Lentiviral vectors for expressing human papilloma virus (HPV) antigens and their use in treatment of HPV-induced cancers

Abstract

The present invention relates to a lentiviral vector, in particular a non-integrative lentiviral vector, or a lentiviral vector particle, in particular a non-integrative lentiviral vector particle, for use in the treatment or prevention of HPV-induced cancers, said lentiviral vector comprising at least four different nucleic acid sequences; the lentiviral vector particles comprise at least one lentiviral vector; the lentiviral vector or lentiviral vector particle is administered in combination with at least one immune checkpoint inhibitor.

Description

Field of the Invention

[0001] The present invention relates to the field of recombinant vaccine technology and to improvements in lentiviral vectors that can be used as therapeutic and preventive vaccines. In particular, the present invention relates to lentiviral vectors expressing human papillomavirus (HPV) antigens and their use in preventing and treating HPV-induced cancers. Background of the Invention

[0003] HPV causes 5.2% of cancers worldwide (Tota et al., Prev Med 2011 Oct; 53 Suppl 1: S12-21). More than 100 HPV types have been identified and divided into three groups based on their association with cancer: high-risk types with high carcinogenic potential (including HPV 16 (HPV-16) and HPV 18 (HPV-18)), low-risk types associated with benign lesions (HPV-6, -11) and skin types (including HPV-1, -2, -3, -4...) (Chen et al. Virology Vol. 516 (2018): 86-101). The proportion of HPV-related cancers varies by cancer type and geographic setting, but it is estimated that 90% of cervical cancers, 91% of anal cancers, 75% of vaginal cancers, 70% of oropharyngeal cancers, 69% of vulvar cancers, and 63% of penile cancers are associated with HPV infection (Saraiya et al., J Natl Cancer Inst. 2015 Apr 29;107(6)).

[0004] The two most common HPV types found in cancer are HPV 16 and HPV 18. For example, HPV 16 and HPV 18 are thought to be involved in 70-75% of all cervical cancers (de Sanjose et al., Eur J Cancer. 2013 Nov; 49(16): 3450-61). HPV 16 and 18 are also involved in most anal cancers (91%), oropharyngeal cancers (70%), vaginal cancers (75%), penile cancers (63%), and vulvar cancers (68%) (Saraiya et al., J Natl Cancer Inst. 2015 Apr 29; 107(6)).

[0005] Therefore, therapeutic vaccines targeting HPV16 / 18 could potentially be used to treat and prevent related cancers regardless of their location.

[0006] Human papillomavirus (HPV) is a non-enveloped double-stranded DNA virus. Their genome encodes six nonstructural proteins (early proteins E1, E2, E4, E5, E6 and E7) and two structural proteins (late proteins L1 and L2) (Chen et al. Virology Vol. 516 (2018): 86-101).

[0007] Of these proteins, the oncogenicity of E6 and E7 has been well characterized. They are known to inactivate the p53 and pRb tumor suppressor proteins, thereby promoting cell proliferation. The E6 / E7 oncogenes are crucial for the induction of HPV-associated malignant cell transformation and the maintenance of the oncogenic phenotype of HPV-positive cancer cells (Yim and Park, Cancer Res Treat. 2005 Dec;37(6):319-24.). The observation that E6 and E7 proteins are expressed by all HPV-positive cells throughout the infection process makes them perfect targets for vaccines.

[0008] Recombinant viral vectors have been extensively developed for vaccination purposes. Modification of the viral genome enables the production of non-toxic and non-infectious viral particles that can be used as a tool to introduce genetic material into target cells. The use of recombinant viral vectors to induce T cell-mediated immunity is a very promising vaccination approach. A variety of viral vectors have been evaluated for vaccination purposes, including retroviral vectors, adenoviral vectors, and vaccinia virus vectors (Milone and O'Doherty, Leukemia (2018) 32: 1529-1541 and Ku et al., Expert Review of Vaccines (2021). Lentiviruses are part of the retroviridae family, which includes the human immunodeficiency virus (HIV). Lentiviral vectors are primarily derived from HIV-1. Their safety has been improved by removal of the LTR U3 sequence, resulting in a "self-inactivating" vector that is completely devoid of viral promoter and enhancer sequences. Lentiviral vectors have become a promising tool because they show several advantages over other viral systems. In particular, lentiviral vectors are non-toxic and, unlike other retroviruses, are able to transduce non-dividing cells, particularly dendritic cells (He et al. 2007, Expert Rev vaccines, 6(6): 913-24), allowing for sustained antigen presentation through endogenous pathways.

[0009] Contrary to other commonly used viral vectors, lentiviral vectors have the ability to transduce non-dividing cells. Efficient transduction in non-dividing cells requires the formation of a three-stranded DNA structure known as a central DNA "flap" that maximizes the efficiency of gene input into the nucleus of non-dividing cells (including dendritic cells (DCs)) (Arhel et al., EMBO J. 2007 June 20; 26 (12): 3025-3037) (Zennou et al., Cell. 2000 April 14; 101 (2): 173-85).

[0010] Dendritic cells (DC) are essential for antigen presentation because they constitute the main category of antigen presenting cells (APC), whose main function is to present antigens and initiate immune responses (Steinman, R., Banchereau, J. Nature 449, 419-426 (2007)). Mature DCs migrate to draining lymph nodes, where they present short peptides derived from antigens on the surface through major histocompatibility complex (MHC) molecules. Antigen-specific T cells present in the lymph nodes can then interact with the peptide-MHC complex through TCR (T cell receptor). The recognition of peptide-MHC by specific TCR in conjunction with costimulatory signals initiates T cell activation (Steinman, R., Banchereau, J. Nature 449, 419-426 (2007)).

[0011] It is an object of the present invention to provide therapeutic and prophylactic vaccines for the prevention and treatment of HPV-induced cancers.

[0012] Therapeutic vaccination against high-risk human papillomaviruses using an integrase-deficient lentiviral vector expressing non-oncogenic HPV16 E7 fused to calreticulin (CRT) has been described (Grasso et al., Int J Cancer. 2013 Jan 15; 132(2):335-44). The construct was tested against early-stage tumors and demonstrated the ability to eradicate the tumor in a reasonable but not perfect number of vaccinated mice.

[0013] Thus, there remains a need in the art for treatments for the more aggressive and / or well-implanted HPV-induced tumors, which are known in the art to be more difficult to eliminate than small, early-stage tumors.

[0014] There is also a need for treatments of HPV-induced resistant tumors, ie tumors characterized by their intense infiltration by regulatory T cells (Tregs).

[0015] In addition, there is a need for therapeutic vaccines that allow CD8 + and CD4 + The cells infiltrate the HPV-induced tumor to be treated, while simultaneously reducing Tregs in the tumor.

[0016] There is also a need for the generation of strong immune memory against HPV, particularly against PDHPV antigens, and more particularly against HPV16 and HPV18 antigens.

[0017] There is also a need for the development of new safe, non-oncogenic, preventive and therapeutic vaccines against HPV-induced cancers.

[0018] There is also a need for vaccines that allow complete elimination of primary tumors after administration of a single dose and provide strong protection against recurrence.

[0019] The object of the present invention is to meet the above needs. Summary of the invention

[0020] Therefore, the present invention relates to the following items:

[0021] Project 1: A A lentiviral vector, in particular a non-integrating lentiviral vector, comprising at least four different nucleic acid sequences selected from the group consisting of:

[0022] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen,

[0023] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen,

[0024] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen, and

[0025] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen.

[0026] As shown in the examples, the lentiviral vector of the present invention has strong therapeutic and preventive activity against HPV-induced tumors.

[0027] Project 2: The lentiviral vector according to item 1, wherein the nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E6 antigen encodes an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0028] Project 3: The lentiviral vector according to item 1 or 2, wherein the nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen encodes an amino acid sequence having at least 68% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO: 14 and SEQ ID NO: 15.

[0029] Project 4:A lentiviral vector according to any one of items 1 to 3, wherein the nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen encodes an amino acid sequence having at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 24, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

[0030] Project 5: The lentiviral vector according to any one of items 1 to 4, wherein the nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen encodes an amino acid sequence having at least 83% sequence identity with the amino acid sequence shown in SEQ ID NO: 33, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.

[0031] Project 6: The lentiviral vector according to any one of items 1 to 5, wherein the at least four different nucleic acid sequences encoding antigens are fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence.

[0032] Item 7: The lentiviral vector according to any one of items 1 to 6, wherein the at least four different nucleic acid sequences are selected from the group consisting of:

[0033] (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen;

[0034] (b) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen;

[0035] (c) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen; and

[0036] (d) A nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E6 antigen.

[0037] Item 8: The lentiviral vector according to any one of items 1 to 7, wherein the order of the at least four different nucleic acid sequences from the 5' end to the 3' end is: (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen.

[0038] Item 9: The lentiviral vector according to any one of items 1 to 8, comprising a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:42, in particular the nucleic acid sequence SEQ ID NO:41.

[0039] Item 10: The lentiviral vector according to any one of items 1 to 9, which is chosen from the group consisting of the non-integrating lentiviral vector submitted to the CNCM under accession number I-5759, I-5760, I-5761 and I-5762, in particular the non-integrating lentiviral vector submitted to the CNCM under accession number I-5759.

[0040] Item 11: The lentiviral vector according to any one of items 1 to 10, wherein the lentiviral vector comprises an MHC class I promoter, in particular a β2-microglobulin promoter.

[0041] Item 12: The lentiviral vector according to any one of items 1 to 11, wherein the lentiviral vector comprises a cPPT / CTS sequence, in particular a cPPT / CTS sequence as shown in SEQ ID NO: 37.

[0042] Item 13: The lentiviral vector according to any one of items 1 to 12, wherein the lentiviral vector comprises a 3' long terminal repeat (LTR) lacking its U3 promoter sequence.

[0043] Item 14: The lentiviral vector according to any one of items 1 to 13, wherein the lentiviral vector does not comprise a constitutive enhancer sequence.

[0044] Item 15: The non-integrating lentiviral vector according to any one of items 1 to 14, wherein the lentiviral vector comprises a mutated form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), and specifically has a sequence as shown in SEQ ID NO: 38.

[0045] Project 16: A lentiviral vector particle, in particular a non-integrating lentiviral vector particle, comprising at least one lentiviral vector as defined in any one of items 1 to 15.

[0046] Item 17: The lentiviral vector particle according to item 16, wherein the lentiviral vector particle comprises a functional lentiviral integrase protein.

[0047] Item 18: The lentiviral vector particle according to item 16 or 17, wherein the lentiviral vector particle comprises vesicular stomatitis virus glycoprotein (VSVG), in particular VSV-G Indiana serotype or VSV-G New Jersey serotype.

[0048] Item 19: The lentiviral vector particle according to any one of items 16 to 18, wherein the lentiviral vector particle comprises HIV-1 subtype D Gag and Pol proteins.

[0049] Item 20: An isolated cell comprising the lentiviral vector according to any one of items 1 to 14 or the lentiviral vector particle according to any one of items 16 to 19.

[0050] Item 21: A vaccine composition comprising the lentiviral vector according to any one of items 1 to 14, the lentiviral vector particle according to any one of items 16 to 19, or the cell according to item 19.

[0051] Item 22: A vaccine composition according to item 21, for use in treating or preventing HPV-induced cancer, in particular a cancer selected from the group consisting of cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, oropharyngeal cancer and their metastases, in particular their lung metastases.

[0052] Item 23: A lentiviral vector according to any one of items 1 to 15, a lentiviral vector particle according to any one of items 16 to 19 or a cell according to item 20 for use as a medicament or vaccine.

[0053] Item 24: A lentiviral vector, lentiviral vector particle or cell according to item 23, for use in treating or preventing HPV-induced cancer, in particular a cancer selected from the group consisting of cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, oropharyngeal cancer and their metastases, in particular their lung metastases.

[0054] Item 25: A vaccine composition for use according to item 22, or a lentiviral vector, lentiviral vector particle or cell for use according to item 23 or 24, wherein the vaccine composition, lentiviral vector, lentiviral vector particle or cell is administered in combination with at least one immune checkpoint inhibitor, in particular at least one monoclonal antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-NKG2A, anti-TIM-3, anti-TIGIT and anti-LAG-3 monoclonal antibodies, more particularly with at least one anti-PD monoclonal antibody.

[0055] Item 26: The vaccine composition, lentiviral vector, lentiviral vector particle or cell according to item 25, wherein the at least one immune checkpoint inhibitor is administered simultaneously or separately, in particular at least 2 days, especially at least 4 days after the administration of the vaccine composition, lentiviral vector, lentiviral vector particle or cell.

[0056] The present invention also relates to the use of a lentiviral vector (particularly a non-integrating lentiviral vector) or a lentiviral vector particle (particularly a non-integrating lentiviral vector particle) for treating or preventing HPV-induced cancer.

[0057] The lentiviral vector comprises at least four different nucleic acid sequences selected from the group consisting of:

[0058] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen,

[0059] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen,

[0060] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen, and

[0061] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen;

[0062] The lentiviral vector particle comprises at least one lentiviral vector;

[0063] The lentiviral vector or lentiviral vector particle is administered in combination with at least one immune checkpoint inhibitor, in particular at least one monoclonal antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-NKG2A, anti-TIM-3, anti-TIGIT and anti-LAG-3 monoclonal antibodies.

[0064] The HPV-induced cancer may be selected from the group consisting of cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, oropharyngeal cancer and metastases thereof, in particular lung metastases thereof.

[0065] The nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E6 antigen can encode an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0066] The nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen can encode an amino acid sequence having at least 68% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO: 14 and SEQ ID NO: 15.

[0067] The nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen can encode an amino acid sequence having at least 60% sequence identity with the amino acid sequence as described in SEQ ID NO:24, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

[0068] The nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen can encode an amino acid sequence having at least 83% sequence identity with the amino acid sequence shown in SEQ ID NO:33, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0069] The at least four different nucleic acid sequences encoding antigens may be fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence.

[0070] The order of the at least four different nucleic acid sequences (from 5' end to 3' end) can be selected from the group consisting of:

[0071] (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen;

[0072] (b) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen;

[0073] (c) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen; and

[0074] (d) A nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E6 antigen.

[0075] The order of the at least four different nucleic acid sequences from the 5' end to the 3' end can be: (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen.

[0076] The lentiviral vector or lentiviral vector particle for use according to the present invention may comprise a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:42, in particular the nucleic acid sequence SEQ ID NO:41.

[0077] The lentiviral vector or lentiviral vector particle for use according to the invention can be selected from the group consisting of the non-integrating lentiviral vectors submitted to the CNCM under accession numbers I-5759, I-5760, I-5761 and I-5762, in particular the non-integrating lentiviral vector submitted to the CNCM under accession number I-5759.

[0078] The lentiviral vector for use according to the present invention may comprise an MHC class I promoter, in particular a β2-microglobulin promoter.

[0079] The lentiviral vector for use according to the present invention may comprise a cPPT / CTS sequence, in particular a cPPT / CTS sequence as shown in SEQ ID NO:37.

[0080] The lentiviral vector for use according to the present invention may comprise a 3' long terminal repeat sequence (LTR) lacking a U3 promoter sequence.

[0081] The lentiviral vector for use according to the invention in particular does not comprise a constitutive enhancer sequence.

[0082] The lentiviral vector for use according to the present invention may comprise a mutated form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), in particular having the sequence shown in SEQ ID NO:38.

[0083] The lentiviral vector particles for use according to the present invention may comprise a functional lentiviral integrase protein.

[0084] The lentiviral vector particles for use according to the invention may comprise vesicular stomatitis virus glycoprotein (VSVG), in particular VSV-G Indiana serotype or VSV-G New Jersey serotype.

[0085] The lentiviral vector particles for use according to the present invention may contain HIV-1 subtype D Gag and Pol proteins.

[0086] The lentiviral vector or lentiviral vector particle for use according to the present invention may be contained in an isolated cell.

[0087] The lentiviral vector or lentiviral vector particle for use according to the present invention may be comprised in a vaccine composition.

[0088] The at least one immune checkpoint inhibitor can be selected from the group consisting of anti-PD-1, anti-NKG2A and anti-TIM-3 monoclonal antibodies.

[0089] The at least one immune checkpoint inhibitor may be administered simultaneously or separately, in particular, the at least one immune checkpoint inhibitor may be administered at least 2 days, in particular at least 4 days, after administration of the vaccine composition, lentiviral vector, lentiviral vector particles or cells.

[0090] The details, embodiments and preferred solutions provided relevant to any specific one or more described aspects of the present invention will be further described herein, and the details, embodiments and preferred solutions are equally applicable to all aspects of the present invention. Unless otherwise specified in this article, or obviously contradictory with the context, any combination of the embodiments, embodiments and preferred solutions described herein in all possible variations thereof is encompassed by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 This indicates that the HPV vaccine of the present invention has immunogenicity in vivo. 7 TU of lentiviral vector particles or 50 μL of diluent, the lentiviral vector particles containing the lentiviral vector submitted to CNCM under accession number I-5759, the lentiviral vector submitted to CNCM under accession number I-5760, the lentiviral vector submitted to CNCM under accession number I-5761, or the lentiviral vector submitted to CNCM under accession number I-5762. After 14 days, splenocytes were prepared and re-stimulated overnight with 4 different peptide pools to obtain IFNg ELISPOT.

[0092] Horizontal axis: from left to right: results obtained with the lentiviral vector submitted to the CNCM under accession number I-5759, the lentiviral vector submitted to the CNCM under accession number I-5760, the lentiviral vector submitted to the CNCM under accession number I-5761 or the lentiviral vector submitted to the CNCM under accession number I-5762 or with 50 μL of diluent (control).

[0093] Vertical axis: spot forming cells (SFC) / 10 6 cell.

[0094] Figure 2 The vaccine of the present invention completely eliminated well-implanted tumors in vivo. TC-1 cells were injected subcutaneously and tumor volumes were measured every other day (caliper measurement). When the average tumor volume was 70 mm 3 At 1 pm, the mice were randomly divided into groups and intramuscularly inoculated with 1×10 8 TU's LV-GFP Indiana (as a control), Indiana lentiviral vector particles containing I-5759, Indiana lentiviral vector particles containing I-5760, Indiana lentiviral vector particles containing I-5761, or Indiana lentiviral vector particles containing I-5762.

[0095] Horizontal axis: days.

[0096] Vertical axis: tumor volume (mm 3 ).

[0097] Figure 3 The ability of lentiviral vectors expressing designs I-5759, I-5760, I-5761, I-5762 according to the present invention or diluents as controls to generate persistent immunity to prevent relapse was demonstrated. Mice that had eliminated the primary tumor were challenged again on the other flank at day 60. Control mice (untreated) were also injected subcutaneously to examine tumor cell growth in mice that were naive to the experiment.

[0098] Horizontal axis: days.

[0099] Vertical axis: tumor volume (mm 3 ).

[0100] Figure 4 represents the dose / response in mice. 6 TC-1 cells were injected into the flank of the animals, and tumor volume was measured twice a week (caliper measurement). 3 The mice were randomly divided into groups and treated with diluent (control), 1×10 7 TU I-5759 vaccine or 1×10 8 I-5759 vaccination at TU(im).

[0101] Horizontal axis: days.

[0102] Vertical axis: tumor volume (mm 3 ).

[0103] Figure 5 Figure 2 shows the lymphocyte tumor infiltration after inoculation with the lentiviral vector according to the present invention. 6 TC1 tumor cells were injected (sc) into the flank of the animals, and tumor volume was measured twice a week (caliper measurement). 3 At 1 , mice were randomly divided into groups and inoculated with diluent (control) or 1 × 10 7 TU I-5759 vaccine or 1×10 8 TU(im)'s I-5759 vaccine.

[0104] Tumors were collected 10 days after inoculation, digested and analyzed by flow cytometry. FACS staining was performed and data were acquired on Macsquant facs according to methods well known in the art.

[0105] Horizontal axis: from left to right: diluent (control); 1×10 8 TU's I-5759 vaccine.

[0106] Vertical axis: upper left graph: percentage of CD8+T cells (in live cells); upper right graph: percentage of CD4+T cells (in live cells); lower graph: percentage of Treg cells (in live cells).

[0107] Figure 6 The ability of the vector according to the present invention to eliminate large, well-established tumors was demonstrated. 6 TC1 cells were injected (subcutaneously) into the flank of the animals. When the average tumor volume was approximately 300 mm 3 The mice were randomly divided into groups and treated with diluent (control) or 1×10 8 Vaccination according to the invention of TU (im), said vaccine comprising lentiviral vector particles comprising the lentiviral vector filed with the CNCM under accession number I-5759.

[0108] Horizontal axis: days.

[0109] Vertical axis: tumor volume (mm 3 ).

[0110] Figure 7 T cell responses in human PBMCs labeled with CFSE and cultured in the absence (unstimulated conditions) or presence of a vaccine according to the invention (I-5759) are described. Cell proliferation and activation were measured after 2 weeks of culture (n=3). CD8+T cell and CD4+T cell proliferation (measured by CFSE dilution) (A) and expression of the CD25 activation marker (B) were increased by adding the lentiviral vector of the invention to the culture.

[0111] Abscissa: from left to right: unstimulated (no stimulation - control); I-5759 vaccine.

[0112] Vertical axis: (A) Left: percentage of CFSElow in CD8+ population; right: percentage of CFSElow in CD4+ population. (B) Left: percentage of CD25+ in CD4+ population; right: percentage of CD25+ in CD8+ population.

[0113] Figures 8A to 8DFour examples of antigen constructs of lentiviral vectors according to the present invention are described. Each of these antigen constructs consists of the following four sequences in different orders: a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen, a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen, a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen, and a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen. Fig. 8A Represents the antigenic construct of the lentiviral vector submitted to the CNCM under accession number I-5759. Figure 8B Represents the antigenic construct of the lentiviral vector submitted to the CNCM under accession number I-5760. Figure 8C represents the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5761, Fig.8D Represents the antigenic construct of the lentiviral vector submitted to the CNCM under accession number I-5762.

[0114] Fig. 9 A and Fig. 9 B depicts synthetic peptides (both containing RAHYNIVTF H-2D) with or without ETTDPDRAHYNIVTF (SEQ ID NO:39) and PDRAHYNIVTFCCKC (SEQ ID NO:40). b -Restricted T cell epitopes (bold characters represent H-2D b T cell cytokine responses of splenocytes analyzed by intracellular cytokine staining (ICS) obtained 14 days after intramuscular immunization of C57BL / 6 mice (n=5 / group) with Ctrl Lenti (LV-GFP Indiana) or with a vaccine according to the present invention comprising lentiviral vector particles comprising the lentiviral vector submitted to the CNCM under accession number I-5759, when stimulated with a mixture of 1:154 (LV-GFP Indiana) and 1:154 (LV-GFP Indiana). Fig. 9 A specifically shows the cell counting gating strategy for cytokine-producing CD8+ T cells and IFN-γ-producing CD8+ T cells assessed by surface CD107a staining. + Degranulation activity of T cells. Fig. 9 B indicates each (multi)functional cell subset and CD8 + IFN-γ in T subsets + CD107a + Summary frequency of cells.

[0115] Fig.10Cytometric analysis of tumor-infiltrating innate immune cells (NK) of mice transplanted with tumors and vaccinated with the HPV vaccine according to the invention (I-5759) or mice transplanted with tumors but not vaccinated (control Ctrl Lenti) is described. CD11b and NKp46 were detected.

[0116] Fig.11 The ability of the vector according to the invention to eliminate large, well-established tumors was demonstrated. 6 Cured mice that had eliminated their primary tumor (right picture) were re-challenged with 10 TC-1 tumor cells in the other flank and remained without any treatment. Control mice (untreated - control aged mice - left picture) were also injected subcutaneously to check for tumor cell growth.

[0117] Abscissa: days after tumor rechallenge.

[0118] Vertical axis: tumor volume (mm 3 ).

[0119] Fig.12 A and Fig.12 B illustrates the synergistic effect between vaccination with a suboptimal dose of the vaccine according to the invention (I-5759) and anti-PD-1 therapy (monoclonal antibody anti-PD-1).

[0120] Fig.12 A represents the tumor volume in mice (mm 3- The ordinate) changes with the number of days (abscissa) after tumor implantation (D0). The experiment was performed on three identical groups of mice implanted with tumors. In the first group (10 mice - control group - Fig.12 A, left panel), mice were administered LV-empty Indiana (D13) (as a control) (dates indicated by arrows), and four days later anti-PD-1 (programmed cell death protein-1) monoclonal antibody was administered (D17, then D20, D22, D24, D28, and D31). In the second group (12 mice - control group - Fig.12 A), mice were administered a vaccine according to the invention (I-5759) (D13) (dates indicated by arrows), and four days later a control antibody (isotype control) was administered (D17, then D20, D22, D24, D28 and D31). In the third group (14 mice - Fig.12 A, left panel), mice were administered a vaccine according to the invention (D13) (I-5759) (dates indicated by arrows) and four days later mAb anti-PD-1 (D17, then D20, D22, D24, D28 and D31).

[0121] Fig.12B represents the survival rate (percentage of mice - vertical axis) of mice in each group (control group 1: Ctrl Lenti + anti-PD-1; control group 2: I-5759 + ctrl Ig; group 3: I-5759 + anti-PD-1) over time (days - horizontal axis).

[0122] Fig.13 A and Fig.13 B shows the cure of mice with lung metastases induced by intravenous injection of TC1-nLuc cells after a single infection with Lenti-HPV-07 vaccine.

[0123] Fig.13 A represents the change in luminescence value expressed in photons per second (p / s) (ordinate-total flux) caused by nanoluciferase stably expressed by TC1-nLuc cells injected into different groups of mice over time (abscissa-days) after TC1-nLuc cells were intravenously injected into the mice. Three groups of mice were tested: a negative control group in which mice were not injected with TC1-nLuc cells (n=11) (negative control group), in which mice were injected with TC1-nLuc cells and control Lenti (LV-empty Indiana)-1.10 on day 5 9 TU control group (n=11) (TC1+Ctrl Lenti) And wherein mice were injected with TC1-nLuc cells and injected with the vaccine according to the present invention (Lenti-HPV-07 (I-5759))-1.10 on day 5 9 TU group (n=11) (TC1+Lenti-HPV-07)

[0124] Fig.13 B represents the p / s values ​​of each mouse in the above three experimental groups on day 22 after tumor injection. Vertical axis: luminescence value (total flux) expressed in photons per second (p / s). Horizontal axis, from left to right: negative control group, Ctrl Lenti group, and then Lenti-HPV-07 group.

[0125] Fig.14 A to 14E illustrate the synergistic effect of Lenti-HPV07 and anti-NKG2A mAb in TC-1 tumor eradication.

[0126] Fig.14 A shows the timeline of tumor transplantation and combined treatment with Lenti-HPV-07 and anti-NKG2A mAb in C57BL / 6 mice. 1×10 6TC-1 cells. On the 10th day after implantation, when the tumor volume reached an average of 120-140 mm 3 At 10 s, the mice were randomly divided into groups and vaccinated with a suboptimal dose of 1 × 10 8 TU / mouse Lenti-HPV-07. Mice were then treated 2 to 3 times per week with anti-NKG2A mAb (clone 20D5, Bioxcell) or Ig control (clone 2A3, Bioxcell). A total of 8 injections were given from day 14 to day 31 (200 μg / injection). Fig.14 B shows the spaghetti graph of tumor growth in two test groups: Lenti-HPV-07 (I-5759) + Ctrl Ig group on the left and Lenti-HPV-07 (I-5759) + anti-NKG2A group on the right. Vertical axis: tumor volume (mm 3 ). Horizontal axis: days after tumor transplantation. Fig.14 C represents the treatment response rate according to RECIST criteria in the two test groups. Horizontal axis: Lenti-HPV-07 (I-5759) + Ctrl Ig group on the left; Lenti-HPV-07 (I-5759) + anti-NKG2A group on the right. Vertical axis: percentage of treatment response. Each group from bottom to top represents complete response, then partial response, and finally no response. Fig.14 D represents the progression-free survival time of mice in the two test groups. Horizontal axis: left side Lenti-HPV-07 (I-5759) + Ctrl Ig group; right side Lenti-HPV-07 (I-5759) + anti-NKG2A group. Vertical axis: PFS (days). Fig.14 E stands for Fig.14 B shows the survival curve of the animals. Abscissa: days. Ordinal axis: survival rate (percentage of mice). The curve with a value of about 80% on day 40 represents the Lenti-HPV-07 (I-5759) + anti-NKG2A group, and the curve with a value of about 40% on day 40 represents the Lenti-HPV-07 (I-5759) + Ctrl Ig group.

[0127] Fig.15 A to 15E illustrate the synergistic effect of Lenti-HPV07 and anti-TIM-3 mAb in TC-1 tumor eradication.

[0128] Fig.15 A shows the timeline of tumor implantation and combined treatment with Lenti-HPV-07 and anti-TIM-3 mAb. 1×10 6 TC-1 cells. On day 13 after implantation, when the tumor volume reached an average of 130 mm 3At 10 s, the mice were randomly divided into groups and vaccinated with a suboptimal dose of 1 × 10 8 Lenti-HPV-07 or Ctrl Lenti in TU / mouse. Mice were then treated 2 to 3 times per week with anti-TIM-3 mAb (clone RMT3-23, Bioxcell) or Ig control (clone 2A3, Bioxcell). A total of 8 injections (200 μg / injection) were given from day 16 to day 36. Fig.15 B shows a spaghetti graph of tumor growth in two test groups: left group Lenti-HPV-07 (I-5759) + Ctrl Ig. Right group Lenti-HPV-07 (I-5759) + anti-TIM-3. Vertical axis: tumor volume (mm 3 ). Horizontal axis: days after tumor transplantation. Fig.15 C represents the treatment response rate drawn according to the RECIST criteria in the two test groups. Horizontal axis: groups from left to right: CtrlLenti+Ctrl Ig; Ctrl Lenti+anti-TIM-3; Lenti-HPV-07(I-5759)+Ctrl Ig; Lenti-HPV-07(I-5759)+anti-TIM-3. Vertical axis: percentage of treatment response. Each group represents complete response from bottom to top, then partial response, and finally no response. The two groups on the left part of the horizontal axis have only no response results. Fig.15 D represents the progression-free survival time of mice in the two test groups. Horizontal axis: left group Lenti-HPV-07 (I-5759) + Ctrl Ig; right group Lenti-HPV-07 (I-5759) + anti-TIM-3. Vertical axis: PFS (days). Fig.15 E stands for Fig.15 Survival curves of the animals shown in B. Abscissa: number of days. Ordinal axis: survival rate (percentage of mice). The curve with a value of about 75% on day 40 represents the Lenti-HPV-07 (I-5759) + anti-TIM-3 group. The curve with a value of about 35% on day 40 represents the Lenti-HPV-07 (I-5759) + CtrlIg group. The curve in which all animals died on day 33 represents the Ctrl Lenti + anti-TIM-3 group. The curve in which all animals died on day 34 represents the Ctrl Lenti + Ctrl Ig group.

[0129] Sequence Overview

[0130] SEQ ID NO:1 is a nucleic acid sequence encoding the E6 protein from HPV 16

[0131] SEQ ID NO:2 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16

[0132] SEQ ID NO: 3 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16

[0133] SEQ ID NO:4 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16

[0134] SEQ ID NO:5 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16

[0135] SEQ ID NO:6 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16

[0136] SEQ ID NO:7 is the amino acid sequence of the E6 protein from HPV 16

[0137] SEQ ID NO:8 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0138] SEQ ID NO:9 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0139] SEQ ID NO:10 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0140] SEQ ID NO:11 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0141] SEQ ID NO:12 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0142] SEQ ID NO:13 is a nucleic acid sequence encoding the E7 protein from HPV 16

[0143] SEQ ID NO:14 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 16

[0144] SEQ ID NO:15 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 16

[0145] SEQ ID NO:16 is the amino acid sequence of the E7 protein from HPV 16

[0146] SEQ ID NO:17 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 16

[0147] SEQ ID NO:18 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 16

[0148] SEQ ID NO:19 is a nucleic acid sequence encoding the E6 protein from HPV 18

[0149] SEQ ID NO:20 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18

[0150] SEQ ID NO:21 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18

[0151] SEQ ID NO:22 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18

[0152] SEQ ID NO:23 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18

[0153] SEQ ID NO:24 is the amino acid sequence of the E6 protein from HPV 18

[0154] SEQ ID NO:25 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18

[0155] SEQ ID NO:26 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18

[0156] SEQ ID NO:27 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18

[0157] SEQ ID NO:28 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18

[0158] SEQ ID NO:29 is a nucleic acid sequence encoding the E7 protein from HPV 18

[0159] SEQ ID NO:30 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18

[0160] SEQ ID NO:31 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18

[0161] SEQ ID NO:32 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18

[0162] SEQ ID NO:33is the amino acid sequence of the E7 protein from HPV 18

[0163] SEQ ID NO:34 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18

[0164] SEQ ID NO:35 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18

[0165] SEQ ID NO:36 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18

[0166] SEQ ID NO:37 Is the nucleic acid sequence encoding the cPPT / CTS sequence

[0167] SEQ ID NO:38 is a nucleic acid sequence encoding a mutated form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE)

[0168] SEQ ID NO:39 Contains RAHYNIVTF H-2D b -Synthesis of T-cell-restricted epitopes E7 HPV16 -Derivatized Peptides

[0169] SEQ ID NO:40 Contains RAHYNIVTF H-2D b -Synthesis of T-cell-restricted epitopes E7 HPV16 -Derivatized Peptides

[0170] SEQ ID NO: 41 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5759

[0171] SEQ ID NO: 42 is the amino acid sequence of the antigen construct encoded by the lentiviral vector submitted to CNCM under accession number I-5759

[0172] SEQ ID NO:43 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5760

[0173] SEQ ID NO:44 is the amino acid sequence of the antigen construct encoded by the lentiviral vector submitted to CNCM under accession number I-5760

[0174] SEQ ID NO:45 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5761

[0175] SEQ ID NO:46is the amino acid sequence of the antigen construct encoded by the lentiviral vector submitted to CNCM under accession number I-5761

[0176] SEQ ID NO:47 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5762

[0177] SEQ ID NO:48 is the amino acid sequence of the antigen construct encoded by the lentiviral vector submitted to CNCM under accession number I-5762

[0178] SEQ ID NO:49 is H-2D b -Restricted T cell epitopes The amino acid sequence DETAILED DESCRIPTION OF THE INVENTION

[0180] The present inventors have discovered that administration of a lentiviral vector encoding at least four different human papillomavirus (HPV) antigens, in particular at least four HPV antigens selected from proteins E6 and E7 of at least two different HPV subtypes (in particular HPV16 and HPV18 subtypes), to an individual in need thereof can result in a high degree of preventive and therapeutic activity against HPV-induced cancers.

[0181] The lentiviral vector according to the present invention is capable of inducing a strong, durable and extensive cell-mediated response against tumors induced by HPV infection.

[0182] The present specification describes throughout the present invention lentiviral vectors, as well as lentiviral vector particles comprising them, isolated cells comprising the lentiviral vectors or lentiviral vector particles, and vaccine compositions comprising them.

[0183] definition

[0184] Unless otherwise defined, all scientific and technical terms used in this application have the commonly used meanings in the art.

[0185] As used herein, "transgene" means a polynucleotide that can be expressed in a non-native environment or heterologous cells by recombinant technology under appropriate conditions.

[0186] As used herein, the term "recombinant", when used to refer to a cell of the invention, means that the cell has been modified by introducing endogenous and / or heterologous nucleic acids or proteins into the cell or by altering a native cell, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes or nucleic acids that are not present in the native (non-recombinant) form of the cell, or expresses the native (e.g., endogenous) gene at a level that is different from the native level of the native (e.g., endogenous) gene, or expresses an additional or supplementary copy of the native (e.g., endogenous) gene at a level that is different from the native level of the native (e.g., endogenous) gene. An isolated cell according to the invention is recombinant because it comprises at least one lentiviral vector according to the invention and / or at least one lentiviral vector particle according to the invention.

[0187] As used herein, the term "recombinant", when used to refer to a vector, is a sequence formed / obtained by genetic engineering techniques well known to those skilled in the art.

[0188] As used herein, the term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and containing more than five amino acid residues. Amino acids can be identified by single-letter or three-letter names. The term "protein" as used herein is synonymous with the term "polypeptide" and can also refer to two or more polypeptides. Therefore, the terms "protein", "peptide" and "polypeptide" can be used interchangeably. Polypeptides can be optionally modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, etc.) to increase functionality. Polypeptides that show activity can be referred to as enzymes. It should be understood that due to the degeneracy of the genetic code, a large number of nucleotide sequences encoding a given polypeptide can be generated.

[0189] The term "operably linked" as used herein refers to two or more nucleic acid sequence elements that are physically linked and in a functional relationship with each other. For example, in a lentiviral vector according to the present invention, a promoter is operably linked to a coding sequence (also referred to herein as an "antigen construct") because the promoter is able to initiate or regulate the transcription or expression of the antigen construct, in which case the antigen construct is understood to be "under the control" of the promoter. Typically, when two nucleic acid sequences are operably linked, they will be in the same orientation and typically also in the same reading frame. They are typically substantially contiguous, although this may not be required.

[0190] The term "encoding" or "coding for" refers to the process by which a polynucleotide produces an amino acid sequence through transcription and translation mechanisms.

[0191] For each amino acid sequence or target amino acid sequence, a reference sequence is described herein. The specification also encompasses amino acid sequences having a specific percentage of amino acid identity with a reference amino acid sequence.

[0192] For obvious reasons, throughout this specification, a specific nucleic acid sequence or a specific amino acid sequence that complies with the nucleotide or amino acid identity under consideration, respectively, should further result in a protein (or antigen) that exhibits the desired biological activity. As used herein, the "percentage of identity" between two nucleic acid sequences or between two amino acid sequences is determined by comparing the two optimally aligned sequences via a comparison window.

[0193] Thus, a portion of the nucleotide or amino acid sequence in the comparison window may include additions or deletions (eg, "gaps") compared to the reference sequence (which does not include additions or deletions) in order to obtain optimal alignment between the two sequences.

[0194] The terms "sequence homology" or "sequence identity" or "homology" or "identity" are used interchangeably herein. For purposes of the present invention, as defined herein, in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences, a gap may be introduced in either of the two sequences being compared. This alignment may be performed over the full length of the compared sequences. Alternatively, the alignment may be performed over a shorter length, such as over about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported alignment region.

[0195] The comparison of sequence and the determination of the percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. Those skilled in the art will appreciate the fact that there are several different computer programs that can be used to compare two sequences and determine the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (eds.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).

[0196] The percent sequence identity between two amino acid sequences or between two nucleotide sequences can be determined by aligning the two sequences using the Needleman and Wunsch algorithm. (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by algorithms. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE.

[0197] For the purposes of the present invention, the NEEDLE program from the EMBOSS package (version 2.8.0 or higher, EMBOSS: European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277, http: / / emboss.bioinformatics.nl / ) is used. For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. The optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5. The end gap penalty is not increased. In the output section, the answer to the question "simple identity and similarity" is "yes" and the output alignment format is "SRS paired".

[0198] After alignment by the program as described above, the sequence identity percentage between the query sequence and the sequence of the present invention is calculated as follows: the number of corresponding positions showing identical amino acids or identical nucleotides in the two sequences in the alignment is divided by the total length of the alignment after deducting the total number of gaps in the alignment. The identity defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked as "longest identity" in the output of the program.

[0199] The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined by using several other algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), using hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or using the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80) (available in, e.g. https: / / www.ebi.ac.uk / Tools / msa / clustalo / The sequence alignment was determined by using the GAP program (a mathematical algorithm from the University of Iowa) or the mathematical algorithm of Myers and Miller (1989-Cabios 4: 11-17) or Clone Manager 9. The preferred parameters used are https: / / www.ebi.ac.uk / Tools / msa / clustalo / The default parameters are set above.

[0200] The rank of sequence identity (sequence match) can be calculated using, for example, BLAST, BLAT or BlastZ (or BlastX). Similar algorithms are introduced in the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed using the BLASTN program, score = 100, word length = 12 to obtain polynucleotide sequences homologous to those nucleic acids encoding related proteins.

[0201] BLAST protein searches were performed using the BLASTP program, score = 50, word length = 3 to obtain amino acid sequences homologous to SHC polypeptides. To obtain gapped alignments for comparison purposes, Gapped BLAST was utilized as described in Altschul et al. (1997) (1997) Nucleic Acids Res. 25, 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs were used. Analysis of sequence matches can be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: 154-162) or Markov random fields. When percentages of sequence identity are mentioned in this application, unless otherwise specifically stated, these percentages are calculated relative to the full length of the longer sequence.

[0202] In a specific embodiment, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0203] As used herein, the term "non-carcinogenic" is used in its conventional sense, i.e. relates to an element, in this case an antigen, that is not capable of causing tumor formation. As described in detail elsewhere, the antigens used in the present invention have been genetically modified so as to be non-carcinogenic. According to the ordinary meaning of these terms, this means that the nucleic acid sequence encoding the antigen used herein does not occur in nature and has been modified by introduction or by deletion or by modification of its nucleic acid sequence, resulting in an encoded amino acid sequence that is also not naturally occurring in nature.

[0204] Various means for making deletions, substitutions or introductions into nucleic acid sequences are known to those skilled in the art for a long time.

[0205] As will be appreciated by those skilled in the art, it may also be advantageous to modify the encoding sequence to enhance its expression in a particular host. The genetic code is redundant, with 64 possible codons, but most organisms typically use a subset of these codons. The most frequently used codons in a species are referred to as optimal codons, and those infrequently used are classified as rare or low-usage codons. In a process sometimes referred to as "codon optimization" or "controlling species codon preference", codons may be replaced to reflect the preferred codon usage of the host. The codon optimization of other host cells may be easily determined using a codon usage table, or commercially available software may be used, such as CodonOp (www.idtdna.com / CodonOptfrom) from Integrated DNA Technologies to carry out. For example, an optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared (Murray et al., 1989, Nucl Acids Res. 17: 477-508) to increase the translation rate or produce a recombinant RNA transcript with a desired property (such as a longer half-life compared to transcripts produced by non-optimized sequences). The translation stop codon can also be modified to reflect host preference. For example, the typical stop codon for monocots is UGA, while insects and Escherichia coli (E. coli) typically use UAA as a stop codon (Dalphin et al., 1996, Nucl Acids Res. 24: 216-8).

[0206] A "non-integrating" lentiviral vector means that when the lentiviral vector is in a cell, it is not integrated into the host cell genome. A non-integrating lentiviral vector particle refers to a lentiviral vector particle comprising a non-integrating lentiviral vector. It may also be referred to as an integration-defective lentiviral vector or a non-integrating lentiviral vector.

[0207] HPV-induced cancers are also known as HPV (human papillomavirus)-associated cancers. In fact, this is when the HPV infection is not successfully controlled by the infected host's immune system. When high-risk HPV infection persists for many years, it causes cellular changes that, if not treated, may worsen over time and become cancer.

[0208] The lentiviral vector according to the present invention

[0209] The present inventors have conceived novel lentiviral vector-based therapeutic and prophylactic vaccines against HPV-induced cancers.

[0210] In particular, the present invention relates to a lentiviral vector comprising at least four different nucleic acid sequences selected from a group of specific non-oncogenic HPV antigens.

[0211] The so-called different nucleic acid sequences refer to that at least four nucleic acid sequences contained in the lentiviral vector are different, that is, each of them is a different member of the specific non-oncogenic HPV antigen group.

[0212] The groups of non-oncogenic HPV antigens are as follows:

[0213] - a nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen;

[0214] - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen;

[0215] - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen; and

[0216] - A nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen.

[0217] Unlike most HPV proteins that are lost after HPV integration, E6 and E7 proteins are continuously expressed in HPV-induced tumors (Ghittoni, Raffaella et al., Virus genes Vol. 40, No. 1 (2010): 1-13; Morrow, Matthew et al., Expert review of vaccines Vol. 12, No. 3 (2013): 271-83). These proteins interfere with cellular functions and are known to play an important role in HPV-related carcinogenesis. ( Vjekoslav. Cancers, Vol. 8, No. 10, 95. October 19, 2016; Ghittoni, Raffaella et al. Virus genes, Vol. 40, No. 1 (2010): 1-13). E6 and E7 are thought to interfere with multiple pathways, but most importantly, E6 protein expression in cells leads to ubiquitin-mediated degradation of the tumor suppressor p53 through direct interaction with the cellular E3 ubiquitin ligase E6AP (Huibregtse, JM et al. The EMBO journal Vol. 10, No. 13 (1991): 4129-35; Martinez-Zapien, Denise et al. Nature Vol. 529, No. 7587 (2016): 541-5), and E7 binds to the Rb protein, thereby disrupting the interaction between Rb and E2F and releasing the E2F factor (Cassetti, M Cristina et al. Vaccine Vol. 22, No. 3-4 (2004): 520-7).

[0218] Since E6 and E7 proteins are expressed in all HPV-induced cancers, it was decided to include antigens of these proteins from the two major subtypes (HPV16 and HPV18) in the lentiviral vector of the present invention. In order to develop a vaccine from E6 and E7 antigens, it is very important to eliminate the carcinogenic risks associated with these proteins.

[0219] Therefore, non-oncogenic E6 and E7 proteins are used in the present invention. "Non-oncogenic E6 and E7 HPV proteins" means that their coding sequences are modified to remove p53, Mi2b and Rb binding sites and PDZ binding motifs. In a specific embodiment, since partial mutations of the E6 and E7 HPV protein binding sites cannot completely eliminate Rb binding, the sites are specifically completely removed from the sequences used in the present invention.

[0220] The lentiviral vector according to the present invention may be single-stranded or double-stranded.The lentiviral vector according to the present invention may be an RNA or DNA molecule.

[0221] In the context of the present invention, "lentiviral vector" refers to a non-replicating vector used to transduce host cells with a transgene comprising a cis-acting lentiviral RNA or DNA sequence and requiring essential lentiviral proteins (e.g., Gag, Pol and / or Env) and accessory proteins (e.g., Tat, Rev) provided in trans. Lentiviral vectors lack expression of all functional HIV proteins. The lentiviral vector genome can exist in the form of an RNA or DNA molecule, depending on the production or development stage of the retroviral vector.

[0222] In a preferred embodiment, the lentiviral vector of the present invention is a non-integrating lentiviral vector.

[0223] Non-integrating lentiviral vectors have been designed to reduce the potential carcinogenic risk associated with insertional mutagenesis events, particularly for vaccination purposes. Examples of non-integrating lentiviral vectors are provided in Coutant et al., PLOS ONE 7(11):e48644(2012), Karwacz et al., J. Virol. 83(7):3094-3103(2009), Negri et al., Molecular Therapy 15(9):1716-1723(2007); and Hu et al., Vaccine 28:6675-6683(2010). Therefore, it is reported that, compared with the integration system, the non-integrating lentiviral vector system can reduce the potential risk of insertional mutagenesis (Hu et al., Vaccine 28:6675-6683(2010)). It is further reported that in some functional analyses, the intensity and quality of the immune response triggered by the integration-defective lentiviral vector (IDLV) directed by DC are comparable to its integration counterpart. Therefore, integration-defective lentiviral vectors (IDLVs) have been considered safer vectors for human administration than integrating vectors, with comparable efficacy.

[0224] In addition, the deletion of the U3 region of the 3'LTR of the viral promoter and enhancer sequence in the self-inactivating lentiviral vector limits the possibility of endogenous promoter activation. These concerns about safety are directly derived from the experience gained in the SCID-X1 gene therapy trial conducted in 1998-1999, which was conducted on children with rare forms of X-linked (SCID-X1 gene) severe immunodeficiency using a retroviral vector based on Moloney virus (Cavazzana-Calvo et al., 2000, Science., 288 (5466): 669-72). During this trial, 4 of the 9 children developed leukemia due to the integration of Moloney virus-derived retroviral vectors in close proximity to the human LM02 proto-oncogene (Hacein-Bey-Abina et al., 2008, J. Clin. Invest., 118 (9): 3132-3142). Malignant tumors have been shown to be the result of the proximity of the viral U3 promoter / enhancer to the LM02 proto-oncogene. Therefore, safety is a major concern for administration of lentiviral vectors to humans.

[0225] Therefore, the lentiviral vector according to the present invention may comprise a cis-long terminal repeat sequence (LTR) known in the art, in particular, a 3'long terminal repeat sequence (LTR) lacking its U3 promoter sequence (Miyoshi H et al., 1998, J Virol. 72(10):8150-7; Zufferey et al., 1998, J V / ro / 72(12):9873-80).

[0226] Enhancers are cis-acting sequences that can act as transcriptional activators at a distance. They have been widely used in virus-derived vectors because they appear to be the most efficient (Chinnasamy et al., 2000, Hum Gene Ther 11 (13): 1901-9 for obtaining strong transgenic expression in a variety of cell types, particularly in DC; Rouas et al., 2008, Cancer Gene Ther 9 (9): 715-24; Kimura et al., 2007, Mol Ther 15 (7): 1390-9; Gruh et al., 2008, J Gene Med 10 (1) 21-32). However, considering the safety issues of insertion mutagenesis, such transcription enhancer sequences should be deleted from lentiviral vector constructs to eliminate the risk of insertion mutagenesis caused by enhancer proximity effects. This enhancer proximity effect is the most common insertion mutagenesis mechanism to date, and is the only effect described in human or animal cases of tumorigenic events after gene transfer.

[0227] Therefore, the lentiviral vector according to the present invention may not comprise a constitutive enhancer sequence.

[0228] Previous studies have reported the replacement of viral promoters with DC-specific promoters derived from major histocompatibility complex class II genes (MHC class II) (Kimura et al., 2007, Mol Ther 15(7):1390-9) and dectin-2 genes (Lopes et al., 2008, J Virol 82(1):86-95). The dectin-2 gene promoter used by Lopes et al. contains a putative enhancer and adenovirus conserved sequences (inverted terminal repeats in adenovirus promoters) (Bonkabara et al., 2001, J. Immunology, 167:6893-6900). The MHC class II gene promoter used by Kimura et al. does not contain any known enhancer.

[0229] However, in the absence of an enhancer, the MHC class II promoter was found to be unable to provide adequate transgene expression in DCs when administered intravenously. In particular, in contrast to the immune responses observed with the CMV promoter / enhancer, lentiviral vectors containing an MHC class II promoter did not elicit an immune response in immunocompetent C57BL / 6 mice. Although integration and sustained transgene expression were observed after injection in mice, lentiviral vectors transcribed via the MHC class II promoter failed to stimulate antigen-specific CD8+ cytotoxic T lymphocyte responses, even after vaccine boosting. Therefore, the authors of these studies concluded that the use of the MHC class II promoter is only of interest for applications seeking sustained expression in gene replacement therapy rather than in the context of immunotherapy. Notably, the MHC class II promoter is poorly expressed in most cell types.

[0230] Therefore, the MHC class II promoter is not a suitable promoter for lentiviral vectors used to induce immune responses to antigens by IV injection. In addition, the dectin-2 promoter is poorly expressed in most cell types and appears to contain enhancers. Therefore, for safety reasons, the dectin-2 promoter is not a good promoter for lentiviral vectors.

[0231] Therefore, the lentiviral vector according to the present invention may comprise an MHC class I promoter, ie, the nucleic acid sequence encoding the antigen of the lentiviral vector according to the present invention may be under the control of the MHC class I promoter.

[0232] A suitable MHC class I promoter may be selected from the group consisting of a β2-microglobulin promoter, an HLA-A2 promoter, an HLA-B7 promoter, an HLA-Cw5 promoter, an HLA-E promoter or an HLA-F promoter, more particularly a β2-microglobulin promoter.

[0233] MHC class I promoters are dendrite-specific (APC) because the expression of the promoters in BDCA+ dendritic cells is higher than in kidney, smooth muscle, liver and heart cells. They also have relatively high expression in other transduced cell types, for example, in BDCA+ dendritic cells, the expression of the promoter is only 12-100 times that of the promoter in skeletal muscle cells, compared to 900 times that of the MHCII HLA-DRα promoter.

[0234] The promoter specifically drives the transcription of the nucleic acid sequence encoding the HPV antigen in the lentiviral vector of the present invention.

[0235] The promoter may be a naturally occurring or synthetic MHC class I promoter, obtained using well-known molecular biology techniques.

[0236] The lentiviral vector according to the present invention may comprise a cPPT / CTS sequence, such as the cPPT / CTS sequence described in EP 2169073. The cPPT / CTS sequence may specifically be a sequence as shown in SEQ ID NO:37.

[0237] In fact, efficient integration and replication in non-dividing cells usually require the presence of two cis-acting sequences in the center of the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS). This leads to the formation of a three-stranded DNA structure called the central DNA "flap", which serves as a signal for uncoating the pre-integration complex at the nuclear pore and efficiently introducing the expression cassette into the nucleus of non-dividing cells such as dendritic cells.

[0238] The lentiviral vector of the present invention may comprise a woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE) that allows for more stable expression of a transgene in vivo, particularly a mutated form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE).

[0239] The mutant woodchuck posttranscriptional regulatory element (mWPRE) was characterized by introducing point mutations to avoid the expression of the X protein contained in the WPRE region, as the X protein may have oncogenic properties (Kingsman et al., Gene Ther. 2005 Jan;12(1):3-4).

[0240] The mutant form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE) contained in the lentiviral vector of the present invention may specifically have a nucleic acid sequence as shown in SEQ ID NO:38.

[0241] In a specific embodiment, the lentiviral vector according to the present invention, in particular the non-integrating lentiviral vector of the present invention:

[0242] (i) comprising at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen, at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen, at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen, and at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen;

[0243] (ii) contains a 3' long terminal repeat (LTR) lacking its U3 promoter sequence;

[0244] (iii) does not contain a constitutive enhancer sequence;

[0245] (iv) comprising an MHC class I promoter, in particular the β2-microglobulin promoter;

[0246] (v) comprising a cPPT / CTS sequence, which in particular has the sequence shown in SEQ ID NO: 37; and

[0247] (vi) comprising a mutated form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), which specifically has the nucleic acid sequence shown in SEQ ID NO:38.

[0248] As mentioned above, the lentiviral vector according to the present invention is characterized in that it comprises:

[0249] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen,

[0250] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen,

[0251] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen, and

[0252] - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen.

[0253] The at least four (particularly the four) different nucleic acid sequences encoding the HPV antigens of the lentiviral vector of the present invention can be particularly fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence, particularly (i) when there is no linker sequence (also referred to herein as a spacer) between each of the at least four different nucleic acid sequences, or (ii) when there is a linker sequence (or spacer) between at least two of the at least four different nucleic acid sequences, more particularly when there is a linker sequence (or spacer) between each of the at least four different nucleic acid sequences.

[0254] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7.

[0255] As described herein, an amino acid sequence having at least 80% amino acid identity to a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity to the reference amino acid sequence.

[0256] In particular, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may in particular have a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0257] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity to the reference nucleic acid sequence.

[0258] In a specific embodiment, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may specifically have a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0259] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of: an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0260] In a specific embodiment, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of: an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0261] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence having at least 68% sequence identity with the amino acid sequence shown in SEQ ID NO:16.

[0262] As described herein, an amino acid sequence having at least 68% amino acid identity to a reference amino acid sequence includes amino acid sequences having at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity to the reference amino acid sequence.

[0263] In particular, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may in particular have a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO:14 and SEQ ID NO:15.

[0264] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity to the reference nucleic acid sequence.

[0265] In a specific embodiment, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may specifically have a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO:14 and SEQ ID NO:15.

[0266] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of: an amino acid sequence as shown in SEQ ID NO:17 and SEQ ID NO:18.

[0267] In a specific embodiment, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of: an amino acid sequence as shown in SEQ ID NO:17 and SEQ ID NO:18.

[0268] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E6 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence having at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO:24.

[0269] As described herein, an amino acid sequence having at least 60% amino acid identity to a reference amino acid sequence includes amino acid sequences having at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity to the reference amino acid sequence.

[0270] In particular, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E6 antigen may in particular have a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

[0271] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity to the reference nucleic acid sequence.

[0272] In a specific embodiment, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E6 antigen may specifically have a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

[0273] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E6 antigen may in particular have a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of: an amino acid sequence as shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

[0274] In a specific embodiment, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E6 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of: an amino acid sequence as shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

[0275] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence having at least 83% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0276] As described herein, an amino acid sequence having at least 83% amino acid identity to a reference amino acid sequence includes amino acid sequences having at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity to the reference amino acid sequence.

[0277] In particular, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may in particular have a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0278] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity to the reference nucleic acid sequence.

[0279] In a specific embodiment, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may specifically have a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0280] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of: an amino acid sequence as shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0281] In a specific embodiment, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may specifically have a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of: an amino acid sequence as shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0282] As described above, the lentiviral vector according to the present invention comprises:

[0283] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen (also referred to herein as noE6-HPV16),

[0284] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen (also referred to herein as noE7-HPV16),

[0285] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen (also referred to herein as noE6-HPV18), and

[0286] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen (also referred to herein as noE7-HPV18).

[0287] In a specific embodiment, the lentiviral vector according to the present invention, in particular the non-integrating lentiviral vector of the present invention:

[0288] (i) comprising a nucleic acid sequence encoding a non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen; a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen; a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen and a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen;

[0289] The nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence, more particularly in the absence of any linker sequence between each of them;

[0290] (ii) contains a 3' long terminal repeat (LTR) lacking its U3 promoter sequence;

[0291] (iii) does not contain a constitutive enhancer sequence;

[0292] (iv) comprising an MHC class I promoter, in particular the β2-microglobulin promoter;

[0293] (v) comprising a cPPT / CTS sequence, which in particular has the sequence shown in SEQ ID NO: 37; and

[0294] (vi) comprising a mutated form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), which specifically has the nucleic acid sequence shown in SEQ ID NO:38.

[0295] The lentiviral vector according to the present invention may more specifically comprise:

[0296] - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen, the nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; the nucleic acid sequence is particularly selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;

[0297] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen, said nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 14 and SEQ ID NO: 15; said nucleic acid sequence is particularly selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 14 and SEQ ID NO: 15;

[0298] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen, said nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; said nucleic acid sequence is particularly selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; and

[0299] - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen, said nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32; said nucleic acid sequence is particularly selected from the group consisting of: a nucleic acid sequence as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32;

[0300] The nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence, particularly in the absence of any linker sequence between them.

[0301] In the lentiviral vector according to the present invention, in particular the non-integrating lentiviral vector of the present invention, the at least four (in particular four) different nucleic acid sequences encoding HPV antigens may be arranged in any order in the conventional 5' to 3' reading direction (from the 5' end to the 3' end).

[0302] Specifically, in the 24 possible combinations in the lentiviral vector according to the present invention, in particular the non-integrating lentiviral vector according to the present invention, the four different nucleic acid sequences encoding the HPV antigens noE6-HPV16, noE7-HPV16, noE6-HPV18 and noE7-HPV18 as defined above can be arranged in any order in the conventional 5' to 3' reading direction.

[0303] In a specific embodiment, the at least four different nucleic acid sequences are selected from the group consisting of:

[0304] (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen;

[0305] (b) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen;

[0306] (c) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen; and

[0307] (d) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen,

[0308] The nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence, particularly in the absence of any linker sequence between each of them.

[0309] These sequences are FIG. 8A to FIG. 8D shown.

[0310] In the lentiviral vector according to the present invention, the order of the at least four different nucleic acid sequences from the 5' end to the 3' end may more specifically be:

[0311] (d) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen,

[0312] The nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence, particularly in the absence of any linker sequence between each of them.

[0313] Preferably, in the lentiviral vector according to the present invention, the order of the at least four different nucleic acid sequences from the 5' end to the 3' end is:

[0314] (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen,

[0315] The nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence, particularly in the absence of any linker sequence between each of them.

[0316] More preferably, in the lentiviral vector according to the present invention, the order of the at least four different nucleic acid sequences from the 5' end to the 3' end is:

[0317] (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen,

[0318] The nucleic acid sequences encoding non-oncogenic HPV antigens are fused together without any linker sequence between them to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence.

[0319] These four different antigen constructs have been used in the following lentiviral vectors in this example:

[0320] - Lentiviral vector submitted to the Collection Nationale de Cultures de Microorganismes (CNCM) under accession number I-5759 on October 21, 2021 (sequence (a) above);

[0321] - Lentiviral vector submitted to the National Collection of Microorganisms (CNCM) under accession number I-5760 on October 21, 2021 (sequence (b) above);

[0322] - a lentiviral vector submitted to the National Collection of Microorganisms (CNCM) on October 21, 2021 under accession number I-5761 (sequence (c) above); or

[0323] - Lentiviral vector submitted to the National Collection of Microorganisms (CNCM) under accession number I-5762 on October 21, 2021 (sequence (d) above);

[0324] Therefore, the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5759 has the following nucleotide sequence as shown in SEQ ID NO:41:

[0325]

[0326] And encodes the following amino acid sequence as shown in SEQ ID NO:42:

[0327] MPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIASQAFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRSA GPGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWVGEPGRTIP YKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGH(SEQ ID NO:42)

[0328] The antigen construct of the lentiviral vector submitted to CNCM under accession number I-5760 has the following nucleotide sequence as shown in SEQ ID NO:43:

[0329]

[0330] And encodes the following amino acid sequence as shown in SEQ ID NO:44:

[0331] MFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRGPDDPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNT GLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIGPDDKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWA(SEQ ID NO:44)

[0332] The antigen construct of the lentiviral vector submitted to CNCM under accession number I-5761 has the following nucleotide sequence as shown in SEQ ID NO: 45:

[0333]

[0334] And encodes the following amino acid sequence as shown in SEQ ID NO:46:

[0335] MRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIA GHFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDL LIRCINCQKPLRFHNIRGRWTGRCMSCCRSAGPGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLAFQQLFLNTLSFVCPWAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIA(SEQ ID NO:46)

[0336] The antigen construct of the lentiviral vector submitted to CNCM under accession number I-5762 has the following nucleotide sequence as shown in SEQ ID NO: 47:

[0337]

[0338] And encodes the following amino acid sequence as shown in SEQ ID NO:48:

[0339] MGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPGEPGRTIPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGPGDT PTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIASQAFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRS(SEQ ID NO:48)

[0340] The lentiviral vector according to the present invention may particularly comprise a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:42, and the nucleic acid sequence is particularly the nucleic acid sequence SEQ ID NO:41.

[0341] As described herein, an amino acid sequence that is at least 90% identical to a reference amino acid sequence includes amino acid sequences that are at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical to the reference amino acid sequence.

[0342] Therefore, the lentiviral vector according to the present invention can more particularly be selected from the group consisting of lentiviral vectors submitted to the CNCM under accession number I-5759, I-5760, I-5761 and I-5762, in particular the lentiviral vector submitted to the CNCM under accession number I-5762. The lentiviral vector according to the present invention can preferably be a lentiviral vector submitted to the CNCM under accession number I-5759, and therefore preferably comprises the nucleic acid sequence SEQ ID NO: 41.

[0343] Lentiviral vector particles according to the present invention

[0344] Another object of the present invention relates to a lentiviral vector particle comprising at least one lentiviral vector according to the present invention, in particular at least one lentiviral vector as defined above.

[0345] The lentiviral vector particles of the present invention comprising the lentiviral vector of the present invention can be produced by recombinant techniques known in the art by transiently transfecting cells (e.g., HEK 293T human cultured cells) with the following different DNA plasmids:

[0346] (i) a packaging plasmid that expresses at least Gag, Pol, Rev, Tat and, in some cases, structural and enzyme proteins necessary for packaging the transfer construct;

[0347] (ii) a lentiviral vector according to the present invention, comprising an expression cassette (antigen) and HIV cis-acting factors necessary for packaging, reverse transcription and integration; and

[0348] (iii) An envelope encoding plasmid, in most cases the glycoprotein of vesicular stomatitis virus (VSV.G), a protein that allows the formation of hybrid particles (pseudotypes) that can target a variety of cells, particularly major histocompatibility (MHC) antigen presenting cells (APCs), including dendritic cells.

[0349] This method allows the production of recombinant vector particles according to the invention and comprises the following steps:

[0350] i) transfecting a suitable host cell with the lentiviral vector according to the present invention;

[0351] ii) transfecting the host cell with a packaging plasmid vector containing at least a viral DNA sequence encoding the structure and polymerase activity of a retrovirus (preferably a lentivirus); such packaging plasmids are described, for example, in the art (Dull et al., 1998, J Virol, 72(11):8463-71; Zufferey et al., 1998, J Virol 72(12):9873-80).

[0352] iii) culturing the transfected host cells to obtain expression of the lentiviral vector and packaging it into lentiviral vector particles; and

[0353] iv) harvesting the lentiviral vector particles produced by the expression and packaging in step iii) in the cultured host cells.

[0354] In order to pseudotype the retroviral particles of the invention, the host cells may be further transfected with one or several envelope DNA plasmids encoding one or more viral envelope proteins, preferably VSV-G envelope proteins.

[0355] This procedure allows the transient production of lentiviral particle vectors by transfecting cells. However, by stably inserting packaging genes, proviral encoding DNA and envelope genes into the cell genome, lentiviral particle vectors can also be continuously produced by cells. This allows cells to continuously produce lentiviral particle vectors without transient transfection. Of course, a combination of these methods can be used, in which some DNA / plasmids are integrated into the cell genome and others are provided by transient transfection.

[0356] The lentiviral vector particle can be a non-integrating lentiviral vector particle. The non-integrating vector particle has one or more mutations that eliminate most or all of the integration ability of the lentiviral vector particle. For example, the non-integrating vector particle can contain one or more mutations in the integrase encoded by the lentiviral pol gene, which mutations result in reduced integration ability.

[0357] The lentiviral vector particle according to the present invention particularly comprises the non-integrating lentiviral vector of the present invention.

[0358] The lentiviral vector particle according to the invention may comprise vesicular stomatitis virus glycoprotein (VSVG), in particular VSV-G Indiana serotype or VSV-G New Jersey serotype.

[0359] In terms of vaccination strategies, pseudotyped lentiviral vector particles are more likely to escape the immune system when the immune system has already developed immunity to the lentivirus. This is particularly useful when serial injections of similar particle vectors are needed to immunize a patient against a disease.

[0360] The lentiviral vector particles may comprise HIV-1 Gag and Pol proteins, in particular HIV-1 subtype D Gag and Pol proteins.

[0361] Another object of the present invention relates to an isolated cell comprising (ie transformed with) a lentiviral vector according to the present invention or a lentiviral vector particle according to the present invention.

[0362] The cells according to the invention are preferably mammalian cells, in particular human cells. Particularly preferred are non-dividing human cells.

[0363] Another object of the present invention relates to a vaccine composition comprising a lentiviral vector according to the present invention, a lentiviral vector particle according to the present invention or a cell according to the present invention.

[0364] The vaccine composition according to the present invention comprises a pharmaceutically acceptable medium.

[0365] "Pharmaceutically acceptable medium" refers to any solution used to dissolve and deliver the lentiviral vector, lentiviral vector particles or cells according to the present invention to an individual. An ideal pharmaceutically acceptable carrier is saline. In an ideal embodiment, the pharmaceutically acceptable medium includes an adjuvant.

[0366] Suitable physiologically acceptable media and their formulation are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, (20th edition), ed. A. Gennaro, 2003, Lippincott Williams & Wilkins.

[0367] According to the embodiment of the present invention

[0368] One object of the present invention relates to the use of the lentiviral vector of the present invention, the lentiviral vector particle of the present invention or the isolated cell of the present invention as a medicament or vaccine.

[0369] In particular, an object of the present invention relates to a lentiviral vector according to the invention, a lentiviral vector particle according to the invention or an isolated cell according to the invention, in particular in the form of a vaccine composition according to the invention, for use in the treatment or prevention of HPV-induced cancers and their metastases, in particular HPV-induced cancers.

[0370] As mentioned above, HPV-induced cancer is a cancer induced by HPV infection. Methods for detecting HPV in cancer are known in the art (Aldo Venuti and Francesca Paolini; Head Neck Pathol. 2012 Jul;6(Suppl 1):63-74).

[0371] The HPV-induced cancer may in particular be selected from the group consisting of cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer and oropharyngeal cancer.

[0372] Metastases of such cancer according to the invention may in particular be lung metastases.

[0373] Such prevention and / or treatment implies the administration of the active substance in question, in particular the vaccine composition of the invention as defined above, to an individual in need thereof.

[0374] The individual in need is an animal, particularly a mammal, more particularly a human.

[0375] The lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the present invention are administered to an individual in need thereof by conventional methods at a dose sufficient to elicit an immune response, which dose can be readily determined by a person skilled in the art.

[0376] Thus, the lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the present invention may be administered intravenously or intramuscularly as shown below.

[0377] The lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the invention can also be administered intranasally. This route of administration is particularly suitable for treating or preventing oropharyngeal cancer and / or lung metastasis.

[0378] The lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the present invention are administered in a therapeutically effective amount, and in particular may be administered in an amount corresponding to at least 1×10 6 , 2×10 6 , 5×10 6 , 10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 or at least 1×10 9 The dose of the lentiviral vector of the invention of TU (transducing unit), in particular, corresponds to at least 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 TU or at least 1×10 9 In a preferred embodiment, the lentiviral vector, lentiviral vector particles, cells and vaccine compositions according to the present invention are administered at a dose corresponding to at least 1×10 7 TU of the present invention, more particularly at least 1×10 8 TU of the present invention, particularly at least 1×10 9 TU of lentiviral vector of the present invention is administered at a dose of 100 TU.

[0379] A "therapeutically effective amount" refers, for example, to the amount of a lentiviral vector or lentiviral vector particle, cell or vaccine composition of the invention required to produce one or more of the following effects in a subject: an immune response against an HPV-induced tumor; a reduction in the size of an HPV-induced tumor (i.e., a reduction in tumor size of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% within 15 to 45 days compared to the tumor size at the time of administration); an increase in CD8+ and / or CD4+ infiltration in HPV-induced tumors 5 to 45 days after administration; a decrease in CD25+FoxP3+CD4+ regulatory T cells (Tregs) in HPV-induced tumors 5 to 45 days after administration.

[0380] Administration can be carried out using known routes including, for example, intravenous, intramuscular, intranasal, intraperitoneal or subcutaneous injection, particularly intravenous, intranasal or intramuscular injection, and may be intravenous or intramuscular injection.

[0381] Appropriate doses and regimens will vary significantly between species and individuals, depending on many factors. For example, higher doses are generally required to achieve an effective immune response in humans compared to mice.

[0382] The lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the invention can be administered, for example, in a single dose (as shown in the examples), or in two or more doses. The practitioner will determine in each case the appropriate regimen and dosage for administering the active substances of the invention.

[0383] The lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the present invention may advantageously be administered in combination with at least one immune checkpoint inhibitor (ICI).

[0384] The immune checkpoint inhibitor (ICI) according to the present invention can be an antibody, in particular, anti-PD-1, anti-PD-L1 (PD-1 ligand), anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4), anti-NKG2A, anti-TIM-3 (containing T cell immunoglobulin and mucin domain-3), anti-TIGIT (T cell immune receptor with Ig and ITIM domains) or anti-LAG-3 (lymphocyte activation gene 3) antibody. More particularly, the at least one immune checkpoint inhibitor according to the present invention can be a monoclonal antibody selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-NKG2A, anti-TIM-3, anti-TIGIT and anti-LAG-3 monoclonal antibodies. Even more particularly, at least one immune checkpoint inhibitor according to the present invention can be a monoclonal antibody selected from the group consisting of: anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-NKG2A, anti-TIM-3 and anti-TIGIT monoclonal antibodies.

[0385] The immune checkpoint inhibitor (ICI) according to the present invention can more particularly be an antibody, in particular an anti-PD-1, anti-PD-L1 (PD-1 ligand), anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4), anti-NKG2A, anti-TIM-3 (T cell immunoglobulin and mucin domain-3), anti-TIGIT (T cell immunoreceptor with Ig and ITIM domains) or anti-LAG-3 (lymphocyte activation gene 3) antibody, and even more particularly an antibody, in particular an anti-PD-1, anti-PD-L1 (PD-1 ligand), anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4), anti-NKG2A, anti-TIM-3 (T cell immunoglobulin and mucin domain-3) or anti-TIGIT (T cell immunoreceptor with Ig and ITIM domains) antibody. More particularly, at least one immune checkpoint inhibitor according to the present invention may be a monoclonal antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-NKG2A, anti-TIM-3, anti-TIGIT and anti-LAG-3 monoclonal antibodies, and in particular may be a monoclonal antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-NKG2A, anti-TIM-3 and anti-TIGIT monoclonal antibodies.

[0386] The anti-PD-1 monoclonal antibody may, for example, be selected from the group consisting of nivolumab, pembrolizumab and cemiplizumab.

[0387] The anti-PD-L1 monoclonal antibody may, for example, be selected from the group consisting of atezolizumab, avelumab and durvalumab.

[0388] The anti-CTLA-4 monoclonal antibody can, for example, be selected from the group consisting of ipilimumab, tesimumab and quavonlimab.

[0389] NKG2A is a receptor with ITIM (intracytoplasmic tyrosine-based inhibitory motif) expressed on the surface of 50% of peripheral blood NK cells and 5% of human peripheral blood CD8+T cells. This cell surface molecule is expressed as a heterodimer with CD94 and interacts with non-classical molecules of major histocompatibility complex class I (MHC-I), namely human leukocyte antigen (HLA)-E in and mouse Qa-1b. This interaction inhibits T effector anti-tumor function and NK effector anti-tumor function (Andre et al., Cell. 2018 December 13; 175 (7): 1731-1743.e13). It has been described that anti-NKG2A mAbs can be used as checkpoint inhibitors and can promote anti-tumor cellular immunity by releasing not only NK cells but also CD8+T cells in mice (Andre et al., Cell. 2018 December 13; 175 (7): 1731-1743.e13). The anti-NKG2A monoclonal antibody can be, for example, monalizumab.

[0390] T cell immunoglobulin and mucin domain-3 (TIM-3) is a negative regulatory immune checkpoint. TIM-3 is expressed by various immune cells (especially T cells). TIM-3 has four ligands, including galectin-9 (Gal-9), carcinoembryonic antigen cell adhesion molecule 1 (CEACAM-1), high mobility group protein B1 (HMGB1) and phosphatidylserine (PS) (He et al., OncoTargets Ther. 2018; 11: 7005-7009). TIM-3 / Gal-9 inhibits cancer immunity by negatively regulating T cell immunity. TIM-3 plays an important role in T cell exhaustion. In cancer immunotherapy, anti-TIM-3mAb treatment shows beneficial effects comparable to anti-PD-1mAb therapy. Anti-TIM-3 monoclonal antibodies can be selected, for example, from the group consisting of Sym023 and sabatolimab.

[0391] The anti-TIGIT monoclonal antibody can be, for example, tiragolumab.

[0392] The anti-LAG-3 monoclonal antibody can be, for example, relatlimab.

[0393] In particular, the ICI may be an anti-PD-L1 or anti-PD-1 monoclonal antibody, in particular an anti-PD-1 monoclonal antibody.

[0394] In particular, the ICI can be selected from the group consisting of anti-PD-L1, anti-NKG2A, anti-TIM-3 and anti-PD-1 monoclonal antibodies, especially the group consisting of anti-NKG2A, anti-TIM-3 and anti-PD-1 monoclonal antibodies.

[0395] The vaccine composition, lentiviral vector, lentiviral vector particle or cell, and immune checkpoint inhibitor for use according to the present invention may be administered simultaneously or separately.

[0396] Taking into account the unexpected synergistic properties obtained when combining the lentiviral vector according to the present invention with immune checkpoint inhibitors (as demonstrated in the Examples), it can be expected that vaccination with the lentiviral vector according to the present invention can increase the number of patients eligible for immune checkpoint inhibitor therapy (especially anti-PD-1, anti-NKG2A or anti-TIM-3).

[0397] "Simultaneously" should be understood as (i) the vaccine composition, lentiviral vector, lentiviral vector particles or cells and (ii) the immune checkpoint inhibitor can be administered at the same time or up to the same day or days. In this case, they can be administered in the same composition or in separate compositions.

[0398] Separate is to be understood as: (i) the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the present invention and (ii) the immune checkpoint inhibitor may be administered at least a few days (eg at least two days) apart.

[0399] In particular, when (i) a vaccine composition, a lentiviral vector, a lentiviral vector particle or a cell and (ii) an immune checkpoint inhibitor are administered separately, the vaccine composition, the lentiviral vector, the lentiviral vector particle or a cell according to the present invention can be administered before the immune checkpoint inhibitor.

[0400] Advantageously, the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the present invention can be administered at least 2 days, in particular at least 4 days, before the administration of the immune checkpoint inhibitor. Therefore, the immune checkpoint inhibitor can be advantageously administered at least 2 days, in particular at least 4 days, after the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the present invention. The immune checkpoint inhibitor can more particularly be administered 4 days to 1 month after the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the present invention, in particular 4 days to 15 days, more particularly 4 days to 10 days after the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the present invention.

[0401] The vaccine composition, lentiviral vector, lentiviral vector particle or cell, and immune checkpoint inhibitor for use according to the present invention may be administered by the same route or different routes.

[0402] At least one immune checkpoint inhibitor described herein is administered in a therapeutically effective dose (i.e., a dose that produces the effect of its administration). The exact dosage of the immune checkpoint inhibitor will depend on the purpose of treatment and can be determined by those skilled in the art using known techniques.

[0403] The present invention also relates to a method for treating and / or preventing HPV-induced cancer in an individual in need thereof, comprising administering to said individual at least one lentiviral vector of the invention, a lentiviral vector particle of the invention or an isolated cell of the invention, in particular in the form of a vaccine composition according to the invention.

[0404] The present invention also relates to the use of at least one lentiviral vector of the invention, the lentiviral vector particle of the invention or the isolated cell of the invention for treating and / or preventing HPV-induced cancer in an individual in need thereof, in particular in the form of a vaccine composition according to the invention.

[0405] The following examples and figures are given by way of illustration and are not meant to limit the present invention. Example

[0406] Materials and methods

[0407] Mouse

[0408] C57BL6jRj mice were purchased from Janvier Laboratories (Le Genest-Saint-Isle, France). All animals were kept under specific pathogen-free conditions, and all procedures were performed according to approved animal protocols and recommendations for the proper use and care of laboratory animals. All animal experiments were performed according to the guidelines established by French and European regulations on the care and use of laboratory animals.

[0409] Peptides, antibodies and reagents

[0410] To test the reactivity of the vaccine according to the present invention, 15-mer overlapping peptides with a purity of ≥80% were ordered from GenScript Biotech (Netherlands). Anti-CD4-VioBlue (clone REA604), anti-CD45-VioGreen (clone REA737), anti-FoxP3-Vio515 (clone REA788), anti-CD279 (PD1)-PE (clone REA802), anti-CD8a-PE-Vio770 (clone REA601), anti-CD25-APC (clone REA568), anti-CD11c-FITC (clone REA754), anti-CD11b-APC-Vio770 (clone REA592) were purchased from Miltenyi Biotec. Anti-anti-mouse H-2kb (clone AF6-88.5), anti-CD274 (PD-L1)-APC (clone MIH5) and anti-CD16 / CD32 (clone 2.4G2) were purchased from BD Biosciences.

[0411] The antibodies were mixed with PBS containing 1% FCS (Gibco).

[0412] Cyclophosphamide was purchased from Sigma, resuspended in PBS (Gibco), and stored at -20°C before use.

[0413] cell

[0414] TC-1 tumor cells expressing HPV-16 E6 and E7 were generated as described previously (Lin et al. Cancer Res. 1996 Jan 1; 56(1): 21-6): primary lung cells of C57BL6 mice were transformed with HPV-16 E6 and E7 genes and activated human c-Ha-ras oncogene expressing pVEJB. TC-1 cell line was cultured in Glutamax RPMI medium (Gibco supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin and 10% fetal bovine serum).

[0415] Lentiviral vector construction

[0416] The antigen (Ag) construct is cloned into the pFlap-B2m-Ag-WPREMutee backbone (for the backbone, see, e.g., WO2016012623). The antigen plasmid contains a cPPT / CTS sequence (sequence SEQ ID NO: 37), which is essential for the transduction of non-mitotic cells. The U3 promoter sequence is deleted from the 3' long terminal repeat (LTR) to avoid vector replication. The β-2 microglobulin (β2m) promoter controls vaccine antigen expression in all transduced cells, so the antigen will be preferentially expressed in APCs (antigen presenting cells). In addition, it lacks any known enhancer sequences that may induce mutagenic and / or genotoxic effects. The antigen plasmid contains a mutant form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE) (sequence SEQID NO: 38). The wild-type WPRE region contains a truncated form of the WHV X protein that may have oncogenic properties (Kingsman et al., Gene Ther. 2005 Jan;12(1):3-4). The mutant form of WPRE used in our constructs precludes expression of the truncated X protein by including a point mutation in the X protein start codon. This mutant WPRE sequence does not appear to be oncogenic (Themis et al., Mol Ther. 2005 Oct;12(4):763-71).

[0417] The packaging plasmid (pNDK) contains the gag-pol sequence from HIV-1 subtype NDK (GenBank accession number: A34828). Nef, vif, vpr and env proteins are not expressed. In addition, the replacement of aspartic acid (D) at position 64 in the HIV-1 integrase protein sequence (pol gene) with valine (V) (D64V) is sufficient to inhibit integration without interfering with the in vitro expression of the transgene. The lentiviral particles according to the present invention are non-integrating particles.

[0418] Envelope plasmids: pCMV-VSV-G INDco (Indiana) and pCMV-VSV-G Njco (New Jersey) vectors were constructed by subcloning the vesicular stomatitis virus (VSV) G protein (VSV-G) Indiana (GenBank Accession No. J02428) and New Jersey (GenBank Accession No. P04882) serotype inserts into the pVAX1 expression vector (Invitrogen). Mammalian codon-optimized synthetic genes (GeneArt) encoding glycoproteins from the following vesicular viruses were cloned into the pVAX1 plasmid (Invitrogen): vesicular stomatitis virus Indiana serotype (GenBank FW591952), New Jersey serotype (GenBank FW591956), and Cocal virus (GenBank: AF045556.1).

[0419] Production of lentiviral vector particles

[0420] After HEK 293T cells (ATCC) were amplified in DMEM containing 1% penicillin / streptomycin and 10% FCS, HEK 293T cells (ATCC) were transiently co-transfected with three plasmids (viral antigen plasmid, envelope expression plasmid, and packaging plasmid) by calcium phosphate to produce non-integrated lentiviral particles according to methods well known in the art. After 24 hours, the culture medium was replaced with serum-free medium. 48 hours after transfection, the supernatant was collected and clarified by centrifugation at 2500 rpm. The viral particles were concentrated by ultracentrifugation (1 hour at 22000 rpm / 88250g 4°C) and resuspended in preservative buffer (20 mM Pipes, 75 mM NaCl and 2.5% sucrose).

[0421] Carrier titration

[0422] After transduction of cells (HEK 293T), lentiviral vector titers were determined by quantitative PCR. Aphidicolin was added to HEK293T cells 24h before transduction and maintained throughout the titration process. The cells were incubated with lysis buffer (200mM Tris, 1% NP40 and 1% Tween20) containing 50μg / ml RNase A (sigma) for 30min. Proteinase K (0.2mg / ml) was added to the suspension and incubated at 56°C for 4h. Quantitative PCR was performed using primer pairs specific for RRE (element of the Ag vector) and GAPDH (in the host cell). Titers are expressed in transduction units (TU) / mL of vector.

[0423] HPV vaccine design

[0424] The implemented non-oncogenic immunogenic E6 and E7 protein sequences were selected and modified as previously described herein.

[0425] In particular, 4 different vaccines were designed, which contained the following lentiviral vectors:

[0426] - Lentiviral vector submitted to the National Collection of Microorganisms (CNCM) under accession number I-5759 on October 21, 2021;

[0427] - Lentiviral vector submitted to the National Collection of Microorganisms (CNCM) under accession number I-5760 on October 21, 2021;

[0428] - a lentiviral vector submitted to the National Collection of Microorganisms (CNCM) under accession number I-5761 on October 21, 2021; or

[0429] -Lentiviral vector submitted to the National Collection of Microorganisms (CNCM) on October 21, 2021 under accession number I-5762.

[0430] Lentiviral vector particles containing these functional lentiviral vectors were utilized in the following examples, which were quantified by vector titration and expressed in transducing units (TU).

[0431] In vivo immunogenicity of lentiviral vector vaccines (LV vaccines)

[0432] Naive C57BL6 female mice were vaccinated with the LV vaccine according to the invention (i.e., the lentiviral vector particles of the invention containing the functional lentiviral vector of the invention) by intramuscular (im) injection in 50 μL of diluent. After 14 days, splenocytes were prepared and restimulated overnight with 4 different HPV peptide pools (finally 2 μg / mL for each peptide) to obtain IFNγ ELISPOT. Each peptide pool corresponds to one of the following non-oncogenic antigen variants: a non-oncogenic variant of the E6 protein of HPV16, a non-oncogenic variant of the E7 protein of HPV16, a non-oncogenic variant of the E6 protein of HPV17, and a non-oncogenic variant of the E7 protein of HPV17. They consist of overlapping 15-mers (with an overlap of 11 amino acids) corresponding to the complete selected antigen.

[0433] In vivo tumor vaccination therapy

[0434] For in vivo tumor experiments, 1.10 6TC-1 cells were injected subcutaneously (sc) into the right flank of 7-9 week old C57BL / 6 mice (the mice were shaved with an electric razor before injection). When the mean tumor volume reached the expected range, the mice were randomized and injected with the LV vaccine of the invention by intramuscular (im) injection. The mice were monitored for tumor growth by measuring the tumor diameter with a caliper 3 times per week. For ethical reasons, tumors > 1500 mm 3 The mice were killed painlessly.

[0435] Immunogenicity of the LV vaccine of the present invention in human PBMC

[0436] Frozen human PBMC (StemCell) were gently thawed and stained with 0.5 μM CFSE (Thermofischer) at 37°C for 10 min. 6 Cells were cultured in complete RPMI: 10% FCS, 10 mM Hepes (Gibco), 100 U / ml penicillin, 100 μg / ml streptomycin, 0.1 mM non-essential amino acids (Gibco) and 1 mM sodium pyruvate (Gibco) in 1% 10 cells. After 7 days, the cells were centrifuged and added to new complete RPMI (pre-warmed). After another 7 days (14 days in total), the cells were stained with fluorescent antibodies and data were obtained by flow cytometry (MACSQuant analyzer).

[0437] Cytometric analysis of tumor immune infiltrates

[0438] Tumors were treated with the Mouse Tumor Dissociation Kit (Miltenyi). The cell suspension was then filtered through a 70 μm pore filter, treated with red blood cell lysis buffer (Sigma), then washed and centrifuged at 1200 rpm for 5 minutes. The recovered cells were stained as follows.

[0439] For detection of NK, Near IR LD (Invitrogen), FcγII / III receptor blocking anti-CD16 / CD32 (clone 2.4G2, BD Biosciences), APC anti-CD11b (clone N418, BD Biosciences), BV421 anti-NKp46 (clone 29A1.4, Biolegend) were used.

[0440] Samples were acquired in an Attune NxT cell counter (Invitrogen), and data were analyzed by FlowJo software (Treestar, OR, USA).

[0441] Intracellular cytokine staining

[0442] Splenocytes from immunized mice were obtained by homogenization and passage through a 100-μm nylon filter (Cell Strainer, BD Biosciences) and plated at 4 × 10 6 Cells / well were plated in 24-well plates. Splenocytes were stimulated for 6 h in the presence of 10 μg / mL homologous or control peptides, 1 μg / mL anti-CD28 (clone 37.51) and 1 μg / mL anti-CD49d (clone 9C10-MFR4.B) mAbs (BD Biosciences). In the last 3 h of culture, cells were treated with a mixture of Golgi Plug and Golgi Stop, both from BD Biosciences. PE-Cy7-anti-CD107a (clone 1D4B, BioLegend) mAbs were also added to the culture in this step. The cells were then collected, washed with PBS containing 3% FBS and 0.1% NaN3 (FACS buffer), and incubated with a mixture of Near IR Live / Dead (Invitrogen), FcγII / III receptor blocking anti-CD16 / CD32 (clone 2.4G2), PerCP-cy5.5-anti-CD3ε (clone 145-2C11), PE-Cy7-anti-CD4 (clone RM4-5) and BV711-anti-CD8 (clone 53-6.7) mAbs (BD Biosciences or eBioscience) for 25 min at 4° C. The cells were washed twice in FACS buffer and then permeabilized by using a Cytofix / Cytoperm kit (BD Bioscience). The cells were then washed twice with PermWash 1X buffer from the Cytofix / Cytoperm kit and incubated with a mixture of BV421-anti-IL-2 (clone JES6-5H4), FITC-anti-TNF (MP6-XT22), APC-anti-IFN-γ (clone XMG1.2), and BV605-anti-IL-17A (clone TC11-18H10) mAbs (BD Biosciences) or a mixture of appropriate control Ig isotypes at 4°C for 30 min. The cells were then washed twice in PermWash and once in FACS buffer before being fixed overnight with Cytofix (BD Biosciences) at 4°C. Cells were acquired in an Attune NxT cell counter system (Invitrogen) and data analysis was performed using FlowJo software (Treestar, OR, USA).

[0443] Example 1: The HPV vaccine of the present invention is immunogenic in vivo

[0444] To measure the ability of the vaccines of the present invention to induce an immune response, recipient mice (a group of 5 mice per test vaccine group and control group) were immunized with the four vaccines.

[0445] Mice were injected intramuscularly with 1×10 7 TU of lentiviral vector particles or 50 μL of diluent, the lentiviral vector particles containing the lentiviral vector submitted to CNCM with accession number I-5759, the lentiviral vector submitted to CNCM with accession number I-5760, the lentiviral vector submitted to CNCM with accession number I-5761, or the lentiviral vector submitted to CNCM with accession number I-5762. After 14 days, splenocytes were prepared and restimulated overnight with 4 different peptide pools (each peptide was 2 μg / mL in the end) to obtain IFNg ELISPOT. The results obtained are as follows Figure 1 shown.

[0446] Example 2: The HPV vaccine of the present invention completely eliminates fully implanted tumors in vivo

[0447] TC-1 tumor cells have been widely used as a preclinical model for studying HPV-induced tumors (Kim, JW et al. Genetherapy Vol. 11 No. 12 (2004): 1011-8). These lung tumor cells were modified to express E6 and E7 from HPV16 (Lin et al. Cancer Res. 1996 Jan 1; 56(1): 21-6).

[0448] After sc (subcutaneous) injection of TC-1 cells into mice, solid tumors were rapidly found at the injection site, and tumors in untreated animals grew to ethical endpoints within 30-40 days. To test the efficacy of the lentiviral vector particles of the present invention, TC-1 cells were injected sc and tumor volumes were measured every other day (caliper measurements). When the average tumor volume was 70 mm 3 At 1 pm, the mice were randomly divided into groups and intramuscularly inoculated with 1×10 8 TU's LV-GFP Indiana (as a control), Indiana lentiviral vector particles containing I-5759, Indiana lentiviral vector particles containing I-5760, Indiana lentiviral vector particles containing I-5761, or Indiana lentiviral vector particles containing I-5762.

[0449] The results obtained are as follows Figure 2 shown.

[0450] Rapid and very efficient tumor elimination was observed in 100% of animals vaccinated with lentiviral vector particles (including the lentiviral vector submitted to the CNCM under Accession No. I-5762 and the lentiviral vector submitted to the CNCM under Accession No. I-5759), 87.5% of animals vaccinated with the lentiviral vector submitted to the CNCM under Accession No. I-5760, and 75% of animals vaccinated with the lentiviral vector submitted to the CNCM under Accession No. I-5761.

[0451] Vaccines containing lentiviral vector particles containing the lentiviral vector submitted to the CNCM under accession number I-5759 and lentiviral vector particles containing the lentiviral vector submitted to the CNCM under accession number I-5762 showed equivalent tumor elimination rates (higher than I-5760 and I-5761), but I-5759 vaccination was able to completely eliminate tumors in an average of 37.5 days (+ / - 7.4SD), while it took 54.7 days after I-5762 vaccination.

[0452] We surprisingly observed that the most immunogenic vectors were not the most protective and that the ranking of immunogenicity was not applicable to antitumor efficacy. In fact, when measuring IFN-γ production, the lentiviral vector particles containing the lentiviral vector submitted to the CNCM under accession number I-5759 were the most efficient antitumor vaccine, while the lentiviral vector particles containing the lentiviral vectors submitted to the CNCM under accession numbers I-5760 and I-5762 were more immunogenic vectors.

[0453] Example 3: Administration of a single HPV vaccine of the present invention is highly effective in combating tumor recurrence

[0454] Relapse is common in most cancer types and is defined as the return of the disease after a period of improvement. This is usually due to some tumor cells surviving the initial treatment and forming new tumors weeks, months or even years after treatment.

[0455] A. To mimic relapse in our model, mice that had their primary tumors eliminated were re-challenged in the other flank on day 60. Control mice (untreated) were also injected sc to examine tumor cell injection.

[0456] The results obtained are as follows Figure 3 shown.

[0457] This figure shows that sc injection of TC-1 cells in control mice was able to form solid tumors reaching ethical size limits in less than 30 days. Tumor growth was significantly reduced in re-challenged mice (which had previously eliminated right flank tumors after vaccination). Tumor growth was observed in the first 6 days, after which tumor elimination began.

[0458] Tumors were completely eliminated even 13-16 days after implantation. A single dose of vaccination in tumor-bearing mice completely eliminated primary tumors and was highly protective against recurrence, as these mice rapidly eliminated secondary tumors.

[0459] B. By using 1.10 119 days after the first transplant 6 Additional experiments were performed by re-challenging mice that had their primary tumors eliminated in the other flank with TC-1 tumor cells and remaining without any treatment. Control mice (untreated) were also injected sc to examine tumor cell injection.

[0460] The results obtained are as follows Fig.11 shown.

[0461] All re-challenged mice were still alive 145 days after the initial tumor challenge, demonstrating that a single injection of the vaccine according to the invention effectively promoted a strong anti-tumor memory protective immune response that efficiently shaped the T cell response to a new challenge.

[0462] Example 4: The therapeutic effect of the anti-HPV vaccine of the present invention is dose-dependent

[0463] To determine the effect of a lower dose of the vaccine, an efficacy study was performed in mice bearing TC1 tumors. 8 or 1×10 7 TU / Mice were vaccinated with a vaccine according to the invention comprising lentiviral vector particles comprising the lentiviral vector submitted to the CNCM under accession number I-5759.

[0464] Specifically, 1×10 6 TC-1 cells were injected into the flank of the animals, and the tumor volume was measured twice a week (caliper measurement). When the average tumor volume was 80 mm3, the mice were randomly divided into groups and treated with diluent (control), 1×10 7 TU or 1×10 8 I-5759 vaccination at TU(im).

[0465] The results obtained are as follows Figure 4 shown.

[0466] Use 1×10 8 TU vaccination resulted in complete and rapid tumor elimination (less than 20 days after vaccination), whereas we observed 7 The vaccination dose of TU had a partial effect on tumor growth. 3 / 6 (50%) mice were tumor-free 22 days after vaccination, and the remaining mice showed a first decrease 15-18 days after vaccination (within 5-10 days), but after that tumor growth was uncontrollable.

[0467] A single low dose (1×10 7 ) showed a partial inhibitory effect, which is comparable to the inhibitory effect observed for three injections of adenoviral vector-based vaccines (Rice, AE et al. Cancer gene therapy Vol. 22 No. 9 (2015): 454-62). This suggests that the optimal dose of the vaccine according to the present invention will be more efficient than the adenoviral platform. In addition, the second injection of the vaccine is likely to improve the efficacy of the low dose.

[0468] Example 5: Vaccination according to the invention increases CD4 + and CD8 + T cell infiltration and reduced the Tregs in tumors

[0469] Tumor infiltration was studied to further understand the anti-tumor mechanism induced after vaccination with the lentiviral vector according to the present invention. 6 TC1 tumor cells were injected (sc) into the flank of the animals, and tumor volume was measured twice a week (caliper measurement). 3 At 1 , mice were randomly divided into groups and inoculated with diluent (control) or 1 × 10 7 TU I-5759 or 1×10 8 I-5759 from TU(im).

[0470] Ten days after vaccination, tumors were harvested, digested and analyzed by flow cytometry. FACS staining was performed and data were acquired on Macsquant facs according to methods well known in the art.

[0471] The results obtained are as follows Figure 5 shown.

[0472] Compared with control tumors, tumors from vaccinated mice were infiltrated with more CD8 + T cells and CD4 + T cells. CD8 in tumors + T cells and CD4 + The percentage of T cells increased by about 4-fold and 3-fold, respectively. On the other hand, the percentage of CD25+FoxP3+CD4+ regulatory T cells (Treg) in the tumors was significantly reduced in the treated animals.

[0473] These observations are very important because they show that vaccines comprising the lentiviral vector of the present invention increase CD8 + T cells and CD4 + This reduced the recruitment of T cells to tumors and decreased the percentage of Tregs in tumors.

[0474] Example 6: The HPV vaccine of the present invention completely eliminated large tumors in vivo

[0475] Well-established tumors are known to be more difficult to eliminate than small and early-stage tumors. Most vaccines tested on the TC1 model have weaker effects when administered at later time points (Rice, AE et al. Cancer gene therapy Vol. 22 No. 9 (2015): 454-62; Berraondo, Pedro et al. Cancer research Vol. 67 No. 18 (2007): 8847-55). 6 TC1 tumors were injected (sc) into the flank of the animals. When the average tumor volume was approximately 300 mm 3 The mice were randomly divided into groups and treated with diluent (control) or 1×10 8 TU(im) vaccination according to the invention, said vaccine comprising lentiviral vector particles comprising the lentiviral vector submitted to the CNCM under accession number 1-5759. Tumor volume was measured twice a week with a caliper.

[0476] The results obtained are as follows Figure 6 shown.

[0477] It can be seen that the vaccine according to the invention is highly effective in completely eliminating well-established HPV-induced tumors.

[0478] Example 7: HPV vaccine of the present invention induces human PBMC activation in vitro

[0479] In order to confirm that the vaccine according to the present invention can induce T cell response in human cells, human PBMC (StemCell) were labeled with CFSE and cultured in the absence (unstimulated condition) or presence of the vaccine according to the present invention (I-5759). Cell proliferation and activation were measured after 2 weeks of culture.

[0480] By adding the lentiviral vector of the present invention to the culture, CD8 + T cells and CD4 + T cell proliferation (measured by CFSE dilution) and expression of the CD25 activation marker were increased.

[0481] The results obtained are as follows Figure 7 shown.

[0482] It can be concluded that antigen-presenting cells from PBMCs can be transduced by the HPV vaccine according to the present invention and process antigens to activate T cells.

[0483] Example 8: HPV vaccines of the present invention induce systemic T cell immunity and phenotypes of effector T cells

[0484] To gain further insights into the quality of the induced T cell responses, splenocytes from mice injected with Ctrl Lenti (LV-empty Indiana) or the vaccine according to the invention (comprising lentiviral vector particles containing the lentiviral vector submitted to the CNCM under accession number I-5759) were either left untreated or in vitro treated with ETTDPD containing the immunodominant H-2Db-restricted RAHYNIVTF epitope. RAHYNIVTF and PD RAHYNIVTF CCKC E7 HPV16 Cells were stimulated with a mixture of peptide derivatives (Feltkamp MC et al. Eur J Immunol 1993; 23: 2242-9) and analyzed by intracellular staining (ICS) for IL-2, TNF-α and IFN-γ.

[0485] The results obtained are as follows Fig. 9 As shown in A.

[0486] In mice vaccinated with the lentiviral vector according to the present invention, CD8 + T cell response. Functional CD8 + T cell effectors are mainly distributed in IFN-γ + (single positive), TNF-α + IFN-γ + or IL-2 + IFN-γ + (double positive), IL-2 + TNF-α + IFN-γ + (Triple positive) subgroup (see Fig. 9 B).

[0487] Most IFN-γ + CD8 + T cells also express the surface CD107a degranulation marker, indicating the effector properties of these T cells (see Fig. 9 B).

[0488] Example 9: Tumor cells and tumor-infiltrating innate immune cells in mice vaccinated with the HPV vaccine of the present invention Features

[0489] Characterization of the intratumoral infiltrates by cytometry on day 11 after vaccination and thus during the tumor regression phase showed a significant increase in the proportion of natural killer (NK) cells in the regressed tumors from mice vaccinated with the vaccine of the invention comprising lentiviral vector particles comprising the lentiviral vector submitted to the CNCM under accession number I-5759.

[0490] The results are as follows Fig.10 shown.

[0491] Example 10: Suboptimal Lenti-HPV-07 vaccination synergizes with anti-PD1 immunotherapy

[0492] The present inventors further investigated the potential synergy between suboptimal doses of Lenti-HPV-07 (I-5759) vaccination and anti-PD-1 therapy.

[0493] Anti-PD-1 treatment was started four days after injection of the vaccine according to the invention (D13, i.e. 13 days after sc administration of TC-1 cells to mice) (D17, i.e. 17 days after sc administration of TC-1 cells to mice), the vaccine comprising lentiviral vector particles comprising the lentiviral vector submitted to CNCM under accession number I-5759. Several injections of anti-PD-1 were performed (D17 as described above, then D20, D22, D24, D28 and D31).

[0494] In particular, three groups of identical mice with tumors transplanted were tested. In the first group (10 mice-group control), mice were administered LV-empty Indiana (D13) (as a control), and anti-PD-1 monoclonal antibodies were administered four days later (D17, followed by D20, D22, D24, D28 and D31). In the second group (12 mice-control group), mice were administered a vaccine according to the present invention (I-5759) (D13), and control antibodies (isotype ctrl) were administered four days later (D17, followed by D20, D22, D24, D28 and D31). In the third group (14 mice), mice were administered a vaccine according to the present invention (D13) (I-5759), and mAb anti-PD-1 was administered four days later (D17, followed by D20, D22, D24, D28 and D31).

[0495] The results are as follows Fig.12 A and Fig.12 As shown in B.

[0496] Suboptimal doses of the vaccine induced insufficient antitumor T cell responses, which synergized with anti-PD-1 to increase tumor regression rates.

[0497] Six of the 14 mice achieved complete tumor regression, and another two showed partial tumor regression. In the latter, tumor volume was reduced by 67%, and then the tumor recurred 6 to 7 days after the end of anti-PD-1 treatment, which highlights the need for repeated injections of anti-PD-1 until the tumor disappears completely. Only 3 of the 12 mice treated with a suboptimal dose of the vaccine of the present invention alone showed partial tumor regression. Therefore, the survival rate of mice in the combined treatment group was significantly increased compared with mice treated with a suboptimal dose of Lenti-HPV-07 ( Fig.12B). Therefore, when the Lenti-HPV-07 vaccine candidate is combined with anti-PD1 checkpoint inhibition therapy, a synergistic anti-tumor effect can be achieved.

[0498] Example 11: A single injection of the vaccine according to the present invention cured the patient with TC1-nLuc cells injected intravenously induced lung metastases in mice

[0499] The TC1 parental cell line was stably transduced with an integrative lentiviral vector encoding a nanoluciferase reporter protein and puromycin N-acetyltransferase (for selection) under the ubiquitin (UBC) promoter. After selection on puromycin, cells were subcloned to obtain the TC1-nLuc cell line.

[0500] Six-week-old C57BL / 6JRj mice purchased from Janvier Laboratory were injected intravenously with 150,000 TC1-nLuc cells. On day 5, mice were injected intramuscularly with a single dose of 1.10 9 TU / Lenti-HPV-07 or control Lenti (empty vector) in mice.

[0501] Bioluminescent imaging was performed on live animals using an IVIS imaging system (IVIS Spectrum, Perkin Elmer) coupled to a charge-coupled device camera. Prior to bioluminescent imaging, mice were anesthetized with 2% isoflurane in oxygen and maintained in a controlled flow of 1.5% isoflurane in oxygen through a nose cone during imaging. The substrate furimazine (Z108) (provided by Dr. Yves Janin of the Pasteur Institute) was dissolved in acidic ethanol at 2 mg / ml. Prior to intravenous injection, Z108 was further diluted to the desired concentration (0.4 mg / kg) in sterile D-PBS. The mice were then immediately placed in an imaging chamber and imaged. Continuous images were taken at an automatic exposure setting with a maximum exposure time of 2 minutes.

[0502] Images from each experimental setup were analyzed using live Image software (Ver. 2.50.1 Xenogen). Measurements of the region of interest were performed and the luminescence values ​​were evaluated as total flux (photons / second). The hair of the abdomen and trunk of the mice was shaved to improve the signal-to-noise ratio. The baseline signal was obtained from untreated mice (i.e., mice that were neither injected with TC1-nLuc cells nor injected with lentiviral vectors, but injected with Z108).

[0503] The results obtained are as follows Fig.13 A and Fig.13 As shown in B.

[0504] The inventors have determined that a single intramuscular injection of the Lenti-HPV-07 vaccine (I-5759) according to the invention completely eradicated subcutaneously implanted TC1 tumors in 100% of the animals.However, in humans, many cancers, including HPV-induced cancers, are located in mucosal sites.

[0505] Therefore, this experiment evaluated the ability of Lenti-HPV-07 to inhibit tumor growth in mucosal sites.

[0506] To address this issue, a TC1 cell line stably expressing a nanoluciferase reporter gene (TC1-nLuc) was developed. After intravenous injection of TC1-nLuc, mice readily developed lung metastases.

[0507] Longitudinal tumor outgrowth in living animals was tracked by bioluminescence imaging.

[0508] Five days after tumor injection, mice received a single intramuscular injection of Lenti-HPV-07 (1.109 TU / mouse) or control Lenti (empty vector) ( Fig.13 A).

[0509] All mice that received Lenti-HPV-07 were cured by day 22 after tumor injection, while lung metastases continued to grow in the control group. The differences observed between the mean values ​​of bioluminescent signals in the two groups were largely statistically significant ( Fig.13 A and Fig.13 B).

[0510] This observation demonstrates in a clear manner that the vaccine according to the invention is able to eradicate lung tumors as efficiently as it is able to eradicate tumors established subcutaneously.

[0511] Example 12: Lenti-HPV-07 vaccination synergizes with anti-NKG2A immunotherapy

[0512] Using monoclonal antibody (mAb) clone 20D5 (Catalog No. BE0321, BioXCell) in C57BL / 6 mice implanted subcutaneously with HPV-induced TC-1 tumor cells, we demonstrated antitumor synergy between suboptimal doses of Lenti-HPV-07 (I-5759) vaccination and anti-NKG2A therapy ( Fig.14 A).

[0513] Since the dose used in the previous experiment was 1 × 10 9 TU Lenti-HPV-07 induced complete eradication of tumors in 100% of mice. To evaluate the possible beneficial effects of additional anti-NKG2AmAb treatment, we used 1×10 8Suboptimal dose of TU. Anti-NKG2A mAb treatment was started four days after Lenti-HPV-07 injection. Control animals received 1×10 8 Lenti-HPV-07 and control Ig (Ctrl Ig) at TU.

[0514] It is known that anti-NKG2A mAb treatment has no effect on TC1 tumor cell growth (van Montfoort et al. Cell. 2018 Dec 13;175(7):1744-1755.e15). This is why the group with only anti-NKG2A mAb was not included.

[0515] result

[0516] In the Lenti-HPV-07+Ctrl Ig group, 5 of 12 mice achieved tumor regression, while 7 of 12 mice could not control tumor growth ( Figure 1 B). In contrast, in the Lenti-HPV-07+anti-NKG2A mAb group, 11 of 12 mice achieved tumor regression, while 1 of 12 mice failed to control tumor growth ( Fig.14 B).

[0517] Notably, after stopping anti-NKG2A mAb treatment, tumors recurred in 6 of 12 mice, strongly suggesting that repeated injections of anti-NKG2A are required to maintain tumors under immune control. 80% of mice treated with Lenti-HPV-07 + anti-NKG2A mAb showed complete or partial regression, while only 40% of mice treated with Lenti-HPV-07 alone showed complete or partial regression ( Fig.14 C).

[0518] Compared with mice treated with Lenti-HPV-07+Ctrl Ig, mice receiving the combination of Lenti-HPV-07 and anti-NKG2A mAb had a doubled progression-free survival (PFS) time ( Fig.14 D).

[0519] Therefore, the survival of the combination treatment group was significantly increased ( Fig.14 E).

[0520] By using Fig.14 Log-rank Mantel-Cox test in E, *p=0.0134) or Fig.14 Unpaired t-test in D was used to determine statistical significance.

[0521] Because treatment with anti-NKG2A mAb alone had no effect on TC-1 tumor growth (van Montfoort et al. Cell. 2018 Dec 13;175(7):1744-1755.e15) and the effect of the combination treatment was higher than that of Lenti-HPV-07+Ctrl Ig, the effect of the combination treatment was synergistic rather than additive.

[0522] Therefore, when Lenti-HPV-07 is combined with anti-NKG2A checkpoint inhibition therapy, a synergistic anti-tumor effect can be achieved.

[0523] Example 13: Lenti-HPV-07 vaccination synergizes with anti-TIM-3 immunotherapy

[0524] By using the monoclonal antibody (mAb) clone RMT3-23 (Cat. No. BE0115 Bioxcell) in C57BL / 6 mice implanted subcutaneously with HPV-induced TC-1 tumor cells, we demonstrated potential antitumor synergy between suboptimal doses of Lenti-HPV-07 vaccination and anti-TIM-3 therapy ( Fig.15 A). As mentioned above, due to the optimal dose of 1×10 9 TU's Lenti-HPV-07 induced complete tumor eradication in 100% of the mice. To evaluate the possible beneficial effects of additional anti-TIM-3 mAb treatment, 1×10 8 Suboptimal dose of TU. Anti-TIM-3 mAb treatment was started four days after Lenti-HPV-07 injection. Control animals received 1×10 8 Lenti-HPV-07 and control Ig (Ctrl Ig) at TU. Other groups received CtrlLenti+Ctrl Ig or Ctrl Lenti+anti-TIM-3 mAb.

[0525] No tumor regression was recorded in the Ctrl Lenti + Ctrl Ig group and the Ctrl Lenti + anti-TIM-3 mAb group. Therefore, anti-TIM-3 mAb treatment alone had no effect on TC-1 tumor growth ( Fig.15 B). In the Lenti-HPV-07+Ctrl Ig group, 4 out of 12 mice achieved tumor regression, and 8 out of 12 mice could not control tumor growth ( Fig.15 B). In contrast, in the Lenti-HPV-07+anti-TIM-3 mAb group, 8 out of 12 mice achieved tumor regression, while 4 out of 12 mice failed to control tumor growth.

[0526] 60% of mice treated with Lenti-HPV-07 + anti-TIM-3 mAb showed complete or partial remission, while 40% of mice treated with Lenti-HPV-07 alone showed complete or partial remission ( Fig.15 C). Mice receiving the combination of Lenti-HPV-07 and anti-TIM-3 mAb had a longer progression-free survival (PFS) compared to mice treated with Lenti-HPV-07+Ctrl Ig, although the difference did not reach statistical significance ( Fig.15 D). Survival was significantly increased in the combined treatment group ( Fig.15 E). Statistical significance was determined by using the log-rank Mantel-Cox test, **p=0.0465, ****p<0.0001).

[0527] Because treatment with anti-TIM-3 mAb alone had no effect on TC-1 tumor growth and the effect of the combination treatment was higher than that of Lenti-HPV-07+Ctrl Ig, the effect of the combination treatment was synergistic rather than additive.

[0528] Therefore, when Lenti-HPV-07 is combined with anti-TIM-3 checkpoint inhibition therapy, a synergistic anti-tumor effect can be achieved.

[0529] sequence

[0530] SEQ ID NO:1 is a nucleic acid sequence encoding the E6 protein from HPV 16 (NC_001526.4)

[0531] ATGCACCAAAAGAGAACTGCAATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTTTCGGGATTTATGCATAGTATATAGAGATGGGAATCCATATGCTGTATGTGATAAATGTTTAAAGTTTTATTCTAAAATTAGTGAGTATAGACATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGATTTGTTAATTAGGTGTATTAACTGTCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGCAGATCATCAAGAACACGTAGAGAAACCCAGCTGTAA

[0532] SEQ ID NO:2 It is a nucleic acid sequence encoding a non-carcinogenic variant of the E6 protein from HPV 16

[0533] GACCCCCAAGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACCGGAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGCCTGTACGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACTGCCAGAAGCCCCTGCGGTTCCACAACATCCGGGGCAGATGGACCGGCCGGTGCATGAGCTGCTGCAGA

[0534] SEQ ID NO:3 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16

[0535] ATGGGCACCCTGGGCATCGTGTGCCCCATCGACCCCCAAGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACCGGAACCCCTACGCCG TGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCCTACAGCCTGTACGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACTGCCAGAAGCCCCTGCGGTTCCACAACATCCGGGGCAGATGGACCGGCCGGTGCATGAGCTGCTGCAGA

[0536] SEQ ID NO:4 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16

[0537] ATGGACCCCCAAGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACCGGAACCCCTACGCCGTGTGCGACAAGTGC CTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCCTACAGCCTGTACGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACTGCCAGAAGCCCTGCGGTTCCACAACATCCGGGGCAGATGGACCGGCCGGTGCATGAGCTGCTGCAGA

[0538] SEQ ID NO:5 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16

[0539] TTTCAGGACCCCCAGGAAAGGCCCAGGAAGTTGCCCCAGCTCTGCACCGAACTGCAGACCACCATTCATGACATCATCCTCGAATGCGTGTACTGCAAGCAGCAGCTCCTGAGGAGGGAGGTGTACGATTTCGCCTTCAGAGACGGCTGTATCGTCTACAGGAACCCCTATGCCGTCTGCGACAAATGCCTGAAGTTTTATTCCAAGATCTCCGAGTACAGGCACTATTGCTACAGCCTGTATGGGACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTCCTGATCAGGTGCATCAACTGCCAGAAGCCCCTGAGGTTCCACAACATCCGCGGCAGGTGGACCGGAAGGTGCATGTCCTGCTGCAGG

[0540] SEQ ID NO:6 It is a nucleic acid sequence encoding a non - oncogenic variant of the E6 protein from HPV 16

[0541] TTCCAGGACCCCCAGGAGAGGCCCAGGAAACTGCCCCAGTTGTGCACCGAGCTCCAGACAACCATCCACGACATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGTTGCTGAGGAGAGAGGTGTATGACTTCGCCTTCAGAGACGGATGCATTGTCTATAGGAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACTCCAAGATCAGTGAGTACAGGCATTACTGCTACAGCCTGTATGGAACCACACTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTCCTGATTAGGTGCATCAACTGCCAGAAGCCCCTCAGGTTCCACAACATCCGGGGCAGGTGGACCGGAAGGTGCATGTCCTGCTGCAGGTCC

[0542] SEQ ID NO:7 It is the amino acid sequence of the wild - type (WT) E6 protein from HPV 16

[0543] MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL

[0544] SEQ ID NO:8 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0545] DPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR

[0546] SEQ ID NO:9 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0547] MGTLGIVCPIDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR

[0548] SEQ ID NO:10 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0549] MDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR

[0550] SEQ ID NO:11 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0551] FQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR

[0552] SEQ ID NO:12 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16

[0553] FQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRS

[0554] SEQ ID NO:13 is a nucleic acid sequence encoding the E7 protein from HPV 16 (NP-041326.1)

[0555] ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAG CCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA

[0556] SEQ ID NO:14 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 16

[0557] ACCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCCGAGACAACCGACCCCGACCGGGCCCACTACAATATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGCGGCTGTGCGTGCAGAGCACCCACGTGGACATCCGGACCCTGGAAGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCCATT

[0558] SEQ ID NO:15 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 16

[0559] CCCGGAGACACCCCCACCCTGCACGAATACATGCTGGACCTGCAGCCCGAAACCACCGACCCCGACCGCGCTCACTACAACATCGTTACATTCTGTTGTAAATGCGACTCCACCCTGAGAAGATGCGTGCAGTCCACCCACGTGGACATCAGGACCCTGGAGGACCTCCTCATGGGAACCCTGGGTATCGTCTGCCCCATC

[0560] SEQ ID NO:16 is the amino acid sequence of the wild-type (WT) E7 protein from HPV 16

[0561] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

[0562] SEQ ID NO:17 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 16

[0563] TPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPI

[0564] SEQ ID NO:18 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 16

[0565] PGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPI

[0566] SEQ ID NO:19 is the nucleic acid sequence encoding the E6 protein from HPV 18 (MF288727.1)

[0567] ATGGCGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGAACACTTCACTGCAAGACATAGAAATAACCTGTGTATATTGCAAGACAGTATTGGAACTTACAGAGGTATTTGAATTTGCATTTAAAGATTTATTTGTGGTGTATAGAGACAGTATACCGCATGCTGCATGCCATAAATGTATAGATTTTTATTCTAGAATTAGAGAATTAAGACATTATTCAGACTCTGTGTATGGAGACACATTGGAGAAACTAACTAACACTGGGTTATACAATTTATTAATAAGGTGCCTGCGGTGCCAGAAACCGTTGAATCCAGCAGAAAAACTTAGACACCTTAATGAAAAACGACGATTCCACAACATAGCTGGGCACTATAGAGGCCAGTGCCATTCGTGCTGCAACCGAGCACGACAGGAAAGACTCCAACGACGCAGAGAAACACAAGTATAA

[0568] SEQ ID NO:20 is the nucleic acid sequence encoding a non - oncogenic variant of the E6 protein from HPV 18

[0569] CCCTACAAGCTGCCTGACCTGTGTACAGAGCTGAACACCTCCCTGCAGGACATCGAGATCACCTGTGTGTATTGCAAGACCGTGCTGGAACTGACCGAGGTGTTCGAGTTTGCCTTCAAGGATCTGTTCGTGGTGTACCGGGACAGCATCCCCCACGCCGCCTGCCACAAGCTGGAAAAGCTGACCAACACCGGCCTGTACAACCTGCTGATTCGGTGCCTGCGGTGTCAGAAGCCTCTGAACCCCGCCGAGAAGCTGCGGCACCTGAACGAGAAGCGGAGATTCCACAATATCGCC

[0570] SEQ ID NO:21 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18

[0571] CCCTACAAGCTGCCTGACCTGTGTACAGAGCTGAACACCTCCCTGCAGGACATCGAGATCACCTGTGTGTATTGCAAGACCGTGCTGGAACTGACCGAGGTGTTCGAGTTTGCCTTCAAGGATCTGTTCGTGGTGTACCGGGACAGCATCCCCCACGCCGCCTGCCACAAG

[0572] SEQ ID NO:22 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18

[0573] CCCTACAAGCTGCCCGATCTGTGCACAGAGCTGAACACCTCCCTGCAGGACATCGAGATCACCTGCGTCTACTGCAAGACCGTGCTGGAACTGACCGAGGTGTTCGAATTCGCCTTCAAGGACGGCTTCGTGGTGTACAGGGACAGCA TTCCCCACGCCGCCTGCCATAAGCTGGAGAAACTGACCAACACCGGACTGTATAACCTGCTGATCAGGTGTCTGAGGTGCCAGAAGGCAGAGAAACTGAGACATCTGAACGAGAAAAGGAGGTTCCACAATATTGCCGGGCACTGATAA

[0574] SEQ ID NO:23 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18

[0575] ATGAGGCGGCCCTACAAGCTGCCCGACCTGTGCACCGAGCTGAACACCTCCCTGCAGGACATCGAGATCACCTGCGTGTACTGCAAGACCGTGCTGGAGCTGACCGAGGTGTTCGAATTCGCATTCAAGGACGGATTCGTCGTGTATAGG GACAGCATTCCACACGCCGCCTGCCACAAGCTGGAGAAATTGACTAACACCGGACTGTATAATCTGCTGATCCGGTGCCTGAGGTGTCAGAAGGCCGAGAAGCTGAGGCATCTGAACGAGAAAAGGAGATTCCACAATATCGCCGGACAC

[0576] SEQ ID NO:24 is the amino acid sequence of the wild-type (WT) E6 protein from HPV 18

[0577] MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV

[0578] SEQ ID NO:25 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18

[0579] PYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIA

[0580] SEQ ID NO:26 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18

[0581] PYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHK

[0582] SEQ ID NO:27 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18

[0583] PYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGH

[0584] SEQ ID NO:28 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18

[0585] MRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGH

[0586] SEQ ID NO:29 is a nucleic acid sequence encoding the E7 protein from HPV 18 (NC_001357.1)

[0587] ATGCATGGACCTAAGGCAACATTGCAAGACATTGTATTGCATTTAGAGCCCCAAAATGAAATTCCGGTTGACCTTCTATGTCACGAGCAATTAAGCCGACTCAGAGGAAGAAAACGATGAAATAGATGGAGTTAATCATCAACATTTACCAGCCCGACGA GCCGAACCACAACGTCACACAATGTTGTGTATGTGTTGTAAGTGTGAAGCCAGAATTGAGCTAGTAGTAGAAAGCTCAGCAGACGACCTTCGAGCATTCCAGCAGCTGTTTCTGAACACCCTGTCCTTTGTGTGTCCGTGGTGTGCATCCCAGCAGTAA

[0588] SEQ ID NO:30 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18

[0589] AAGGCCACACTGCAGGATATCGTGCTGCACCTGGAACCCCAGAACGAGATCCCCGTGGACAGCGAGGAAGAGAACGACGAGATCGACGGCGTGAACCACCAGCATCTGCCCGCCAGAAGGGCCGAGCCCCAGAGACACACCATGCTGTGCATGTGTTGCAAATGCGAGGCCCGGATCAAGCTGGTGGTGGAAAGCAGCGCCGACGACCTGCGGGCCTTCCAGCAGCTGTTCCTGAACACCCTGTCCTTCGTGTGCCCTTGG

[0590] SEQ ID NO:31 It is a nucleic acid sequence encoding a non - oncogenic variant of the E7 protein from HPV 18

[0591] GGACCTAAAGCCACCCTCCAGGACATCGTGCTGCACCTGGAGCCCCAGAACGAGATCCCCGTCGACTCAGAGGAGGAGAACGACGAAATTGACGGCGTCAACCACCAGCACCTGCCCGCTCGCAGAGCCGAACCCCAGAGACACACCATGCTCTGCATGTGCTGCAAATGCGAGGCCCGGATTAAGCTGGTGGTGGAGAGCTCCGCCGACGATCTGAGAGCCTTCCAGCAGCTCTTCCTGAACACCCTGTCCTTCGTGTGCCCCTGG

[0592] SEQ ID NO:32 It is a nucleic acid sequence encoding a non - oncogenic variant of the E7 protein from HPV 18

[0593] GGACCTAAAGCCACCCTCCAGGACATCCGTCTGGAGCCCCAGA

[0594] ACGAGATCCCCGTCGACTCAGAGGAGGAGAACGACGAAATTGACGGCAACCACCAGCACCTGCCCGCTCGCAGAGCCGAACCCCAGAGACACACCATGCTCTGCATGTGCTGCAAATGCGAGGCCCGGATTAAGCTGGTGGTGGAGAGCTCCGCCGACGATCTGAGAGCCTTCCAGCAGCTCTTCCTGGATTCCTTCGTGTGCCCCTGG

[0595] SEQ ID NO:33 is the amino acid sequence of the wild-type (WT) E7 protein from HPV 18

[0596] MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ

[0597] SEQ ID NO:34 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18

[0598] KATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPW

[0599] SEQ ID NO:35 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18

[0600] GPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPW

[0601] SEQ ID NO:36 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18

[0602] GPKATLQDIRLEPQNEIPVDSEEENDEIDGNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLAFQQLFLDSFVCPW

[0603] SEQ ID NO:37 The nucleic acid sequence encoding the cPPT / CTS sequence is AATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTT

[0604] SEQ ID NO:38 is a nucleic acid sequence encoding a mutated form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE)

[0605] TTCCCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCC TTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTT CCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCGCGGCTGCTCGCCTGTGTTGCCACCTGGA TTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC

[0606] SEQ ID NO:39 Contains RAHYNIVTF H-2D b -Synthesis of T-cell-restricted epitopes E7 HPV16 -Derivatized Peptides

[0607] ETTDPDRAHYNIVTF

[0608] SEQ ID NO:40 Contains RAHYNIVTF H-2D b -Synthesis of T-cell-restricted epitopes E7 HPV16 -Derivatized Peptides

[0609] PDRAHYNIVTFCCKC

[0610] SEQ ID NO:41 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5759

[0611]

[0612] SEQ ID NO:42 is the amino acid sequence of the antigen construct encoded by the lentiviral vector submitted to CNCM under accession number I-5759

[0613] MPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIASQAFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR SAGPGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWVGEPGRTIPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGH

[0614] SEQ ID NO:43 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5760

[0615]

[0616] SEQ ID NO:44 is the amino acid sequence of the antigen construct encoded by the lentiviral vector submitted to CNCM under accession number I-5760

[0617] MFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRGPDDPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLT NTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIGPDDKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWA

[0618] SEQ ID NO:45 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5761

[0619]

[0620] SEQ ID NO:46 is the amino acid sequence of the antigen construct encoded by the lentiviral vector submitted to CNCM under accession number I-5761

[0621] MRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNI AGHFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLC DLLIRCINCQKPLRFHNIRGRWTGRCMSCCRSAGPGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIA

[0622] SEQ ID NO:47 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector submitted to CNCM under accession number I-5762

[0623]

[0624] SEQ ID NO:48 is the amino acid sequence of the antigen construct encoded by the lentiviral vector submitted to CNCM under accession number I-5762

[0625] MGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPGEPGRTIPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGPG DTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIASQAFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRS

[0626] SEQ ID NO: 49 is H-2D b -Amino acid sequence of the restricted T cell epitope

[0627] RAHYNIVTF

Claims

1. A lentiviral vector, in particular a non-integrating lentiviral vector, or a lentiviral vector particle, in particular a non-integrating lentiviral vector particle, for treating or preventing HPV-induced cancer, The lentiviral vector comprises at least four different nucleic acid sequences selected from the group consisting of: - at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen, - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen, - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen, and - at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen; The lentiviral vector particle comprises at least one lentiviral vector; The lentiviral vector or lentiviral vector particle is administered in combination with at least one immune checkpoint inhibitor, in particular at least one monoclonal antibody selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-NKG2A, anti-TIM-3, anti-TIGIT and anti-LAG-3 monoclonal antibodies.

2. The lentiviral vector or lentiviral vector particle for use according to claim 1, wherein the HPV-induced cancer is selected from the group consisting of cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, oropharyngeal cancer and metastases thereof, in particular lung metastases thereof.

3. The lentiviral vector or lentiviral vector particle for use according to claim 1 or 2, wherein the nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E6 antigen encodes an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:

6.

4. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen encodes an amino acid sequence having at least 68% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO: 14 and SEQ ID NO:

15.

5. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 4, wherein the nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen encodes an amino acid sequence having at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 24, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO:

23.

6. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 5, wherein the nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen encodes an amino acid sequence having at least 83% sequence identity with the amino acid sequence shown in SEQ ID NO: 33, and the nucleic acid sequence is specifically selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO:

32.

7. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 6, wherein the at least four different nucleic acid sequences encoding antigens are fused together to form a single antigenic nucleic acid sequence encoding a single antigenic fusion protein under the control of a single promoter sequence.

8. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 7, wherein the at least four different nucleic acid sequences are selected from the group consisting of: (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen; (b) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen; (c) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen; and (d) A nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E6 antigen.

9. The lentiviral vector or lentiviral vector particle used according to any one of claims 1 to 8, wherein the order of the at least four different nucleic acid sequences from the 5' end to the 3' end is: (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen - a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen.

10. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 9, comprising a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:42, in particular the nucleic acid sequence SEQ ID NO:

41.

11. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 10, chosen from the group consisting of the non-integrating lentiviral vector submitted to the CNCM under accession number I-5759, I-5760, I-5761 and I-5762, in particular the non-integrating lentiviral vector submitted to the CNCM under accession number I-5759.

12. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 11, wherein the lentiviral vector comprises an MHC class I promoter, in particular the β2-microglobulin promoter.

13. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 12, wherein the lentiviral vector comprises a cPPT / CTS sequence, in particular a cPPT / CTS sequence as shown in SEQ ID NO:

37.

14. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 13, wherein the lentiviral vector comprises a 3' long terminal repeat (LTR) lacking its U3 promoter sequence.

15. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 14, wherein the lentiviral vector does not comprise a constitutive enhancer sequence.

16. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 15, wherein the lentiviral vector comprises a mutated form of the Woodchuck Hepatitis B Virus (WHV) post-transcriptional regulatory element (WPRE), and in particular has the sequence shown in SEQ ID NO:

38.

17. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 16, wherein the lentiviral vector particle comprises a functional lentiviral integrase protein.

18. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 17, wherein the lentiviral vector particle comprises vesicular stomatitis virus glycoprotein (VSVG), in particular VSV-G Indiana serotype or VSV-G New Jersey serotype.

19. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 18, wherein the lentiviral vector particle comprises HIV-1 subtype D Gag and Pol proteins.

20. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 19, wherein the lentiviral vector or lentiviral vector particle is contained in an isolated cell.

21. The lentiviral vector or lentiviral vector particle according to any one of claims 1 to 20, wherein the lentiviral vector or lentiviral vector particle is comprised in a vaccine composition.

22. A lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 21, wherein the at least one immune checkpoint inhibitor is selected from the group consisting of anti-PD-1, anti-NKG2A and anti-TIM-3 monoclonal antibodies.

23. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 22, wherein the at least one immune checkpoint inhibitor is administered simultaneously or separately, and in particular at least 2 days, in particular at least 4 days after administration of the vaccine composition, lentiviral vector, lentiviral vector particle or cell.

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