Lentiviral vectors for the expression of human papillomavirus (HPV) antigens and their implementation in the treatment of HPV-induced cancers

Abstract

The present invention relates to a lentiviral vector, particularly a non-integrating lentiviral vector, or a lentiviral vector particle, particularly a non-integrating lentiviral vector particle, for use in the treatment or prevention of HPV-induced cancer, wherein the lentiviral vector comprises at least four different nucleic acid sequences; the lentiviral vector particle comprises at least one of the lentiviral vectors; and the lentiviral vector or lentiviral vector particle is administered in combination with at least one immune checkpoint inhibitor.

Description

[Technical Field]

[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 prophylactic vaccines. In particular, the present invention relates to lentiviral vectors that express Human Papillomavirus (HPV) antigens and their use in the prevention and treatment of HPV-induced cancers. [Background technology]

[0002] HPV accounts for 5.2% of cancers worldwide (Tota et al., Prev Med 2011 Oct;53 Suppl 1:S12-21). Over 100 HPV types have been identified and classified into three groups based on their association with cancer: high-risk types (especially HPV types 16 (HPV-16) and 18 (HPV-18)), low-risk types associated with benign lesions (HPV-6 and -11), and cutaneous types (especially HPV-1, -2, -3, and -4) (Chen et al. Virology vol.516 (2018):86-101). The proportion of cancers associated with HPV varies by cancer type and region, 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)).

[0003] The two most common types of HPV in cancer are HPV16 and HPV18. For example, HPV16 and HPV18 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). HPV16 and HPV18 are also significantly involved in anal cancer (91%), oropharyngeal cancer (70%), vaginal cancer (75%), penile cancer (63%), and vulvar cancer (68%) (Saraiya et al., J Natl Cancer Inst. 2015 Apr 29;107(6)).

[0004] Thereby, therapeutic vaccines targeting HPV16 / 18 could potentially be used to treat and prevent associated cancers, regardless of their site.

[0005] Human papillomaviruses (HPVs) are non-enveloped, double-stranded DNA viruses whose genomes encode 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).

[0006] Among these proteins, E6 and E7 have been well characterized for their oncogenic potential. E6 and E7 are known to inactivate p53 and pRb tumor suppressor proteins, thereby promoting cell proliferation. The E6 / E7 oncogenes are important for both the induction of HPV-linked 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). E6 and E7 proteins are expressed by all HPV-positive cells throughout infection, making them perfect targets for vaccines.

[0007] Recombinant viral vectors have been widely developed for vaccination purposes. Modification of viral genomes has enabled the production of nontoxic and noninfectious viral particles, which can be used as a tool to introduce genetic material into target cells. Inducing T cell-mediated immunity using recombinant viral vectors is a very promising approach for vaccination. Various viral vectors, including retroviral, adenoviral, and vaccinia viral vectors, have been evaluated for vaccination purposes (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 human immunodeficiency viruses (HIV). Lentiviral vectors are primarily derived from HIV-1. Lentiviral vectors have been improved in terms of their safety by removing the U3 sequence of the long terminal repeat (LTR), resulting in a "self-inactivating" vector completely devoid of viral promoter and enhancer sequences. Lentiviral vectors have emerged as promising tools because they offer several advantages over other viral systems: in particular, they are nontoxic and, unlike other retroviruses, can transduce non-dividing cells, especially dendritic cells (He et al. 2007, Expert Rev vaccines, 6(6):913-24), allowing sustained antigen presentation via the endogenous pathway.

[0008] In contrast to other commonly used viral vectors, lentiviral vectors have the ability to transduce non-dividing cells. Efficient transduction into non-dividing cells requires the formation of a triple-stranded DNA structure called the central DNA "flap," which maximizes the efficiency of gene import into the nuclei of non-dividing cells, including dendritic cells (DCs) (Arhel et al., EMBO J. 2007 Jun 20;26(12):3025-3037) (Zennou et al., Cell. 2000 Apr 14;101(2):173-85).

[0009] Dendritic cells (DCs) are important for antigen presentation because they constitute a major class of antigen-presenting cells (APCs) whose primary 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 antigen-derived peptides on their surface via major histocompatibility complex (MHC) molecules. Antigen-specific T cells present in the lymph nodes can then interact with the peptide-MHC complexes via their TCRs (T cell receptors). Recognition of peptide-MHC by specific TCRs, coupled with costimulatory signals, initiates T cell activation (Steinman, R., Banchereau, J. Nature 449, 419-426 (2007)). Summary of the Invention [Problem to be solved by the invention]

[0010] It is an object of the present invention to provide therapeutic and prophylactic vaccines for the prevention and treatment of HPV-induced cancers.

[0011] 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 study was performed on early-stage tumors, and this construct demonstrated the ability to eradicate the tumors in a reasonable, but not complete, number of vaccinated mice.

[0012] Thus, there remains a need in the art for treatments for the more aggressive and / or well-implanted tumors caused by HPV, which are known in the art to be more difficult to remove than small and early stage tumors.

[0013] There is also a need for treatment of HPV-induced resistant tumors, tumors characterized by a strong infiltration of regulatory T cells (Tregs).

[0014] Furthermore, it is possible to increase CD8 in HPV-induced cancers while decreasing Tregs in the HPV-induced cancers. + and CD4 + There is a need for therapeutic vaccines that allow for the treatment of cellular infiltration.

[0015] There is also a need for the generation of strong immune memory against HPV, particularly PDHPV antigens, and more particularly HPV16 and HPV18 antigens.

[0016] There is also a need for the development of new, safe, non-carcinogenic preventative and therapeutic vaccines against HPV-induced cancers.

[0017] There is a further need for a vaccine that can completely eliminate the primary tumor after a single administration and provide strong protection against recurrence.

[0018] The present invention aims to meet the above-mentioned needs. [Means for solving the problem]

[0019] Therefore, the present invention relates to the following items:

[0020] Item 1 Lentiviral vectors, in particular non-integrative lentiviral vectors, The lentiviral vector comprises: at least one nucleic acid sequence encoding the 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 nucleic acid sequence comprises at least four different nucleic acid sequences selected from the group consisting of:

[0021] As shown in the Examples, the lentiviral vectors of the present invention enable potent therapeutic and preventive activity against HPV-induced cancers.

[0022] Item 22. 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 set forth as SEQ ID NO: 7, wherein the nucleic acid sequence is particularly 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.

[0023] Item 3 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 set forth as SEQ ID NO: 16, wherein the nucleic acid sequence is in particular selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 15.

[0024] Item 4 4. The 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 set forth as SEQ ID NO: 24, wherein the nucleic acid sequence is particularly selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

[0025] Item 5 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 set forth as SEQ ID NO: 33, wherein the nucleic acid sequence is particularly selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.

[0026] Item 66. 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 antigen nucleic acid sequence encoding a single antigen fusion protein under the control of a single promoter sequence.

[0027] Item 7 the at least four different nucleic acid sequences are in the order from the 5' end to the 3' end: (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 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. 7. The lentiviral vector according to any one of items 1 to 6, selected from the group consisting of:

[0028] Item 8 8. The lentiviral vector according to any one of Items 1 to 7, wherein the at least four different nucleic acid sequences, in order from the 5' end to the 3' end, are: (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen; (b) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen; (c) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen; and (d) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen.

[0029] Item 9 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 set forth as SEQ ID NO: 42, wherein the nucleic acid sequence is in particular the nucleic acid sequence SEQ ID NO: 41.

[0030] Item 10 10. The lentiviral vector according to any one of items 1 to 9, which is selected from the group consisting of the non-integrative lentiviral vectors deposited at the CNCM under accession numbers I-5759, I-5760, I-5761 and I-5762, in particular the non-integrative lentiviral vector deposited at the CNCM under accession number I-5759.

[0031] Item 11 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.

[0032] Item 1212. The lentiviral vector according to any one of items 1 to 11, wherein the lentiviral vector comprises a cPPT / CTS sequence, in particular the cPPT / CTS sequence set forth as sequence SEQ ID NO: 37.

[0033] Item 13 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 a U3 promoter sequence.

[0034] Item 14 14. The lentiviral vector according to any one of items 1 to 13, wherein the lentiviral vector does not contain a constitutive enhancer sequence.

[0035] Item 15 15. The non-integrative 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), in particular having the sequence set out as SEQ ID NO: 38.

[0036] Item 16 16. A lentiviral vector particle, in particular a non-integrative lentiviral vector particle, comprising at least one lentiviral vector according to any one of items 1 to 15.

[0037] Item 17 17. The lentiviral vector particle according to item 16, wherein the lentiviral vector particle comprises a functional lentiviral integrase protein.

[0038] Item 1818. The lentiviral vector particle according to item 16 or 17, wherein the lentiviral vector particle comprises a vesicular stomatitis virus glycoprotein (VSVG), in particular a VSV-G Indiana serotype or a VSV-G New Jersey serotype.

[0039] Item 19 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.

[0040] 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.

[0041] Item 21 19. A vaccine composition 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.

[0042] Item 22 22. The vaccine composition according to item 21, for use in the treatment or prevention of HPV-induced cancers, in particular HPV-induced cancers 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.

[0043] Item 23 20. The lentiviral vector according to any one of items 1 to 15, the lentiviral vector particle according to any one of items 16 to 19, or the cell according to item 20, for use as a pharmaceutical or vaccine.

[0044] Item 2424. The lentiviral vector, the lentiviral vector particle, or the cell according to item 23 for use in the treatment or prevention of HPV-induced cancer, in particular HPV-induced cancer selected from cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, oropharyngeal cancer and metastases thereof, in particular lung metastases thereof.

[0045] Item 25 25. The vaccine composition for use according to item 22, or the 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, particularly at least one monoclonal antibody selected from the group consisting of an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, an anti-NKG2A monoclonal antibody, an anti-TIM-3 monoclonal antibody, an anti-TIGIT monoclonal antibody, and an anti-LAG-3 monoclonal antibody, more particularly at least one anti-PD-1 monoclonal antibody.

[0046] Item 26 26. The vaccine composition, the lentiviral vector, the lentiviral vector particle or the cell for use according to item 25, wherein the at least one immune checkpoint inhibitor is administered simultaneously or separately, in particular the at least one immune checkpoint inhibitor is administered at least 2 days, in particular at least 4 days, after administration of the vaccine composition, the lentiviral vector, the lentiviral vector particle or the cell.

[0047] The present invention also relates to a lentiviral vector, particularly a non-integrative lentiviral vector, or a lentiviral vector particle, particularly a non-integrative lentiviral vector particle, for use in the treatment or prevention of HPV-induced cancer, the lentiviral vector comprising: at least one nucleic acid sequence encoding the 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; at least four different nucleic acid sequences selected from the group consisting of The lentiviral vector particle comprises at least one of the lentiviral vectors, The lentiviral vector or the 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 an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, an anti-NKG2A monoclonal antibody, an anti-TIM-3 monoclonal antibody, an anti-TIGIT monoclonal antibody, and an anti-LAG-3 monoclonal antibody; The present invention relates to the lentiviral vector or the lentiviral vector particle.

[0048] 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, particularly lung metastases thereof.

[0049] The nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E6 antigen may encode an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth as SEQ ID NO:7, wherein the nucleic acid sequence is particularly 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.

[0050] The nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen may encode an amino acid sequence having at least 68% sequence identity with the amino acid sequence set forth as SEQ ID NO:16, wherein the nucleic acid sequence is particularly selected from the group consisting of SEQ ID NO:14 and SEQ ID NO:15.

[0051] The nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen may encode an amino acid sequence having at least 60% sequence identity with the amino acid sequence set forth as SEQ ID NO:24, wherein the nucleic acid sequence is particularly selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

[0052] The nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen may encode an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth as SEQ ID NO:33, wherein the nucleic acid sequence is particularly selected from the group consisting of SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0053] The at least four different nucleic acid sequences encoding antigens may be fused together to form a single antigen nucleic acid sequence that encodes a single antigen fusion protein under the control of a single promoter sequence.

[0054] The at least four different nucleic acid sequences are in the following order from the 5' end to the 3' end: (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 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. may be selected from the group consisting of:

[0055] The at least four different nucleic acid sequences may be in the following order from the 5' end to the 3' end: (a) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen; (b) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen; (c) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen; and (d) a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen.

[0056] 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 to the amino acid sequence set forth as SEQ ID NO:42, wherein the nucleic acid sequence is in particular the nucleic acid sequence SEQ ID NO:41.

[0057] The lentiviral vector or lentiviral vector particle for use according to the present invention may be selected from the group consisting of the non-integrative lentiviral vectors deposited at the CNCM under accession numbers I-5759, I-5760, I-5761 and I-5762, in particular the non-integrative lentiviral vector deposited at the CNCM under accession number I-5759.

[0058] The lentiviral vector for use according to the present invention may comprise an MHC class I promoter, in particular the β2-microglobulin promoter.

[0059] The lentiviral vector for use according to the present invention may comprise a cPPT / CTS sequence, in particular the cPPT / CTS sequence set out as sequence SEQ ID NO:37.

[0060] The lentiviral vector for use according to the present invention may comprise a 3' long terminal repeat (LTR) that lacks a U3 promoter sequence.

[0061] The lentiviral vectors for use according to the present invention are particularly free of constitutive enhancer sequences.

[0062] 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 set out as SEQ ID NO:38.

[0063] The lentiviral vector particles for use according to the present invention may comprise a functional lentiviral integrase protein.

[0064] The lentiviral vector particles for use according to the present invention may comprise vesicular stomatitis virus glycoprotein (VSVG), in particular VSV-G Indiana serotype or VSV-G New Jersey serotype.

[0065] The lentiviral vector particles for use according to the present invention may comprise HIV-1 subtype D Gag and Pol proteins.

[0066] The lentiviral vector or lentiviral vector particle for use according to the present invention may be contained in an isolated cell.

[0067] The lentiviral vector or lentiviral vector particle for use according to the present invention may be comprised in a vaccine composition.

[0068] The at least one immune checkpoint inhibitor may be selected from the group consisting of an anti-PD-1 monoclonal antibody, an anti-NKG2A monoclonal antibody, and an anti-TIM-3 monoclonal antibody.

[0069] The at least one immune checkpoint inhibitor may be administered simultaneously or separately, and 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, the lentiviral vector, the lentiviral vector particle, or the cell.

[0070] The details, examples, and preferred aspects provided with respect to any particular one or more of the described aspects of the invention are further described herein and apply equally to all aspects of the invention. Any combination in all possible variations of the embodiments, examples, and preferred aspects described herein is encompassed by the present invention unless otherwise stated herein or clearly contradicted by context. [Brief explanation of the drawings]

[0071] [Figure 1]Figure 1 demonstrates that the HPV vaccine of the present invention is immunogenic in vivo. Mice were injected intramuscularly (i.m.) with 1 x 107 TU of lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759, the lentiviral vector deposited at the CNCM under accession number I-5760, the lentiviral vector deposited at the CNCM under accession number I-5761, or the lentiviral vector deposited at the CNCM under accession number I-5762, or 50 μL of a dilution. 14 days later, splenocytes were prepared and restimulated overnight with four different peptide pools for IFNg ELISPOT assays. Horizontal axis: from left to right: results obtained with the lentiviral vector deposited at the CNCM under accession number I-5759, the lentiviral vector deposited at the CNCM under accession number I-5760, the lentiviral vector deposited at the CNCM under accession number I-5761, the lentiviral vector deposited at the CNCM under accession number I-5762, or results obtained with 50 μL of diluent (control). Vertical axis: Spot forming cells (SFC) / 106 cells. [Figure 2] Figure 2 shows that the vaccine of the present invention successfully and completely eliminated transplanted tumors in vivo. TC-1 cells were injected subcutaneously (sc), and tumor volume was measured every other day (using a caliper). When the average tumor volume reached 70 mm, mice were randomized and vaccinated intramuscularly (im) with 1 x 10 TU of LV-GFP Indiana (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. Horizontal axis: days. Vertical axis: tumor volume (mm). [Figure 3]Figure 3 shows the ability of lentiviral vectors according to the present invention expressing designs I-5759, I-5760, I-5761, I-5762, or diluent as a control, to generate long-lasting immunity to prevent recurrence. Mice from which primary tumors were removed were rechallenged in the other flank on day 60. Control mice (untreated) were also injected subcutaneously (sc) to confirm tumor cell growth in naive mice. Horizontal axis: number of days. Vertical axis: tumor volume (mm3). [Figure 4] Figure 4 shows the dose / response in mice. 1x106 TC-1 cells were injected into the flank of the animals, and tumor volumes were measured twice a week (caliper measurement). When the average tumor volume reached 80mm3, the mice were randomized and vaccinated intramuscularly (im) with diluent (control), 1x107 TU of I-5759 vaccine, or 1x108 TU of I-5759 vaccine. Horizontal axis: days. Vertical axis: tumor volume (mm3). [Figure 5] Figure 5 shows lymphocyte tumor infiltration after vaccination with lentiviral vectors according to the present invention. 1x106 TC1 tumor cells were injected subcutaneously (sc) into the flank of animals, and tumor volumes were measured twice weekly (by caliper measurement). When the mean tumor volume reached 80 mm3, mice were randomized and vaccinated intramuscularly (im) with diluent (control), 1x107 TU of I-5759 vaccine, or 1x108 TU of I-5759 vaccine. 10 days after vaccination, tumors were harvested, digested, and analyzed by flow cytometry. FACS staining was performed, and data were acquired on a Macsquant FACS system according to methods well known in the art. Horizontal axis: from left to right: diluent (control); 1x108 TU of I-5759 vaccine. Vertical axis: Top left: % CD8+ T cells (in live cells); Top right: % CD4+ T cells (in live cells); Bottom: % Treg cells (in live cells). [Figure 6]Figure 6 shows the ability of a vector according to the invention to eliminate large, established tumors. 1 x 10 TC1 cells were injected subcutaneously (sc) into the flank of the animals. When the average tumor volume reached approximately 300 mm , the mice were randomized and vaccinated intramuscularly (im) with 1 x 10 TU of a vaccine according to the invention containing diluent (control) or lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759. Horizontal axis: number of days. Vertical axis: tumor volume (mm ). [Figure 7] Figure 7 shows T cell responses in human PBMCs labeled with CFSE and cultured in the absence (unstimulated condition) or presence of a vaccine according to the invention (I-5759). Cell proliferation and activation were measured after 2 weeks of culture (n=3). CD8+ 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 cultures. Horizontal axis: from left to right: Unstimulated (Unstim-control); I-5759 vaccine. Vertical axis: (A) % CFSE low in the CD8+ population; right panel: % CFSE low in the CD4+ population. (B) Left panel: % CD25+ in the CD4+ population; right panel: % CD25+ in the CD8+ population. [Figure 8A] Figure 8A illustrates four examples of lentiviral vector antigen constructs according to the present invention. Each of these antigen constructs consists of four sequences in various orders: a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) 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 (HPV18) protein E6 antigen, and a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7. Figure 8A represents the lentiviral vector antigen construct deposited at the CNCM under accession number I-5759. [Figure 8B]Figure 8B illustrates four examples of lentiviral vector antigen constructs according to the present invention. Each of these antigen constructs consists of four sequences in various orders: a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) 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 (HPV18) protein E6 antigen, and a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7. Figure 8B represents the lentiviral vector antigen construct deposited at the CNCM under accession number I-5760. [Figure 8C] Figure 8C illustrates four examples of lentiviral vector antigen constructs according to the present invention. Each of these antigen constructs consists of four sequences in various orders: a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) 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 (HPV18) protein E6 antigen, and a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7. Figure 8C represents the lentiviral vector antigen construct deposited at the CNCM under accession number I-5761. [Figure 8D] Figure 8D illustrates four examples of lentiviral vector antigen constructs according to the present invention. Each of these antigen constructs consists of the following four sequences in various orders: a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) 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 (HPV18) protein E6 antigen, and a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7. Figure 8D represents the lentiviral vector antigen construct deposited at the CNCM under accession number I-5762. [Figure 9]Figure 9 (Figures 9A and 9B) show the T cell cytokine responses of splenocytes analyzed by intracellular cytokine staining with or without stimulation with a mixture of ETTDPDRAHYNIVTF (SEQ ID NO: 39) and PDRAHYNIVTFCCKC (SEQ ID NO: 40) synthetic peptides, where both synthetic peptides contain the RAHYNIVTF H-2Db-restricted T cell epitope (bold indicates H-2Db anchor residues). Splenocytes were obtained 14 days after vaccination of C57BL / 6 mice (n = 5 mice / group) immunized by intramuscular (i.m.) injection with control Lenti (LV-GFP Indiana) or with a vaccine according to the present invention comprising lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759. Figure 9A specifically depicts the cytometric gating strategy implemented on cytokine-producing CD8+ T cells and the degranulation activity of IFN-γ-producing CD8+ T cells assessed by surface CD107a staining. Figure 9B illustrates the T cell cytokine responses of splenocytes analyzed by intracellular cytokine staining with or without stimulation with a mixture of the ETTDPDRAHYNIVTF (SEQ ID NO: 39) and PDRAHYNIVTFCCKC (SEQ ID NO: 40) synthetic peptides, where both synthetic peptides contain the RAHYNIVTF H-2Db-restricted T cell epitope (bold indicates the H-2Db anchor residues). Splenocytes were obtained 14 days after intramuscular (i.m.) vaccination of immunized C57BL / 6 mice (n = 5 mice / group) with control Lenti (LV-GFP Indiana) or a vaccine according to the present invention containing lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759. Figure 9B shows the recapitulation frequencies of each (multifunctional) cell subset and IFN-γ CD107a cells within the CD8 T subset. [Figure 10]Figure 10 shows a cytometric analysis of tumor-infiltrating innate immune cells (NK) in mice that had been tumor-implanted and vaccinated with an HPV vaccine according to the present invention (I-5759), or in mice that had been tumor-implanted and not vaccinated (control - control Lenti). CD11b and NKp46 were detected. [Figure 11] Figure 11 shows the ability of the vector according to the present invention to eliminate large established tumors. Cured mice (right panel) from which the primary tumor had been eliminated were rechallenged with 1.106 TC-1 tumor cells in the other flank 119 days after the first implantation and maintained without any treatment. To confirm tumor cell growth, control mice (untreated - control aged mice - left panel) were also injected subcutaneously (sc). Horizontal axis: days after tumor rechallenge. Vertical axis: tumor volume (mm3). [Figure 12]Figure 12 (Figures 12A and 12B) shows the synergistic effect between suboptimal vaccination with a vaccine according to the present invention (I-5759) and anti-PD-1 therapy (anti-PD-1 monoclonal antibody). Figure 12A shows the change in tumor volume (mm3 - vertical axis) over days (horizontal axis) after tumor implantation in mice (D0). The experiment was performed in three identical groups of tumor-implanted mice. In the first group (10 mice - control group - left panel of Figure 12A), the mice were administered LV-Empty Indiana (D13) (as a control) (day indicated by the arrow), and four days later, anti-PD-1 (Programmed Cell Death Protein-1) monoclonal antibody was administered (D17, followed by D20, D22, D24, D28, and D31). In the second group (12 mice—control group—center panel of FIG. 12A ), the mice were administered a vaccine according to the present invention (I-5759) (D13) (day indicated by the arrow) followed four days later by a control antibody (isotype control) (D17, followed by D20, D22, D24, D28, and D31). In the third group (14 mice—right panel of FIG. 12A ), the mice were administered a vaccine according to the present invention (I-5759) (D13) (day indicated by the arrow) followed four days later by mAb anti-PD-1 (D17, followed by D20, D22, D24, D28, and D31). Figure 12B shows the survival rate (% of mice - vertical axis) over time (days - horizontal axis) of mice in each group (control group 1: control Lenti + anti-PD-1; control group 2: I-5759 + control Ig; control group 3: I-5759 + anti-PD-1). [Figure 13A]Figure 13A shows the cure of mice bearing lung metastases induced by intravenous injection of TC1-nLuc cells after a single infection with the Lenti-HPV-07 vaccine. Figure 13A shows the change in luminescence (photons per second) due to TC1-nLuc cells stably expressing nanoluciferase injected into different groups of mice over time (horizontal axis - days) after iv injection of TC1-nLuc cells into the mice (vertical axis - total flux). Three groups of mice were tested: (Description of Figure 13A follows below) [Figure 13B] Figure 13B shows the individual p / s values for each mouse in the three experimental groups detailed above on day 22 after tumor injection. Vertical axis: luminescence values (total flux) expressed as photons per second (p / s). Horizontal axis, from left to right: negative group, control group, Lenti group, Lenti-HPV-07 group. [Figure 14]Figures 14A-14E illustrate the synergistic effect of Lenti-HPV07 and anti-NKG2A mAb in eradicating TC-1 tumors. Figure 14A shows the timeline of tumor implantation and combined treatment with Lenti-HPV-07 and anti-NKG2A mAb in C57BL / 6 mice. Mice (n = 12 per group) were implanted sc in the flank with 1 x 10 TC-1 cells. Ten days after implantation, when tumor volumes reached an average of 120-140 mm , mice were randomized and vaccinated with a suboptimal dose of Lenti-HPV-07 at 1 x 10 TU / mouse. Mice were then treated with anti-NKG2A mAb (clone 20D5, Bioxcell) or an Ig control (clone 2A3, Bioxcell) two to three times weekly. A total of eight injections (200 μg / injection) were administered from day 14 to day 31. Figure 14B shows spaghetti plots of tumor growth in the two tested groups: left group, Lenti-HPV-07 (I-5759) + control Ig; right group, Lenti-HPV-07 (I-5759) + anti-NKG2A. Vertical axis: tumor volume (mm3). Horizontal axis: number of days after tumor implantation. Figure 14C shows plotted therapy response rates according to RECIST criteria in the two tested groups. Horizontal axis: left group, Lenti-HPV-07 (I-5759) + control Ig; right group, Lenti-HPV-07 (I-5759) + anti-NKG2A. Vertical axis: % of therapy response. From bottom to top, each group represents a complete response, then a partial response, and finally, no response. Figure 14D shows the progression-free survival time of mice in the two tested groups. Horizontal axis: left group Lenti-HPV-07 (I-5759) + control Ig; right group Lenti-HPV-07 (I-5759) + anti-NKG2A. Vertical axis: PFS (days). Figure 14E shows the survival curve of the animals shown in Figure 14B. Horizontal axis: days. Vertical axis: survival rate (% of mice).The curve with a value of approximately 80% at day 40 represents the Lenti-HPV-07(I-5759) + anti-NKG2A group, while the curve with a value of approximately 40% at day 40 represents the Lenti-HPV-07(I-5759) + control Ig group. [Figure 15]Figures 15A-15E illustrate the synergistic effect of Lenti-HPV07 and anti-TIM-3 mAb in eradicating TC-1 tumors. Figure 15A shows the timeline of tumor implantation and combined treatment with Lenti-HPV-07 and anti-TIM-3 mAb. C57BL / 6 mice (n = 11-12 per group) were implanted sc in the flank with 1 x 106 TC-1 cells. 13 days after implantation, when tumor volume reached a mean of 130 mm3, mice were randomized and vaccinated with a suboptimal dose of 1 x 108 TU / mouse of Lenti-HPV-07 or control Lenti. Mice were then treated with anti-TIM-3 mAb (clone RMT3-23, Bioxcell) or Ig control (clone 2A3, Bioxcell) two to three times weekly. A total of eight injections (200 μg / injection) were administered from day 16 to day 36. Figure 15B shows spaghetti plots of tumor growth in the two tested groups: left group, Lenti-HPV-07 (I-5759) + control Ig; right group, Lenti-HPV-07 (I-5759) + anti-TIM-3. Vertical axis: tumor volume (mm3). Horizontal axis: number of days after tumor implantation. Figure 15C shows plotted treatment response rates according to RECIST criteria in the two tested groups. Horizontal axis: groups from left to right: control Lenti + control Ig; control Lenti + anti-TIM-3; Lenti-HPV-07 (I-5759) + control Ig; Lenti-HPV-07 (I-5759) + anti-TIM-3. Vertical axis: % treatment response. From bottom to top, each group represents a complete response, then a partial response, and finally no response. The two groups on the left side of the horizontal axis are non-responders only. Figure 15D shows the progression-free survival time of mice in the two tested groups. Horizontal axis: left group Lenti-HPV-07 (I-5759) + control Ig; right group Lenti-HPV-07 (I-5759) + anti-TIM-3. Vertical axis: PFS (days). Figure 15E shows the survival curve of the animals shown in Figure 15B. Vertical axis: survival rate (% of mice).The curve with a value of approximately 75% at day 40 represents the Lenti-HPV-07(I-5759) + anti-TIM-3 group. The curve with a value of approximately 35% at day 40 represents the Lenti-HPV-07(I-5759) + control Ig group. The curve in which all animals died by day 33 represents the control Lenti + anti-TIM-3 group. The curve in which all animals died by day 34 represents the control Lenti + control Ig group.

[0072] (Figure 13A continued) JPEG2025527186000001.jpg48170

[0073] Sequence Summary SEQ ID NO:1 is the nucleic acid sequence encoding the E6 protein from HPV 16. SEQ ID NO:2 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16. Sequence number: 3 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:4 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:5 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:6 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:7 is the amino acid sequence of the E6 protein from HPV 16. SEQ ID NO:8 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:9 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:10 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:11is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:12 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16. SEQ ID NO:13 is the nucleic acid sequence encoding the E7 protein from HPV 16. SEQ ID NO:14 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 16. SEQ ID NO:15 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 16. SEQ ID NO:16 is the amino acid sequence of the E7 protein from HPV 16. SEQ ID NO:17 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 16. SEQ ID NO:18 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 16. SEQ ID NO:19 is the nucleic acid sequence encoding the E6 protein from HPV 18. SEQ ID NO:20 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18. SEQ ID NO:21 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18. SEQ ID NO:22 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18. SEQ ID NO:23 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18. SEQ ID NO:24 is the amino acid sequence of the E6 protein from HPV 18. SEQ ID NO:25 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18. SEQ ID NO:26 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18. SEQ ID NO:27 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18. SEQ ID NO:28 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18. SEQ ID NO:29 is the nucleic acid sequence encoding the E7 protein from HPV 18. SEQ ID NO:30 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18. SEQ ID NO:31 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18. SEQ ID NO:32 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18. SEQ ID NO:33 is the amino acid sequence of the E7 protein from HPV 18. SEQ ID NO:34 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18. SEQ ID NO:35 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18. SEQ ID NO:36 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18. SEQ ID NO:37 is the nucleic acid sequence encoding the cPPT / CTS sequence. SEQ ID NO:38 is a nucleic acid sequence encoding a variant of the woodchuck hepatitis B virus (WHV) posttranscriptional regulatory element (WPRE). SEQ ID NO:39 RAHYNIVTF H-2D b -Contains restricted T cell epitopes, E7 HPV16 It is a synthetic peptide derived from SEQ ID NO:40 RAHYNIVTF H-2D b -Contains restricted T cell epitopes, E7 HPV16 It is a synthetic peptide derived from SEQ ID NO:41is the nucleic acid sequence encoding the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5759. SEQ ID NO: 42 is the amino acid sequence of the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5759. SEQ ID NO:43 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5760. SEQ ID NO:44 is the amino acid sequence of the antigen construct encoded by the lentiviral vector deposited at the CNCM under accession number I-5760. SEQ ID NO:45 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5761. SEQ ID NO:46 is the amino acid sequence encoded by the lentiviral vector deposited at the CNCM under accession number I-5761. SEQ ID NO:47 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5762. SEQ ID NO:48 is the amino acid sequence of the antigen construct encoded by the lentiviral vector deposited at the CNCM under accession number I-5762. SEQ ID NO:49 is H-2D b -The amino acid sequence of a restricted T cell epitope. DETAILED DESCRIPTION OF THE INVENTION

[0074] The present inventors have discovered that administering to an individual in need thereof a lentiviral vector encoding at least four different human papillomavirus (HPV) antigens, in particular a lentiviral vector encoding at least four HPV antigens selected from among the E6 and E7 proteins of at least two different HPV subtypes, in particular HPV16 and HPV18 subtypes, results in high prophylactic and therapeutic activity against HPV-induced cancers.

[0075] The lentiviral vector according to the invention can make it possible to induce a strong, sustained and broad cell-mediated response against tumors induced by HPV infection.

[0076] Lentiviral vectors according to the invention, as well as lentiviral vector particles comprising them, isolated cells comprising said lentiviral vectors or said lentiviral vector particles, and vaccine compositions comprising them, are described throughout this specification.

[0077] definition

[0078] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

[0079] As used herein, "transgene" means a polynucleotide that can be expressed in a non-native environment or heterologous cell under appropriate conditions via recombinant techniques.

[0080] As used herein, the term "recombinant," when used in reference to a cell of the invention, indicates that the cell has been modified by the introduction of endogenous and / or heterologous nucleic acids or proteins into the cell, or by modification of 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 not found within the native (non-recombinant) form of the cell, expresses native (e.g., endogenous) genes at levels different from their native levels, or expresses additional or supplemental copies of native (e.g., endogenous) genes at levels different from their native levels. Isolated cells according to the invention are recombinant in that they comprise at least one lentiviral vector according to the invention and / or at least one lentiviral vector particle according to the invention.

[0081] As used herein, the term "recombinant" when used in reference to a vector is a sequence formed / obtained by genetic engineering techniques well known to those skilled in the art.

[0082] As used herein, the term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and comprising more than five amino acid residues. Amino acids are identified by one-letter or three-letter designations. As used herein, the term "protein" is synonymous with the term "polypeptide" and may also refer to two or more polypeptides. Thus, the terms "protein," "peptide," and "polypeptide" can be used interchangeably. Polypeptides may optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, etc.) to add functionality. A polypeptide that exhibits activity is sometimes referred to as an enzyme. It will be understood that, as a result of the degeneracy of the genetic code, numerous nucleotide sequences encoding a given polypeptide can be produced.

[0083] As used herein, the term "operably linked" refers to two or more nucleic acid sequence elements that are physically linked and functionally related to each other. For example, in a lentiviral vector according to the present invention, a promoter is operably linked to a coding sequence, and since the promoter can initiate or regulate the transcription or expression of the antigen construct, it is also referred to herein as an "antigen construct." In this case, the antigen construct should be understood as being "under the control" of the promoter. Generally, when two nucleic acid sequences are operably linked, they are in the same orientation and also usually in the same reading frame. They are usually essentially contiguous, although this may not be required.

[0084] The term "encoding" or "coding for" refers to the process by which a polynucleotide produces an amino acid sequence through the mechanisms of transcription and translation.

[0085] For each amino acid sequence or amino acid sequence of interest, a reference sequence is described herein. The present specification also encompasses amino acid sequences that have a specified percentage of amino acid identity with the reference amino acid sequence.

[0086] For obvious reasons, throughout this specification, a specific nucleic acid sequence or a specific amino acid sequence that meets the nucleotide identity or amino acid identity, respectively, considered should further lead to obtaining a protein (or antigen) that exhibits the desired biological activity. As used herein, the "percentage of identity" between two nucleic acid sequences or two amino acid sequences is determined by comparing both optimally aligned sequences over a comparison window.

[0087] Thus, the portion of the nucleotide or amino acid sequence within the comparison window may contain additions or deletions (e.g., "gaps") compared to the reference sequence (which does not include these additions or these deletions) to obtain optimal alignment between the two sequences.

[0088] The terms "sequence homology" or "sequence identity" or "homology" or "identity" are used interchangeably herein. For purposes of the present invention, to determine the percentage of sequence homology or sequence identity between two amino acid sequences or two nucleic acid sequences, it is defined herein that the sequences are aligned for optimal comparison purposes. To optimize the alignment between the two sequences, gaps may be introduced into either of the two sequences being compared. Such alignment can be performed over the entire length of the sequences being compared. Alternatively, the alignment can be performed over a shorter length, for example, about 20, about 50, about 100 or more nucleic acids / bases or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported alignment region.

[0089] The comparison of sequences and determination of the percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. Those skilled in the art will recognize the fact that several different computer programs are available for aligning two sequences and determining the identity between the two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).

[0090] The percent sequence identity between two amino acid sequences or two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for aligning two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned using this algorithm. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE.

[0091] For the purposes of this invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 was used for the substitution matrix. For nucleotide sequences, EDNAFULL was used. The optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5. No end gap penalty is added. In the output section, the question "Brief identity and similarity" is answered with "Yes", and "SRS pairwise" is indicated as the output alignment format.

[0092] After alignment by the program NEEDLE as described above, the percentage of sequence identity between the query sequence and a sequence of the invention is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is displayed as "longest-identity" in the program output.

[0093] Nucleotide and amino acid sequence similarity, i.e., percentage of sequence identity, can be determined using several other art-known algorithms, preferably using 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 the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), such as the CLUSTAL algorithm available at https: / / www.ebi.ac.uk / Tools / msa / clustalo / , or the GAP program (a mathematical algorithm from University of Iowa), or the mathematical algorithm of Myers and Miller (1989-Cabios 4:11-17), or Clone Manager. The number of sequences can be determined via sequence alignments using the Clustalo Genetics Toolkit. 9. The preferred parameters used are the default parameters set at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0094] The grade of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). A similar algorithm is incorporated in Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score=100, word length=12, to obtain polynucleotide sequences homologous to nucleic acids encoding related proteins.

[0095] BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to the SHC polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis can be supplemented with 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 referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless otherwise specified.

[0096] In certain embodiments, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0097] The term "non-oncogenic" as used herein is used in its traditional sense, i.e., to refer to an element, in this case an antigen, that is incapable of causing tumor formation. As detailed elsewhere, the antigens embodied in the present invention have been genetically modified to be non-oncogenic. According to the normal meaning of these terms, it means that the nucleic acid sequences encoding the antigens embodied herein do not occur in nature and have been altered, either by introduction, deletion, or modification of those nucleic acid sequences, resulting in an encoded amino acid sequence that also does not occur in nature.

[0098] Those skilled in the art have long known various means for making deletions, substitutions or introductions in nucleic acid sequences.

[0099] As will be appreciated by those skilled in the art, it may be further advantageous to modify a coding sequence to enhance its expression in a particular host. While the genetic code is redundant with 64 possible codons, most organisms typically use a subset of these codons. The most frequently utilized codons in a given species are referred to as optimal codons, while less frequently utilized codons are classified as rare or low-usage codons. Codons can be substituted to reflect the host's preferred codon usage, a process also known as "codon optimization" or "controlling for species codon bias." Codon optimization for other host cells can be readily determined using codon usage tables or can be performed using commercially available software, such as CodonOp from Integrated DNA Technologies (www.idtdna.com / CodonOptfrom). Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res. 17:477-508) can be prepared, for example, to increase the translation rate or to produce recombinant RNA transcripts with desirable properties, such as a longer half-life, compared to transcripts produced from non-optimized sequences. Translation stop codons can also be modified to reflect host preferences. For example, the typical stop codon for monocotyledonous plants is UGA, while insects and E. coli generally use UAA as the stop codon (Dalphin et al., 1996, Nucl Acids Res. 24:216-8).

[0100] A "non-integrative" lentiviral vector means that the lentiviral vector does not integrate into the host cell genome when the lentiviral vector is in a cell. A non-integrative lentiviral vector particle relates to a lentiviral vector particle comprising a non-integrative lentiviral vector. Non-integrative lentiviral vectors are also called integration-defective lentiviral vectors or non-integrating lentiviral vectors.

[0101] HPV-induced cancer is also known as HPV (Human Papillomavirus)-associated cancer. In fact, HPV infection is not well controlled by the immune system of the infected host. If high-risk HPV infection persists for many years, it can cause cellular changes that, if not treated, can worsen over time and turn into cancer.

[0102] Lentiviral vectors according to the present invention

[0103] The present inventors have devised a novel therapeutic and preventative lentiviral vector-based vaccine against HPV-induced cancers.

[0104] In particular, the present invention relates to lentiviral vectors comprising at least four different nucleic acid sequences selected from a group of specific non-oncogenic HPV antigens.

[0105] By different nucleic acid sequences is meant that the at least four nucleic acid sequences contained in the lentiviral vector are all different, i.e., each of the nucleic acid sequences is a different member of a group of specific non-oncogenic HPV antigens.

[0106] The group of non-carcinogenic HPV antigens is as follows: a nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen; a nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen; Encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen; and A nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen.

[0107] Unlike most HPV proteins that disappear after HPV integration, E6 and E7 proteins are continuously expressed in HPV-induced tumors (Ghittoni, Raffaella et al. Virus genes vol. 40, 1 (2010): 1-13; Morrow, Matthew P et al. Expert review of vaccines vol. 12, 3 (2013): 271-83). These proteins are known to inhibit cellular functions and play an important role in HPV-associated carcinogenesis (Tomaic, Vjekoslav. Cancers vol. 8, 10 95. 19 Oct. 2016; Ghittoni, Raffaella et al. Virus genes vol. 40, 1 (2010): 1-13). E6 and E7 are thought to interfere with multiple pathways, but most importantly, expression of E6 protein in cells leads to ubiquitin-mediated degradation of the tumor suppressor p53 through direct interaction with the intracellular E3 ubiquitin ligase E6AP (Huibregtse, JM et al: 4129-35; Martinez-Zapien, Denise et al. Nature 529,7587 (2016): 541-5), and E7 binds to Rb protein, thereby disrupting the interaction between Rb and E2F and releasing E2F factors (Cassetti, M Cristina et al: 520-7).

[0108] Because E6 and E7 proteins are expressed in all HPV-induced cancers, it was decided to include antigens from these proteins for the two major subtypes (HPV16 and HPV18) in the lentiviral vectors of the present invention. In order to develop a vaccine from E6 and E7 antigens, it is important to eliminate the carcinogenic risk associated with these proteins.

[0109] Non-oncogenic E6 and E7 proteins are thus implemented 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 certain embodiments, partial mutation of the binding sites of E6 and E7 HPV proteins cannot completely eliminate Rb binding, so these sites are particularly completely removed from the sequences implemented in the present invention.

[0110] 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 a DNA molecule.

[0111] In the context of the present invention, a "lentiviral vector" refers to a non-replicating vector for introducing a transgene into a host cell that contains cis-acting lentiviral RNA or DNA sequences and requires essential lentiviral proteins (e.g., Gag, Pol, and / or Env) and accessory proteins (e.g., Tat, Rev) provided in trans. The lentiviral vector lacks expression of all functional HIV proteins. The genome of the lentiviral vector can be in the form of an RNA or DNA molecule, depending on the stage of production or development of the retroviral vector.

[0112] In a preferred embodiment, the lentiviral vector of the present invention is a non-integrative lentiviral vector.

[0113] Non-integrating lentiviral vectors have been designed to mitigate the potential oncogenic risks 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). As a result, it has been reported that non-integrating lentiviral vector systems can reduce the potential risk of insertional mutagenesis compared to integrating systems (Hu et al., Vaccine 28:6675-6683 (2010)). Furthermore, several functional analyses have shown that the magnitude and quality of immune responses elicited by DC-directed integration-defective lentiviral vectors (IDLVs) are comparable to those of integrating vectors. Therefore, integration-defective lentiviral vectors (IDLVs) have been considered safer and equally effective than integrating vectors for human administration.

[0114] Additionally, the deletion of the U3 region of the 3'LTR of the viral promoter and enhancer sequences in self-inactivating lentiviral vectors limits the potential for endogenous promoter activation. These safety concerns directly relate to the experience gained from the SCID-X1 gene therapy trial conducted in 1998-1999, which used a Moloney virus-based retroviral vector in children with a rare form of X-linked (SCID-X1) severe immunodeficiency disease (Cavazzana-Calvo et al., 2000, Science., 288 (5466):669-72). During this study, four of nine children developed leukemia as a result of integration of the Moloney-derived retroviral vector close to the human LM02 proto-oncogene (Hacein-Bey-Abina et al., 2008, J. Clin. Invest., 118(9):3132-3142). The malignancy was demonstrated to be the result of the proximity of the viral U3 promoter / enhancer to the LM02 proto-oncogene. Consequently, safety is a major concern for the administration of lentivectors to humans.

[0115] Thus, lentiviral vectors according to the invention may contain long terminal repeat (LTR) sequences in cis, as known in the art, and in particular contain a 3' long terminal repeat (LTR) lacking its U3 promoter sequence (Miyoshi H et al, 1998, J Virol. 72(10):81 50-7; Zufferey et al., 1998, JV / ro / 72(12):9873-80).

[0116] Enhancers are cis-acting sequences that can act as transcriptional activators at a distance. Enhancer sequences have been widely adopted in virus-derived vectors because they are believed to be the most efficient for achieving strong transgene expression in various cell types, especially DCs (Chinnasamy et al., 2000, Hum Gene Ther 11(13):1901-9; 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 issue of insertional mutagenesis, such transcriptional enhancer sequences should be deleted from lentiviral vector constructs to eliminate the risk of insertional mutagenesis due to the enhancer proximity effect. This enhancer proximity effect is the most frequent mechanism of insertional mutagenesis and the only effect described in human or animal cases of tumorigenic events after gene transfer.

[0117] Therefore, a lentiviral vector according to the present invention may not contain a constitutive enhancer sequence.

[0118] Previous studies have reported replacing viral promoters with DC-specific promoters derived from the major histocompatibility complex class II (MHC class II) and dectin-2 genes (Lopes et al., 2008, J Virol 82(1):86-95) (Kimura et al., 2007, Mol Ther 15(7):1390-9). The dectin-2 gene promoter used by Lopes et al. contains a putative enhancer and an adenoviral conserved sequence (inverted terminal repeats in adenoviral 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 enhancers.

[0119] However, without an enhancer, the MHC class II promoter was found to not provide sufficient transgene expression in DCs when administered intravenously. Notably, lentiviral vectors containing the MHC class II promoter did not elicit an immune response in immunocompetent C57BL / 6 mice, in contrast to the immune response observed with the CMV promoter / enhancer. Although integration and persistent transgene expression were observed after injection into mice, lentiviral vectors transcribed via the MHC class II promoter did not elicit antigen-specific CD8+ cells, even after a vaccination boost. + These studies failed to stimulate a cytotoxic T lymphocyte response. Therefore, the authors of these studies concluded that the use of MHC class II promoters is only of interest in applications requiring sustained expression, such as gene replacement therapy, and not in the context of immunotherapy. Of note, MHC class II promoters are poorly expressed in most cell types.

[0120] Therefore, the MHC class II promoter is not suitable as a promoter for lentiviral vectors to induce immune responses to antigens via intravenous (IV) injection. Furthermore, the dectin-2 promoter is poorly expressed in most cell types and appears to contain an enhancer. Therefore, the dectin-2 promoter is not a good promoter for lentiviral vectors for safety reasons.

[0121] Thus, a lentiviral vector according to the invention may comprise an MHC class I promoter, i.e. the nucleic acid sequence encoding the antigen of a lentiviral vector according to the invention may be under the control of an MHC class I promoter.

[0122] Suitable MHC class I promoters may be selected from the group consisting of β2-microglobulin promoter, HLA-A2 promoter, HLA-B7 promoter, HLA-Cw5 promoter, HLA-E promoter or HLA-F promoter, more particularly β2-microglobulin promoter.

[0123] The MHC class I promoter is dendritic-specific (APC), and promoter expression in BDCA+ dendritic cells is higher than expression in kidney, smooth muscle, liver, and cardiac cells. Other transduced cell types show relatively high expression, e.g., BDCA + Expression of a promoter in dendritic cells is only 12-100 times higher than that of that promoter in skeletal muscle cells, whereas the MHCII HLA-DRα promoter is 900 times higher.

[0124] This promoter specifically drives transcription of the nucleic acid sequence encoding the HPV antigen in the lentiviral vector of the present invention.

[0125] The promoter can be a naturally occurring MHC class I promoter or a synthetic MHC class I promoter obtained using well-known molecular biology techniques.

[0126] A lentiviral vector according to the present invention may comprise a cPPT / CTS sequence as described in EP 2169073. This cPPT / CTS sequence may in particular be the sequence set out as SEQ ID NO: 37.

[0127] In fact, efficient integration and replication in non-dividing cells generally 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 triple-stranded DNA structure called the central DNA "flap," which acts as a signal for the uncoating of the pre-integration complex at the nuclear pore and the efficient uptake of the expression cassette into the nuclei of non-dividing cells, such as dendritic cells.

[0128] The lentiviral vectors of the present invention may contain a woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), which allows for more stable expression of transgenes in vivo, particularly variants of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE).

[0129] The mutated Woodchuck Posttranscriptional Regulatory Element (mWPRE) has been characterized in that a point mutation was introduced to prevent expression of the X protein contained in the WPRE region, which may have oncogenic properties (Kingsman et al. 2005 Jan;12(1):3-4).

[0130] The variant of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE) contained in the lentiviral vector of the invention may in particular have the nucleic acid sequence set forth as sequence SEQ ID NO:38.

[0131] In a particular embodiment, the lentiviral vector according to the invention, in particular the non-integrative lentiviral vector of the invention, (i) comprising at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen, wherein at least one nucleic acid sequence encodes a non-oncogenic human papillomavirus (HPV16) protein E7 antigen, at least one nucleic acid sequence encodes a non-oncogenic human papillomavirus (HPV18) protein E6 antigen, and at least one nucleic acid sequence encodes a non-oncogenic human papillomavirus (HPV18) protein E7 antigen; (ii) contains a 3' long terminal repeat (LTR) lacking the U3 promoter sequence; (iii) does not contain constitutive enhancer sequences; (iv) an MHC class I promoter, particularly a β2-microglobulin promoter; (v) comprises a cPPT / CTS sequence, particularly the cPPT / CTS sequence set forth as SEQ ID NO: 37; and (vi) A variant of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), particularly having the sequence set forth as SEQ ID NO:38.

[0132] As described above, the lentiviral vector according to the present invention comprises: at least one nucleic acid sequence encoding the 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 It is characterized by including

[0133] At least four nucleic acid sequences, in particular four different nucleic acid sequences, encoding HPV antigens of the lentiviral vector of the present invention may be fused together and under the control of a single promoter sequence to form a single antigen nucleic acid sequence encoding a single antigen fusion protein, in particular when (i) there is no linking sequence (also referred to herein as a spacer) between each of the at least four different nucleic acid sequences, or (ii) there is a linking sequence (or spacer) between at least two of the at least four different nucleic acid sequences, more in particular when there is a linking sequence (or spacer) between each of the at least four different nucleic acid sequences.

[0134] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may in particular have a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth as SEQ ID NO:7.

[0135] As used herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% amino acid identity with the reference amino acid sequence.

[0136] 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 the nucleic acid sequences set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0137] As used 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%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and 99% nucleotide identity to the reference nucleic acid sequence.

[0138] In certain embodiments, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may in particular have a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0139] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may in particular comprise 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 the amino acid sequences set forth as SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0140] In certain embodiments, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E6 antigen may in particular have a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of the amino acid sequences set forth as SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0141] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may in particular have a nucleic acid sequence encoding an amino acid sequence having at least 68% sequence identity with the amino acid sequence set forth as SEQ ID NO:16.

[0142] As used 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%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and 99% amino acid identity to the reference amino acid sequence.

[0143] In particular, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may have a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO: 14 and SEQ ID NO: 15.

[0144] As used 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%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and 99% nucleotide identity to the reference nucleic acid sequence.

[0145] In certain embodiments, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may have a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:14 and SEQ ID NO:15.

[0146] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may in particular comprise 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 the amino acid sequences set forth as SEQ ID NO: 17 and SEQ ID NO: 18.

[0147] In certain embodiments, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 16 (HPV16) protein E7 antigen may in particular have a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of the amino acid sequences set forth as SEQ ID NO:17 and SEQ ID NO:18.

[0148] 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 60% sequence identity with the amino acid sequence set forth as SEQ ID NO:24.

[0149] As used 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%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and 99% amino acid identity to the reference amino acid sequence.

[0150] 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 the nucleic acid sequences set forth as SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

[0151] As used 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%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and 99% nucleotide identity to the reference nucleic acid sequence.

[0152] In certain embodiments, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E6 antigen may have a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

[0153] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E6 antigen may in particular comprise 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 the amino acid sequences set forth as SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

[0154] In certain embodiments, 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 selected from the group consisting of the amino acid sequences set forth as SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

[0155] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may in particular comprise a nucleic acid sequence encoding an amino acid sequence having at least 83% sequence identity with the amino acid sequence set forth as SEQ ID NO:33.

[0156] As used herein, an amino acid sequence having at least 83% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and 99% amino acid identity with the reference amino acid sequence.

[0157] 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 the nucleic acid sequences set forth as SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0158] As used 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%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and 99% nucleotide identity to the reference nucleic acid sequence.

[0159] In certain embodiments, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may in particular have a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

[0160] The nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may in particular comprise 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 the amino acid sequences set forth as SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0161] In certain embodiments, the nucleic acid sequence encoding the non-oncogenic human papillomavirus 18 (HPV18) protein E7 antigen may in particular have a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of the amino acid sequences set forth as SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0162] As indicated above, the lentiviral vector according to the present invention comprises: at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E6 antigen (also referred to herein as noE6-HPV16), at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV16) protein E7 antigen (also referred to herein as noE7-HPV16), at least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E6 antigen (also referred to herein as noE6-HPV18); and At least one nucleic acid sequence encoding the non-oncogenic human papillomavirus (HPV18) protein E7 antigen (also referred to herein as noE7-HPV18) Includes.

[0163] In a particular embodiment, the lentiviral vector according to the invention, in particular the non-integrative lentiviral vector of the invention, comprises: (i) 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; wherein nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigen nucleic acid sequence encoding a single antigen fusion protein under the control of a single promoter sequence, more particularly in the absence of any linking sequence between each of them; (ii) containing a 3′ long terminal repeat (LTR) lacking the U3 promoter sequence; (iii) does not contain constitutive enhancer sequences; (iv) an MHC class I promoter, particularly a β2-microglobulin promoter; (v) comprises a cPPT / CTS sequence, particularly the cPPT / CTS sequence set forth as SEQ ID NO: 37; and (vi) A variant of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), particularly having the sequence set forth as SEQ ID NO:38.

[0164] The lentiviral vector according to the invention is more particularly A nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E6 antigen, wherein the nucleic acid sequence has at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and in particular the nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; at least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV16) protein E7 antigen, wherein said nucleic acid sequence has at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO: 14 and SEQ ID NO: 15, and in particular said nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO: 14 and SEQ ID NO: 15; At least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen, which has at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, in particular the nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23; and At least one nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E7 antigen, wherein said nucleic acid sequence has at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, and in particular the nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32. and Here, nucleic acid sequences encoding non-oncogenic HPV antigens are fused together to form a single antigen nucleic acid sequence encoding a single antigen fusion protein under the control of a single promoter sequence, more particularly in the absence of any linking sequences between each of them.

[0165] In a lentiviral vector according to the invention, in particular a non-integrative lentiviral vector of the invention, the at least four, in particular four, different nucleic acid sequences encoding HPV antigens may be in any order in the conventional 5' to 3' reading direction (from the 5' end to the 3' end).

[0166] In particular, the four different nucleic acid sequences encoding the HPV antigens noE6-HPV16, noE7-HPV16, noE6-HPV18 and noE7-HPV18 defined above may be in any order in the conventional 5' to 3' reading direction among the 24 possible combinations in a lentiviral vector according to the invention, in particular a non-integrative lentiviral vector according to the invention, reading from the 5' to 3' end.

[0167] In certain embodiments, the at least four different nucleic acid sequences are, in order from the 5' end to the 3' end: (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 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. is selected from the group consisting of Here, the nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigen nucleic acid sequence encoding a single antigen fusion protein under the control of a single promoter sequence, more particularly in the absence of any linking sequence between each of them.

[0168] These sequences are shown in Figures 8A-8D.

[0169] In the lentiviral vector according to the invention, the order of the at least four different nucleic acid sequences from the 5' end to the 3' end is in particular: (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. may be Here, the nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigen nucleic acid sequence encoding a single antigen fusion protein under the control of a single promoter sequence, more particularly in the absence of any linking sequence between each of them.

[0170] Preferably, the order of the at least four different nucleic acid sequences in the lentiviral vector according to the invention 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; and Here, the nucleic acid sequences encoding non-oncogenic HPV antigens are particularly fused together to form a single antigen nucleic acid sequence encoding a single antigen fusion protein under the control of a single promoter sequence, more particularly in the absence of any linking sequence between each of them.

[0171] More preferably, the order of the at least four different nucleic acid sequences from the 5' end to the 3' end in the lentiviral vector according to the invention 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; and a nucleic acid sequence encoding a non-oncogenic human papillomavirus (HPV18) protein E6 antigen. and Here, the nucleic acid sequences encoding non-oncogenic HPV antigens are fused together and under the control of a single promoter sequence, in the absence of any linking sequences between each of them, to form a single antigen nucleic acid sequence encoding a single antigen fusion protein.

[0172] Each of these four different groups of antigen constructs is implemented in this example: The lentiviral vector deposited at the Collection Nationale de Cultures de Microorganisme (CNCM) on October 21, 2021, under accession number I-5759 (order (a) above); Lentiviral vector deposited with the Collection Nationale de Cultures de Microorganisme (CNCM) on October 21, 2021, under accession number I-5760 (order (b) above); a lentiviral vector deposited with the Collection Nationale de Cultures de Microorganisme (CNCM) under accession number I-5761 on October 21, 2021 (order (c) above); or Lentiviral vector (order (d) above) deposited with the Collection Nationale de Cultures de Microorganisme (CNCM) on October 21, 2021, under accession number I-5762.

[0173] Thus, the lentiviral vector antigen construct deposited at the CNCM under accession number I-5759 has the following nucleotide sequence, set forth as SEQ ID NO:41: and encoding the following amino acid sequence set forth as SEQ ID NO:42: MPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIASQAFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRSAGPGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWVGEPGRTIPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGH (SEQ ID NO: 42).

[0174] The lentiviral vector antigen construct deposited at the CNCM under accession number I-5760 has the following nucleotide sequence, set forth as SEQ ID NO:43: and encoding the following amino acid sequence set forth as SEQ ID NO:44: MFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRGPDDPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIGPDDKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWA (SEQ ID NO: 44).

[0175] The lentiviral vector antigen construct deposited at the CNCM under accession number I-5761 has the following nucleotide sequence, set forth as SEQ ID NO:45: and encoding the following amino acid sequence set forth as SEQ ID NO:46: MRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGHFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRSAGPGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIA (SEQ ID NO: 46).

[0176] The lentiviral vector antigen construct deposited at the CNCM under accession number I-5762 has the following nucleotide sequence, set forth as SEQ ID NO:47: and encoding the following amino acid sequence set forth as SEQ ID NO:48: MGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPGEPGRTIPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIASQAFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRS (SEQ ID NO: 48).

[0177] A lentiviral vector according to the present invention may in particular comprise a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth as SEQ ID NO:42, wherein said nucleic acid sequence is in particular nucleic acid sequence SEQ ID NO:41.

[0178] As used herein, an amino acid sequence having at least 90% identity to a reference amino acid sequence includes amino acid sequences having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and 100% identity to the reference amino acid sequence.

[0179] Thus, the lentiviral vector according to the invention may more particularly be chosen from the lentiviral vectors deposited at the CNCM under accession numbers I-5759, I-5760, I-5761 and I-5762, and in particular the lentiviral vector deposited at the CNCM under accession number I-5762. The lentiviral vector according to the invention may preferably be the lentiviral vector deposited at the CNCM under accession number I-5759, and therefore preferably comprises the nucleic acid sequence SEQ ID NO: 41.

[0180] Lentiviral vector particles according to the present invention

[0181] Another object of the invention relates to a lentiviral vector particle comprising at least one lentiviral vector according to the invention, in particular at least one lentiviral vector as defined above.

[0182] Lentiviral vector particles according to the invention comprise a lentiviral vector according to the invention and can be produced by recombinant techniques known in the art upon transient transfection of cells, e.g., HEK 293T human cultured cells, with different DNA plasmids: (i) a packaging plasmid, which expresses at least Gag, Pol, Rev, Tat, and optionally the structural and enzymatic proteins required for packaging of the transfer construct; (ii) a lentiviral vector according to the invention, wherein the lentiviral vector comprises an expression cassette (antigen) and HIV cis-acting factors necessary for packaging, reverse transcription and integration; and (iii) Plasmids encoding an envelope, most often the glycoprotein of vesicular stomatitis virus (VSV.G), which allows the formation of mixed particles (pseudotypes) that can target a wide variety of cells, particularly major histocompatibility (MHC) antigen-presenting cells (APCs), including DCs.

[0183] Such a method can be used to produce a recombinant vector particle according to the invention, which method comprises: i) transfecting a suitable host cell with a lentiviral vector according to the invention; ii) transducing the host cells with a packaging plasmid vector comprising viral DNA sequences encoding at least the structural and polymerase activities of a retrovirus (preferably a lentivirus), where such packaging plasmids have been 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); iii) culturing the transfected host cells to obtain expression of the lentiviral vector and packaging of the lentiviral vector into lentiviral vector particles; and iv) harvesting the lentiviral vector particles resulting from expression and packaging of step iii) in the cultured host cells. The process includes the steps of:

[0184] To pseudotype the retroviral particles of the present invention, the host cells can be further transfected with one or several envelope DNA plasmids encoding one or more viral envelope proteins, preferably VSV-G envelope proteins.

[0185] This procedure allows for transient production of lentiviral particle vectors by transfected cells. However, by stably inserting the packaging gene, proviral coding DNA, and envelope gene into the cell genome, lentiviral particle vectors can also be continuously produced by the cells. This allows for continuous production of lentiviral particle vectors by the cells without the need for transient transfection. Of course, a combination of these procedures can be used, where part of the DNA / plasmid is integrated into the cell genome and the other is provided by transient transfection.

[0186] The lentiviral vector particle may be a non-integrating lentiviral vector particle. A non-integrating vector particle has one or more mutations that eliminate most or all of the integrating capacity of the lentiviral vector particle. For example, a non-integrating vector particle can contain one or more mutations in the integrase encoded by the lentiviral pol gene that result in a reduction in integrating capacity.

[0187] Lentiviral vector particles according to the invention in particular include non-integrating lentiviral vectors of the invention.

[0188] Lentiviral vector particles according to the invention may comprise vesicular stomatitis virus glycoprotein (VSVG), in particular VSV-G Indiana serotype or VSV-G New Jersey serotype.

[0189] With regard to vaccination strategies, pseudotyped lentiviral vector particles have a high potential to evade the immune system if it has already acquired immunity to the lentivirus, which is particularly useful when serial injections of the same particle vector are required to immunize a patient against a disease.

[0190] The lentiviral vector particle may comprise HIV-1 Gag and Pol proteins, particularly HIV-1 subtype D Gag and Pol proteins.

[0191] A further object of the present invention relates to an isolated cell comprising a lentiviral vector according to the invention or a lentiviral vector particle of the invention (i.e. transformed with a lentiviral vector according to the invention or a lentiviral vector particle of the invention).

[0192] The cells according to the invention are preferably mammalian cells, in particular human cells, particularly preferred are human non-dividing cells.

[0193] Another object of the invention relates to a vaccine composition comprising a lentiviral vector according to the invention, a lentiviral vector particle according to the invention, or a cell according to the invention.

[0194] The vaccine composition according to the present invention comprises a pharmaceutically acceptable vehicle.

[0195] "Pharmaceutically acceptable vehicle" refers to any solution used to solubilize and deliver to an individual a lentiviral vector, lentiviral vector particle, or cell according to the present invention. A preferred pharmaceutically acceptable carrier is saline. In a preferred embodiment, the pharmaceutically acceptable vehicle includes an adjuvant.

[0196] Suitable physiologically acceptable vehicles and their formulations 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.

[0197] Implementation in accordance with the present invention

[0198] An object of the present invention relates to a lentiviral vector, a lentiviral vector particle of the invention or an isolated cell of the invention for use as a medicament or a vaccine.

[0199] In particular, the object of the present invention relates to a 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, for use in the treatment or prevention of HPV-induced cancers and their metastases, in particular HPV-induced cancers.

[0200] As previously indicated, HPV-induced cancers are cancers induced by infection with HPV. Methods for detecting HPV in cancers are known in the art (Aldo Venuti and Francesca Paolini; Head Neck Pathol. 2012 Jul;6(Suppl 1):63-74).

[0201] 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.

[0202] Such cancer metastases according to the present invention may in particular be lung metastases.

[0203] Such prevention and / or treatment means the administration of the considered active agent, in particular the vaccine composition of the invention as defined above, to an individual in need of such prevention and / or treatment.

[0204] Individuals in need of such prevention and / or treatment are animals, particularly mammals, and especially humans.

[0205] The lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the present invention are administered by conventional methods to individuals in need of such prevention and / or treatment in doses sufficient to elicit an immunological response that can be readily determined by one of skill in the art.

[0206] Thus, lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the present invention may be administered intravenously or intramuscularly as indicated below.

[0207] Alternatively, lentiviral vectors, lentiviral vector particles, cells, and vaccine compositions according to the invention may be administered intranasally, which route of administration is particularly useful in the treatment or prevention of oropharyngeal cancer and / or lung metastases.

[0208] The lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the present invention are administered in therapeutically effective amounts, in particular at least 1 x 10 6 , 2 × 10 6 , 5×10 6 , 1×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 or at least 1 x 10 of a lentiviral vector according to the invention. 9 In particular at a dose corresponding to TU (Transduction units), at least 1 x 10 of the lentiviral vector according to the invention 7, 2 × 10 7 , 5×10 7 , 1×10 8 TU or at least 1 x 10 9 In a preferred embodiment, the lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the invention are administered in a dose equivalent to at least 1 x 10 TU of a lentiviral vector according to the invention. 7 TU, more particularly at least 1 x 10 of a lentiviral vector according to the invention 8 TU, in particular at least 1 x 10 of a lentiviral vector according to the invention 9 TU, is administered at a dose equivalent to

[0209] A "therapeutically effective amount" refers, for example, to the amount of a lentiviral vector or lentiviral vector particle, cell, or vaccine composition according to the invention that is necessary to produce one or more of the following effects in a subject: an immune response against HPV-induced tumors; a reduction in the size of HPV-induced tumors, i.e., a reduction of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% in tumor size within 15 to 45 days compared to the tumor size at the time of administration; an increase in CD8+ T cell counts in HPV-induced tumors within 5 to 45 days after administration; + and / or CD4 + Increase in infiltration; CD25 in HPV-induced tumors 5 to 45 days after administration + FoxP3 + CD4 + Decrease in regulatory T cells (Tregs).

[0210] Administration can be carried out using well-known routes, such as those mentioned above, including intravenous, intramuscular, intranasal, intraperitoneal or subcutaneous injection, and in particular may be intravenous, intranasal or intramuscular, and may be intravenous or intramuscular.

[0211] It will be apparent that appropriate dosages and regimens will vary between species and individuals depending on many factors, for example, higher doses will generally be required to achieve an effective immune response in humans compared to mice.

[0212] The lentiviral vectors, lentiviral vector particles, cells and vaccine compositions according to the invention can be administered in a single dose or in two or more doses, for example as illustrated in the Examples. A person skilled in the art will determine in each case the appropriate regimen and dosage for the administration of the active ingredients according to the invention.

[0213] 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).

[0214] The immune checkpoint inhibitor (ICI) according to the present invention may in particular be an antibody, in particular an anti-PD-1 antibody, an anti-PD-L1 (PD-1 ligand) antibody, an anti-CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) antibody, an anti-NKG2A antibody, an anti-TIM-3 (T-cell immunoglobulin and mucin domain-containing 3) antibody, an anti-TIGIT (T-cell immunoreceptor having an Ig domain and an ITIM domain) antibody or an anti-LAG-3 (lymphocyte activation gene 3) antibody. More in particular, the at least one immune checkpoint inhibitor according to the present invention may be a monoclonal antibody selected from the group consisting of an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, an anti-NKG2A monoclonal antibody, an anti-TIM-3 monoclonal antibody, an anti-TIGIT monoclonal antibody and an anti-LAG-3 monoclonal antibody. Even more particularly, the at least one immune checkpoint inhibitor according to the present invention may be a monoclonal antibody selected from the group consisting of an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, an anti-NKG2A monoclonal antibody, an anti-TIM-3 monoclonal antibody, and an anti-TIGIT monoclonal antibody.

[0215] Immune checkpoint inhibitors (ICIs) according to the invention are more particularly antibodies, in particular anti-PD-1 antibodies, anti-PD-L1 (PD-1 ligand) antibodies, anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) antibodies, anti-NKG2A antibodies, anti-TIM-3 (T cell immunoglobulin and mucin domain-containing-3) antibodies, anti-TIGIT (T cell immunoreceptor having an Ig domain and an ITIM domain) antibodies or anti-LAG-3 (lymphocyte-activation gene 3) antibodies, and even more particularly anti-PD-1 antibodies, anti-PD-L1 (PD-1 ligand) antibodies, anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) antibodies, anti-NKG2A antibodies, anti-TIM-3 (T cell immunoglobulin and mucin domain-containing-3) antibodies or anti-TIGIT (T cell immunoreceptor having an Ig domain and an ITIM domain) antibodies. More particularly, the at least one immune checkpoint inhibitor according to the present invention may be an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, an anti-NKG2A monoclonal antibody, an anti-TIM-3 monoclonal antibody, an anti-TIGIT monoclonal antibody and an anti-LAG-3 monoclonal antibody, and in particular may be a monoclonal antibody selected from the group consisting of an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, an anti-NKG2A monoclonal antibody, an anti-TIM-3 monoclonal antibody and an anti-TIGIT monoclonal antibody.

[0216] The anti-PD-1 monoclonal antibody may, for example, be selected from the group consisting of Nivolumab, Pembrolizumab, and Cemiplimab.

[0217] The anti-PD-L1 monoclonal antibody may, for example, be selected from the group consisting of Atezolizumab, Avelumab, and Durvalumab.

[0218] The anti-CTLA-4 monoclonal antibody may, for example, be selected from the group consisting of ipilimumab, tremelimumab, and quavonlimab.

[0219] NKG2A is expressed in 50% of peripheral blood NK cells and in human peripheral blood CD8 + NKG2A is an ITIM (intracytoplasmic tyrosine-based inhibitory motif)-bearing receptor expressed on the surface of 5% of T cells. This cell surface molecule is expressed as a heterodimer with CD94 and interacts with non-classical molecules of the major histocompatibility complex class I (MHC-I), namely, human leukocyte antigen (HLA)-E and mouse Qa-1b. This interaction inhibits the antitumor functions of both T and NK effectors (Andre et al., Cell. 2018 Dec 13;175(7):1731-1743.e13). Anti-NKG2A mAb can be used as a checkpoint inhibitor and inhibits not only NK but also CD8 in mice. + It has been reported that anti-tumor cell-mediated immunity can also be promoted by releasing T cells (Andre et al., Cell. 2018 Dec 13;175(7):1731-1743.e13). An example of an anti-NKG2A monoclonal antibody is monalizumab.

[0220] T-cell immunoglobulin and mucin domain-3 (TIM-3) is a negative regulatory immune checkpoint. It is expressed by various immune cells, particularly 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., Onco Targets Ther. 2018;11:7005-7009). TIM-3 / Gal-9 can inhibit cancer immunity by negatively regulating T cell immunity. TIM-3 plays an important role in T cell exhaustion. In cancer immunotherapy, anti-TIM-3 mAb treatment shows beneficial effects comparable to those of anti-PD-1 mAb treatment. The anti-TIM-3 monoclonal antibody can be selected from the group consisting of Sym023 and sabatolimab.

[0221] The anti-TIGIT monoclonal antibody can be, for example, tiragolumab.

[0222] The anti-LAG-3 monoclonal antibody can be, for example, relatlimab.

[0223] In particular, the ICI may be an anti-PD-L1 monoclonal antibody or an anti-PD-1 monoclonal antibody, in particular an anti-PD-1 monoclonal antibody.

[0224] In particular, the ICI may be selected from the group consisting of anti-PD-L1, anti-NKG2A, anti-TIM-3 and anti-PD-1 monoclonal antibodies, more particularly from the group consisting of anti-NKG2A, anti-TIM-3 and anti-PD-1 monoclonal antibodies.

[0225] The vaccine composition, lentiviral vector, lentiviral vector particle or cell and the immune checkpoint inhibitor for use according to the present invention may be administered simultaneously or separately.

[0226] Considering the unexpected synergistic advantageous properties obtained when combining a lentiviral vector according to the invention with an immune checkpoint inhibitor, as demonstrated in the examples, it may be expected that vaccination with a lentiviral vector according to the invention may increase the number of patients eligible for immune checkpoint inhibitor therapy, in particular anti-PD-1 therapy, anti-NKG2A therapy or anti-TIM-3 therapy.

[0227] By "concurrently," it is understood that (i) the vaccine composition, lentiviral vector, lentiviral vector particles or cells, and (ii) the immune checkpoint inhibitor can be administered at the same moment, or up to the same day or days, in which case they can be administered in the same composition or in separate compositions.

[0228] By "separately", it is understood that (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 several days apart, for example at least two days apart.

[0229] In particular, when (i) the vaccine composition, lentiviral vector, lentiviral vector particle or cell, and (ii) the immune checkpoint inhibitor are administered separately, the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the invention may be administered before the immune checkpoint inhibitor.

[0230] Advantageously, the vaccine composition, lentiviral vector, lentiviral vector particle, or cell according to the present invention can be administered at least two days, particularly at least four days, before the administration of the immune checkpoint inhibitor. Accordingly, the immune checkpoint inhibitor can be advantageously administered at least two days, particularly at least four days, after the administration of the vaccine composition, lentiviral vector, lentiviral vector particle, or cell according to the present invention. More particularly, the immune checkpoint inhibitor can be administered 4 days to 1 month, particularly 4 days to 15 days, and more particularly 4 days to 10 days after the administration of the vaccine composition, lentiviral vector, lentiviral vector particle, or cell according to the present invention.

[0231] The vaccine composition, lentiviral vector, lentiviral vector particle or cell and the immune checkpoint inhibitor for use according to the present invention may be administered by the same route or by different routes.

[0232] The at least one immune checkpoint inhibitor herein is administered in a therapeutically effective dose, i.e., a dose that produces the effect for which it is administered. The exact dose of the immune checkpoint inhibitor will depend on the purpose of treatment and will be ascertainable by one skilled in the art using known techniques.

[0233] The present invention further relates to a method for the treatment and / or prevention of 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.

[0234] The present invention further 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, in particular 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 in the form of a vaccine composition according to the invention, for the treatment and / or prevention of HPV-induced cancer in an individual in need thereof.

[0235] The following examples and figures are offered for illustrative purposes and are not intended to implicitly limit the invention.

[0236] Example

[0237] Materials and Methods

[0238] mouse

[0239] C57BL6jRj mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). All animals were maintained under specific pathogen-free conditions, and all procedures were performed according to approved animal protocols and in accordance with the recommendations for the proper use and care of laboratory animals. All animal experiments were performed in accordance with the guidelines established by French and European regulations on the care and use of laboratory animals.

[0240] Peptides, antibodies, and reagents

[0241] To test the reactivity of the vaccine according to the present invention, 15-mer overlapping peptides with a purity of 80% or higher were ordered from GenScript Biotech (The Netherlands). 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), and anti-CD11b-APC-Vio770 (clone REA592) were purchased from Miltenyi Biotec. 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.

[0242] Antibodies were mixed with PBS containing 1% FCS (Gibco).

[0243] Cyclophosphamide was purchased from Sigma, resuspended in PBS (Gibco), and stored at −20° C. before use.

[0244] cell

[0245] HPV-16 E6 and E7-expressing TC-1 tumor cells were generated as previously described (Lin et al. Cancer Res. 1996 Jan 1;56(1):21-6): Primary lung cells from C57BL6 mice were transformed with the HPV-16 E6 and E7 genes and pVEJB, which expresses the activated human c-Ha-ras oncogene. TC-1 cell lines were cultured in Glutamax RPMI medium (Gibco, supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum).

[0246] Lentiviral vector construction

[0247] The antigen (Ag) construct was cloned into the pFlap-B2m-Ag-WPRE Mutee backbone (see, for example, International Publication No. WO 2016 / 012623). The antigen plasmid contains the cPPT / CTS sequence (SEQ ID NO: 37) required for transduction of non-mitotic cells. The U3 promoter sequence was deleted from the 3' long terminal repeat (LTR) to avoid vector replication. The beta-2 microglobulin (β2m) promoter controls vaccine antigen expression in all transduced cells, thereby preferentially expressing the antigen in antigen-presenting cells (APCs). In addition, it does not contain any known enhancer sequences that may induce mutagenesis and / or genotoxic effects. The antigen plasmid contains a mutated version of the woodchuck hepatitis B virus (WHV) posttranscriptional regulatory element (WPRE) (SEQ ID 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 construct contains a point mutation within the start codon of the X protein, thereby preventing expression of the truncated X protein. Such mutant WPRE sequences do not appear to have oncogenic properties (Themis et al., Mol Ther. 2005 Oct;12(4):763-71).

[0248] The packaging plasmid (pNDK) contains the gag-pol sequence of HIV-1 subtype NDK (GenBank accession number: A34828). The proteins nef, vif, vpr, and env are not expressed. Furthermore, a substitution of aspartic acid (D) to valine (V) at position 64 (D64V) in the HIV-1 integrase protein sequence (pol gene) is sufficient to inhibit integration without interfering with transgene expression in vitro. The lentiviral particles of the present invention are non-integrative.

[0249] Envelope plasmids: pCMV-VSV-G INDco (Indiana) and pCMV-VSV-G NJco (New Jersey) vectors were constructed by subcloning vesicular stomatitis virus (VSV) G protein (VSV-G) Indiana (GenBank accession number J02428) and New Jersey (GenBank accession number P04882) serotype inserts into the pVAX1 expression vector (Invitrogen). Mammalian codon-optimized synthetic genes (GeneArt) encoding glycoproteins from the following vesiculoviruses 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).

[0250] Generation of lentiviral vector particles

[0251] Non-integrative lentiviral particles were produced by transient calcium phosphate co-transfection of HEK 293 T cells (ATCC) with three plasmids (viral antigen plasmid, envelope expression plasmid, and packaging plasmid) following methods well known in the art after expansion of HEK 293 T cells (ATCC) in DMEM supplemented with 1% penicillin / streptomycin and 10% FCS. The culture medium was replaced with serum-free medium after 24 hours. 48 hours after transfection, the supernatant was collected and clarified by centrifugation at 2500 rpm. The viral particles were concentrated by ultracentrifugation (22000 rpm / 88250 g, 4°C for 1 hour) and resuspended in storage buffer (20 mM Pipes, 75 mM NaCl, and 2.5% sucrose).

[0252] Vector Titration

[0253] Lentiviral vector titers were determined by quantitative PCR after transduction of HEK 293 T cells. Aphidicolin was added to HEK 293 T cells 24 h before transduction and maintained throughout the titration process. Cells were incubated for 30 min in lysis buffer (200 mM Tris, 1% NP40, and 1% Tween 20) containing 50 μg / ml RNase A (Sigma). Proteinase K (0.2 mg / ml) was added to the suspension, which was then incubated at 56°C for 4 h. For quantitative PCR, a primer pair specific for RRE (an element of the Ag vector) and GAPDH (in the host cell) was used. Titers were expressed as vector transduction units (TU) / mL.

[0254] HPV vaccine design

[0255] The non-oncogenic, immunogenic E6 and E7 protein sequences employed were selected and modified as previously described herein.

[0256] In particular, four different vaccines have been designed containing the following lentiviral vectors: Lentiviral vector deposited at the Collection Nationale de Cultures de Microorganisme (CNCM) on October 21, 2021, under accession number I-5759; Lentiviral vector deposited at the Collection Nationale de Cultures de Microorganisme (CNCM) on October 21, 2021, under accession number I-5760; a lentiviral vector deposited with the Collection Nationale de Cultures de Microorganisme (CNCM) under accession number I-5761 on October 21, 2021; or Lentiviral vector deposited at the Collection Nationale de Cultures de Microorganisme (CNCM) on October 21, 2021, under accession number I-5762.

[0257] These lentiviral vector particles contain these functional lentiviral vectors, which are quantified by vector titration and expressed as transducing units (TU), as performed in the examples below.

[0258] In vivo immunogenicity of lentiviral vector vaccines (LV vaccines)

[0259] Naive C57BL6 female mice were vaccinated intramuscularly (i.e., with lentiviral vector particles of the present invention containing a functional lentiviral vector of the present invention) in 50 μL of diluent. 14 days later, splenocytes were prepared and restimulated overnight for IFNg ELISPOT with four different HPV peptide pools (each peptide at a final concentration of 2 μg / mL). Each peptide pool corresponds to one of the following non-oncogenic antigen variants: a non-oncogenic variant of the HPV16 E6 protein, a non-oncogenic variant of the HPV16 E7 protein, a non-oncogenic variant of the HPV17 E6 protein, and a non-oncogenic variant of the HPV17 E7 protein. These pools consist of overlapping 15-mers (with an 11 a.a. overlap) corresponding to the full selected antigens.

[0260] In vivo tumor vaccination treatment

[0261] 1.10 for in vivo tumor experiments 6 TC-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 shaver before injection). When the mean tumor volume reached the expected range, the mice were randomized and injected intramuscularly (im) with the LV vaccine of the present invention. Mice were monitored for tumor growth by measuring tumor diameter with a vernier caliper three times a week. For ethical reasons, tumors were limited to 1500 mm 3 Super mice had to be euthanized.

[0262] Immunogenicity of the LV vaccine of the present invention in human PBMCs

[0263] Frozen human PBMCs (StemCell) were gently thawed and stained with 0.5 μM CFSE (Thermofischer) for 10 minutes at 37°C. Cells were then 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), 1 mM sodium pyruvate (Gibco)) in round-bottom 96-well plates (0.2 × 10 cells per well). After 7 days, cells were centrifuged and fresh prewarmed complete RPMI was added. After another 7 days (14 days total), cells were stained with fluorescent antibodies and data were acquired by flow cytometry (MACSQuant analyzer).

[0264] Cellular analysis of tumor immune infiltrate

[0265] Tumors were treated with a Mouse Tumor Dissociation kit (Miltenyi). The cell suspension was filtered through a 70 μm pore filter, treated with red blood cell lysis buffer (Sigma), washed, and centrifuged at 1200 rpm for 5 minutes. The collected cells were stained as described below.

[0266] Near IR LD (Invitrogen), FcγII / III receptor-blocking anti-CD16 / CD32 (clone 2.4G2, BD Biosciences), APC-anti-CD11b (clone N418, BD Biosciences), and BV421-anti-NKp46 (clone 29A1.4, Biolegend) were used to detect NK.

[0267] Samples were acquired on an Attune NxT cytometer (Invitrogen) and data were analyzed with FlowJo software (Treestar, Oregon, USA).

[0268] Intracellular cytokine staining

[0269] Spleen cells from immunized mice were obtained by tissue homogenization and passage through a 100 μm nylon filter (Cell Strainer, BD Biosciences) and plated at 4 × 10 in a 24-well plate. 6Cells were seeded at 1000 cells / well. Spleen cells were stimulated for 6 hours in the presence of 10 μg / mL homologous or control peptide, 1 μg / mL anti-CD28 (clone 37.51), and 1 μg / mL anti-CD49d (clone 9C10-MFR4.B) mAbs (BD Biosciences). For the final 3 hours of incubation, cells were treated with a mixture of Golgi Plug and Golgi Stop (both BD Biosciences). PE-Cy7-anti-CD107a (clone 1D4B, BioLegend) mAb was also added to the cultures at this time. Next, cells were harvested, washed with PBS containing 3% FBS and 0.1% NaN (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. Cells were washed twice in FACS buffer and then permeabilized using a Cytofix / Cytoperm kit (BD Bioscience). Next, cells were washed twice with PermWash 1X buffer from the Cytofix / Cytoperm kit and incubated with a mixture of BV421-anti-IL-2 (clone JES6-5H4) and FITC-anti-TNF (MP6-XT22). Cells were then contacted with a mixture of APC-anti-IFN-γ (clone XMG1.2) and BV605-anti-IL-17A (clone TC11-18H10) mAbs (BD Biosciences) or appropriate control Ig isotypes for 30 minutes at 4°C. Next, cells were washed twice in PermWash and once in FACS buffer, and then fixed overnight with Cytofix (BD Biosciences) at 4°C. Cells were acquired using an Attune NxT cytometer system (Invitrogen), and data analysis was performed using FlowJo software (Treestar, Oregon, USA).

[0270] Example 1: The HPV vaccine of the present invention is immunogenic in vivo

[0271] To determine the ability of the vaccines of the present invention to elicit an immune response, recipient mice were immunized with the four vaccines (five mice per tested vaccine and control group).

[0272] Mice are injected with 1 x 10 lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759, the lentiviral vector deposited at the CNCM under accession number I-5760, the lentiviral vector deposited at the CNCM under accession number I-5761, or the lentiviral vector deposited at the CNCM under accession number I-5762. 7 TU or 50 μL of diluent was injected intramuscularly (i.m.). 14 days later, splenocytes were prepared and restimulated overnight in an IFNg ELISPOT assay with four different peptide pools (each peptide at a final concentration of 2 μg / mL). The results are shown in Figure 1.

[0273] Example 2: The HPV vaccine of the present invention successfully and completely eliminates transplanted tumors in vivo

[0274] TC-1 tumor cells are widely used as a preclinical model to study HPV-induced tumors (Kim, JW et al. Gene therapy vol. 11, 12 (2004): 1011-8). These lung tumor cells were engineered to express E6 and E7 from HPV16 (Lin et al. Cancer Res. 1996 Jan 1;56(1):21-6).

[0275] After TC-1 cells were injected subcutaneously (sc) into the mice, solid tumors were rapidly found at the injection site, and tumors in untreated animals grew and reached the ethical endpoint in 30-40 days. To test the efficacy of the lentiviral vector particles of the present invention, TC-1 cells were injected subcutaneously (sc), and tumor volumes were measured (caliper measurements) every other day. The average tumor volume was 70 mm 3At 10 days, mice were randomized and administered 1 x 10 of 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. 8 TU was vaccinated by intramuscular (im) injection.

[0276] The results obtained are shown in FIG.

[0277] Rapid and highly efficient elimination of tumors is observed in 100% of animals vaccinated with lentiviral vector particles comprising the lentiviral vector deposited at the CNCM under accession number I-5762 and lentiviral vector particles comprising the lentiviral vector deposited at the CNCM under accession number I-5759, in 87.5% of animals vaccinated with lentiviral vector particles comprising the lentiviral vector deposited at the CNCM under accession number I-5760, and in 75% of animals vaccinated with lentiviral vector particles comprising the lentiviral vector deposited at the CNCM under accession number I-5761.

[0278] The vaccine comprising lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759 and the vaccine comprising lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5762 show comparable tumor elimination rates (higher than I-5760 and I-5761), however, complete tumor elimination occurs on average in 37.5 days (±7.4 SD) with I-5759 vaccination, whereas complete tumor elimination occurs 54.7 days after vaccination with I-5762 vaccination.

[0279] The inventors were surprised to observe that the most immunogenic vectors were not necessarily the most effective prophylactically, and that the ranking of immunogenicity did not translate to antitumor efficacy. Indeed, lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759 were the most efficient antitumor vaccines, whereas lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5760 and lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5762 were more immunogenic vectors as measured by IFN-γ production.

[0280] Example 3: A single dose of the HPV vaccine of the present invention is effective against tumor recurrence

[0281] Relapse is common in most cancer types and is defined as the return of disease after a period of improvement, often caused by a small number of tumor cells that survive the initial treatment and form new tumors weeks, months, or even years after treatment.

[0282] A. To mimic recurrence in our model, mice that had cleared the primary tumor were re-challenged on the other flank on day 60. Control mice (naive) were also injected subcutaneously (sc) to confirm tumor cell injection.

[0283] The results obtained are shown in FIG.

[0284] This figure shows that subcutaneous (sc) injection of TC-1 cells into control mice resulted in the formation of solid tumors that reached the ethical limit in less than 30 days. Tumor growth was strongly reduced in rechallenged mice (mice whose right flank tumors disappeared after vaccination). Tumor growth was observed during the first 6 days, after which tumor elimination began.

[0285] The tumors completely disappeared between 13 and 16 days after implantation. A single vaccination of tumor-bearing mice resulted in complete disappearance of the primary tumor, and these mice rapidly cleared secondary tumors, providing strong protection against recurrence.

[0286] B. 119 days after the first implantation, the primary tumor on the other flank disappeared in the mouse. 6 Additional experiments were performed in which mice were re-challenged with TC-1 tumor cells and maintained without any treatment. Control mice (untreated) were also injected subcutaneously (sc) to confirm tumor cell transfer.

[0287] The results obtained are shown in FIG.

[0288] All of the re-challenged mice were still alive 145 days after the initial tumor challenge, demonstrating that a single injection of a vaccine according to the present invention effectively promotes a potent anti-tumor memory protective immune response, thereby efficiently generating a T cell response against a new challenge.

[0289] Example 4: The therapeutic effect of the anti-HPV vaccine of the present invention is dose-dependent

[0290] To determine the effectiveness of lower doses of vaccine, efficacy studies were performed on TC1 tumor-bearing mice. Mice were administered 1 x 10 doses of a vaccine according to the present invention comprising lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759. 8 or 1x10 7 TU / mice were vaccinated.

[0291] In particular, 1x10 6 TC-1 cells were injected into the flank of the animals, and tumor volumes were measured twice weekly (caliper measurements). The average tumor volume was 80 mm 3 When the mice reached 1x10 sera, they were randomized and given either diluent (control), 1x10 sera, or 1x10 sera. 7 TU or 1x10 8Vaccination was administered with TU's I-5759 vaccine via intramuscular (im) injection.

[0292] The results obtained are shown in FIG.

[0293] 1x10 8 Vaccination with TU allows for complete and rapid disappearance of tumors (less than 20 days after vaccination), whereas 1x10 7 We observed that the TU vaccination dose had a partial effect on tumor growth: 3 / 6 (50%) mice were tumor-free by day 22 after vaccination, and the other mice showed initial reductions between days 15 and 18 (5–10 days) after vaccination, but tumor growth could not be controlled thereafter.

[0294] A single low dose (1x10 7 ) showed partial inhibition comparable to that observed with three injections of an adenoviral vector-based vaccine (Rice, AE et al. Cancer gene therapy vol. 22, 9 (2015): 454-62). This suggests that optimal doses of the vaccine according to the present invention are more efficient than adenoviral platforms. Furthermore, efficacy at low doses is most likely enhanced by a second injection of the vaccine.

[0295] Example 5: Vaccination according to the present invention increases CD4 + and CD8 + Increases T cell infiltration and reduces Tregs in treated tumors

[0296] To further understand the antitumor mechanisms induced after vaccination with lentiviral vectors according to the present invention, tumor infiltration was investigated. 6 TC1 tumor cells were injected subcutaneously (sc) into the flank of the animals, and tumor volumes were measured (caliper measurements) twice weekly. The mean tumor volume was 80 mm 3 When the mice reached 100 mg / mL, they were randomized to receive either diluent (control) or 1x10 mL of I-5759. 7TU or I-5759 1x10 8 Patients were vaccinated with either TU via intramuscular (im) injection.

[0297] Ten days after vaccination, tumors were harvested, digested, and analyzed by flow cytometry. FACS staining was performed and data was acquired on a Macsquant facs according to methods well known in the art.

[0298] The results obtained are shown in FIG.

[0299] Tumors from vaccinated mice contained more CD8 + T cells and CD4 + T cells were infiltrating. CD8 + T cells and CD4 + The ratios of CD25 and T cells in the tumor increased approximately four-fold and three-fold, respectively. + FoxP3 + CD4 + The proportion of regulatory T cells (Treg) was strongly reduced in treated animals.

[0300] These observations suggest that vaccines containing the lentiviral vectors of the present invention can target CD8 + T cells and CD4 + This is very important as it suggests that it not only improves T cell recruitment but also reduces the proportion of Tregs within the tumor.

[0301] Example 6: The HPV vaccine of the present invention completely eliminates large tumors in vivo

[0302] Well-established tumors are known to be more difficult to eliminate than small and early-stage tumors. Most vaccines tested in the TC1 model have weak efficacy when administered at later time points (Rice, AE et al. Cancer gene therapy vol. 22, 9 (2015): 454-62; Berraondo, Pedro et al. Cancer research vol. 67, 18 (2007): 8847-55). 1x10 6 TC1 tumors were injected subcutaneously (sc) into the flank of the animals. The average tumor volume was approximately 300 mm 3 When the mice reached 100 mg / mL, they were randomized and administered 1 x 10 mL of a vaccine according to the invention containing diluent (control) or lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759. 8 Mice were vaccinated with TU by intramuscular (im) injection. Tumor volumes were measured twice weekly with calipers.

[0303] The results obtained are shown in FIG.

[0304] Vaccines according to the present invention prove to be highly effective in completely eliminating established HPV-induced tumors.

[0305] Example 7: The HPV vaccine of the present invention induces activation of human PBMCs in vitro

[0306] To confirm that the vaccine according to the present invention can induce T cell responses in human cells, human PBMCs (stem cells) were labeled with CFSE and cultured in the absence (unstimulated condition) or presence of the vaccine according to the present invention (I-5759). After two weeks of culture, cell proliferation and activation were measured.

[0307] CD8 + T cells and CD4 + T cell proliferation (measured by CFSE dilution) and expression of the CD25 activation marker were increased by adding the lentiviral vector of the invention during culture.

[0308] The results obtained are shown in FIG.

[0309] Thereby it can be concluded that PBMC-derived antigen-presenting cells are transduced by the HPV vaccine according to the invention and are able to process antigens and activate T cells.

[0310] Example 8: Systemic T cell immunity and effector T cell phenotypes induced by the HPV vaccine of the present invention

[0311] To gain further insight into the quality of the elicited T cell response, mice injected with control Lenti (LV-empty Indiana) or with a vaccine according to the present invention comprising lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759 were either left untreated or injected with the immunodominant H-2D b ETTDPD containing the restricted RAHYNIVTF epitope RAHYNIVTF and PD RAHYNIVTF CCKC E7 HPV16 The cells were stimulated in vitro with a mixture of derived peptides (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.

[0312] The results obtained are shown in FIG. 9A.

[0313] Stimulation with these peptides increased the CD8 + T cell responses were detected. Functional CD8 + T cell effectors are IFN-γ + (single positive), TNF-α + IFN-γ + or IL-2 + IFN-γ +(double positive) and IL-2 + TNF-alpha + IFN-γ + (triple positive) subset (see Figure 9B).

[0314] IFN-γ + CD8 + The majority of T cells also expressed the surface CD107a degranulation marker, indicating the effector properties of these T cells (see Figure 9B).

[0315] Example 9: Characteristics of tumor cells and tumor-infiltrating innate immune cells in mice vaccinated with the HPV vaccine of the present invention

[0316] Cytometric characterization of intra-tumoral infiltrates 11 days after vaccination, thus during the tumor regression phase, showed a significant increase in the proportion of natural killer (NK) cells in regressing tumors of mice vaccinated with a vaccine according to the present invention comprising lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759.

[0317] The results are shown in FIG.

[0318] Example 10: Suboptimal Lenti-HPV-07 Vaccination Acts Synergistically with Anti-PD1 Immunotherapy

[0319] The inventors further investigated the possible synergistic effect of suboptimal Lenti-HPV-07 (I-5759) vaccination and anti-PD-1 therapy.

[0320] Anti-PD-1 treatment was initiated 4 days (D17, i.e., 17 days after subcutaneous (sc) administration of TC-1 cells to mice) after injection of a vaccine according to the present invention (D13, i.e., 13 days after subcutaneous (sc) administration of TC-1 cells to mice) containing lentiviral vector particles containing the lentiviral vector deposited at the CNCM under accession number I-5759. Several injections of anti-PD-1 were performed (D17 as described above, followed by D20, D22, D24, D28, and D31).

[0321] Specifically, the experiment was conducted in three identical groups of tumor-implanted mice. In the first group (10 mice—control group), mice were administered LV-empty Indiana (D13) (as a control) and, four days later, an anti-PD-1 monoclonal antibody (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, four days later, an isotype-controlled control antibody (D17, followed by D20, D22, D24, D28, and D31). In a third group (14 mice), mice were administered a vaccine according to the invention (D13) (I-5759) followed 4 days later by mAb anti-PD-1 (D17, then D20, D22, D24, D28 and D31).

[0322] The results are shown in Figures 12A and 12B.

[0323] Suboptimal doses of the vaccine, which elicited insufficient anti-tumor T cell responses, acted synergistically with anti-PD-1 to increase tumor regression rates.

[0324] Six of 14 mice achieved complete tumor regression, and the other two showed partial tumor regression. In the latter, tumor volume decreased by 67% and then recurred 6–7 days after the end of anti-PD-1 treatment, highlighting the need for repeated anti-PD-1 injections until the tumor completely disappeared. Of the 12 mice treated with the vaccine of the present invention alone, only three showed partial tumor regression. Accordingly, mouse survival rates were significantly increased in the combo-treatment group compared with mice treated with a suboptimal dose of Lenti-HPV-07 (Figure 12B). Therefore, synergistic anti-tumor effects can be obtained when the Lenti-HPV-07 vaccine candidate is combined with anti-PD1 checkpoint blockade treatment.

[0325] Example 11: A single injection of a vaccine according to the invention cures mice with pulmonary metastatic lesions induced by intravenous injection of TC1-nLuc cells

[0326] The TC1 parental cell line was stably transduced with an integrative lentiviral vector encoding a nanoluciferase reporter and puromycin N-acetyltransferase (for selection) under the ubiquitin (UBC) promoter. After puromycin selection, the cells were subcloned to obtain the TC1-nLuc cell line.

[0327] Six-week-old C57BL / 6JRj mice purchased from Janvier Laboratory were intravenously injected with 150,000 TC1-nLuc cells. 9 TU / mouse were injected intramuscularly with Lenti-HPV-07 or control Lenti (empty vector).

[0328] Bioluminescence imaging of live animals was performed using an IVIS Imaging System (IVIS Spectrum, Perkin Elmer) connected to a charge-coupled device camera. Prior to bioluminescence 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 at Institut Pasteur) was dissolved at 2 mg / ml in acidified ethanol. Z108 was further diluted with sterile D-PBS to the desired concentration (0.4 mg / kg) before intravenous injection. Mice were then immediately placed in the imaging chamber and imaged. Sequential images were taken under autoexposure settings with a maximum exposure time of 2 min.

[0329] Images from each experimental set were analyzed using Living Image Software (Ver. 2.50.1 Xenogen). Measurements were taken from regions of interest, and luminescence values were evaluated as total luminous flux (photons / second). The abdomen and torso of the mice were shaved to increase the signal-to-noise ratio. Baseline signals were obtained from untreated mice injected with Z108, i.e., mice not injected with TC1-nLuc cells or lentiviral vectors.

[0330] The results obtained are shown in Figures 13A and 13B.

[0331] The present inventors have confirmed that a single intramuscular injection of the Lenti-HPV-07 vaccine (I-5759) according to the present invention completely eradicates subcutaneously established TC1 tumors in 100% of animals. However, in humans, many cancers, including those induced by HPV, occur at mucosal sites.

[0332] Therefore, the present experiment evaluated the ability of Lenti-HPV-07 to inhibit tumor growth at mucosal sites.

[0333] To address this question, a TC1 cell line (TC1-nLuc) stably expressing a nanoluciferase reporter gene was developed. After intravenous injection of TC1-nLuc, mice readily developed lung metastases.

[0334] Longitudinal tumor progression was followed in live animals by bioluminescence imaging.

[0335] Five days after tumor injection, mice were infected with Lenti-HPV-07 (1.10 9 TU / mouse) or control Lenti (empty vector) was injected intramuscularly once (Figure 13A).

[0336] All mice treated with Lenti-HPV-07 were cured 22 days after tumor injection, whereas lung metastases continued to grow in the control group. The observed difference between the mean bioluminescence signals in the two groups was nearly statistically significant (Figures 13A and 13B).

[0337] This observation clearly demonstrates that the vaccine according to the invention is able to eradicate lung tumors as efficiently as tumors formed subcutaneously.

[0338] Example 12: Lenti-HPV-07 vaccination acts synergistically with anti-NKG2A immunotherapy

[0339] Antitumor synergy between suboptimal Lenti-HPV-07 (I-5759) vaccination and anti-NKG2A therapy was demonstrated using monoclonal antibody (mAb) clone 20D5 (catalog no. BE0321, BioXCell) in C57BL / 6 mice subcutaneously implanted with HPV-induced TC-1 tumor cells (Figure 14A).

[0340] 1x10 used in previous experiments 9Because administration of TU Lenti-HPV-07 completely eradicated tumors in 100% of mice, we administered a suboptimal dose of 1x10 8 A dose of 1x10 TU was used. Anti-NKG2A mAb treatment was initiated 4 days after Lenti-HPV-07 injection. Control animals received 1x10 8 TU Lenti-HPV-07 and control Ig (Ctrl Ig) were administered.

[0341] It is well known that anti-NKG2A mAb treatment has no effect on the proliferation of TC1 tumor cells (van Montfoort et al., Cell. 2018 Dec 13;175(7):1744-1755.e15), because an anti-NKG2A mAb monotherapy group was not included.

[0342] result

[0343] In the Lenti-HPV-07 + control Ig group, 5 of 12 mice achieved tumor regression, while 7 of 12 mice failed to control tumor growth (Figure 1B). 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 (Figure 14B).

[0344] It should be noted that after cessation of anti-NKG2A mAb treatment, tumors recurred in 6 of 12 mice, strongly suggesting the need for repeated injections of anti-NKG2A to maintain the tumor under immune control. 80% of mice treated with Lenti-HPV-07 plus anti-NKG2A mAb showed a complete or partial regression response, compared with only 40% of mice treated with Lenti-HPV-07 alone (Figure 14C).

[0345] Progression-free survival (PFS) was doubled in mice receiving Lenti-HPV-07 in combination with anti-NKG2A mAb compared to mice treated with Lenti-HPV-07 plus control Ig (Figure 14D).

[0346] Thus, survival time was significantly increased in the combined treatment group (FIG. 14E).

[0347] Statistical significance was determined by Log-rank Mantel-Cox test in Figure 14E, *p=0.0134, and unpaired t-test in Figure 14D.

[0348] 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 therapy was greater than that of Lenti-HPV-07 plus control Ig, suggesting that the effect of the combination therapy was synergistic rather than additive.

[0349] Therefore, when Lenti-HPV-07 is combined with anti-NKG2A checkpoint blockade treatment, a synergistic anti-tumor effect can be achieved.

[0350] Example 13: Lenti-HPV-07 vaccination acts synergistically with anti-TIM-3 immunotherapy

[0351] Potential antitumor synergy between suboptimal Lenti-HPV-07 vaccination and anti-TIM-3 therapy was demonstrated using monoclonal antibody (mAb) clone RMT3-23 (catalog no. BE0115, Bioxcell) in C57BL / 6 mice subcutaneously implanted with HPV-induced TC-1 tumor cells (Figure 15A). As described above, to assess the possible beneficial effect of additional administration of anti-TIM-3 mAb, a suboptimal dose of 1x10 Lenti-HPV-07 was administered. 9 TU induced complete tumor eradication in 100% of mice, so 1x108 A suboptimal dose of TU was used. Anti-TIM-3 mAb treatment was initiated 4 days after Lenti-HPV-07 injection. Control animals received 1x10 8 TU received Lenti-HPV-07 and control Ig (Ctrl Ig). Other groups received control Lenti + control Ig or control Lenti + anti-TIM-3 mAb.

[0352] No tumor regression was recorded in the control Lenti + control Ig group or the control Lenti + anti-TIM-3 mAb group. Therefore, anti-TIM-3 mAb treatment alone had no effect on TC-1 tumor growth (Figure 15B). In the Lenti-HPV-07 + control Ig group, 4 of 12 mice achieved tumor regression, whereas 8 of 12 mice failed to control tumor growth (Figure 15B). In contrast, in the Lenti-HPV-07 + anti-TIM-3 mAb group, 8 of 12 mice achieved tumor regression, whereas 4 of 12 mice failed to suppress tumor growth.

[0353] Sixty percent of mice treated with Lenti-HPV-07 plus anti-TIM-3 mAb showed complete or partial regression responses, compared with 40% of mice treated with Lenti-HPV-07 alone (Figure 15C). Progression-free survival (PFS) was longer in mice treated with Lenti-HPV-07 plus anti-TIM-3 mAb compared with mice treated with Lenti-HPV-07 plus control Ig, although the difference did not reach statistical significance (Figure 15D). Survival was significantly increased in the combined treatment group (Figure 15E). Statistical significance was determined using the log-rank Mantel-Cox test (*p=0.0465, ****p<0.0001).

[0354] Because treatment with anti-TIM-3 mAb alone had no effect on TC-1 tumor growth, the effect of the combination therapy was greater than that of Lenti-HPV-07 plus control Ig, suggesting that the effect of the combination therapy was synergistic rather than additive.

[0355] Therefore, when Lenti-HPV-07 is combined with anti-TIM-3 checkpoint blockade treatment, a synergistic anti-tumor effect can be achieved.

[0356] array

[0357] SEQ ID NO:1 is the nucleic acid sequence encoding the E6 protein from HPV 16 (NC_001526.4): ATGCACCAAAAGAGAACTGCAATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTGCAAACAACTATACATGATATAATATTAGAATGTGTACTGCAAGCAACAGTTACTGCGACGTGAGGTATATGACTTTGCTTTTCGGGATTTATGCATAGTATAGAGATGGGAATCCATATGCTGTATGTGATAAATGTTTAAAGTTTTATTCTAAAA TTAGTGAGTATAGACATTATTGTTATAGTTTGTATGGAACAACATTAGAACAGCAATACAACAAACCGTTGTGTGATTTGTTAATTAGGTGTATTAACTGTCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACAAAAAGCAAAGATTCCATAATATAAGGGGTCGGTGGACCGGTCGATGTATGTCTTGTTGGAATCATCAAGAACACGTAGAGAAACCCAGCTGTAA

[0358] SEQ ID NO:2 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16: GACCCCCAAGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACCGGAACCCCTACGCCGTGTGCGACAAGTGCC TGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGCCTGTACGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACTGCCAGAAGCCCCTGCGGTTCCACAACATCCGGGGCAGATGGACCGGCCGGTGCATGAGCTGCTGCAGA

[0359] SEQ ID NO:3 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16: ATGGGCACCCTGGGCATCGGTGTGCCCCATCGACCCCCAAGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACCGGAACCCCTACGCCG TGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGCCTGTACGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACTGCCAGAAGCCCCTGCGGTTCCACAACATCCGGGGCAGATGGACCGGCCGGTGCATGAGCTGCTGCAGA

[0360] SEQ ID NO:4 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16: ATGGACCCCCAAGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGCTGAGAAGAGAGGTGTACGACTTCGCCTTCCGGGACCTGTGCATCGTGTACCGGAACCCCTACGCCGTGTGCGACAAGTGC CTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGCCTGTACGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCAGATGCATCAACTGCCAGAAGCCCCTGCGGTTCCACAACATCCGGGGCAGATGGACCGGCCGGTGCATGAGCTGCTGCAGA

[0361] SEQ ID NO:5 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16: TTTCAGGACCCCCAGGAAAGGCCCAGGAAGTTGCCCCAGCTCTGCACCGAACTGCAGACCACCATTCATGACATCATCCTCGAATGCGTGTACTGCAAGCAGCAGCTCCTGAGGAGGGAGGTGTACGATTTCGCCTTCAGAGACGGCTGTATCGTCTACAGGAACCCCTATGCCGTCTGCGACAAAT GCCTGAAGTTTTATTCCAAGATCTCCGAGTACAGGCACTATTGCTACAGCCTGTATGGGACCACCCTGGAGCAGCAGTACAACAAGCCCCTGTGCGACCTCCTGATCAGGTGCATCAACTGCCAGAAGCCCCTGAGGTTCCACAACATCCGCGGCAGGTGGACCGGAAGGTGCATGTCCTGCTGCAGG

[0362] SEQ ID NO:6 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 16: TTCCAGGACCCCCAGGAGAGGCCCAGGAAACTGCCCCAGTTGTGCACCGAGCTCCAGACAACCATCCACGACATCATCCTGGAGTGCGTGTACTGTAAGCAGCAGTTGCTGAGGAGAGAGGTGTATGACTTCGCCTTCAGAGACGGATGCATTGTCTATAGGAACCCCTACGCCGTGTGCGACAAGTGC CTGAAGTTCTACTCCAAGATCAGTGAGTACAGGCATTACTGCTACAGCCTGTATGGAACCACACTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTCCTGATTAGGTGCATCAACTGCCAGAAGCCCCTCAGGTTCCACAACATCCGGGGCAGGTGGACCGGAAGGTGCATGTCCTGCTGCAGGTCC

[0363] SEQ ID NO:7 is the amino acid sequence of the wild-type (WT) E6 protein from HPV 16: MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL

[0364] SEQ ID NO:8 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16: DPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR

[0365] SEQ ID NO:9 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16: MGTLGIVCPIDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR

[0366] SEQ ID NO:10 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16: MDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR

[0367] SEQ ID NO:11 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16: FQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR

[0368] SEQ ID NO:12 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 16: FQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRS

[0369] SEQ ID NO:13 is the nucleic acid sequence encoding the E7 protein from HPV 16 (NP-041326.1): ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAG CCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA

[0370] SEQ ID NO:14 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 16: ACCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCCGAGACAACCGACCCCGACCGGGCCCACTACAATATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGCGGCTGTGCGTGCAGAGCACCCACGTGGACATCCGGACCCTGGAAGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCCATT

[0371] SEQ ID NO:15 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 16: CCCGGAGACACCCCCACCCTGCACGAATACATGCTGGACCTGCAGCCCGAAACCACCGACCCCGACCGCGCTCACTACAACATCGTTACATTCTGTTGTAAATGCGACTCCACCCTGAGAAGATGCGTGCAGTCCACCCACGTGGACATCAGGACCCTGGAGGACCTCCTCATGGGAACCCTGGGTATCGTCTGCCCCATC

[0372] SEQ ID NO:16 is the amino acid sequence of the wild-type (WT) E7 protein from HPV 16: MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

[0373] SEQ ID NO:17 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 16: TPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPI

[0374] SEQ ID NO:18 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 16: PGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPI

[0375] SEQ ID NO:19 is the nucleic acid sequence encoding the E6 protein from HPV 18 (MF288727.1): ATGGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGAACACTTCACTGCAAGACATAGAAATAACCTGTGTATATTGCAAGACAGTATTGGAACTTACAGAGGTATTTGAATTTGCATTTAAAGATTTATTTGTGGTGTATAGAGACAGTATACCGCATGCTGCATGCCATAAATGTATAGATTTTTATTCTAGAATTAGAGAATTAAGAC ATTATTCAGACTCTGTGTATGGAGACACATTGGAGAAACTAACTAACACTGGGTTATACAATTTATTAATAAGGTGCCTGCGGTGCCAGAAACCGTTGAATCCAGCAGAAAAACTTAGACACCTTAATGAAAAACGACGATTCCACAACATAGCTGGGCACTATAGAGGCCAGTGCCATTCGTGCTGCAACCGAGCACGACAGGAAAGACTCCAACGACGCAGAGAAACACAAGTATAA

[0376] SEQ ID NO:20 is the nucleic acid sequence encoding the E6 protein from HPV 18: CCCTACAAGCTGCCTGACCTGTGTACAGAGCTGAACACCTCCCTGCAGGACATCGAGATCACCTGTGTGTATTGCAAGACCGTGCTGGAACTGACCGAGGTGTTCGAGTTTGCCTTCAAGGATCTGTTCGTGGTGTACCGGGACAGCA TCCCCCAGCCGCCTGCCACAAGCTGGAAAAGCTGACCAACACCGGCCTGTACAACCTGCTGATTCGGTGCCTGCGGTGTCAGAAGCCTCTGAACCCCGCCGAGAAGCTGCGGCACCTGAACGAGAAAGCGGAGATTCCACAATATCGCC

[0377] SEQ ID NO:21 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18: CCCTACAAGCTGCCTGACCTGTGTACAGAGCTGAACACCTCCCTGCAGGACATCGAGATCACCTGTGTGTATTGCAAGACCGTGCTGGAACTGACCGAGGTGTTCGAGTTTGCCTTCAAGGATCTGTTCGTGGTGTACCGGGACAGCATCCCCCACGCCGCCTGCCACAAG

[0378] SEQ ID NO:22 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18: CCCTACAAGCTGCCCGATCTGTGCACAGAGCTGAACACCTCCCTGCAGGACATCGAGATCACCTGCGTCTACTGCAAGACCGTGCTGGAACTGACCGAGGTGTTCGAATTCGCCTTCAAGGACGGCTTCGTGGTGTACAGGGACAGCA TTCCCCACGCCGCCTGCCATAAGCTGGAGAAACTGACCAACACCGGACTGTATAACCTGCTGATCAGGTGTCTGAGGTGCCAGAAGGCAGAGAAACTGAGACATCTGAACGAGAAAAGGAGGTTCCACAATATTGCCGGGCACTGATAA

[0379] SEQ ID NO:23 is a nucleic acid sequence encoding a non-oncogenic variant of the E6 protein from HPV 18: ATGAGGCGGCCCTACAAGCTGCCCGACCTGTGCACCGAGCTGAACACCTCCCTGCAGGACATCGAGATCACCTGCGTGTACTGCAAGACCGTGCTGGAGCTGACCGAGGTGTTCGAATTCGCATTCAAGGACGGATTCGTCGTGTATAGG GACAGCATTCCACACGCCGCCTGCCACAAGCTGGAGAAATTGACTAACACCGGACTGTATAATCTGCTGATCCGGTGCCTGAGGTGTCAGAAGGCCGAGAAGCTGAGGCATCTGAACGAGAAAAGGAGATTCCACAATATCGCCGGACAC

[0380] SEQ ID NO:24 is the amino acid sequence of the wild-type (WT) E6 protein from HPV 18: MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV

[0381] SEQ ID NO:25 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18: PYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIA

[0382] SEQ ID NO:26 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18: PYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHK

[0383] SEQ ID NO:27 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18: PYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGH

[0384] SEQ ID NO:28 is the amino acid sequence of a non-oncogenic variant of the E6 protein from HPV 18: MRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGH

[0385] SEQ ID NO:29 is the nucleic acid sequence encoding the E7 protein from HPV 18 (NC_001357.1): ATGCATGGACCTAAGGCAACATTGCAAGACATTGTATTGCATTTAGAGCCCAAAATGAAATTCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACTCAGAGGAAGAAAACGATGAAATAGATGGAGTTAATCATCAACATTTACCAGCCCGACGA GCCGAACCACAACGTCACACAATGTTGTGTATGTGTTGTAAGTGTGAAGCCAGAATTGAGCTAGTAGTAGAAAGCTCAGCAGACGACCTTCGAGCATTCCAGCAGCTGTTTCTGAACACCCTGTCCTTTGTGTGTCCGTGGTGTGCATCCCAGCAGTAA

[0386] SEQ ID NO:30 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18: AAGGCCACACTGCAGGATATCGTGCTGCACCTGGAACCCCAGAACGAGATCCCCGTGGACAGCGAGGAAGAGAACGACGAGATCGACGGCGTGAACCACCAGCATCTGCCCGCCAGAAGGGCCGAGCCCC AGAGACACACCATGCTGTGCATGTGTTGCAAATGCGAGGCCCGGATCAAGCTGGTGGTGGAAAGCAGCGCCGACGACCTGCGGGCCTTCCAGCAGCTGTTCCTGAACACCCTGTCCTTCGTGTGCCCTTGG

[0387] SEQ ID NO:31 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18: GGACCTAAAGCCACCCTCCAGGACATCGTGCTGCACCTGGAGCCCCAGAACGAGATCCCCGTCGACTCAGAGGAGGAGAACGACGAAATTGACGGCGTCAACCACCAGCACCTGCCCGCTCGCAGAGCCGAAC CCCAGAGACACACCATGCTCTGCATGTGCTGCAAATGCGAGGCCCGGATTAAGCTGGTGGTGGAGAGCTCCGCCGACGATCTGAGAGCCTTCCAGCAGCTCTTCCTGAACACCCTGTCCTTCGTGTGCCCCTGG

[0388] SEQ ID NO:32 is a nucleic acid sequence encoding a non-oncogenic variant of the E7 protein from HPV 18: GGACCTAAAGCCACCCTCCAGGACATCCGTCTGGAGCCCCAGAACGAGATCCCCGTCGACTCAGAGGAGGAGAACGACGAAATTGACGGCAACCACCAGCACCTGCCCGCTCGCAGAGCCGAACCC CAGAGACACACCATGCTCTGCATGTGCTGCAAATGCGAGGCCCGGATTAAGCTGGTGGTGGAGAGCTCCGCCGACGATCTGAGAGCCTTCCAGCAGCTCTTCCTGGATTCCTTCGTGTGCCCCTGG

[0389] SEQ ID NO:33 is the amino acid sequence of the wild-type (WT) E7 protein from HPV 18: MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ

[0390] SEQ ID NO:34 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18: KATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPW

[0391] SEQ ID NO:35 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18: GPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPW

[0392] SEQ ID NO:36 is the amino acid sequence of a non-oncogenic variant of the E7 protein from HPV 18: GPKATLQDIRLEPQNEIPVDSEEENDEIDGNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLDSFVCPW

[0393] SEQ ID NO:37 is the nucleic acid sequence encoding the cPPT / CTS sequence: AATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTT

[0394] SEQ ID NO:38 is a nucleic acid sequence encoding a variant of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE): TTCCCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCC TTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTT CCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCGCGGCTGCTCGCCTGTGTTGCCACCTGGA TTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC

[0395] SEQ ID NO:39 RAHYNIVTF H-2D b -Contains restricted T cell epitopes, E7 HPV16 is a synthetic peptide derived from: ETTDPDRAHYNIVTF

[0396] SEQ ID NO:40 RAHYNIVTF H-2D b -Contains restricted T cell epitopes, E7 HPV16 is a synthetic peptide derived from: PDRAHYNIVTFCCKC

[0397] SEQ ID NO:41is the nucleic acid sequence encoding the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5759:

[0398] SEQ ID NO:42 is the amino acid sequence of the antigen construct encoded by the lentiviral vector deposited at the CNCM under accession number I-5759: MPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIASQAFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCR SAGPGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWVGEPGR TIPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGH

[0399] SEQ ID NO:43 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5760:

[0400] SEQ ID NO:44 is the amino acid sequence of the antigen construct encoded by the lentiviral vector deposited at the CNCM under accession number I-5760: MFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRGPDDPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLT NTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIGPDDKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWA

[0401] SEQ ID NO:45 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5761:

[0402] SEQ ID NO:46 is the amino acid sequence of the antigen construct encoded by the lentiviral vector deposited at the CNCM under accession number I-5761: MRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNI AGHFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLC DLLIRCINCQKPLRFHNIRGRWTRGRCMSCCRSAGPGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWAGPGDTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIA

[0403] SEQ ID NO:47 is the nucleic acid sequence encoding the antigen construct of the lentiviral vector deposited at the CNCM under accession number I-5762:

[0404] SEQ ID NO:48 is the amino acid sequence of the antigen construct encoded by the lentiviral vector deposited at the CNCM under accession number I-5762: MGPKATLQDIVLHLEPQNEIPVDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPGEPGRTIPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDGFVVYRDSIPHAACHKLEKLTNTGLYNLLIRCLRCQKAEKLRHLNEKRRFHNIAGPG DTPTLHEYMLDLQPETTDPDRAHYNIVTFCCKCDSTLRRCVQSTHVDIRTLEDLLMGTLGIVCPIASQAFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDGCIVYRNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLRFHNIRGRWTGRCMSCCRS

[0405] SEQ ID NO:49 is H-2D b - the amino acid sequence of the restricted T cell epitope: RAHYNIVTF

[0406] JPEG2025527186000002.jpg232170

[0407] JPEG2025527186000003.jpg235170

[0408] JPEG2025527186000004.jpg233170

[0409] JPEG2025527186000005.jpg234170

Claims

1. 1. A lentiviral vector, particularly a non-integrating lentiviral vector, or a lentiviral vector particle, particularly a non-integrating lentiviral vector particle, for use in the treatment or prevention of HPV-induced cancer, comprising: The lentiviral vector comprises: at least one nucleic acid sequence encoding the 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; at least four different nucleic acid sequences selected from the group consisting of the lentiviral vector particle comprises at least one of the lentiviral vectors; the lentiviral vector or the 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 an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, an anti-NKG2A monoclonal antibody, an anti-TIM-3 monoclonal antibody, an anti-TIGIT monoclonal antibody, and an anti-LAG-3 monoclonal antibody; The lentiviral vector or the lentiviral vector particle.

2. 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. 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 set forth as SEQ ID NO: 7, wherein the nucleic acid sequence is particularly 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. 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 set forth as SEQ ID NO: 16, wherein the nucleic acid sequence is in particular selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO:

15.

5. 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 set forth as SEQ ID NO: 24, wherein the nucleic acid sequence is in particular selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO:

23.

6. 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 set forth as SEQ ID NO: 33, wherein the nucleic acid sequence is in particular selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO:

32.

7. 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 antigen nucleic acid sequence encoding a single antigen fusion protein under the control of a single promoter sequence.

8. The at least four different nucleic acid sequences are in the following order from the 5' end to the 3' end: (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. The lentiviral vector or the lentiviral vector particle for use according to any one of claims 1 to 7, which is selected from the group consisting of:

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

10. 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 set forth as SEQ ID NO: 42, wherein said nucleic acid sequence is in particular the nucleic acid sequence SEQ ID NO:

41.

11. 11. The lentiviral vector or lentiviral vector particle for use according to any one of claims 1 to 10, which is selected from the group consisting of the non-integrative lentiviral vectors deposited at the CNCM under accession numbers I-5759, I-5760, I-5761 and I-5762, in particular the non-integrative lentiviral vector deposited at 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 a β2-microglobulin promoter.

13. 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 the cPPT / CTS sequence set out as sequence 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 a 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. 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 version of the Woodchuck Hepatitis B Virus (WHV) post-transcriptional regulatory element (WPRE), in particular having the sequence set out as sequence 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 the lentiviral vector particle for use according to any one of claims 1 to 17, wherein the lentiviral vector particle comprises a vesicular stomatitis virus glycoprotein (VSVG), in particular a VSV-G Indiana serotype or a 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 for use according to any one of claims 1 to 20, wherein the lentiviral vector or lentiviral vector particle is comprised in a vaccine composition.

22. 22. The 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 an anti-PD-1 monoclonal antibody, an anti-NKG2A monoclonal antibody, and an anti-TIM-3 monoclonal antibody.

23. 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, in particular the at least one immune checkpoint inhibitor is administered at least 2 days, in particular at least 4 days, after administration of the vaccine composition, the lentiviral vector, the lentiviral vector particle, or the cell.

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