural protein is a fusion protein further comprising a second protein or peptide domain.

8. Parvovirus mutated structural protein according to claim 1, wherein at least one of the mimotopes is not present in a wild type parvoviral structural protein, or wherein the wild type parvoviral structural protein is not capable of specifically binding to Trastuzumab.

9. A multimeric structure comprising parvovirus mutated structural proteins according to claim 1, wherein the structure is an aggregate of at least 5, at least 10, at least 30, or at least 60 mutated structural proteins.

10. The multimeric structure according to claim 9, wherein the multimeric structure is a capsomeric structure, capsomer, a capsid, a virus-like particle, or a virus.

11. The multimeric structure according to claim 9, wherein the one or more HER2 mimotopes are located on the surface of the multimeric structure.

12. The multimeric structure according to claim 9, wherein the spacing of at least two HER2 mimotopes on the surface of one multimeric structure is 10-500 angstroms, 50-300 angstroms, or 80 to 120 angstroms.

13. A medicament for treating or preventing a HER2-related disease, the medicament comprising at least one parvovirus mutated structural protein according to claim 1, and at least one suitable excipient, carrier, and/or stabilizer.

14. A vaccine comprising at least one parvovirus mutated structural protein according to claim 1, and at least one suitable adjuvant, excipient, carrier, and/or stabilizer.

15. The vaccine according to claim 14, wherein the adjuvant is selected from the group consisting of mineral oil-based adjuvants, oil in water emulsion adjuvants, syntax adjuvant formulation containing muramyl dipeptide, and aluminum salt adjuvants.

16. The vaccine according to claim 15, wherein the adjuvant is selected from the group consisting of Freund's complete or incomplete adjuvant, CpG, imidazoquinoline, MPL, MDP, MALP, flagellin, LPS, LTA, cholera toxin, a cholera toxin derivative, HSP60, HSP70, HSP90, saponins, QS21, ISCOMs, CFA, SAF, MF59, admamantane, aluminum hydroxide, aluminum phosphate, and a cytokine.

17. The vaccine according to claim 14, wherein the vaccine comprises a combination of more than one, adjuvants.

18. A medicament comprising at least one parvovirus mutated structural protein according to claim 1, and at least one suitable excipient, carrier, and/or stabilizer.

19. A method of inducing a B-cell response against HER2, the method comprising administering the Parvovirus mutated structural protein according to claim 1, in an effective dose to a mammal.

20. The method of inducing a B-cell response according to claim 19, wherein the parvovirus mutated structural protein is administered parenterally.

21. The method according to claim 19, wherein the Parvovirus mutated structural protein is administered multiple times.

22. A method of treating a HER2-related disease, the method comprising administering the Parvovirus mutated structural protein according to claim 1, in an effective dose to a mammal.

23. The method according to claim 22, wherein the HER2-related disease is cancer.

24. A nucleic acid coding for a parvovirus mutated structural protein according to claim 1.

25. An expression cassette, construct, or vector comprising the nucleic acid according to claim 24.

26. A cell comprising the expression cassette, construct, or vector according to claim 24.

27. The cell according to claim 26, wherein the cell is a bacterium, a yeast cell, an insect cell, or a mammalian cell.

28. A method of preparing a structural protein, the method comprising: a) expressing a nucleic acid coding for a parvovirus mutated structural protein by cultivating a the cell according to claim 26 under suitable conditions, and b) isolating the expressed parvovirus mutated structural protein of step a).

29. A composition for inducing a B-cell response comprising: a) a support capable of presenting peptides in a repetitive array; and b) at least three peptides, identical and/or different, each having an amino acid sequence independently selected from SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99, the peptides being joined to the support so as to form a HER2 mimotope-presenting support.

30. The composition according to claim 29, wherein the support is selected from the group consisting of a bead, a lipid membrane, a protein, or an inorganic carrier.

31. The composition according to claim 30, wherein the bead is selected from a polyacrylamide bead, an agarose bead, a polystyrene bead, a magnetic bead, a latex particle, a carbohydrate assembly.

32. The composition according to claim 30, wherein the lipid membrane is selected from a lipid assembly and a liposome.

33. The composition according to claim 30, wherein the protein is a protein assembly comprising a structural protein of a virus or phage, a virus-like particle or a virus; a polymer; KLH (Keyhole limpet hemocyanin); or LPH (Hemocyanin from Limulus polyphemus hemolymph).

34. The composition according to claim 30, wherein the inorganic carrier is selected from silica material and wherein the one or more HER2 mimotopes are covalently linked through a hydroxy, carboxy, or amino group and with a reactive group on the carrier.

35. Parvovirus mutated structural protein for inducing a B-cell response against human epidermal growth factor receptor (HER2) which comprises one or more mimotopes of HER2 capable of specifically binding to an antibody directed against HER2, wherein at least one of the mimotopes comprises an amino acid sequence of any one of SEQ ID NO: 100-166 or a sequence thereof having one or two amino acid substitutions.

Description

FIGURES

(1) FIG. 1: Image of analytical agarose gel (1%) of PCR products from AAV 2 library screen. Line 1: marker, line 2: 5 μl of 20 μl PCR product, line 3: 1 μl of 20 μl PCR product, line 4: negative control.

(2) FIG. 2: Sandwich ELISA of HER2 mimotope AAVs DMD01 to DMD06 from cell lysates. AAVs were immobilized by A20 mAb to ELISA plate and detected by trastuzumab (100 μl of 10.0 μg/ml) or an HER2 unspecific IgG mAb. As negative control AAV containing a HER2 unspecific epitope was used. OD.sub.450 after subtraction of the negative value is shown.

(3) FIG. 3: Indirect ELISA of HER2 mimotope AAVs DMD01 to DMD07, DMD15, DMD18, DDD19, DDD21, DDD30 and DDD31 and wild type AAV. AAV were bound to the ELISA plate and trastuzumab or a different humanized IgG-1 monoclonal antibody as a negative control added.

(4) FIG. 4: Sandwich ELISA of HER2 mimotope AAVs DMD01 to DMD07, DMD11, DMD15, DMD18, DDD19, DDD21, DDD30, DDD31, DMM44 and wild type AAV. A20 monoclonal antibodies coated ELISA plates were used to immobilize the AAV particles and subsequently trastuzumab (100 μl of a 10 μg/ml dilution in PBS/0.01% Tween-20) or a different humanized IgG-1 monoclonal antibody was added as a negative control.

(5) FIG. 5: Sandwich ELISA of HER2 mimotope AAVs DMD01 to DMD07, DMD11, DMD18, DDD19, DDD30 and DMM44. AAVs were immobilized to ELISA plate in duplets and detected by trastuzumab at two different, predetermined, optimal concentrations with or without washing under chaotropic conditions (5M urea). Avidity indices in % were calculated as a ration between OD.sub.450 with or without the chaotropic wash step.

(6) FIG. 6: ELISA for screening of sera of immunized mice (PIS=pre immune sera, MIS=mouse immune sera after respective immunization/boost) for specific IgG1 antibodies against HER2. Mice were immunized with indicated AAV clones. VP3-TTSN, an AAV containing an unrelated mimotope insert served as negative control.

(7) FIG. 7: EZ4U cell proliferation assay for mBT474 human breast cancer cells (passaged once through SCID mice) after incubation with (A) trastuzumab, an isotype control or (B) DMD01, DMD02, DMD04 or DMD06 compared each to untreated cells.

(8) FIG. 8a) shows HER-2 specific total IgG levels induced by DMD2 in combination with different adjuvants (a) and HER-2 specific total IgG levels induced by DMD2 in combination with different adjuvants (b).

EXAMPLES

(9) 1. Screening of AAV Library for Trastuzumab Binders

(10) A phenotye and genotype coupled AAV2 library comprising an (NNK).sub.15 insert (with N=A, G, C or T and K=G or T) and an upstream AAAGGG linker and a downstream GGGSG linker inserted after amino acid N.sub.587 of the VP proteins was constructed with slight modifications as described in Perabo et al. (2003) and Büning et al. (2008, examples 1 and 2), leading to a diversity of about 3.6×10.sup.6 viruses. Accordingly, the sequence of the library at N.sub.587 was:

(11) TABLE-US-00047 .sub.3949                     NotI ctc cag gca ggc aac gcg gcc gca gga ggt gga  L   Q   A.sub.585 G   N.sub.587 A   A   A   G   G   G                     BspEI (NNK).sub.15 ggt ggc ggt tcc gga gca caa gca gct   x.sub.15    G   G   G   S   G   A.sub.588 Q   A   A acc gca (SEQ ID NO: 167)  T   C  (SEQ ID NO: 168)

(12) Screening for trastuzumab binding AAV2 insertion mutants was carried out similar to the methods described in Perabo et al. (2003) and Büning et al. (2008). In brief, commercially available monoclonal anti-HER2 antibody trastuzumab (Roche, Basel) was immobilized through its Fc-part to Protein G coupled Dynabeads (Immunprecipitation Kit, Invitrogen) and subsequently incubated with the AAV2 library from above. Alternatively, Protein A coupled MagnaBind beads (Thermo Scientific) were used as a matrix. Non-bound and unspecifically bound viruses were washed away under stringent conditions, whereas DNA from bound viruses was isolated. Such DNA was used as a template for a subsequent PCR amplification (Phusion High-Fidelity PCR Kit, Finnzymes) of the cap fragment comprising the insertion. The band of correct size was isolated (a representative agarose gel of a PCR amplification is shown in FIG. 1) and cloned back into the AAV2 encoding plasmid pUCAV2 (Hörer and Hallek 2005). Resulting clones were on the one hand sequenced; on the other hand they were used to generate a new library (library of the first selection round) by transfecting the DNA into 293-T cells and producing viruses as described in example 0. Each time 95 clones of the selection round were sequenced with respect to their respective inserted sequence at amino acid N.sub.587 of the VP proteins. The generated library of first round had a diversity of about 10.sup.6. As expected, the first selection round did not yet lead to a marked enrichment of sequences.

(13) Therefore, selection was repeated using the library of the first selection round, resulting in the library of the second selection round. After the second selection round, 9 clones had been enriched (at least two times present in the sequenced sample), the most frequent clone was found 8 times. Accordingly, diversity of the library was still sufficient to make a further selection round and the library of the second selection round was subjected to a third selection round. Again, 95 clones were sequenced with 23 sequences not being evaluated due to not being readable, having one or more inserts, containing mixed sequences (more than one sequence was sequenced) or incomplete sequences (linker missing or stop codon). From the 72 sequences that were evaluated more than half of the sequences had similarities with each other resulting in the definition of a first consensus sequence (see below).

(14) A number of selections was carried out according to the above scheme with slight variations e.g. with different or alternating matrices (Dyna beads versus MagnaBind beads) or negative preselection against an antibody of the same isotype to avoid selection of binders to beads or antibody outside of the idiotype determining region of trastuzumab as generally suggested earlier (Büning et al. 2008).

(15) The various screenings lead to the identification of inserted amino acid sequences as shown in Table 1.

(16) TABLE-US-00048 TABLE 1 Frequency of identified amino acid insertions in third screening rounds Sum of 3. Internal and 4. Ref. AA Sequence SEQ ID NO rounds DMD01 RLVPVGLERGTVDWV SEQ ID NO: 1 108 DMD02 TRWQKGLALGSGDMA SEQ ID NO: 2 24 DMD03 RTWQSGMADGEEIGR SEQ ID NO: 3 25 DMD04 QVSHWVSGLAEGSFG SEQ ID NO: 4 6 DMD05 SSWAAGTAAGDFKGY SEQ ID NO: 5 37 DMD06 LSHTSGRVEGSVSLL SEQ ID NO: 6 55 DMD07 SLWLLGRADGVSSGH SEQ ID NO: 7 3 DMD08 QGRAKNLPNCGSGQR SEQ ID NO: 8 1 DMD09 RSFEKFGGMKERLHC SEQ ID NO: 9 1 DMD10 AVEPCITAHKSYMRV SEQ ID NO: 10 1 DMD11 STLWHRGLAAGDVSR SEQ ID NO: 11 1 DMD12 SESGVFVLQSCAWEY SEQ ID NO: 12 1 DMD13 WGNCPLSSGGPKTFR SEQ ID NO: 13 1 DMD14 WVGSRSMWAECGLDE SEQ ID NO: 14 1 DMD15 LDSTSLAGGPYEAIE SEQ ID NO: 15 4 DMD16 LSRCGKPMDVEAALN SEQ ID NO: 16 1 DMD17 FPKSQVSRGEMRLGG SEQ ID NO: 17 1 DMD18 FFSGRWSEGTALGSS SEQ ID NO: 18 1 DDD19 HVVMNWMREEFVEEF SEQ ID NO: 19 6 DDD20 GVAWSSGQAHGSRTE SEQ ID NO: 20 7 DDD21 WDSGDAVGNEVLLVG SEQ ID NO: 21 5 DDD22 WKMGTAQGSGQDGEY SEQ ID NO: 22 27 DDD23 GWNSGKVDGGAGRSM SEQ ID NO: 23 5 DDD24 LVGVWPVMVETVYET SEQ ID NO: 24 2 DDD25 QWLEGLAEGMVHTLG SEQ ID NO: 25 5 DDD26 DPYESSRSTLLRAAR SEQ ID NO: 26 3 DDD27 GDEEMWPIVRELQSL SEQ ID NO: 27 2 DDD28 LKWYSGELEGSKELL SEQ ID NO: 28 1 DDD29 NPGTWERGVAAGDIE SEQ ID NO: 29 1 DDD30 QTKWPIATEVWRETV SEQ ID NO: 30 4 DDD31/ VGAAAQWPEVREYLM SEQ ID NO: 31 3 DDM51 DDD32 YFSGKAEGREAPSWD SEQ ID NO: 32 1 DMM33 CDVQLASAKKRYLGV SEQ ID NO: 33 1 DMM34 CMGVVKAWDSIRLVE SEQ ID NO: 34 1 DMM35 DVGQGRRKSLNLECF SEQ ID NO: 35 1 DMM36 DVTWRHKHIVTKGGL SEQ ID NO: 36 1 DMM37 GGYEVRKHFQSREVV SEQ ID NO: 37 1 DMM38 KNCDRLSWSGARNLS SEQ ID NO: 38 1 DMM39 LVEHWSRSKKSSFEF SEQ ID NO: 39 1 DMM40 LVSVQKKWERKAESM SEQ ID NO: 40 1 DMM41 MFLSDKYSREPHKGK SEQ ID NO: 41 1 DMM42 RRTLCGTGSEMVLFK SEQ ID NO: 42 1 DMM43 SVRLSSAQGCINMVV SEQ ID NO: 43 1 DMM44 SWASGMAVGSVSFEE SEQ ID NO: 44 1 DMM45 VTGNCKGSRQQHVLG SEQ ID NO: 45 1 DMM46 WWAHGEDITGHSLCL SEQ ID NO: 46 1 DDM47 ASQGSWKLGTARGSG SEQ ID NO: 47 3 DDM48 MQLICRTSLREERII SEQ ID NO: 48 2 DDM49 RFSCAVGKECSHKQC SEQ ID NO: 49 1 DDM50 RSNDVLGCKLRVVGC SEQ ID NO: 50 1 DDM52 WAFGLALGSLETIDL SEQ ID NO: 52 1 DDM53 WGEPYSGKGSHGKIG SEQ ID NO: 53 1 DDD54 ASAWLLGNVEGSEIR SEQ ID NO: 54 2 DDD55 CQWRAGTAVGSSVGN SEQ ID NO: 55 1 DDD56 HEVLWGEMDAPWVVP SEQ ID NO: 56 2 DDD57 IASGWSVGWADGDDS SEQ ID NO: 57 1 DDD58 PYEELVATRSRAGGM SEQ ID NO: 58 2 DDD59 SCLARVHCDMPREWE SEQ ID NO: 59 1 DDD60 VATKGVTLWETGLAE SEQ ID NO: 60 1 DDD61 VTLMKISKWDAGLAE SEQ ID NO: 61 1 DDD62 WMSGQSDGSSGGGPK SEQ ID NO: 62 4 DDDD63 REAGQWARGLAVGSC SEQ ID NO: 63 1 DDDD64 YVIGPYERECELGMG SEQ ID NO: 64 1 DDDD65 WKMGMAQGSGQDGEY SEQ ID NO: 65 1

(17) 2. Alignment Analysis of Identified Sequences

(18) Sequence alignments using the MultAlin algorithm (Corpet 1988) of all identified sequences and subgroups of identified sequences lead to the identification of consensus sequences. The most frequently found motif is the amino acid sequence WxxGxAxGS (consensus 1, SEQ ID NO: 51), which was found in 21 individual sequences (Table 2). More specifically, this consensus sequence can be defined as

(19) TABLE-US-00049 (SEQ ID NO: 66) (W/H/Y/V)xxGx(A/L/V/C/E)xG(S/M/D/E/V/N/G/R/T), (SEQ ID NO: 67) WxxGx(A/V/L)xG(S/T/M/D/E), (SEQ ID NO: 68) Wx(S/T/K/R/M/L/A/V)Gx(A/V)xG(S/M/D), (SEQ ID NO: 69) Wx(K/R/E/S/T/F)G(L/M/T/V)A(A/V/L/E/D)G (S/T/D/E/M), (SEQ ID NO: 70) (S/T/G)(S/T/R/Q/H/L)Wx(K/R/S/E/F)G(L/M/T/V)A (A/V/L/E)G(S/D/M)(G/V/L/I/S/C/F), (SEQ ID NO: 71) (S/T/L/Q)Wx(M/A/L/V)G(T/A/S/M)A(Q/A/V/H/K/R) G(S/T/D/E), (SEQ ID NO: 72) (S/T/G)(S/T/Q)W(K/R/A)(M/A/L)G(T/A/M)A(Q/A/V/R) G(S/D)(G/F/S)(Q/K/V)(D/G), (SEQ ID NO: 73) Wx(S/T/A/L/V)Gx(A/V)(E/D/A/V/H/K/R)G (S/T/D/E/N/G/V), (SEQ ID NO: 74) (S/R/V)xWx(S/L/V)G(Q/R/N/M/W/D)(A/V/S)(E/D/V/H) G(S/D/E/N/V)(E/D/S/R).

(20) TABLE-US-00050 TABLE 2 Alignment of amino acid insertion of consensus 1 Internal Ref. AA Sequence SEQ ID NO DMD02 TRWQKGLALGSGDMA SEQ ID NO: 2 DMD18 FFSGRWSEGTALGSS SEQ ID NO: 18 DDDD63 REAGQWARGLAVGSC SEQ ID NO: 63 DDD29 NPGTWERGVAAGDIE SEQ ID NO: 29 DMD11 STLWHRGLAAGDVSR SEQ ID NO: 11 DMM44 SWASGMAVGSVSFEE SEQ ID NO: 44 DDM52 WAFGLALGSLETIDL SEQ ID NO: 52 DDD25 QWLEGLAEGMVHTLG SEQ ID NO: 25 DMD04 QVSHWVSGLAEGSFG SEQ ID NO: 04 DDD22 WKMGTAQGSGQDGEY SEQ ID NO: 22 DDDD65 WKMGMAQGSGQDGEY SEQ ID NO: 65 DDM47 ASQGSWKLGTARGSG SEQ ID NO: 47 DMD05 SSWAAGTAAGDFKGY SEQ ID NO: 05 DDD55 CQWRAGTAVGSSVGN SEQ ID NO: 55 DMD03 RTWQSGMADGEEIGR SEQ ID NO: 03 DMD07 SLWLLGRADGVSSGH SEQ ID NO: 07 DDD62 WMSGQSDGSSGGGPK SEQ ID NO: 62 DDD20 GVAWSSGQAHGSRTE SEQ ID NO: 20 DDD54 ASAWLLGNVEGSEIR SEQ ID NO: 54 DDD57 IASGWSVGWADGDDS SEQ ID NO: 57 DDD21 WDSGDAVGNEVLLVG SEQ ID NO: 21 Consensus 1 WxxGxAxGS SEQ ID NO: 51

(21) Another six sequences were identified with the same motif but lacking the N-terminal W (Table 3), therefore being defined as the incomplete consensus 1 GxAxGS (SEQ ID NO: 75). More specifically, this consensus sequence can be defined as

(22) TABLE-US-00051 (SEQ ID NO: 76) (W/H/Y/V)x(S/T/A/V/L)Gx(A/V/L/C/E)(E/D/K/R/H) G(S/T/G/V/R), or (SEQ ID NO: 77) (L/R)x(W/H/Y/V)x(S/T/V)G(K/R/N/E/L)(A/V/L/C/E) (E/D/K/R)G(S/T/G/R)xx(L/R/P/Q/W).

(23) TABLE-US-00052 TABLE 3 Alignment of amino acid insertion of incomplete consensus 1 Internal Ref. AA Sequence SEQ ID NO DDD28 LKWYSGELEGSKELL SEQ ID NO: 28 DMD06 LSHTSGRVEGSVSLL SEQ ID NO: 06 DDD23 GWNSGKVDGGAGRSM SEQ ID NO: 23 DDD32 YFSGKAEGREAPSWD SEQ ID NO: 32 DMM45 VTGNCKGSRQQHVLG SEQ ID NO: 45 DMD01 RLVPVGLERGTVDWV SEQ ID NO: 01 Inc. GxAxGS SEQ ID NO: 75 Consensus 1

(24) Seven sequences were grouped to consensus 2 being WxxxxxSRGxxR (SEQ ID NO: 78) as shown in Table 4. More specifically, this consensus sequence can be defined as

(25) TABLE-US-00053 (SEQ ID NO: 79) (W/F/K)x(N/K/H/E/S/C)xxx(S/G/I/V)(S/T/K/R/W) (G/S/E/K), (SEQ ID NO: 80) (W/F)x(N/H/K/E)xxx(S/G/I/V)(S/T/K/R)(G/K) (G/E/S/H)x(R/K/L/G),, or (SEQ ID NO: 81) (F/K)xx(S/D)xxS(R/W)(G/S/E)(G/E/P)x(R/K).

(26) TABLE-US-00054 TABLE 4 Alignment of amino acid insertion of consensus 2 Internal Ref. AA Sequence SEQ ID NO DDM53 WGEPYSGKGSHGKIG SEQ ID NO: 53 DMD13 WGNCPLSSGGPKTFR SEQ ID NO: 13 DMM46 WWAHGEDITGHSLCL SEQ ID NO: 46 DMD17 FPKSQVSRGEMRLGG SEQ ID NO: 17 DMM38 KNCDRLSWSGARNLS SEQ ID NO: 38 DMM41 MFLSDKYSREPHKGK SEQ ID NO: 41 DMM36 DVTWRHKHIVTKGGL SEQ ID NO: 36 Consensus 2 WxxxxxSRGxxR SEQ ID NO: 78

(27) Seven sequences were grouped to consensus 3 being RSxSxxGGPxE (SEQ ID NO: 82) as shown in Table 5. More specifically, this consensus sequence can be defined as

(28) TABLE-US-00055 (SEQ ID NO: 83) (R/D)(S/T/F/L)x(E/D/S/C)xxG(G/C/S/K), (SEQ ID NO: 84) RSx(E/D)xxG(G/C)xx(E/D/R)(V/A/L/R), or (SEQ ID NO: 85) (R/D)(S/T/F/L)(S/T/L)(S/C)xxG(G/C/S/K)(P/E/I) (M/N/C/Y)(E/D/V/S/M)(V/A/L/H).

(29) TABLE-US-00056 TABLE 5 Alignment of amino acid insertion of consensus 3 Internal Ref. AA Sequence SEQ ID NO DMD09 RSFEKFGGMKERLHC SEQ ID NO: 9 DDM50 RSNDVLGCKLRVVGC SEQ ID NO: 50 DMD15 LDSTSLAGGPYEAIE SEQ ID NO: 15 DMM43 SVRLSSAQGCINMVV SEQ ID NO: 43 DMM42 RRTLCGTGSEMVLFK SEQ ID NO: 42 DDM49 RFSCAVGKECSHKQC SEQ ID NO: 49 DMD16 LSRCGKPMDVEAALN SEQ ID NO: 16 Consensus 3 RSxSxxGGPxE SEQ ID NO: 82

(30) Eight sequences were grouped to consensus 4 being VxxxxxREE (SEQ ID NO: 86) as shown in Table 6. More specifically, this consensus sequence can be defined as

(31) TABLE-US-00057 (SEQ ID NO: 87) (K/R/M/G/D)x(W/Y/S/T/F/I)x(R/N/S/T/L)(K/R/E/D/L), (SEQ ID NO: 88) (D/G)x(Y/Q/T/G)(E/W/Q/L)x(H/R/S/C)(K/R)(K/H/S/T) (S/T/F/I)(L/V/Q)(R/N/S/T/L)(K/R/E/L)(E/A/G), or (SEQ ID NO: 89) (V/L/I)(S/V/I)(K/R/M/G/D)(K/H/N/P)(W/Y)x(R/N) (E/D)(E/C/P)

(32) TABLE-US-00058 TABLE 6 Alignment of amino acid insertion of consensus 4 Internal Ref. AA Sequence SEQ ID NO DMM36 DVTWRHKHIVTKGGL SEQ ID NO: 36 DMM37 GGYEVRKHFQSREVV SEQ ID NO: 37 DDD26 DPYESSRSTLLRAAR SEQ ID NO: 26 DMM35 DVGQGRRKSLNLECF SEQ ID NO: 35 DDM48 MQLICRTSLREERII SEQ ID NO: 48 DDD19 HVVMNWMREEFVEEF SEQ ID NO: 19 DDDD64 YVIGPYERECELGMG SEQ ID NO: 64 DMM41 MFLSDKYSREPHKGK SEQ ID NO: 41 Consensus 4 VxxxxxREE SEQ ID NO: 86

(33) Twelve sequences were grouped to consensus 5 being VGxxxxWPxVRE (SEQ ID NO: 90) as shown in Table 7. More specifically, this consensus sequence can be defined as

(34) TABLE-US-00059 (SEQ ID NO: 91) (G/A/S)x(A/V/I/R/E/Q)(K/S/T/A/G/E)x(W/L) (P/D/E/A/G/S)xxx(E/S/L/K/A), (SEQ ID NO: 92) (G/A)x(A/V/R)(K/S)x(W/L)(P/A)(N/E)CG(S/L)x(E/Q), (SEQ ID NO: 93) (G/S)x(A/V/E/Q)(A/K/E)xW(P/D/E)(I/E/R/S/T) (I/V/K)(R/A/G)(E/L)x(E/Q/L/M), (SEQ ID NO: 94) (V/Q/E)(E/G/T/V)(K/L/V/H)W(P/S/D/E/G) (I/A/V/E/R/S/T)x(K/V/T/D)(E/K/A)(V/S/T/P/E) (S/V/W), or (SEQ ID NO: 95) (V/I)(S/T)(L/K)W(D/E)xGLAE.

(35) TABLE-US-00060 TABLE 7 Alignment of amino acid insertion of consensus 5 Internal Ref. AA Sequence SEQ ID NO DMD8 QGRAKNLPNCGSGQR SEQ ID NO: 8 DMD14 WVGSRSMWAECGLDE SEQ ID NO: 14 DMM34 CMGVVKAWDSIRLVE SEQ ID NO: 34 DDD27 GDEEMWPIVRELQSL SEQ ID NO: 27 DDD31/DDM51 VGAAAQWPEVREYLM SEQ ID NO: 31 DMM40 LVSVQKKWERKAESM SEQ ID NO: 40 DMM39 LVEHWSRSKKSSFEF SEQ ID NO: 39 DDD24 LVGVWPVMVETVYET SEQ ID NO: 24 DDD30 QTKWPIATEVWRETV SEQ ID NO: 30 DDD56 HEVLWGEMDAPWVVP SEQ ID NO: 56 DDD60 VATKGVTLWETGLAE SEQ ID NO: 60 DDD61 VTLMKISKWDAGLAE SEQ ID NO: 61 Consensus 5 VGxxxxWPxVRE SEQ ID NO: 90

(36) Four sequences were grouped to consensus 6 being VxLxSAxKxYxxV (SEQ ID NO: 96) as shown in Table 8. More specifically, this consensus sequence can be defined as

(37) TABLE-US-00061 (SEQ ID NO: 97) (E/D/V/A)(P/Q/N/E/RL/C)(I/A/V/S/T)(S/T/A)(A/T) (H/K/R/Q)(S/K/G)(R/S/C)(Y/A/I/V)xx(V/A/M), (SEQ ID NO: 98) (E/V)(P/Q)(L/C)(I/A/V)(S/T/A)A(H/K/R)K(R/S)YxxV, or (SEQ ID NO: 99) (E/V)(E/R/Q)(L/C)(V/S)(S/T/A)(A/T)(R/Q)(S/K/G) (R/S/C)(A/I/V)xx(V/M)

(38) Table 8: Alignment of amino acid insertion of consensus 6

(39) TABLE-US-00062 Internal Ref. AA Sequence SEQ ID NO DMD10 AVEPCITAHKSYMRV SEQ ID NO: 10 DMM33 CDVQLASAKKRYLGV SEQ ID NO: 33 DDD58 PYEELVATRSRAGGM SEQ ID NO: 58 DMM43 SVRLSSAQGCINMVV SEQ ID NO: 43 Consensus 6 VxLxSAxKxYxxV SEQ ID NO: 96

(40) Identified sequences of DMD12 and DDD59 were not grouped into one of these consensus sequences.

(41) 3. Production of AAV and AAV Like Particles

(42) 3.1. Manufacturing of AAV (Virus) in Mammalian Cells

(43) Manufacturing of AAV (virus) in mammalian cells was performed as described by Sonntag et al. (2010a, examples 1.2 to 1.4). Briefly, AAV manufacturing was carried out by co-transfection of 293-T cells with an AAV encoding plasmid (pUCAV2) with a cap gene containing the respective mimotope DNA sequence insertion and the helper plasmid pUCAdV to provide adenoviral helper functions. The construction of pUCAV2 as an AAV encoding plasmids is described in detail in Hörer and Hallek (2005). Plasmid pTAV2.0 is described in Heilbronn (1990), pVP3 is described in Warrington (2004).

(44) 3.2. Manufacturing of AAVLP (Virus-Like Particles) in Mammalian Cells

(45) Transfection of cells was carried out as described by Sonntag et al. (2010a). Briefly, 293-T cells (ATCC, Manassas, USA) were seeded and after 24 h transfected with 36 μg per 145 cm.sup.2 dish pCI-VP2mutACG containing the respective insertion by calcium phosphate precipitation. 293-T cells were harvested 70 to 72 h after transfection with a cell lifter, transferred into plastic tubes (Falcon) and centrifuged. The cell pellet was resuspended in lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5) and objected to freeze and thaw cycles (liquid nitrogen/37° C.). The cell lysate was cleared by centrifugation and the AAV-containing supernatant was used for further purification. Alternatively the whole dishes were objected to three freeze and thaw cycles (−50° C./RT). The remaining supernatant of centrifugation or, alternatively, flow through of filtration was collected and further purified as described in below.

(46) 3.3. Purification of AAV by Density Gradient Centrifugation Using Iodixanol

(47) AAV particles were purified by iodixanol gradient centrifugation according to example 4.3 of (Büning et al. 2008).

(48) 3.4. Purification of AAV Like Particles by Chromatography

(49) Purification of AAV like particles containing HER2 mimotopes was carried out as described by Sonntag et al. (2010a, examples 1.3). Briefly, the freeze-thawed, cleared lysate containing modified AAVLPs was diluted by adding Hepes buffer (pH 6.0) without NaCl until a conductivity of approximately 3 to 9 mS/cm was reached, the pH of the lysate had been adjusted to 5.5-7.5 depending on the modified AAVLPs and the preparation was cleared by a filtration cascade with two filter capsules (Sartopure PP2, 5 μm and Sartopore PP2, 0.65 μm, Sartorius-Stedim, Göttingen, Germany). The filtrate was bound to a Cation exchange chromatography (Fractogel EMD SO.sub.3.sup.− (M) chromatography column, XK16, Merck, Darmstadt, Germany), washed and bound particles were eluted with sodium chloride. A buffer exchange was performed (Sephadex G25 packed chromatography column, XK26, GE Healthcare, Munich, Germany) in order to continue with an anion exchange chromatography (CaptoQ chromatography column; XK16, GE Healthcare, Munich, Germany). After equilibration, the protein fraction obtained after buffer exchange was loaded and the flow-through containing 90% of the particles was collected. The flow-through containing AAVLPs was concentrated using Vivacell 100 units (MWCO 100,000, Sartorius-Stedim) and a swinging-bucket rotor (MULIFUGE L-R; Heraeus, Hanau, Germany). Resulting concentrate was immediately separated through a size exclusion chromatography (Superdex 200, prep grade, XK50, GE Healthcare, Munich, Germany) which was packed and equilibrated using running buffer consisting of 200 mM NaCl, 50 mM HEPES (pH 6.0), 2.5 mM MgCl.sub.2. Particles were loaded onto the column and eluted first in the first SEC fractions. SEC fractions with a particle purity of greater than 95% were pooled, sterile filtered and stored at −80° C.

(50) Exemplary titers yielded by small scale production and purification are shown in Table 9. Interestingly, titers of AAV clones containing a trastuzumab mimotope identified by the screening methods as described herein typically yielded higher titers as compared to wild type AAV2. This was not expected as the identified AAV clones each contain a 26 AA insert that potentially may interfere with the expression and/or assembly of the AAV capsid proteins. The absence of AAV clones showing lower titers (compared to wild type AAV2) documents that the described screening methods not only select for AAVs that bind to trastuzumab but also for AAVs that have an equal or more efficient expression and/or assembly of the capsid proteins. This of course is a welcome effect as high yields are of course very important for vaccine candidates.

(51) TABLE-US-00063 TABLE 9 Titers of AAV clone productions and purifications. Concentration [particle/ml] DMD01 1.12 × 10.sup.13 DMD02 4.13 × 10.sup.13 DMD03 1.51 × 10.sup.13 DMD04 2.29 × 10.sup.13 DMD05 3.63 × 10.sup.13 DMD06 3.80 × 10.sup.13 DMD07 1.63 × 10.sup.13 DMD15 1.91 × 10.sup.13 DMD18 1.60 × 10.sup.13 DDD19 6.30 × 10.sup.13 DDD21 1.98 × 10.sup.13 DDD30 1.50 × 10.sup.13 DDD31 1.92 × 10.sup.13 wtAAV2 9.07 × 10.sup.12

(52) 4. Sandwich ELISA with AAVs from Cell Lysate

(53) In order to quickly determine whether identified AAVs containing putative HER2 mimotopes specifically interact with the trastuzumab (Roche) AAVs directly within cell lysate were tested in a sandwich ELISA by testing whether trastuzumab can be bound to clones immobilized on the ELISA plate. ELISA plates having A20 mAb on the surface of the plates (AAV2 titration ELISA kit, Progen) were used in order to immobilize identified AAV clones from cell lysates (after freeze thaw lysis, 100 μl of 1:10 dilution in sample buffer from ELISA kit) as previously described (Grimm et al. 1999). As a negative control a different humanized IgG-1 monoclonal antibody was used instead of trastuzumab. Additionally, wild type AAV like particles were used in order to detect unspecific binding of trastuzumab to AAV.

(54) Bound AAVs were incubated with trastuzumab (100 μl of a 10.0 μg/ml solution in PBS/0.01 Tween-20) or with the humanized IgG-1 mAb (identical concentration) and detected with HRP-labeled human anti IgG antibodies (Bethyl #A80-319P, 100 μl of a 1:2,500 dilution in PBS/0.1% Tween-20) by OD measurement at 450 nm (OD.sub.450) in an ELISA reader (ER02). Values of blank (empty wells only with buffer) were subtracted from determined values of the individual clones.

(55) FIG. 2 shows an example of the Sandwich ELISA from cell lysates for the AAV clones DMD1 to DMD6. This ELISA was repeated for all identified AAV clones. Results were grouped according to the following scheme:

(56) TABLE-US-00064 +++ stands for OD.sub.450 >1.0, ++ for OD.sub.450 >0.5, + for OD.sub.450 >0.2, +/− for OD.sub.450 >0.1, and − for OD.sub.450 <0.1.
and results for all clones are summarized in Table 11, columns two and three.

(57) A number of clones selected from the AAV library showed high specificity for trastuzumab compared to the humanized IgG-1 monoclonal antibody control (e.g. DMD02, DMD11, DMM44, DDD21, DMM33), whereas only few clones were rather unspecific within this sandwich ELISA from cell extracts—having a higher reactivity with the IgG mAb control compared to the trastuzumab (DMM45, DMM38, DMM37) or a very reactivity to both antibodies (DMM33).

(58) Therefore it is concluded that the selections generally yielded a meaningful set of AAVs containing at the surface more or less specific mimotopes for soluble trastuzumab. For interpreting data from the sandwich ELISA it has to be understood that, while the tested AAV clones each have 60 copies of the inserted mimotope on the surface, the monoclonal antibody in the ELISA test has only two binding sites for the mimotope. During the selection however, when the selection antibody trastuzumab was immobilized on beads, multiple antibody binding sites can cooperate in retracting matching AAV clones. For the use of identified clones in immunization, the situation for binding the AAV may be more comparable to the selection conditions, as a B-cell response is triggered by binding B-cells to the antigen through membrane bound IgM, where also multiple IgM molecules on the surface of the B-cell can cooperatively bind to multiple epitopes on virus—which exactly is the reason why virus/virus-like particles are potent immunogens. Therefore, it can be assumed that the affinity of a screened AAV clone to the soluble antibody is not the only determining feature for activity in immunization experiments.

(59) 5. ELISA with Purified AAVs

(60) In order to validate affinities observed for AAV clones from cell lysates (example 4) purified AAV particles (see example 0) were analyzed again in two different ELISA systems for their affinity to trastuzumab similar to example 4. Clones were tested in two different settings.

(61) In the indirect ELISA AAV particles (100 μl of a 1×10.sup.11 particle/ml dilution) were directly bound to the ELISA plate, unbound binding sites saturated (5% milk powder in PBS/0.1% Tween-20) and trastuzumab (100 μl of a 10 μg/ml dilution in PBS/0.01% Tween-20) or a different humanized IgG-1 monoclonal antibody as a negative control added. Bound antibodies were detected and evaluated as described above. Results of an exemplary ELISA are depicted in FIG. 3.

(62) In the sandwich ELISA A20 monoclonal antibodies coated ELISA plates (Progen #PRATV) were used to immobilize the AAV particles (100 μl of a 1×10.sup.11 particle/ml dilution in sample buffer of the Progen ELISA kit) and subsequently trastuzumab (100 μl of a 10 μg/ml dilution in PBS/0.01% Tween-20) or a different humanized IgG-1 monoclonal antibody as a negative control added. Again, bound antibodies were detected with HRP-coupled anti-human IgG. Wild type AAV particles are used as negative control for unspecific binding of the antibodies to the AAV particles. Bound antibodies were detected and evaluated as described above. Results of an exemplary ELISA are depicted in FIG. 4.

(63) 6. Sandwich ELISA in Presence of Chaotopic Agent

(64) In order to determine the stability of the interaction between AAV clones and trastuzumab a modified sandwich ELISA of example 4 was done in the presence of a chaotropic agent, i.e. urea, as a measure for avidity (strength of a multivalent interaction between antibody and antigen). Determination of the avidity of the AAV/antibody interaction is based on the disruption of weak interactions by chaotropic substances like urea.

(65) First, AAV clones (10.sup.10 particle in 100 μl; exception DMD11 where only half of the amount was coated due to low availability of material) were coated over night/4° C. onto an ELISA plate, washed, remaining/unspecific binding blocked with 5% milk powder solution in PBS/0.1% Tween-20 and two different amounts of trastuzumab (as indicated in Table 10 in 100 μl) added.

(66) In a pre-experiment the optimal concentration of trastuzumab had been determined Optimal conditions for measuring the avidity are if OD.sub.450 values are equal or larger than 0.4 in order to discriminate against background. Therefore, for AAV clones individual trastuzumab concentrations were determined and are shown in Table 10.

(67) TABLE-US-00065 TABLE 10 Trastuzumab concentrations for avidity determination clones trastuzumab concentrations [μg] DMD01 5.0 and 10.0 DMD02 5.5 and 1.0  DMD03 5.0 and 10.0 DMD04 2.0 and 5.0  DMD05 2.0 and 5.0  DMD06 5.0 and 10.0 DMD07 5.0 and 10.0 DMD11 0.1 and 0.2  DMD18 5.0 and 10.0 DDD19 5.0 and 10.0 DDD30 5.0 and 10.0 DMM44 0.5 and 10.sup. 

(68) After incubation with trastuzumab plates were again washed, first with PBS/0.1% Tween-20 and optionally three times for 5 min with 5 M urea in PBS/0.1% Tween-20, which is the wash step under denaturing/chaotropic conditions, followed by washing with PBS/0.1% Tween-20. Detection and OD.sub.450 measurement was carried out as described in example 4.

(69) The avidity index was calculated according to the formula

(70) $\frac{\mathrm{OD}450\mathrm{with}\mathrm{chaotropic}\mathrm{wash}-\mathrm{OD}450\mathrm{blank}}{\mathrm{OD}450\mathrm{without}\mathrm{chaotropic}\mathrm{wash}-\mathrm{OD}450\mathrm{blank}}.Math.100=\%\mathrm{avidity}$

(71) Averages with standard deviations for resulting avidity indices are depicted as an example for clones AAVs DMD01 to DMD07, DMD11, DMD18, DDD19, DDD30 and DMM44 in FIG. 5. This ELISA under chaotropic conditions was repeated for a number of further AAV clones. Results were grouped according to the following scheme:

(72) TABLE-US-00066 +++ avidity index >30% ++ 15% ≦ avidity index ≦30%  + avidity index <15%
and are summarized in Table 11, column four.

(73) Interestingly, avidity indices of the AAV clones do not correlate with the affinities determined both with crude cell lysates and purified AAV particles. For example, DMD02, DMD11 and DMM44 have shown high affinities for trastuzumab, whereas the avidity indices of these clones are rather low. On the other side, DMD04, DMD06 and DDD19 have a low affinity to trastuzumab, whereas their avidity indices are rather high. It should be noted that for DDD19 the correlation between OD.sub.450 and avidity index could not be shown.

(74) 7. Immunization of Mice

(75) BALBc mice were immunized with AAV clones and blood samples were taken after 2 or more immunizations. HER2 specific antibodies in the mice sera were determined by ELISA. Briefly, ELISA plates were coated with recombinant HER2 overnight at 4° C., washed and unspecific binding was blocked with 1% milk powder in PBS/0.05% Tween-20. Subsequently plates were incubated with 1:100 diluted sera or trastuzumab [1 μg/ml] overnight at 4° C. After washing the plates, bound antibodies were detected using a rat anti-mouse IgG followed by a goat anti-rat IgG coupled to HRP by OD measurement at 490 nm. PIS resembles pre-immune sera, MIS mouse sera after the respective immunization/boost. Results for pooled sera of 8 mice per indicated AAV clone are shown in FIG. 6. VP3-TTSN is an AAV control having an unspecific mimotope insert.

(76) 8. Proliferation Assay

(77) Sera of BALBc mice immunized with AAV displaying mimotopes DMD01, DMD02, DMD04 and DMD06 as well as from naïve mice were purified with protein G sepharose (Incubation of mice sera overnight with Protein G-Sepharose beads; elution with 0.1 m Glycine-buffer; afterwards dialysis against PBS to reduce Glycine which acted in this concentration toxic on cell viability assays. Quality control in SDS-PAGE).

(78) To test whether these purified antibodies act tumoricidic/tumoristatic on HER2 overexpressing cells, a cell viability assay with mBT474 human breast cancer cells was established. “m” means these cells were passaged once through SCID mice. This is important to enable better grafting for the planned consecutive SCID graft experiments. The assay using mBT474 cells was established with monoclonal trastuzumab IgG and rendered a 30% growth inhibition after 24 h. mBT474 cells were incubated with trastuzumab as positive control and an isotype control (1 μg/well), and compared to untreated cells alone (FIG. 7A). In the same assay mBT474 cells were incubated with purified IgG from pooled mouse sera from mice immunized with different AAV particles (DMD1, DMD2, DMD4, DMD6, FIG. 7B).). Readout was performed with the EZ4U cell proliferation assay (Biomedica, Vienna).

(79) The different clones elicited various degrees of proliferation inhibition (all at 5 μg/well) compared to untreated cells alone. The effects on mBT474 seem to be most pronounced for DMD1-antibodies and they reach the effects of trastuzumab.

(80) TABLE-US-00067 TABLE 11 Summary of functional characterization of HER2 AAV clones Trastuzumab IgG mAb Trastuzumab rec. HER2 cellular HER2 Reactivity Reactivity Reactivity Avidity Index Reactivity Reactivity Clone Cell lysate Purified AAV (5M urea) (mice sera) (mice sera) Consensus 1 DMD02 +++ − +++ + +++/+ + DMD18 + − +/− + DDDD63 + +/− DDD29 +/− − DMD11 +++ − +++ + DMM44 +++ +/− +++ +  .sup. n.d./++ DDM52 + +/− DDD25 +/− − DMD04 + − + +++   .sup. +/+++ + DDD22 + − DDDD65 +/− − DDM47 + + DMD05 + − ++ ++ DDD55 + − DMD03 + − + ++ DMD07 + − + ++ + + DDD62 +/− − DDD20 +/− +/− + DDD54 − − DDD57 +/− − DDD21 +++ − +/− +++ + Partial Consensus 1 DDD28 +/− − DMD06 − − +/− ++  .sup. n.d./+++ DDD23 + + DDD32 +/− − DMM45 + ++ DMD01 + − + ++ +++/+ + Consensus 2 DDM53 + + DMD13 − − DMM46 +/− + DMD17 − − DMM38 + ++ DMM41 − − DMM36 + + Consensus 3 DMD09 − − DDM50 − − DMD15 + − + +++/+ ++ DMM43 + + DMM42 + + DDM49 +/− + DMD16 − − Consensus 4 DMM36 + + DMM37 + ++ DDD26 +/− − DMM35 + + DDM48 + + DDD19 +/− − + +++   .sup. −/+++ ++ DDDD64 + +/− DMM41 − − Consensus 5 DMD8 − − DMD14 − − DMM34 − − DDD27 +/− − DDD31 + +/− +/− − − DMM40 − − DMM39 − − DDD24 + +/− DDD30 + − − ++ − − DDD56 +/− − DDD60 +/− − DDD61 − − Consensus 6 DMD10 − − DMM33 +++ +++ DDD58 +/− − DMM43 + + No consensus DMD12 +/− − DDD59 +/− −

(81) Background and Aims.

(82) Cancer is one of the major public health problems in western societies, leading to every fourth case of death in Austria. Highest prevalence rates are described for breast cancer, affecting currently more than 50 000 women in Austria. To date, passive immunotherapy with monoclonal antibodies is a well-established option in clinical oncology. In contrast, anti-cancer vaccines are less advanced. The development of therapeutic vaccines is still a great challenge mostly due to the self-nature of tumor antigens. Mimotopes, small peptides from 6-38 amino acids, resembling B-cell epitopes do not need consensus sequence with the natural antigen, because molecular mimicry via e g amino acid charges is sufficient to shape an electron cloud specifically recognized by the immune system. As they are similar, but not identical to the original tumor antigen, vaccination with mimotopes may overcome tumor tolerance. Adeno-associated virus like particles (AAVLP) could serve as novel vectors for displaying mimotopes to the immune system. We suggest that cancer vaccines will especially open up new treatment options in minimal residual disease and early stage disease.

(83) Methods and Results:

(84) Adeno-associated viruses (AAV) are ssDNA viruses being replication defective in the absence of Adenovirus. Their surface consists of 60 capsomers, which can be exploited for high density display of recombinant peptides. AAV-like particles (AAVLP) can be generated via assembling recombinant AAV-2 capsid fusion proteins. In this study different HER-2 derived linear B-cell epitopes, generated in a biopanning with the clinically used anti-HER-2 antibody trastuzumab, were inserted into AAV-2. Mimotope candidates were screened for trastuzumab binding in ELISA. Appropriate candidates were employed for immunization of BALB/c mice Immune response was monitored measuring circulating levels of IgG1, IgG2a and IgG2b antibodies reactive to recombinant HER-2. Molecular mimicry was also proved in immunofluorescence on human HER-2 overexpressing murine mammary carcinoma D2F2-E2 cells. Sera of mice displaying highest HER-2 specific antibody levels were exploited for antibody purification and purified antibodies were tested for their tumoristatic properties in a tetrazolium based cell viability assay. In this assay HER-2 overexpressing human mammary carcinoma cells mBT474 showed significant growth reduction even after 24 h of antibody incubation with purified antibodies of clone DMD1. This effect increased at consecutive measurements after 48 and 72 h.

(85) Conclusion:

(86) In this study we could demonstrate that AAVLP are suitable vectors for mimotope based cancer vaccines. In our system immunized BALBc mice developed anti-HER-2 antibodies with similar biological properties to the clinically used monoclonal antibody trastuzumab. Due to their easy application and economic advantages, cancer vaccines might become important supplementary therapy options in cancer treatment, especially in the minimal residual disease setting.

(87) 9. Memory Effect

(88) A further experiment was carried out to investigate the memory effect elicited by one species of AAV particles comprising a mimotope insert in combination with different adjuvants. 8 mice per groups were immunized subcutaneously according to Table 12 three times with two weeks intervals. For sera samples, blood was collected before starting of the first immunisation (pre-immunsera; PIS), and at days 13 (1. MIS), 27 (2. MIS), 41 (3. MIS), 69 (4. MIS), 97 (5. MIS), 125 (6. MIS) and at sacrifice of mice (7. MIS).

(89) TABLE-US-00068 TABLE 122 Immunization scene Group Adjuvant Antigen A Alum 10 μg DMD2 B MPL 10 μg DMD2 C Alum + MPL 10 μg DMD2 D ODN-1826 10 μg DMD2 E Alum + ODN-1826 10 μg DMD2 F Alum HER-2 G Naiv (Iodixanol)

(90) In a first analysis, HER-2 specific total IgG levels of PIS, 1. MIS, 2. MIS and 3. MIS were determined by ELISA as described above. The results are shown in FIG. 8a). To evaluate a potential memory effect a further analysis samples PIS, 3. MIS, 4. MIS, 5. MIS, 6. MIS and 7. MIS were analyzed accordingly. The results are shown in FIG. 8b).

(91) The data show, that AAV particles comprising a HER2 minotope, especially DMD2, were able to induce a long lasting B-cell memory. The highest effect was achieved with a combination of Alum and MPL as adjuvants.

(92) 10. Immunofluorescense Staining

(93) D2F2 (control) and D2F2-E2 (transfected with human HER-2) were seeded for a concentration of 2×1 cells in 400 pL per well on four-well immunofluorescence chamber slides (Permanox®, Thernio Scientific). After 24 hours they were fixed with 4% paraformaldehyde for 8 minutes at room temperature before they were washed three times with cold PBS. For DAPI staining, cells were permeabilized 5 minutes with 0.5% TritonX-100 in PBS and washed again with cold PBS. DMD001 sera (as obtained in experiment 8) were diluted 1:20 in PBS/0.5% BSA with 200 pL each well, whereas trastuzumab was incubated with 1 pg/ml each. After an incubation of one hour at room temperature, wells were washed four times with cold PBS, before the secondary antibody, diluted 1:200 in PBS/0.5% BSA was added in 400 pl per well. Incubation was performed by covering slides with aluminum foil for 45 minutes, and stopped by washing slides four times with cold PBS. DAPI diluted 1:5000 was incubated for eight minutes at room temperature and then washed three times with cold PBS. Afterwards all slides were washed in distilled aqua before they were covered with mounting medium (Fluoromount™, Sigma) and stored at 4° C.

(94) Immunofluorescense staining of DMD001 sera on D2F2-E2 cells (a), of DMD001 sera on D2F2 cells (b) and trastuzumab on D2F2-E2 cells (c) showed green fluoresce labeling of cells for conditions (a) and (c) but not for (b), indicative for the presence of HER-2 specific in DMD001 sera in comparison to the positive control with trastuzumab (data not shown).

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