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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2710/00011—Details
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  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to a nucleic acid comprising or consisting of nucleic acid sequences encoding papilloma virus proteins E1, E6, and E7; wherein said nucleic acid molecule encodes a single polyprotein.
• a number of documents including patent applications and 15 relevant for the patentability of this invention is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
• HPV16 + cervical cancer patients have impaired memory CD4 + 5 T-helper responses against E2 and E6, which emphasizes the important role of T cell responses in preventing progression and clearing lesions (Brotherton et al, 2016; Lehtinen et al, 2012; Hildesheim et al, 2007; Schiller et al, 2012; Hung et al, 2008; Ho et al, 1998; Xue et al, 2010; Chang et al, 2013; Hibma, 2012; Monnier-Benoit et al, 2006; de Jong, A. et al.
• Macaca fascicularis papillomavirus type 3 (MfPV3) has a close phylogenetic and phenotypic 30 relationship to HPV16 (Ragonnaud et al, 2017; Chen, Z. et al. Non-human Primate Papillomaviruses Share Similar Evolutionary Histories and Niche Adaptation as the Human
• Naturally occurring infections with this virus are associated with long-term persistence and at least LSIL-like lesions in the cervix of breeding female cynomolgus macaques (Macaca fascicularis), making them an ideal non- human primate (NHP) animal model (Chen, Z. et al. Genomic diversity and interspecies host 5 infection of alpha12 Macaca fascicularis papillomaviruses (MfPVs). Virology 393, 304 310, 2009; Wood, C. E., Chen, Z., Cline, J. M., Miller, B. E. & Burk, R. D.
• the present invention provides a nucleic acid comprising or consisting of nucleic acid sequences encoding papilloma virus proteins E1, E6, and E7; wherein said nucleic acid molecule encodes a single polyprotein.
• the nucleic acid in accordance with the first aspect requires presence of sequences encoding 20 three papilloma virus proteins. Preferred is that either exactly three or exactly four papilloma virus proteins are encoded. To the extent a fourth protein is encoded, preference is given to a further protein of the E family of papilloma virus proteins, in particular to E2. Preferred papillomavirus species and strains are disclosed further below. Particularly preferred is human papillomavirus 16 (HPV16). 25 In accordance with the invention, coding sequences encoding the corresponding full-length or substantially full-length proteins are employed.
• the concomitant presence of three or four full-length protein coding sequences provides for the presentation of a multitude of immunogenic epitopes in an organism vaccinated with the nucleic acid of the first aspect or a 30 vector or plasmid comprising said nucleic acid.
• substantially full-length refers to the optional absence of up to 20, up to 15, up to 10, or up to 9, 8, 7, 6, 5, 4, 3, or
• deletions are at the C-terminus, N-terminus and/or in regions where deletions do not remove immunogenic epitopes or do not affect the immunogenicity of epitopes present in other parts of the amino acid sequence of the encoded protein.
• deletions reduce the oncogenic potential of a 5 papillomavirus protein.
• C- terminal modification of E6 C- terminal modification of E6
• substantially full-length applies to E6 only, whereas E1, E7, and, to the extent present E2, are full-length.
• Immune responses such as T cells directed against three or more, preferably four different 10 full-length proteins will unfold synergistic activity against papilloma virus infections and disorders associated therewith.
• successful stimulation of E1/E2-specific cellular immunity primarily clears infections in the LSIL-stage, whereas E6/E7-specific responses mainly targets HSIL and cancer stages.
• Further advantages of the invention include the surprising capability to completely eradicate a papillomavirus infection, and the usefulness of said nucleic acid for not only prophylactic, but also therapeutic purposes. This is particularly advantageous in view of the oncogenic potential of papillomavirus.
• the synergistic effect arising from the concomitant expression of three or four full- 30 length viral proteins is superior to the concomitant use of only fragments of a plurality of proteins inter alia owing to the larger number of immunogenic epitopes being provided.
• a construct of the invention is capable of triggering immune response in non-human primates 15 which are suffering from persistent HPV infection and T cell exhaustion.
• the papillomavirus antigens of the invention i.e., E1, E6, E7, and, to the extent present, E2, 20 are encoded by the nucleic acid of the invention as a single polyprotein.
• the coding sequences therefor are part of a single open reading frame.
• polyprotein includes fusions of said antigens, more specifically direct fusions with no intervening amino acids between the sequences of the respective proteins.
• polyprotein also embraces proteins wherein one or more amino acids are inserted between the sequences of two25 neighboring proteins or placed N-terminal of the most N-Terminal E protein, or placed C- terminal of the most C-terminal E protein.
• Preferred implementations of such inserted sequences are given below and may serve to provide specific functions such as adjuvant function or self-cleaving function, and/or avoid structural interference between adjacent proteins. The latter issue may be addressed by sequences encoding short flexible linkers being 30 located between the nucleic acid sequences encoding the antigens.
• nucleic acid refers to a polycondensate of nucleotides. Preference is given to nucleic acids which are recognized and processed by polymerases and/or a ribosome. Preferred implementations are DNA and RNA; for details see further below.
• nucleic acid also extends to polycondensates comprising one or more modified nucleotides.
• Sites of modification in a nucleotide are those well known in the art, i.e., the sugar, the phosphate and the base.
• Preferred modification in that respect are 2' modifications such 2' O-alkyl including 2' OMe and 2' halogen substitutions such as 2' F.
• Preferred phosphate modifications include thiophosphate.
• a preferred modified base is pseudouridine; see, for 15 example, Nance and Meier, ACS Cent Sci.2021 May 26; 7(5): 748 756.
• RNA vaccine i.e., where the nucleic acid of the invention is implemented as RNA.
• the E family of papillomavirus proteins are proteins referred to as "early" in the lifecycle of 20 the virus.
• E1 has enzymatic function; it is an ATP-dependent helicase; see, e.g., Bergvall et al., Virology 2013 Oct;445(1-2):35-56.
• E6 is capable of inducing cell division in resting cells. It inhibits apoptosis and the immune response; see, e.g.
• said nucleic acid furthermore comprises or further consists of a nucleic acid sequence encoding papilloma virus protein E2.
• E2 is multifunctional and mainly involved in genome replication and transcription; see, e.g. McBride, Virology.2013 Oct;445(1-2):57-79.
• the term "further consists of” refers to a closed listing of a preceding aspect or embodiment 5 and provides for an expansion of said closed list by addition of an explicitly recited feature, wherein the listing remains closed and confined to the features of said preceding aspect or embodiment, augmented by said explicitly recited additional feature.
• adding E2 as a further antigen does not deteriorate immune response; see, e.g. Example 4 and Figure 10.
• Antigen sequences in accordance with the invention are given in the sequence listing as follows: Amino acid sequences of E1, E2, E6 and E7 of MfPV3 are shown in SEQ ID NOs: 2, 4, 6, and 8, respectively. 15 Amino acid sequences of E1, E2, E6 and E7 of HPV16 are shown in SEQ ID NOs: 10, 12, 14, and 16, respectively. Amino acid sequences of E1, E2, E6 and E7 of HPV18 are shown in SEQ ID NOs: 18, 20, 22, and 24, respectively. The encoding nucleic acids for these proteins are shown, in the same order, in SEQ ID NOs: 20 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, respectively.
• the nucleic acid of the invention encodes the four proteins E1, E2, E6 and E7 as a polyprotein, wherein preferably no further proteins of papillomavirus origin are encoded by said nucleic acid.
• the order of the nucleic acids encoding said proteins is, from 5' to 3', (a) to the extent E2 is present (i) E1, E2, E6, E7 or (ii) E1, E6, E7, E2; or (b) E1, E6, E7.
• any of these listings is closed, i.e., no further sequence encoding a further protein of papillomavirus origin is present.
• the embodiment on the other hand extends to implementations where further amino acid sequences, preferably functional amino acid 30 sequences, are present (for details see further below).
• sequences encoding the four proteins E1, E2, E6 and E7 are present and in the indicated order (from 5' to 3'). While also constructs of the invention with a different order perform well, it can be seen e.g. in Figure 3B that the order in the most preferred construct secures even better performance in terms of the E2 response.
• said nucleic acid of the first aspect comprises nucleic acid sequences encoding papilloma virus proteins E6 and/or E7, wherein said nucleic acid sequences are modified as compared to the wild-type counterparts by point mutations and/or deletions, wherein said point mutations and/or deletions (i) abolish colony forming in a soft 10 agar assay, at least to a background level; and (ii) said modified proteins E6 and E7 maintain at least 70 % of predicted high-affinity epitopes (IC50 50 nM) and at least 50 %, at least 55% or at least 58% of predicted low-affinity epitopes (50 nM ⁇ IC50 5 ⁇ M) of the predicted epitopes of the respective wild-type protein, preferably in terms of (predicted) binding to the most frequent HLA-A, -B, and -C alleles which preferably together comprise 75 % of the 15 human HLA diversity for each HLA
• This preferred embodiment allows for the presence of further papillomavirus proteins, E1 and optionally E2 being preferred in this context.
• Said most frequent alleles are as follows: 20 HLA-A*01:01 HLA-A*02:01 HLA-A*02:02 HLA-A*02:03 HLA-A*02:05 25 HLA-A*02:06 HLA-A*02:07 HLA-A*02:11 HLA-A*02:12 HLA-A*02:16 30 HLA-A*02:17 HLA-A*02:19
• said modified E6 protein maintains at least 80%, at least 90%, at least 95%, or at least 98% of predicted high-affinity epitopes (IC50 50 nM) and 10 at least 60 %, at least 70%, at least 80%, at least 90%, or at least 93% of predicted low-affinity epitopes (50 nM ⁇ IC50 5 ⁇ M) of the predicted epitopes of wild-type E6, preferably determined as specified above.
• IC50 50 nM predicted high-affinity epitopes
• 10 at least 60 %, at least 70%, at least 80%, at least 90%, or at least 93% of predicted low-affinity epitopes (50 nM ⁇ IC50 5 ⁇ M) of the predicted epitopes of wild-type E6, preferably determined as specified above.
• the present invention provides a nucleic acid 15 comprising or consisting of nucleic acid sequences encoding papilloma virus proteins E6 and/or E7, wherein said nucleic acid sequences are modified as compared to the wild-type counterparts by point mutations and/or deletions, wherein said point mutations and/or deletions (i) abolish colony forming in a soft agar assay; and (ii) said modified proteins E6 and E7 maintain at least 70 % of predicted high-affinity epitopes (IC 50 50 nM) and at least 50 %, 20 at least 55% or at least 58% of predicted low-affinity epitopes (50 nM ⁇ IC 50 5 ⁇ M) of the predicted epitopes of the respective wild-type protein, preferably in terms of binding to the most frequent HLA-A, -B, and -C alleles which preferably together comprise 75 % of the human HLA diversity for each HLA, respectively.
• IC 50 50 nM predicted high
• said modified E6 protein maintains at least 80%, at least 90%, at least 95%, or at least 25 98% of predicted high-affinity epitopes (IC 50 50 nM) and at least 60 %, at least 70%, at least 80%, at least 90%, or at least 93% of predicted low-affinity epitopes (50 nM ⁇ IC 50 5 ⁇ M) of the predicted epitopes of wild-type E6, preferably determined as specified above.
• This aspect does not require any further nucleic acid sequences encoding papilloma virus proteins to be present.
• proteins E6 and E7 play a role in controlling and modifying host cell behaviour and can unfold oncogenic or transforming potential.
• using these proteins as immunogens is desirable and attractive, especially when it comes to control, eradicate, prevent, mitigate or treat papillomavirus-associated malignant disorders.
• their oncogenic potential entails corresponding risks associated with their use as a vaccine or components of a vaccine.
• the above preferred embodiment provides measures to mitigate or abolish said oncogenic potential.
• a balance is struck by said embodiment in that oncogenicity is controlled while immunogenicity is retained.
• said point mutations and deletions are minimal in 10 number.
• the present invention provides, in a preferred embodiment, a minimum of modifications as compared to the corresponding wild-type sequences, wherein said minimum achieves both the required performance in a soft agar assay as well as in terms of retaining a high percentage of the immunogenic epitopes of the corresponding wild-type sequences.
• a position in an E2, E6 or E7 protein from a given first papillomavirus species corresponding to a position in an E2, E6 or E7 protein, respectively, from a given second papillomavirus species can be determined using the multiple sequence alignment shown in Figure 9. Corresponding positions are one on top of the other.
• the 20 determination of corresponding positions is straightforward in view of the high degree of conservation across different HPV strains, in particular using said multiple sequence alignment to which sequences from any further papillomavirus strain can be added without further ado.
• Such alignments can be prepared with computer programs well known to the skilled person, preferably with CLC Main Workbench version 8.0.1 (QIAGEN, Aarhus, 25 Denmark) using the default settings (very accurate; gap open cost 10.0; gap extension cost 1.0, end gap cost: as any other).
• said nucleic acid furthermore comprises one or both mutations encoding D21X5 and C58X6 in HPV 16 E7, D24X5 and C65X6 in HPV 18 E7, and D21X5 and 30 C62X6 in MfPV3 E7, wherein X5 and X6 are independently a proteinogenic amino acid different from the respective amino acid they substitute.
• X 1 is selected from Q, Y, and N, and preferably is Q;
• X 2 is selected from G, A, V, L, and I, and preferably is G;
• X 3 is selected from R, K, and H, and preferably is R;
• X 4 is selected from A, G, V, L, and I, and preferably 5 is A;
• X 5 is selected from G, A, V, L, and I, and preferably is G;
• X 6 is selected from G, A, V, L, and I, and preferably is G; and/or
• said modification which reduces or abolishes interactions mediated by the PDZ domain is a deletion of 1 to 10 C-terminal amino acids, preferably counted from the C-terminus, more preferably ETQL in HPV16 E6, ETQV in HPV18 E6, and ETEV in MfPV3 E6; or said modification are one, two
• the total number of Cys residues in said polyprotein is an even number. How this is achieved is not particularly limited. Preference is given to implementations which do not entail loss of one or more immunogenic epitopes. Without wishing to be bound by a specific theory, it is envisaged that an even number of Cys residues increases antigen yield. To explain further, antigens, when fused to invariant chain (for details 20 regarding the invariant chain see further below) are directed to the secretory pathway.
• nucleic acid is comprised in said nucleic acid as sequence encoding E2, wherein X 7 is a proteinogenic amino acid other than C, preferably selected from A, S, G, M, T, Y, Q, N, V, L, I, F, W, H, K, R, Q, N, and P, more preferably A; (ii) or a codon encoding Cys is inserted in said nucleic acid.
• said nucleic acid furthermore comprises at least one nucleic acid sequence encoding a genetic adjuvant, preferably T cell adjuvant MHC-II- associated invariant chain. Said invariant chain is also abbreviated as "li" in the following.
• the T cell adjuvant MHC-II-associated invariant chain has successfully been used as a genetic 10 adjuvant (i.e., an adjuvant encoded by a nucleic acid, said nucleic acid optionally comprising also nucleic acid sequences encoding the antigen(s) against which an immune response is to be raised) in several contexts, see, for example, published international patent applications WO 2007/062656, WO 2010/057501 and WO 2018/172259.
• WO 2020/079234 shark invariant chain (see, e.g., WO 2015/082922) and mammalian, preferably human invariant chain; see the above cited three WO documents. Particularly preferred is human invariant chain.
• Further genetic adjuvants which may be used in accordance with the invention are heat-shock 20 proteins, calreticulin and C4b. Presence of said invariant chain entails unexpected performance owing to a synergistic effect. In particular, the combination of the above disclosed modifications of the oncogenic proteins E6 and E7 with li reduces oncogenicity to an unexpected degree.
• said invariant chain (i) is present once and precedes the sequence encoding the first E protein; or (ii) is present twice, one copy preceding the sequence encoding the first E protein, and a second copy preceding the sequence encoding E6
• first E protein 5 refers to the most N-terminal protein selected from E1, E6 and E7, and, if present, E2 in the fusion protein encoded by the nucleic acid of the invention. Preferred is that E1 is the first E protein.
• adjuvants known in the art may be 10 used.
• Such adjuvants include cytokines, adhesion molecules, chemokines, CCR1/5 agonists, MIP-1 alpha, danger signals, LPS and derivatives thereof such as lipid A and lipid A derivatives, and TLR agonists.
• said nucleic is (i) DNA; (ii) a plasmid; or (iii) RNA, 15 preferably mRNA.
• mRNA vaccines are being developed and several have received market approval such as Comirnaty of Biontech/Pfizer and mRNA-1273 of Moderna Therapeutics. In view of its inherent instability, RNAs may be chemically modified in order to enhance their stability; see, e.g.
• mRNA 20 vaccines generally comprise a Kozak sequence at the translation start point, a translation termination codon at the end of the open reading frame, and a poly-adenylation site in the 3'- untranslated region.
• said DNA and said plasmid comprise a promoter, preferably a 25 promoter active in eukaryotic cells.
• promoters include a CMV promoter, preferably with a HTLV-1 enhancer element, UbC promoter, EF1alpha promoter and SV40 promoter.
• said plasmid comprises a bacterial origin of replication such as pUC ori, and more preferably also selection marker such as an antibiotic resistance gene such 30 as a Kanamycin resistance gene.
• said nucleic acid in particular said DNA, comprises an untranslated 5' terminus comprising a Tet operator.
• Tet-Off preference is given to Tet-Off, given that the off-switch would be a constituent of the producer cell and not of the virus product.
• a preferred Tet operator site is shown in SEQ ID 5 NO: 105.
• the Tet system employed by the present invention is the system known as T-REx system. In the T-REx system the gene of interest is flanked by an upstream CMV promoter and two copies of tetracycline operator 2 (TetO2) sites.
• TetR homodimers Expression of the gene of interest is repressed 10 by the high affinity binding of TetR homodimers to each TetO2 sequences in the absence of tetracycline.
• the production cell lines were modified to express the Tet repressor TetR.
• Introduction of tetracycline results in binding of one tetracycline on each TetR homodimer followed by release of the TetR homodimer from the TetO2.
• Unbinding of TetR homodimers form the TetO2 results in de-repression of the gene of interest. See, for 15 example, Hillen W. and Berens.
• Tn10 encoded tetracycline resistance Mechanisms underlying expression of Tn10 encoded tetracycline resistance, Annu Rev Microbiol.1994;48:345-69; W Hillen, C Gatz, L Altschmied, K Schollmeier, I Meier. Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions, J Mol Biol.1983 Sep 25;169(3):707-21; Kathleen Postle, Toai T. Nguyen, Kevin P. Bertrand. Nucleotide sequence of 20 the repressor gene of the TN10 tetracycline resistance determinant.
• said nucleic acid comprises at least one nucleic acid sequence encoding a self-cleaving peptide or a peptide which causes a ribosome not to form a peptide bond while continuing translation, wherein said nucleic acid sequence encoding said peptide preferably (i) precedes at least one nucleic acid sequence encoding a genetic adjuvant; or (ii) 30 is located between and adjacent to two nucleic acid sequences encoding a papilloma virus protein.
• nucleic acid sequence encoding a self-cleaving peptide into a nucleic acid sequence encoding one of the antigens of the invention.
• a preferred peptide sequence is referred to as "p2a" herein.
• nucleic acids in accordance with the invention are those which encode the following polyproteins: 10 li-E1E2E6E7 li-E1E2-p2a-li-E6E7 E1E2E6E7 li-E1E6E7-p2a-li-E2 15 li-E1E6E7
• sequences of nucleic acids encoding these constructs, wherein the encoded proteins are or are based on MfPV3 proteins are, in the same order: SEQ ID NOs: 25, 27, 29, 31 and 33, and the corresponding polyprotein sequences are shown in SEQ ID NOs: 26, 28, 30, 32 and 34, 20 respectively.
• sequences of nucleic acids encoding these constructs, wherein the encoded proteins are or are based on HPV16 proteins are, in the same order: SEQ ID NOs: 35, 37, 39, 41 and 43, and the corresponding polyprotein sequences are shown in SEQ ID NOs: 36, 38, 40, 42 and 44, respectively.
• sequences of nucleic acids encoding these constructs, wherein the encoded proteins are or are based on HPV18 proteins are, in the same order: SEQ ID NOs: 45, 47, 49, 51 and 53, and the corresponding polyprotein sequences are shown in SEQ ID NOs: 46, 48, 50, 52 and 54, respectively.
• SEQ ID NOs: 100 to 104 all relate to HPV18 li-E1E6E7, more specifically as follows: HPV18 li- 30 E1E6E7 nucleic acid sequence (SEQ ID NO: 100) , HPV18 li-E1E6E7 protein sequence (SEQ ID NO: 100)
• HPV18 li-E1E6E7 payload as inserted into various viral vectors (SEQ ID NOs: 102 to 104).
• E antigens are adjacent, they are preferably separated by a short flexible linker, preferably GS (see also below).
• a linker between li and an adjacent antigen is not 5 required, but not excluded.
• a linker connecting p2a to any of the adjacent sequences, be it an antigen or li, is not required, but not excluded.
• said papilloma virus is a primate papilloma virus, preferably a human papilloma virus (HPV) such as HPV16, HPV18, HPV45, HPV31, HPV33, 10 HPV35, HPV52, HPV58, HPV6 and HPV11, more preferably HPV16 or HPV18, or macaque papilloma virus, preferably MfPV3.
• HPV16 human papilloma virus
• Experimental evidence for HPV16 can be found in Example 4.
• neighbouring nucleic acid sequences are connected in one, 15 more or all instances by sequences encoding a flexible linker which is 10 or less amino acids in length such as 2, 3, 4, or 5 amino acids, optionally comprising a Cys residue, wherein GS and GCS are preferred linkers.
• a flexible linker which is 10 or less amino acids in length such as 2, 3, 4, or 5 amino acids, optionally comprising a Cys residue, wherein GS and GCS are preferred linkers.
• linkers are present only between neighbouring antigens; see also the discussion of the most preferred nucleic acids of the invention as given above.
• a linker comprising a Cys residue such as GCS is preferably to be viewed in the context of the above disclosed preferred embodiment requiring an even number of Cys residues in the polyprotein of the invention.
• the present invention provides a viral vector comprising the nucleic acid 30 of any one of the preceding claims.
• said vector is a DNA viral vector, preferably (i) an adenoviral vector, preferably being deficient with regard to adenoviral E1 and E3 encoding sequences, said vector more preferably belonging to class C, D or E and/or being Ad19a/64, Ad5, Ad5 with fiber replacements such as Ad5F35, Ad26, Chimp63, or chAdOx1; or (ii) a Poxvirus 5 vector, preferably MVA, MVA CR19, SCV, Vaccinia, or Fowlpox.
• Ad5 has been described in Schiedner G, Hertel S, Kochanek S. Hum Gene Ther.2000 Oct 10;11(15):2105-16.
• Ad19a is also known as Ad64; see, for example, Zhou X., Robinson C.M., Rajaiya J., Dehghan S., Seto D., Jones M.S., Dyer D.W., Chodosh J. Analysis of human adenovirus type 19 associated with epidemic keratoconjunctivitis and its reclassification as adenovirus type 64, Invest. Ophthalmol. Vis. Sci, 53 (2012), pp.2804-2811. Ad19a is also described in Ruzsics Z, Wagner M, Osterlehner A, Cook J, Koszinowski U, Burgert HG.
• Ad5F35 Chimeric Ad5.F35 vector evades anti-adenovirus serotype 5 neutralization opposing GUCY2C-targeted antitumor immunity, Journal for ImmunoTherapy of Cancer.2020.
• Ad5 and Ad5F35 induces comparable immune responses and tumour control in na ⁇ ve mice, and 25 that Ad5F35 induces better responses than Ad5 in mice pre-exposed to Ad5 infection.
• Ad5F35 is particularly preferred.
• Particularly preferred DNA payloads (inserts; sequences comprising antigen-encoding 30 sequences) in accordance with the invention for various viral vectors are shown in SEQ ID
• MVA sequence GenBank (release 244.0) accession number: U94848, version number: 10 U94848.1, release date 14.04.2003, https://www.ncbi.nlm.nih.gov/nuccore/U94848.1; MVA.CR19 sequence: GenBank (release 244.0) accession number: KY633487, version number KY633487.1, release date 28.03.2017, https://www.ncbi.nlm.nih.gov/nuccore/KY633487.1;
• a cell line preferably a eukaryotic cell line may be employed, wherein said cell line comprises said vector.
• Suitable eukaryotic 15 cell lines include HEK293, PerC6, AGE1.CR.pIX (Jordan I, Vos A, Beilfuss S, Neubert A, Breul S, & Sandig V, 2009. An avian cell line designed for production of highly attenuated viruses. Vaccine 27, 748 756. https://doi.org/10.1016/j.vaccine.2008.11.066. PMID 19071186), HEK293 T-REx and AGE1.CR.pIX-T-REx (derived from HEK293 and AGE1.CR.pIX, respectively, already expressing TetR), A549, and A549 with engineered adenovirus E1 overexpression.
• said cell lines are preferably, but not necessarily modified to express TetR.
• MVA is adapted to replication in avian cells.
• a host is therefore preferred such as primary chicken embryo fibroblasts (CEF) or AGE1.CR.pIX that is derived 25 from duck retina cells.
• an immortalized (or continuous) cell line such as AGE1.CR.pIX has several advantages: the cell substrate can be retrieved out of locally stored cryocultures and thus is resilient to supply constraints.
• An immortal cell line can furthermore be characterized against adventitious agents at the level of the cell bank, well ahead of the actual production processes.
• the AGE1.CR.pIX cell line (as opposed to primary 30 material) furthermore proliferates in suspension in media free of animal derived components. This property allows highly efficient and scalable fed-batch production processes for
• poxviruses of different genera Jordan I, Northoff S, Thiele M, Hartmann S, Horn D, Höwing K, Bernhardt H, Oehmke S, von Horsten H, Rebeski D, Hinrichsen L, Zelnik V, Mueller W, & Sandig V, 2011. A chemically defined production process for highly attenuated poxviruses. Biologicals 39, 50 58. https://doi.org/10.1016/j.biologicals.2010.11.005. PMID 21237672). 5 Vaccinia viruses mature into infectious particles with one or three membranes without the requirement for budding.
• MVA-CR19 is a strain of MVA with a unique genotype (Jordan I, Horn D, Thiele K, Haag L, Fiddeke K, & Sandig V, 2019. A Deleted 15 Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA). Virol Sin. https://doi.org/10.1007/s12250-019-00176-3. PMID 31833037). Point mutations in structural genes and recombination of a large portion of h i d rminal repeat (ITR) at the left side of the linear genomic DNA have profound effects on the phenotype of MVA-CR19.
• ITR h i d rminal repeat
• MVA-CR19 20 releases a larger number of infectious particles into the culture supernatant and replicates to higher infectious titers.
• Viral factors that impact immune responses of the host and the infectious cycle are encoded in the ITRs.
• the recombination event in MVA-CR19 has changed the expression pattern of these factors (some were deleted, for others the gene dosehas been duplicated) with positive effects on efficacy and stability as a vaccine vector.
• MVA-CR19 The potentially enhanced release of MVA-CR19 from host cells can also be seen in the CPE in adherent cells: whereas wild-type MVA tends to induce cell fusion and syncytia with well circumscribed plaques, infection with MVA-CR19 leads to a pattern consisting of large but loosely packed (unfused) plaques surrounded by isolated infected cells scattered at greater 30 distances to the primary plaque or localized in comets.
• MVA-CR19 has advantages for production and vaccine efficacy it can be more complex to purify due to the less confined nature of replication. Furthermore, for both wild-type and MVA-CR19, selection against expression and maintenance of a transgene may occur if the novel sequence impairs the infectious cycle.
• the present invention provides a polyprotein encoded by the nucleic acid or the vector of any of the preceding claims or cleavage products derived therefrom. 15 To the extent reference is made to cleavage products, these are preferably cleavage products arising from the activity of a self-cleaving sequence which in accordance with a preferred embodiment disclosed above may be present in the polyprotein encoded by the nucleic acid of the invention. It is understood that cleavage does not affect the number of epitopes 20 presented by the proteins encoded by the nucleic acid of the invention.
• the present invention provides a pharmaceutical composition, preferably a vaccine, comprising or consisting of (i) a nucleic acid of the first aspect; (ii) a vector of the 25 second aspect; (iii) a polyprotein or cleavage products thereof of the third aspect; and/or (iv) a cell transduced with the nucleic acid of (i) or the vector of (ii), wherein preferably said cell is an ex vivo or in vitro cell and/or a dendritic cell or a monocyte.
• cells may be transformed with an antigen- 30 encoding construct, thereby obtaining a population of cells with prophylactic or therapeutic properties against a disorder associated with said antigen.
• An example in the field of cancer is Maeng et al., Journal of Clinical Oncology 37, no.15_suppl (May 20, 2019) 2639-2639.
• said cells are autologous cells from the individual to be treated.
• Ad19a/64 is especially preferred for item (iv), to the extent a vector is used.
• routes of administration intramuscular, subcutaneous, intradermal, vaginal, 5 rectal, and/or mucosal administration, including to mucosae of the genital tract are envisaged.
• Formulations include gels, in particular for mucosal, preferably vaginal administration. Excipients, diluents and conservants are well known to the skilled person.
• 10 In terms of dosages, 10 9 to 10 11 infectious units are preferred for adenoviral vectors, and 5 x 10 6 to 5 x 10 8 for poxviral vectors.
• suitable dosages can be determined by a clinician or manufacturer without further ado in view of the present disclosure, possibly of clinical studies, and, where necessary, taking into account parameters such as weight, age, sex 15 etc. of the individual to be vaccinated.
• parameters such as weight, age, sex 15 etc. of the individual to be vaccinated.
• said vector, said nucleic acid, said polyprotein or said cell is the only pharmaceutically active agent.
• said pharmaceutical composition comprises two or more pharmaceutically active agents, wherein a second pharmaceutically active agent is selected from (i) a second nucleic acid of the first aspect; (ii) a second vector of the second aspect; (iii) a second polyprotein or cleavage products of the third aspect; (iv) a second cell as 25 defined above; (v) a protein-based vaccine, preferably against papilloma virus; and (vi) a chemotherapeutic, preferably cisplatin.
• a second pharmaceutically active agent is selected from (i) a second nucleic acid of the first aspect; (ii) a second vector of the second aspect; (iii) a second polyprotein or cleavage products of the third aspect; (iv) a second cell as 25 defined above; (v) a protein-based vaccine, preferably against papilloma virus; and (vi) a chemotherapeutic, preferably cisplatin.
• papillomavirus vaccines which
• checkpoints preferably being PD-1, PD-L1 and CTLA- 5 4.
• the components of a combination therapy may be administered together or separate in any order, possibly following different dosage regimens.
• said pharmaceutical composition comprises two pharmaceutically active agents which are (i) an adenoviral vector as defined above; and a Poxvirus vector as defined above, wherein said two pharmaceutically active agents are preferably confectioned as two pharmaceutical compositions which may be administered in any order or concomitantly, preferably the pharmaceutical composition comprising said 15 Poxvirus vector after said pharmaceutical composition comprising said adenoviral vector, preferably after at least 4 weeks have elapsed.
• This preferred embodiment defines preferred prime/boost schemes in accordance with the invention. As such, it is preferred that said two distinct vectors are administered in a manner 20 which allows time to elapse between the two administrations.
• Preferred time spans are at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks or at least 8 weeks. Preferred upper limits for said time span are 6 weeks, 7 weeks, 8 weeks, 3 months, 4 months, 5 months or 6 months. 25
• said vaccine is a therapeutic vaccine.
• the combination of full-length antigens in accordance with the invention provides for eradication of the virus.
• said 30 vaccine has not only prophylactic applications, but is also a therapeutic agent.
• the present invention provides the nucleic acid, the vector, the polyprotein, the cell, or the vaccine of any of the preceding claims for use in a method of treating, ameliorating or preventing papilloma virus infection or papilloma virus induced disorders such as warts, malignant cell changes or cancer. Infection and associated disorders can occur 5 in a variety of sites, e.g. nasal-oral, skin, genitals (inner and outer).
• LSIL low-grade squamous intraepithelial lesions
• HSIL high-grade squamous intraepithelial lesions
• OPSCC oropharyngeal squamous cell carcinoma
• Papillomavirus strains which are frequently involved as causative agents are known in the art and include HPV16, HPV18, HPV45, HPV31, HPV33, HPV35, HPV52, HPV58, HPV6 and HPV11.
• nasal polyposis may arise from infections with HPV 6 or HPV 15 11.
• Table 1 below provides an overview of HPV strains associated with several malignant and non-malignant disorders.
• HNSCC DOI: HPV Cervical (DOI: 10.1016/j.ejca.2020.04.027 ) 20
• the present invention provides an in vitro or ex vivo method of stimulating and/or expanding T cells, said method comprising bringing into contact in vitro or ex vivo T cells with a nucleic acid, a vector, or a polyprotein of any one of the preceding aspects of the invention. 5
• said bringing into contact is effected in the presence of antigen- presenting cells.
• antigen-presenting cells are preferably dendritic cells, monocytes, macrophages or B lymphocytes.
• the presence of antigen-presenting cells provides for the expanded T cells to be MHC-restricted. 10 Preferred is that said T cells are ex vivo, i.e., obtained from an individual or patient.
• the present invention provides in a further aspect T cells, preferably MHC- restricted T cells obtained by the method of the seventh aspect.
• Figure 1 Schematic representation of the conceived MfPV3 antigen variants.
• 5 MfPV3 antigens were designed as fusion proteins comprising either E1, E2, E6 and E7 altogether, or E1 plus E2 and E6 plus E7 fusion proteins linked by a p2a peptide, and fused to the MHC-II invariant chain (Ii), respectively.
• E1, E2, E6, and E7 were fused as single open reading frames without the Ii coding sequence.
• Theoretical molecular weight in kDa calculated from 10 amino acid sequence.
• Ii MHC-II associated invariant chain
• GS glycine-serine-linker
• 2a p2a peptide for cotranslational separation
• Myc myc- tag sequence for antibody detection
• kDa kilo Dalton.
• Figure 2 Expression analysis of MfPV3 antigens. 15 A. Western blot analysis of HEK293T cell lysates 48 h following transfection with equimolar amounts of pURVac DNA vaccines expressing the various MfPV3 antigens. Antigens were detected with anti-myc (upper panel) and anti- p2a-peptide (middle panel) antibodies.
• Tubulin levels were monitored using an anti-tubulin antibody as loading control (lower panel).
• B Flow cytometry 20 analysis of HEK293T cells 48 hours following transfection with pURVac DNA vaccines. Intracellular staining was performed with anti-myc antibody. Depicted is the mean fluorescence intensity (MFI) of the average of 3 independent experiments. Error bars indicate standard error of the mean.
• C Flow cytometry 20 analysis of HEK293T cells 48 hours following transfection with pURVac DNA vaccines. Intracellular staining was performed with anti-myc antibody. Depicted is the mean fluorescence intensity (MFI) of the average of 3 independent experiments. Error bars indicate standard error of the mean.
• C C.
• FIG. 3 T cell responses induced by the various pURVac DNA vaccines encoding E1, E2, E6 and E7 in outbred CD1 mice.
• CD1 mice (5 per group) were immunized 4 times in 1 week intervals with 0.5 ⁇ g DNA of pURVac DNA encoding the indicated MfPV3 early antigens. Mice 5 were sacrificed 7 days post last immunization, spleens were harvested and CD8 (top panels) and CD4 (bottom panels) T-cell immune responses against E1 (A), and E2 (B) were measured using ICS and flow cytometry.
• Negative control groups consist of all mice immunized with a pURVac DNA vaccine encoding antigens not covered by the peptide pools used for in vitro restimulation. 10 Asterisks between groups indicate significant differences in response-levels after subtraction of background responses. Each symbol represents one mouse; the horizontal bar represents the median. Reference samples (mice vaccinated with pURVac DNA vaccine containing only the individual antigen linked to Ii, respectively) are not included in the statistical analysis (multiple comparison 15 adjustment).
• Figure 4 Expression analysis of rAd-shuttled MfPV3 antigen constructs. A.
• MfPV3 antigen variants Ii-E1E6E7-p2a-Ii-E2 and Ii-E1E6E7.
• MfPV3 antigens were designed as fusion proteins comprising 20 both E1, E6 and E7 altogether, optionally fused to the MHC-II invariant chain (Ii) and E2, respectively. Theoretical molecular weight in kDa calculated from amino acid sequence.
• tubulin levels were monitored using an anti-tubulin antibody (lower panel).
• C Flow cytometry analysis of A549 cells 48 hours following transduction with rAds expressing the various MfPV3 antigens, at an MOI of 30. Intracellular staining was performed with anti-myc antibody. Cells were gated on non-infected cells. Depicted is the mean fluorescence 30 intensity (MFI) of the average of 3 independent experiments. Error bars indicate standard error of the mean.
• MFI mean fluorescence 30 intensity
• FIG. 5 Immunization of mice with adenovirus vectors induces potent cellular immune responses.
• CD1 mice A and B
• OF1 mice C and D
• rAd vaccine 5 (2 ⁇ 10 7 IFU) encoding the various MfPV3 early antigens as indicated.
• Mice were sacrificed on day 14, spleens were harvested and CD8 and CD4 T-cell immune responses against E1, E2, E6, and E7 were measured using ICS and flow cytometry.
• Negative control groups consist of all mice immunized with rAd encoding antigens not covered by the peptide pools used for in vitro 10 restimulation. Each symbol represents one mouse; the horizontal bar represents the median.
• Tubulin levels were monitored using an anti-tubulin antibody as loading control (C).
• C anti-tubulin antibody as loading control
• D-E pURVac Ii-E1-SIINFEKL, pURVac E1-SIINFEKL or empty plasmid were transfected into HEK293T cells. 24 h after transfection, cells were treated with MG132 or DMSO for 6 h. The samples were analysed by western blot using anti-myc antibody (D) and anti- 25 tubulin antibody (E).
• FIG. 7 Immune responses in inbred mice and in vivo cytotoxicity Balb/C mice were immunized with rAd vectored vaccine encoding the indicated MfPV3 early antigens.14 days post vaccination, immune responses 30 were analyzed by ICS (A) or in vivo cytotoxicity regarding specific killing of E1-peptide pulsed cells (B). Each symbol represents one mouse.
• FIG. 8 Representative plot of in vivo cytotoxicity.
• Figure 8 Characterization of anchorage-independent growth.
• Figure 9 Amino acid sequence alignments of E2, E6 and E7 proteins from preferred 15 species. The alignment shows the wild-type (wt) amino acid sequence of the preferred papillomavirus species aligned with antigen sequences disclosed herein (without the "wt” qualifier), which possess the preferred modifications in accordance with the invention. The preferred modifications are highlighted with boxes.
• Figure 10 Top panel: Ad19a/64 prime with MfPV3 Ii-E1E2E6E7 induced IFN-producing MfPV3 specific T-cells in NHPs after 14 days after prime, measured by ELISpot.
• B IFN- by ICS and flow cytometry after 12-16 hours of peptide stimulation before boost-immunization and 14 days after boost immunization.
• C Functionality 15 of the CD8+ T cells was measured by the geometric mean fluorescence intensity (MFI) of the IFN- f all vaccinated animals, irrespective of their MfPV3 infection persistency status.
• Figure 12 MfPV3 viral load in the cervix of NHPs over time. Ad19a/64 prime, MVA boost 20 vaccination with MfPV3 Ii-E1E2E6E7 cleared the infection in all vaccinated animals, compared to spontaneous clearance in 3 out of 6 of PBS injected negative ctrl NHPs.
• FIG. 13 CD1 mice (A-E), different outbred mice as indicated (F-I) or C57BL/6 mice (J-P) 25 were immunized with Ad vaccine(s) (2 10 7 IFU) encoding the various HPV16 early antigens as indicated. Mice were sacrificed at the timepoints indicated, spleens were harvested and CD8+ and CD4+ T-cell immune responses against E1, E2, E6, and E7 were measured using intracellular staining and flow cytometry. Each symbol represents one mouse, bars represent the geometric30 mean. For the two E7-responder mice (E), CD44+IFN- populations of TNF- -
• Figure 14 1x10 6 C3 cells were injected s.c. into the flank of C57BL/6 mice, and the mice received Ad19a/64- and Ad5-vectored vaccines i.m. in the same side as the tumor on day 2 and day 20, respectively.
• Figure 15 1x10 6 C3 cells were injected s.c. into the flank of C57BL/6 mice, and the mice 10 received Ad19a/64- and Ad5-vectored vaccines i.m. in the same side as the tumor on day 10 and day 24, respectively.
• the pictures show the rim (bottom left side of the pictures) and a bit of the core (top right side of the pictures).
• J Mice were sacrificed at the time-points indicated, tumors were harvested and the resulting single cell suspension of a mix of tumor and immune cells were25 incubated for 5 hours without the presence of additional HPV16 peptides. IFN- -cells were detected by intracellular staining and flow cytometry.
• K Mice were sacrificed at the time-points indicated, spleens were harvested and CD8+ T-cell immune responses following E1 and E7 peptide stimulation were measured using intracellular staining and flow cytometry.
• L30 and M Mean fluorescence intensity of IFN- - -
• N tumors were harvested at the time indicated, single cell suspensions were prepared, and the fraction of DCs (CD11c+F4/80-CD8-CD4-CD45.2+TER119-live) was assessed by surface staining and flow cytometry. 5 Pink circles represent the therapeutic vaccine group. Gray squares depict negative control vaccinated mice. Green arrows indicate time of vaccination. Green diamonds indicate time-point of cisplatin injections. G-N: Black bars indicate geometric mean.
• Figure A-F shows data pooled from two independent experiments. 10
• 10 Figure 16: T1x106 C3 cells were injected s.c.
• mice received Ad19a/64- and Ad5-vectored vaccines i.m. in the same side as the tumor on day 10 and day 24, respectively.
• Cisplatin treatment was given i.p. on days 10, 17 and 24.
• the gray curve shows neg ctrl vaccinated mice for comparison (note that this is the same data as depicted in Figure 4D, and pooled from two independent experiments). Green arrows indicate time of vaccination.
• a and B mean tumor growth with SEM (A) and survival curves (B) of peptide vaccine (blue,
• Figure 18 C57BL/6 mice were immunized in the lower limb with rAd5 or Ad5F35 10 encoding HPV16 Ii-E1E2E6E7. Both vaccines induced CD8+ T-cell responses against E1 and E7 of similar magnitude and quality, indicating that Ad5F35 are equally efficient in induction of vaccine specific T-cell responses.
• Figure 19 Expression analysis of rAd-shuttled HPV16 and HPV18 antigen constructs. 15 A. Western blot analysis of A549 cell lysates 48 h following transduction with rAds at an MOI of 100.
• Antigens were detected with anti-HPV16-E2 (upper left panel) and anti-HPV16-E6 (lower left panel), anti-HPV16-E7 (upper right panel) antibodies. As loading control, tubulin levels were monitored using an anti-tubulin antibody (lower right panel).
• FIG. 25 Figure 20: Expression analysis of MVA-shuttled MfPV3, HPV16 and HPV18 antigen constructs.
• A Western blot analysis of HEK293T cell lysates 24 h following transduction with recombinant MVA or MVA-CR19 containing MfPV3 antigen in DelIII 30 locus or TK locus at an MOI of 10. Antigens were detected anti-myc antibody (upper panel). As loading control, tubulin levels were monitored using an anti-
• tubulin antibody (lower panel).
• B Western blot analysis of AGE1.CR-pIX cell lysates 24 h following transduction with MVA-CR19 containing HPV16 Ii- E1E2E6E7 in DelIII locus at an MOI of 1. Antigens were detected with anti- HPV16-E2 (upper left panel) and anti-HPV16-E6 (lower left panel), anti- 5 HPV16-E7 (upper right panel) antibodies. As loading control, tubulin levels were monitored using an anti-tubulin antibody (lower right panel).
• C Western blot analysis of AGE1.CR-pIX cell lysates 24 h following transduction with MVA-CR19 containing HPV16 Ii- E1E2E6E7 in DelIII locus at an MOI of 1. Antigens were detected with anti- HPV16-E2 (upper left panel) and anti-HPV16-E6 (lower left panel), anti- 5 HPV16-E7 (upper right panel) antibodies
• Example 1 Constructs of the invention and their performance 5 Methods Antigen sequences Parts of the antigens were synthesized at Geneart/Thermo Fisher (Regensburg, Germany). The 10 gene optimizer algorithm was used to minimize sequence homology and adapt the sequences to human codon usage. All constructs were cloned using standard molecular biology methods.
• antigens were assembled with fusion PCR, type IIs exocutter sites (BsaI-HF v2, New England Biolabs, Ipswich, USA) or NEBuilder HIFI DNA Assembly Kit (New 15 cloned into the different plasmid backbones using AgeI-HF and NotI-HF (New England Biolabs, Ipswich, USA). Sequence correctness was verified by Sanger sequencing. Plasmids were prepared, depending on amount, with alkaline lysis or commercially available kits Qiagen, Hilden, Germany). The sequence of the antigen Ii-E1E2-p2a-Ii-E6E7 (Fig.
• DNA for vaccination purposes was produced using the EndoFree Plasmid Mega Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
• 5 Cell lines, transfection and viral infection HEK293T cells and A549 cells were maintained and grown in Dulbecco's MEM (DMEM) supplemented with 10% Fetal Calf Serum (FCS) and 1% Penicillin/Streptomycin (Pen/Strep).
• 10 9E10 mycl hybridoma cells were cultivated in RPMI supplemented with 10% FCS, 1% Pen/Strep and 2 mM glutamine (Pan).
• HEK293T cells were transfected using the polyethylenimide (PEI) method (Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: 15 polyethylenimine. Proc. Natl. Acad. Sci.92, 7297 LP 7301, 1995).
• PEI polyethylenimide
• 4 ⁇ 10 5 cells were seeded in 6-well plates one day before transfection. The cells were transfected with 2.5 ⁇ g plasmid (equimolar amounts, filled with empty vector) and 7.5 ⁇ g PEI in DMEM without any supplements.
• adenoviral vectors 25 E1/E3 deficient adenoviral vectors of serotype Ad19a/64 were generated as previously described (Ruzsics, Z., Lemnitzer, F. & Thirion, C. Engineering Adenovirus Genome by Bacterial Artificial Chromosome (BAC) Technology BT - Adenovirus: Methods and Protocols.
• BAC Bacterial Artificial Chromosome
• vectors were purified by CsCl gradient ultracentrifugation followed by a buffer exchange to 10 mM Hepes pH 8.0, 2 mM MgCl 2 and 4% Sucrose via PD10 columns (GE Healthcare, Chicago, USA). Titration was performed using the RapidTiter method by detection of infected production cells via 10 immunohistochemical staining with anti-hexon antibody (Novus, Adenovirus Antibody (8C4)). Insert integrity was confirmed by PCR amplification of the GOI in DNA extracted from the purified vectors.
• the antibody against myc (9E10) was obtained from hybridoma cell supernatants.9E10 mycl hybridoma cells were seeded at 5 ⁇ 10 5 cell per ml in RPMI supplemented with 1% FCS, 1% Pen/Strep and 2 mM glutamine. The supernatant was harvested 5 days after seeding and the antibody was purified via a HiTrap Protein G column (GE Healthcare, Chicago, USA). After 20 washing the column with PBS, the antibody was eluted with 0.1 M glycine/HCl (pH 3.2), neutralized with 0.025 volumes of 1 M Tris/HCl (pH 9) and dialyzed against PBS.
• mouse anti-p2a peptide 3H4, 1:2000, Merck, Darmstadt, Germany
• mouse anti- 1:1000, Santa Cruz, Heidelberg, Germany
• mouse anti-ubiquitin-Biotin eBioP4D1, 1:1000, Invitrogen, Carlsbad, USA
• goat anti-mouse-HRP 25 115-036-003, 1:5000, Jackson, West Grove, USA
• goat anti-rabbit-HRP P0448, 1:2000, Dako, Santa Clara, USA
• Streptavidin-HRP 11089153001, 1:5000, Roche, Basel, Swiss
• rat anti- mouse-PE A85-1, 1:100, BD, Franklin Lakes, USA.
• the proteins were separated on SDS-PAGE under reducing conditions and blotted on a nitrocellulose membrane for western blot analysis.
• Targets were probed with primary and secondary antibodies as listed above.
• HRP- 10 labeled secondary antibodies and enhanced chemiluminescence substrate or Femto ECL (Thermo Fisher, Waltham, USA) were used for detection in a Chemilux Pro device (Intas, Göttingen, Germany).
• the membrane was reprobed with an antibody against tubulin. 15
• Intracellular staining of antigens was performed using standard methods (Kiener et al., 2018).
• Cells were fixed and permeabilized with Cytofix/Cytoperm-Buffer (4% PFA, 1% saponine, in PBS). All washing steps were done with Perm/Wash-Buffer (PBS containing 0.1% saponine). 20 The cells were stained with anti-myc antibody (5 ⁇ g/ml, diluted in Perm/Wash-Buffer) and rat anti-mouse-PE (1:100 diluted in Perm/Wash-Buffer) each for 30 min. The flow cytometry assay was performed using an Attune NxT device (Thermo Fisher, Waltham, USA) with a 488 nm excitation and a 574/26 nm emission filter. Cells were gated on stained, mock-transfected cells.
• Cytofix/Cytoperm-Buffer 4% PFA, 1% saponine, in PBS. All washing steps were done with Perm/Wash-Buffer (PBS containing 0.1% saponine). 20 The cells were stained with anti-myc
• Cells were incubated for 5 hours in 3 ⁇ M monensin with or without 1 ⁇ g/mL of relevant peptides.
• the cells were stained against surface markers: 30 APC-Cy7 or BV421 CD8 (53-6.7, 1:200, BioLegend, San Diego, USA), PE-Cy7 CD4 (RM4-5, 1:800, BD), FITC CD44 (IM7, 1:100, BioLegend, San Diego, USA) and PerCP-Cy5.5 B220 (RA3-
• Flow cytometry was performed on the Fortessa 3 (BD Biosciences, Franklin Lakes, USA) flow cytometer and data analysis was performed using FlowJo V10 software.
• Epitope-specific CD8 + 10 T-cell responses were measured as B220-, CD8 + or CD4 + , CD44 + , IFN- + cells and are presented in total number of cells per organ.
• the quality of the IFN- + responses were evaluated by MFI of IFN- cells (expressing both IFN- - + CD8 + or CD4 + populations.
• 15 In vivo cytotoxicity The assay was performed similarly to what was previously described (Nielsen, K. N., Steffensen, M. A., Christensen, J. P. & Thomsen, A. R.
• Pulsed and stained 25 splenocytes were mixed at a 1:1:1:1:1 ratio, and a total of 2.5 ⁇ 10 7 cells were injected intravenously into Ad19a/64-vaccinated recipient Balb/C mice. 5 hours later, spleens were harvested, and target cells were identified on the Fortessa 3 (BD Biosciences, Franklin Lakes, USA) flow cytometer by CFSE/CTV staining. The percentage of killing was calculated using the following equation: 30
• E1 SIINFEKL presentation on MHC-I HEK293T cells were transfected with 2.5-3.5 ⁇ g DNA using PEI transfection.48 hours post 5 transfection, cells were stained with PE anti-H2KB-SIINFEKL (25-D1.16, 1:160, Invitrogen, Carlsbad, USA) and presence of SIINFEKL-H2Kb presentation on cell surfaces was detected on the LSRII or Fortessa 3 (BD Biosciences, Franklin Lakes, USA) flow cytometers, as a proxy for E1 presentation. All samples were run in biological 6-plicates, and the experiment was repeated at least two times.
• antigen constructs comprising E1, E2, E6, 5 and E7 of MfPV3 linked to the intrinsic T-cell adjuvant Ii were designed. Eight different configurations were conceived (figure 1) to select the one eliciting the most potent and broadest response for further development.
• Ii_E6E7E1E2 While in the first composite antigen the order was Ii-E1E2E6E7, the order of E1/E2 and E6/E7 was reversed in Ii_E6E7E1E2 (i) to test the influence of the antigen order, and (ii) to determine the impact of positioning relative to Ii on the level 10 of transgene expression.
• all proteins were expressed as artificial fusion polypeptides, whereas the individual proteins were separated by a short and flexible GS linker to promote the stability and accumulation of the fusion proteins (Chen, X., Zaro, J. L. & Shen, W.-C. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev.
• Ii was fused to the N-terminus as a T cell adjuvant (Holst, P. J. et al. 15 MHC Class II-Associated Invariant Chain Linkage of Antigen Dramatically Improves Cell- Mediated Immunity Induced by Adenovirus Vaccines. J. Immunol.180, 3339 3346, 2008).
• T cell adjuvant effects of Ii the variant E1E2E6E7 without Ii was designed.
• E1E2 were separated from E6E7 by a p2a peptide in Ii-E1E2-p2a-Ii-E6E7 (Kim, J. H. 20 et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, 2011).
• the nucleotide sequence homology between the two invariant chains was minimized by using divergent codons to hinder homologous recombination.
• each of the four viral proteins was generated on its own as Ii- fusion to serve as benchmark and reference.
• E7 was inactivated by introducing the substitutions C24G, L71R, and C95A, which inactivate the central LxCxE motif to reduce 5 respectively (Wieking, 2013; Edmonds, C. & Vousden, K. H. A Point Mutational Analysis of Human Papillomavirus Type 16 E7 Protein.63, 2650 2656, 1989).
• the conserved DNA binding domain of E2 was modified by C297A to generate polyproteins with an even number of cysteins and reduce DNA binding.
• MHC class I-restricted SIINFEKL epitope is processed from E1 fusion protein and abundantly presented on MHC-I molecules in vitro 25 It has been reported that bovine papillomavirus type 1 E1 is itself an unstable protein which is ubiquitinylated and rapidly degraded in papillomavirus-infected cells (Malcles, M.-H., Cueille, N., Mechali, F., Coux, O. & Bonne-Andrea, C. Regulation of bovine papillomavirus replicative helicase e1 by the ubiquitin-proteasome pathway. J.
• SIINFEKL-peptide could be detected on MHC-I on cell surfaces, both when linked to Ii and when delivered alone. This supports the choice of E1 as a relevant antigen to include in the 15 therapeutic vaccine, as E1-derived peptides can be expected to be presented on the surface of infected cells.
• DNA vaccination of antigen constructs induces CD4 + and CD8 + T cell responses against MfPV3 early antigens 20
• Intradermal (i.d.) DNA immunization of outbred CD1 mice confirmed that all pURVac DNA vaccines encoding E1 and/or E2 induced E1- and E2-specific IFN- + CD8 + and CD4 + T cells measured by ICS and flow cytometry analysis (figure 3.
• the vaccine with two p2a-separated Ii-linked antigen sequences (Ii-E1E2-p2a-Ii-E6E7) and Ii- E6E7E1E2 appeared to elicit slightly better CD4 responses against E1 shown by the significant response and significant difference to the negative control group (figure 3A).
• 10 Characterization of adenoviral vectors To increase the cellular immune response against the antigens, viral vectors were subsequently used for antigen delivery.
• Adenoviral vectors from serotype 19a/64 had 15 been shown to be a suitable vector for the delivery of MfPV3 antigens that were capable of inducing high CD8 + T cell response in cynomolgus macaques (Ragonnaud, E. et al. Replication deficient human adenovirus vector serotype 19a/64: Immunogenicity in mice and female cynomolgus macaques. Vaccine 36, 6212 6222, 2018).
• a refined panel of rAd vectors was generated comprising a modified set of recombinant MfPV3 antigens. Ii-E6E7E1E2 was 20 excluded because it was not superior to the other polyproteins.
• vaccines including Ii showed a trend towards higher magnitude responses than the vaccine not encoding Ii (figure 5 A and B: E1E2E6E7 compared to other vaccines).
• a tendency of Ii-mediated enhancement of responses was observed for both CD4 and CD8 T cell responses for some vaccine configurations, but the difference was 15 only significant for E2-specific CD8 responses induced by Ii-E1E2-p2a-Ii-E6E7 compared to E1E2E6E7 (figure 5B, upper panel). This underlines the ability of Ii to boost T cell responses in the context of adenoviral vaccine delivery.
• mice had CD8 + T-cell responses and these responses were of low magnitude, it could be questioned whether these were true responses or merely due to high background.
• a representative mouse depicted in figure 5C shows a CD44 + IFN- + population behaving like a true response. Furthermore, the majority of the IFN- + cells were producing TNF as well, 30 confirming the activation phenotype.
• Ii-E1E2E6E7- and E1E2E6E7-transfected cells were cultivated in absence or presence of the proteasome inhibitor MG132.
• Anti-ubiquitin western blot analysis 15 of the immunoprecipitated myc-tagged polypeptides revealed higher ubiquitin-depending signal intensity for Ii-E1E2E6E7 compared to E1E2E6E7 without Ii (figure 6A). This effect was even more pronounced when MG132 was present.
• IP 20 procedure Myc-specific signals confirmed the fidelity of the immunoprecipitation (IP) procedure, and analysis of the IP supernatants by an anti- tubulin western blot revealed that comparable amounts of cell lysate were used in the IP 20 procedure (figure 6B and C).
• a higher level of ubiquitination is commonly associated with faster degradation. This could exemplarily be demonstrated by transiently expressing Ii-E1 and E1, both fused with the C-terminal SIINFEKL peptide, in absence or presence of MG132, respectively (figure 6D and E). Without MG132, Ii-E1-SIINFEKL was hardly detectable whereas a prominent signal could be visualized when MG132 was added. Lower molecular 25 weight signals are indicative of degradation products.
• Example 2 Assessment of oncogenic properties of E6 and E7 as modified in accordance with the invention 15 Material and Methods Cell lines NIH-3T3 cells and HEK293T cells were cultivated at 37°C with 5% CO 2 in DMEM 20 supplemented with 10% fetal calf serum (FCS) and 1% Pen/Strep (PS). The NIH-3T3 cells were maintained between 10% and 70% confluence to prevent maturation and differentiation.
• FCS fetal calf serum
• PS Pen/Strep
• HPV16 antigens and HPV16 E6wt were cloned into pLV-MCS-IRES-Puro
• HPV16 E7wt and GFP were cloned into pLV-MCS-IRES-Neo, respectively, by using AgeI-HF and NotI-HF (NEB, Ipswich, USA) restriction enzymes.
• Both pLV vectors contain a CMV promoter followed by an MCS, an internal ribosome entry site (IRES) and antibiotic resistance gene against either puromycin or neomycin.
• HEK293T cells were seeded in T75 flasks.24 h post seeding, the medium was changed to DMEM without additives and the cells were transfected with 3.75 ⁇ g of pLV containing the antigen and 3.75 ⁇ g of lentivirus packaging mix (1:1:1; pMDL-Gag:pVSV-G:pREV) using the PEI method ( 5 B., & Behr, J. P. (1995).
• PEI method 5 B., & Behr, J. P. (1995).
• NIH-3T3 cells and antibiotic selection 1 ⁇ 10 5 NIH-3T3 cells were seeded in T25 flasks.24h post seeding, the medium was aspirated, 15 and the cells were transduced with 2 ml of each supernatant containing the respective lentiviral vector in presence of 10 ⁇ g/ml polybrene (TR-1003-G, Sigma Aldrich, St. Louis, USA).
• transduced cells were selected via 1 mg/ml G418 (CP11.3, Roth, Düsseldorf, Germany) and/or 2 ⁇ g/ml puromycin (ant-pr-1, InVivoGen, San Diego, USA), depending on the lentiviral vector 20 used for transduction. The cells were under antibiotic selection for at least 2 weeks, until all untransduced cells had died.
• Antibodies 25 The following antibodies were used: anti-E2 (TVG-271, 1:200, Santa Cruz Biotechnology, Heidelberg, Germany), anti-E6 (GTX132686, 1:2000, Biozol, Eching, Germany), anti-E7 (NM2, 1:200, Santa Cruz Biotechnology, Heidelberg, Germany), anti- Cruz Biotechnology, Heidelberg, Germany), goat anti-mouse-HRP (115-036-003, 1:5000, Jackson, West Grove, USA) and goat anti-rabbit-HRP (P0448, 1:2000, Dako, Santa Clara, USA). 30 Western blot analysis
• TDLB buffer 50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate
• protease inhibitors Complete Mini, Roche, Basel, Switzerland.
• Total protein concentration of the supernatants was measured by the Bradford method (Protein 10 Assay, BioRad, Feldmün, Germany).
• the proteins were separated on SDS-PAGE under reducing conditions and blotted on a nitrocellulose membrane for western blot analysis. Targets were probed with primary and secondary antibodies as listed above.
• NIH-3T3 cells were transduced with VSV-G-pseudotyped lentiviral vectors encoding the following HPV16 25 antigens: Ii-E1E2E6E7, Ii-E1E2-p2a-Ii-E6E7, E1E2E6E7, Ii-E2, Ii-E1E6E7-p2a-Ii-E2, Ii-E1E6E7, E6wt, and E7wt, respectively (figure 1A).
• NIH-3T3 cells were transduced with VSV-G-pseudotyped lentiviral vectors encoding GFP and lentiviral vectors lacking a transgene, respectively. Additionally, one polyclonal NIH-3T3 cell line was generated by transduction with both, HPV16-E6wt- and HPV16-E7wt-encoding lentiviral 30 vectors.
• Example 3 Immunogenicity of MfPV3 Ii-E1E2E6E7 in rhesus macaque monkeys 15 Materials and methods Cell lines, transfection and viral infection 20 AGE1.CR.pIX and AGE1.CR.pIX-T-REx cells grown in suspension were maintained in chemically-defined CD-U6 medium (ProBioGen AG) supplemented with 2 mM L-glutamine (Sigma) and recombinant insulin-like growth factor (LONG-R 3 IGF, 50 ng/mL final concentra- tion, Sigma) and were incubated in an orbital shaker (Multitron Pro,Infors HT) at 8% CO 2 with a shaking speed of 180 rpm.
• CD-U6 medium ProBioGen AG
• L-glutamine Sigma
• LONG-R 3 IGF insulin-like growth factor
• AGE1.CR.pIX and AGE1.CR.pIX-T-REx cells 25 were cultivated in Dulbecco's Modified Eagle Medium DMEM/F12 supplemented with 5% Fetal Calf Serum (FCS). Adherent cell lines were maintained at 37°C and 8% CO 2 in a humidified incubator. Generation of recombinant MVA vectors 30
• Recombinant MVA encoding the different GOIs were generated by homologous recombination in adherent AGE1.CR.pIX-T-REx cells that prevent the undesirable expression of the GOI during generation and propagation of the recombinant MVA by taking benefit of the Tet system (s. above). Therefore, the culture monolayers seeded in a six-well plate were infected with MVA or MVA-CR19 with a MOI of 10 0.05 and transfected with 2 ⁇ g of the individual shuttle vector using the Effectene Transfection Reagent (Qiagen, Germany) .
• the infected/transfected culture was harvested 2 -3 days post infection/transfection, sonicated, and used for infection of a cell monolayer in a six-well plate format. Resulting plaques were validated by PCR and an iterative plaque purification procedure was initiated until MVAs 15 without the correct GOI expression cassette were not present any more (usually within 5 8 rounds of plaque purification).
• the cell harvest material was sonicated using a Vial Tweeter (set to 20 s of 100% cycle and 90% amplitude, Hielscher, Germany), and 20 AGE1.CR.pIX-T-REx (grown in suspension at 2x 10 6 cells per ml in a 1:1 mixtures of CD-U4 and CD-VP4 (Merck-Millipore) were inoculated with the individual recombinant MVA vectors at MOI 0.05. Finally, MVAs were harvested 48 h 72 h post infection and the TCID 50 titer was determined.
• DNA Purification from Cervical Swabs and MfPV3 detection by qPCR Briefly, cervical samples were obtained using a small cervix cytobrush and placed in a 15 preservative solution called TEN buffer (Tris HCl 10 mM pH 7.5; EDTA 5 mM, NaCl 50 mM). DNA was extracted from the cervical samples with the DNA extraction kit (QIAmp DNA blood mini kit, procedure.
• PBMCs Peripheral blood mononuclear cells
• Ficoll-Paque Ficoll-Paque
• Vaccine antigen specific T-cells from the fresh purified PBMCs were measured by IFN- - 5 ELISPOTplus kit (MABTECH, #3421M-4HST-2) after 18-20 hours incubation at 37 C and 5% CO2 in presence of 2 mg/mL of the relevant peptide pools.
• Spot-forming units (SFU) were read on an IRIS instrument and counted using the ELISpot Big emphasis algorithm. Background responses from unstimulated samples were subtracted prior to graphical representation.
• PBMCs Peripheral blood mononuclear cells
• BDcytofix/Cytoperm After surface staining, cells were fixed and permeabilized in BDcytofix/Cytoperm, and stained intracellularly using FITC-IFN- -1, UCy-Tech) 20 and PE/Cy7-TNF- mAb11, BD biosciences) antibodies.
• the peptides used were 16-mers overlapping by 11 amino acids covering the entire Ii- E1E2E6E7 antigen.
• the peptides were pooled in 5 separate pools containing Ii, E1, E2, E6 and E7 peptides respectively.
• Peptides were obtained from KareBay, Town, China. 25 Flow cytometry was performed on FACS CANTO flow cytometer and data analysis was performed using FlowJo V10 software.
• Epitope-specific CD8 + T-cell responses were measured as viable CD3 + , CD8 + or CD4 + , IFN- + cells. Background (from unstimulated samples) were subtracted before plotting and analysis. All responses below 0.01% were regarded as below 30 detection limit, and manually adjusted to 0.01 for optimal visual representation.
• All vaccinated monkeys show strong CD8 and CD4 responses against at least one of the vaccine antigens (including E1) in 15 contrast to the PBS controls, and CD8 responses against E2 and E7 are detected in a number of vaccinated animals as well ( Figure 10, bottom panels).
• the viral vectored MfPV3 Ii-E1E2E6E7 vaccine is immunogenic in MfPV3 infected monkeys, this also provides a strong indication that the vaccine can cure the 20 pre-existing MfPV3 infections in the NHPs.
• Vaccine-induced T cell responses appear functional in animals with persistent MfPV3 infection. Exhaustion of HPV-specific T cells can occur in people with persistent HPV infection, and is characterized by loss of functionality and eventually by elimination of antigen-specific T cells. 5 Thus, it is important that a therapeutic vaccine can induce antigen-specific immune responses in the context of a persistent presence of the infection.
• Prime immunization with hAd19a/64 Ii-E1E2E6E7 did indeed induce IFN- roducing T cells against the vaccine antigens in all vaccinated animals (Figure 11A). Notably, there was no 10 observed difference between vaccinated animals with persistent MfPV3 infection and animals where the duration of the HPV infection was not known.
• Example 4 properties of the different construct designs with HPV16 E1, E2, E6 and E7 antigens, and tumour protective efficacy of HPV16 Ii-E1E2E6E7 5
• Materials and methods Animals, immunizations, cisplatin injections and tumour challenges C57BL/6 and CD1 mice were obtained from Envigo (Horst, The Netherlands), OF1 mice from 10 Charles River (France) and HSd-Ola mice from Envigo (UK). All animals were females, 6-8 weeks old, and were housed at the Panum Institute, University of Copenhagen. All experiments were initiated after allowing the mice to acclimatize for at least 1 wk.
• tumour cell line C3 was developed by transfection of mouse embryonic cells with the HPV16 genome and an activated ras oncogene (Feltkamp et al, Eur J Immunol 1993). Cell line authentication was carried out by flow cytometry detection of HPV16 E7 presence.
• 1E+06 C3 tumor cell in 200 uL PBS + 0.2% BSA were injected sub- cutaneous (s.c.) into the flank of C57BL/6 mice.
• Tumor size was measured in length and width three times weekly and the tumor volume was calculated as: length * width 2 * 0.5236 (Janik et al, Cancer Res 1975). Animals were sacrificed by cervical dislocation once the tumor exceeded 1000 mm 3 or at the time-points indicated. 25 For treatment of established tumors (d10 immunizations), mice were assigned to treatment groups based on their tumor size just prior to treatment, to ensure equal average tumor sizes for all treatment groups at the start-point of treatment. 30 Immunizations with adenoviral vectors were performed intramuscular (i.m.) in thigh in the same site as tumor injection with 2 ⁇ 10 7 IFU rAd diluted in 50 ⁇ L PBS, except for Figure 18,
• mice were immunized in the lower limb, a mixed systemic and cutaneous route, with 2 ⁇ 10 7 IFU Ad5 or Ad5F35 in 30 uL PBS.
• Immunization with SLP vaccine (HPV16 E743-77 and HPV16 E641-65, Schafer) was performed s.c. in the opposite flank of tumor injection with 50 ug of each peptide.
• the SLP vaccine was prepared by dissolving lyophilized peptide to 100 5 mg/mL in DMSO and further dilution to 0.5 mg/mL in PBS.
• Cisplatin (Sigma-Aldrich) was dissolved to 1 mg/mL in 0.9% NaCl2 solution and 3 mg/kg 10 mouse body-weight was injected intra-peritoneal once weekly three times. Mice were euthanized when tumours exceeded 1000 mm 3 , necrotic wounds emerged or mobility of the mice was markedly reduced.
• the cells were stained against surface markers (all antibodies were from BioLegend, San Diego, USA unless otherwise noted): APC-Cy7 or BV421 CD8 (53- 6.7, 1:200, BD Biosciences, Franklin Lakes, USA), PE-Cy7 CD4 (RM4-5, 1:800, BD Biosciences, Franklin Lakes, USA), FITC CD44 (IM7, 1:100), PerCP-Cy5.5 B220 (RA3-6B2, 1:200), Fixable 30 and BV510 PD-1 (29F.1A12, 1:100). After surface markers (all antibodies were from BioLegend, San Diego, USA unless otherwise noted): APC-Cy7 or BV421 CD8 (53- 6.7, 1:200, BD Biosciences, Franklin Lakes, USA), PE-Cy7 CD4 (RM4-5, 1:800, BD Biosciences, Franklin Lakes, USA), FITC CD44 (IM7, 1:100), PerCP-Cy5.5 B220 (RA3-6B2, 1:200
• the quality of the IFN- + responses were evaluated by MFI of IFN- fraction of double positive cells (expressing both IFN- - - + CD8 + or 10 CD4 + populations.
• Lineage staining and flow cytometry analysis of intratumoral immune cells Tumor tissue was weighed after harvesting, and processed using the Miltenyi mouse tumor15 dissociation kit (cat no 130-096- protocol, and stained against surface markers (all 1:100, BD Biosciences, Franklin Lakes, USA unless otherwise noted): BV650 CD8 (53-6.7), PerCP/Cy5.5 CD4 (RM4-5), PE/Cy7 CD45.2 (104), APC F4/80 (BM8, BioLegend, San Diego, USA), AF488 CD11c (N418, BioLegend, San Diego, USA), BV421 PD-L1 (10F.9G2, BioLegend, San Diego, USA), BV510 20 PD1 (29F.1A12, BioLegend, San Diego, USA), AF700 TER119 (
• Tumor tissue was carefully harvested at the time of sacrifice and fixated in 4% cold PFA (Alfa Aesar). The fixative was exchanged with 70% ethanol after approximately 24 hours. Fixated tissues were embedded in paraffin and slides were stained with PE and with DAB 15 Tissues were scanned using Axio Scan.Z1 (Zeiss) and analysed using Zen 3.4 software (cut- off area: 10-180 um 2 . Cut-off circularity: 0.5-1.0).
• Ad19a/64-vectored vaccination induces robust CD8+ and CD4+ T-cell responses in both outbred and inbred mice which can be boosted by Ad5
• Intramuscular immunization of outbred CD1 mice with a single dose of Ad19a/64-vectored vaccines confirmed immunogenicity of the different antigen constructs in vivo, as CD8+ T- cell responses were detected against E1 ( Figure 13A) and CD4+ responses were measured 25 against E1 ( Figure 13B) and E2 ( Figure 13C).
• a heterologous prime-boost immunization was applied delivering human Ad5 encoded Ii-E1E2E6E7 as a booster vaccine.
• Ii-E1E2E6E7 was further supported when looking at the integrated mean15 fluorescence intensity (iMFI) of IFN- the induced T-cell response (Shoostari et al.2010).
• Ii-E1E2E6E7 significantly enhanced the response against E7 compared to the vaccine not encoding Ii and the vaccine encoding E7 directly linked to the p2a-Ii ( Figure 13M).
• Ad19a/64 and Ad5 vectors containing no antigen were included to confirm that the responses were vaccine specific.
• Ad-vector vaccination shortly after tumor inoculation provides single-agent tumor control and 5 increased survival
• C57BL/6 mice were injected with syngeneic C3 tumor cells.
• the C3 cell line was established by transfection of mouse embryonic cells with the HPV16 genome and an activated ras oncogene (Feltkamp et al. 1993), and the tumors can express all HPV16 antigens (Schmitt and Pawlita et al.2011) , in 10 contrast to the commonly used TC-1 tumor model containing only HPV16 E6 and E7 (Lin et al.1996).
• mice were treated with A19a/64 vectors encoding Ii-E1E2E6E7 or an irrelevant antigen (neg ctrl) 2 days after tumor challenge. On day 20, another treatment with Ad5 vectors containing the same antigens was done to boost responses.
• Ii-E1E2E6E7 treatment significantly reduced tumor growth (Figure 14A), completely cleared tumors in 3 20 out of 10 mice ( Figure 14B) and significantly increased survival (Figure 14C, 3/10 vs 10/10 dead at day 41), indicating that the treatment is effective against small tumors.
• Ad-vectored vaccination provides single-agent control of established tumors and works in synergy with cisplatin co-treatment 25 Having confirmed that the early treatment of C3 tumors with therapeutic vaccination had good effect as vaccine regimen monotherapy, we went on to investigate the potency on established tumors. Once palpable tumors were present (day 10), mice were treated with therapeutic vaccination, which showed a reduction of tumor growth in some animals (Figure 15A and B) and significantly increased survival (Figure 15C).
• Cisplatin is standard30 treatment of HPV+ cancers in the clinic and has previously shown synergy with HPV-
• the Ad-vectored Ii-E1E2E6E7 vaccine provides enhanced survival compared to two E6/E7 synthetic 20 long reference peptides in combination with cisplatin
• E7 peptide E743-77
• This peptide includes a well-established E7 epitope which is immunodominant in C57BL/6 mice (Feltkamp et al.1993).
• Another epitope 25 is also reported in E6 34 , although it is less dominant.
• CD8+ T-cell responses against E1 were detected after vaccination with Ii-E1E2E6E7 and expectedly not after vaccination with the two E6/E7 peptides ( Figure 17C). Both vaccines induced a similar 10 level of E7 specific CD8+ T-cells ( Figure 17D) and no detectable CD4+ T-cell responses (data not shown).
• Vaccination with Ad5F35 vectored HPV16 Ii-E1E2E6E7 induces CD8+ T-cells of similar magnitude and quality as Ad5 in mice 15
• Ad5F35 is a useful alternative to Ad5 as a booster vector
• inbred C57BL/6 mice were immunized with 2 ⁇ 10 7 IFU of either an Ad5 or an Ad5F35 vector encoding HPV16 Ii-E1E2E6E7.
• Splenic CD8+ T-cell responses against HPV16 E1 and E7 were analysed 14 days after immunization by intracellular cytokine staining and flow cytometry.
• Ad5F35 is capable of generating E1 and E7 reactive CD8+ T-cells and20 the magnitude ( Figure 18A and B) and quality, assessed by intensity of IFN- 18E and F) and capability of producing both TNF- - nd D), was similar to the responses induced by and Ad5 vectored vaccine. This provides evidence, that Ad5F35 can replace Ad5 in the therapeutic prime-boost immunization regimen, without affecting the therapeutic efficacy. 25
• Example 5 Additional viral vectors of the invention and their expression control Material and Methods 5
• HEK293T cells and A549 cells were maintained and grown in Dulbecco's MEM (DMEM) supplemented with 10% Fetal Calf Serum (FCS) and 1% Penicillin/Streptomycin (Pen/Strep).
• DMEM Dulbecco's MEM
• FCS Fetal Calf Serum
• Pen/Strep Penicillin/Streptomycin
• 9E10 mycl hybridoma cells were cultivated in RPMI supplemented with 10% FCS, 1% 10 Pen/Strep and 2 mM glutamine (Pan). These cell lines were maintained at 37°C and 5% CO2 in a non-humidified incubator.
• Adherent AGE1.CR.pIX cells were cultivated in Dulbecco's Modified Eagle Medium DMEM/F12 supplemented with 5% Fetal Calf Serum (FCS) and were maintained at 37°C and 8% CO2 in a non-humidified incubator. 15 Subconfluent A549 cells were infected with Ad19a/64 vectors at an MOI of 100 in DMEM without any supplements.2 h post infection, medium was exchanged to DMEM with 10% FCS and 1% Pen/Strep.
• FCS Fetal Calf Serum
• Subconfluent HEK293T cells were infected with MVA and MVA-CR19 vectors at an MOI of 20 10 in DMEM without any supplements.2 h post infection, medium was exchanged to DMEM with 10% FCS and 1% Pen/Strep. Subconfluent AGE1.CR.pIX cells were infected with MVA and MVA-CR19 vectors at an MOI of 1 in DMEM/F12 without any supplements.2 h post infection, medium was exchanged to DMEM with DMEM/F12 supplemented with 5% FCS.
• Residual free DNA was digested by DNase I. Afterwards, vectors were purified by CsCl gradient ultracentrifugation followed by a buffer exchange to 10 mM Hepes pH 8.0, 2 10 mM MgCl2 and 4% Sucrose via PD10 columns (GE Healthcare, Chicago, USA). Titration was performed using the RapidTiter method by detection of infected production cells via immunohistochemical staining with anti-hexon antibody (Novus, Adenovirus Antibody (8C4)). Insert integrity was confirmed by PCR amplification of the GOI in DNA extracted from the purified vectors.
• Recombinant MVA encoding the different GOIs 25 were generated by homologous recombination in adherent AGE1.CR.pIX-T-REx cells that prevent the undesirable expression of the GOI during generation and propagation of the recombinant MVA by taking benefit of the Tet system (s. above). Therefore, the culture monolayers seeded in a six-well plate were infected with MVA or MVA-CR19 with a MOI of 0.05 and transfected with 2 ⁇ g of the individual shuttle vector using the Effectene Transfection 30 infected/transfected culture was harvested 2 -3 days post infection/transfection, sonicated,
• the antibody against myc (9E10) was obtained from hybridoma cell supernatants.9E10 mycl hybridoma cells were seeded at 5 ⁇ 10 5 cell per ml in RPMI supplemented with 1% FCS, 1% Pen/Strep and 2 mM glutamine. The supernatant was harvested 5 days after seeding and the antibody was purified via a HiTrap Protein G column (GE Healthcare, Chicago, USA). After washing the column with PBS, the antibody was eluted with 0.1 M glycine/HCl (pH 3.2), 20 neutralized with 0.025 volumes of 1 M Tris/HCl (pH 9) and dialyzed against PBS.
• anti-HPV16-E2 (TVG-271, 1:200, Santa Cruz Biotechnology, Heidelberg, Germany), anti-HPV16-E6 (GTX132686, 1:2000, Biozol, Eching, Germany), anti- HPV16-E7 (NM2, 1:200, Santa Cruz Biotechnology, Heidelberg, Germany), anti-HPV18-E2 25 (2E7, 1:1000, Abcam, Cambridge, England), anti-HPV18-E6 (C1P5, 1:200, Santa Cruz Biotechnology, Heidelberg, Germany), anti-HPV18-E7 (F-7, 1:200, Santa Cruz Biotechnology, Heidelberg, Germany), anti- Germany), goat anti-mouse-HRP (115-036-003, 1:5000, Jackson, West Grove, USA) and goat anti-rabbit-HRP (P0448, 1:2000, Dako, Santa Clara, USA. 30 Western blot analysis

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