Lentiviral vectors targeting KRAS neoepitopes for cancer immunotherapy

Abstract

The present invention relates to a lentiviral vector, in particular a non-integrative lentiviral vector, encoding a KRAS1-23 N-terminal segment, said KRAS1-23 N-terminal segment comprising at least one amino acid substitution in position 12 or 13, in particular in position 12, compared to the amino acid sequence set forth as SEQ ID NO: 1 It further relates to lentiviral vector particle, an isolated cell or a pharmaceutical composition comprising a lentiviral vector according to the invention. The invention further provides a combinatory treatment comprising at least one lentiviral vector, a lentiviral vector particle, an isolated cell or a pharmaceutical composition according to the invention, and at least one chemotherapy agent or treatment and / or at least one immunotherapy agent or treatment. Finally, the invention provides any of the above for their use in the treatment and / or prevention of cancers, in particular of cancers wherein the KRAS1-23 N-terminal segment is mutated.

Description

[0001] TITLE OF THE INVENTION

[0002] LENTIVIRAL VECTORS TARGETING KRAS NEOEPITOPES FOR CANCER IMMUNOTHERAPY

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of recombinant vaccine technology and relates to improvements of lentiviral vectors, which can be used as therapeutic and prophylactic vaccines. In particular, the present invention relates to lentiviral vectors expressing mutated Kirsten rat sarcoma virus (KRAS) antigens and to their implementation in the prevention and treatment of cancers wherein the KRAS 1-23 N-terminal segment is mutated, more particularly of pancreatic, colorectal, and / or pulmonary cancers.

[0005] BACKGROUND OF THE INVENTION

[0006] Cancer is a complex and multistep disease that however arises from single genetic modification that favors cell proliferation. Each subsequent division increases the malignancy of cancer cells, making them resistant to apoptosis after DNA damage, harsh metabolic conditions and immune system attacks (Hanahan & Weinberg, 2011, Hallmarks of cancer: the next generation. Cell 144: 646-674). These events contribute to a rapid growth of the tumor, yet at the same time, cause accumulation of somatic mutations in the tumor cell DNA, leading to generation of cancer- specific neoantigens. The latter are absent in normal tissues but are expressed by tumor cells and can be presented at their surface in the context of major histocompatibility complex (MHC) molecules (Schumacher & Schreiber, 2015, Neoantigens in cancer immunotherapy. Science 348: 69-74; Ward et al., 2016, The Role of Neoantigens in Naturally Occurring and Therapeutically Induced Immune Responses to Cancer. Adv Immunol 130: 25-74; Borden et al., 2022, Cancer Neoantigens: Challenges and Future Directions for Prediction, Prioritization, and Validation. Front Oncol 12: 836821).

[0007] Neoepitopes are well distinguished targets for cytotoxic immune T cell effectors. The vast majority of somatic mutations occurring in tumors and leading to generation of neoantigens are random and patient specific. Individual neoantigens arise relatively late during the malignant transformation and can be clonal, generating a mosaicism in terms of neoantigen repertoire presented by various cells of a tumor, leading to tumor escape from the effector functions neoantigen- specific T cells. During the last decade, individual neoantigens have been used for personalized immunotherapy, and some of them reached clinical trials, for instance in pancreatic or head and neck cancers and other solid tumors (Stemirna Therapeutics, 2022, A Phase 1 Clinical Study to Evaluate the Tolerability, Safety, Immunogenicity and Efficacy of the Neoantigen mRNA Personalised Cancer Vaccine SW 1115C3 in Patients With Advanced Malignant Solid Tumours clinicaltrials.gov; Rojas et al., 2023, Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer(Nature 618: 144-150; Washington University School of Medicine, 2023, Neoantigen Vaccines in Pancreatic Cancer in the Window Prior to Surgery clinicaltrials.gov). However, the time patients can wait before receiving immunotherapy is generally limited to a few weeks. During this short post-biopsy / surgery period, neoantigens need to be identified by sequencing and data processing, and neoantigen-based personalized GMP vaccines need to be produced, all of which presents a challenge (Shemesh et al., 2021, Personalized Cancer Vaccines: Clinical Landscape, Challenges, and Opportunities. Mol Ther 29: 555-570; Tiireci et al., 2018, Challenges towards the realization of individualized cancer vaccines. Nat Biomed Eng 2: 566-569).

[0008] In contrast to personal neoantigens, a number of somatic mutations, notably in oncogenes such as KRAS (Kirsten rat sarcoma viral oncogene), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) or ERBB2 (human epidermal growth factor receptor 2) are expressed in cancer stem cells, drive tumor initiation and maintenance, are shared by all cells of the tumor, and harbor shared neoantigens which can be common to patient cohorts (J anku et al. , 2011 , PIK3CA Mutations Frequently Coexist with RAS and BRAF Mutations in Patients with Advanced Cancers. PLoS ONE 6; Sholl et al., 2015, Multi-institutional Oncogenic Driver Mutation Analysis in Lung Adenocarcinoma: The Lung Cancer Mutation Consortium Experience. J Thorac Oncol 10: 768-777). In contrast to tumor associated antigens, these recurring tumor specific oncogenic mutations have low potential to induce autoimmunity since they are not or are barely expressed in healthy tissues. On the other hand, the T cell repertoire able to recognize them has not been subjected to thymic editing and central or peripheral tolerance induction and it is thus potentially possible to induce T cell responses against them (Sim & Sun, 2022, T Cell Recognition of Tumor Neoantigens and Insights Into T Cell Immunotherapy. Front Immunol 13; Schumacher & Schreiber, 2015). Generation of vaccines targeting this type of more universal neo-antigens does not need to be initiated after the cancer diagnosis and does not require personalized antigen design and vaccine manufacturing. So far, immunotherapy targeting such common shared neoantigens showed encouraging, yet partial, results in pre- clinical and clinical trials (Washington University School of Medicine, 2023; Rojas et al., 2023; Corulli et al., 2021, Multi-Epitope-Based Vaccines for Colon Cancer Treatment and Prevention. Front Immunol 12; Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 2023, Pooled Mutant KRAS-Targeted Long Peptide Vaccine Combined With Nivolumab and Ipilimumab for Patients With Advanced KRAS Mutated Non-Small Cell Lung Cancer clinicaltrials.gov).

[0009] The small guanosine triphosphatase (GTPase) KRAS switches between its inactive GDP-bound and active GTP-bound forms (Hobbs et al., 2016, RAS isoforms and mutations in cancer at a glance. J Cell Sci 129: 1287-1292). The active KRAS form triggers the mitogen- activated protein kinase (MAPK) pathway that allows transduction of extracellular signals through the cell and activates cell survival, proliferation and differentiation (Nan et al., 2015). Somatic missense mutations in KRAS, including KRAS G12D, KRAS G12V, result in accumulation of its active GTP-bound form and GTPase hyperactivity which ultimately leads to uncontrolled cell proliferation and malignant transformation. In 98% of cases, such gain-of-function KRAS mutations are located at G12, G13 or Q61 position (Nan et al., 2015, Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway. Proc Natl Acad Sci 112: 7996-8001; Hobbs et al., 2016; Guo et al., 2020, ERK / MAPK signaling pathway and tumorigenesis. Exp Ther Med 19: 1997-2007) which are detected in 27% of all human cancers, 85-95% of pancreatic cancers, 30-50% of colorectal cancers, 35% of pulmonary cancers — often correlated with tobacco smoking (Dogan et al., 2012, Molecular Epidemiology of EGFR and KRAS Mutations in 3026 Lung Adenocarcinomas: Higher Susceptibility of Women to Smoking-related KRAS- mutant Cancers. Clin Cancer Res Off J Am Assoc Cancer Res 18: 6169-6177; Riely et al., 2008, Frequency and Distinctive Spectrum of KRAS Mutations in Never Smokers with Lung Adenocarcinoma. Clin Cancer Res Off J Am Assoc Cancer Res 14: 5731-5734) — and 10% of endometrial cancers (Sideris et al., 2019, The Role of KRAS in Endometrial Cancer: A Mini-Review. Anticancer Res 39: 533-539). Even though KRAS is altered in only 2% of breast carcinoma patients (The AACR Project GENIE Consortium et al., 2017, AACR Project GENIE: Powering Precision Medicine through an International Consortium. Cancer Discov 7: 818-831), engagement of RAS functions has been reported, in the progression and metastases of experimental breast cancer studies (Laszld et al., 2021, Recent Updates on the Significance of KRAS Mutations in Colorectal Cancer Biology. Cells 10: 667). A single codon substitution at the position 12 or 13 stabilizes KRAS in an active state, amplifying downstream signaling pathways (Laszlo et al., 2021). KRAS G12D and KRAS G12V represent respectively > 40% and 22-33% of these mutations. The KRAS G13D has been reported in 11% of colorectal cancers. The KRAS G12C is found in about 13% of non- small cell lung cancer (Isaksson et al., 2023, KRAS G12C Mutant Non-Small Cell Lung Cancer Linked to Female Sex and High Risk of CNS Metastasis: Population-based Demographics and Survival Data From the National Swedish Lung Cancer Registry. Clin Lung Cancer), 4% of colorectal cancer (Schirripa et al., 2020, KRAS G12C Metastatic Colorectal Cancer: Specific Features of a New Emerging Target Population. Clin Colorectal Cancer 19: 219- 225) and 1-3% of other solid tumors (Strickler et al., 2023, Sotorasib in KRAS p.G12C- Mutated Advanced Pancreatic Cancer. N Engl J Med 388: 33-43). The KRASG12A mutation is present in 2.3% of colorectal cancers (AACR Project GENIE Consortium, 2017) but only in 0.5% of pancreatic cancers and is associated with poor prognosis (Fiala et al., 2016, G12V and G12A KRAS mutations are associated with poor outcome in patients with metastatic colorectal cancer treated with bevacizumab. Tumour Biol J Int Soc Oncodevelopmental Biol Med 37: 6823-6830). The KRAS G12R mutation is rare in colon and lung cancer but is the third most common KRAS mutation in pancreatic ductal adenocarcinoma (Hobbs et al., 2020, Atypical KRAS G12R Mutant Is Impaired in PI3K Signaling and Macropinocytosis in Pancreatic Cancer. Cancer Discov 10: 104-123).

[0010] The present invention aims at providing an effective method to induce specific and durable immune responses against tumor cells expressing specific oncogenic mutations of KRAS.

[0011] Compared to the vaccination strategies based on peptides or proteins, either adjuvanted or packaged in nanoparticles, mRNA-based or various viral vectors (DeMaria & Bilusic, 2019, Cancer Vaccines. Hematol Oncol Clin North Am 33: 199-214; Arbelaez et al., 2020, A nanoparticle vaccine that targets neoantigen peptides to lymphoid tissues elicits robust antitumor T cell responses. NPJ Vaccines 5: 106; Pan et al., 2017, Immunoprevention of KRAS-driven lung adenocarcinoma by a multipeptide vaccine. Oncotarget 8: 82689- 82699; Remy-Ziller et al., 2018, Sequential administration of MVA-based vaccines and PD- 1 / PD-Ll-blocking antibodies confers measurable benefits on tumor growth and survival: Preclinical studies with MVA-PGal and MVA-MUC1 (TG4010) in a murine tumor model. Hum Vaccines Immunother 14: 140-145; Morse et al., 2013, Novel Adenoviral Vector Induces T Cell Responses Despite Anti-Adenoviral Neutralizing Antibodies in Colorectal Cancer Patients. Cancer Immunol Immunother CII 62: 1293-1301), lentiviral vectors are more efficient at T cell triggering since they induce endogenous expression of the transgenic antigens directly in dendritic cells, the only antigen presenting cells able to activate naive T cells (Ku et al., 2021c, Use of lentiviral vectors in vaccination. Expert Rev Vaccines 20: 1571-1586; Nemirov et al., 2023, Lentiviral Vectors as a Vaccine Platform against Infectious Diseases. Pharmaceutics 15: 846). Lentiviral vectors induce high quality and longterm memory T cells and have the advantages to be non-inflammatory, self-inactivating and non-cytopathic. Non-integrative variants of lentiviral vectors are suitable for vaccination and immuno-oncotherapy (Ward et al., 2016; Nemirov et al., 2023; Gurumoorthy et al., 2022, Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through. Biomedicines 10: 107).

[0012] The pre-clinical proof of concept for the use of lentiviral vectors in numerous bacterial and viral infectious diseases has been established (Vesin et al., 2022, Lull-Lung Prophylaxis against SARS-CoV-2 by One-Shot or Booster Intranasal Lentiviral Vaccination in Syrian Golden Hamsters. Vaccines 11: 12; Anna et al., 2022, A lentiviral vector expressing a dendritic cell-targeting multimer induces mucosal anti-mycobacterial CD4+ T- cell immunity. Mucosal Immunol 15: 1389-1404; Lopez et al., 2022, A lentiviral vector encoding fusion of light invariant chain and mycobacterial antigens induces protective CD4+ T cell immunity. Cell Rep 40: 111142; Ku et al., 2021c, 2021b, 2021a (Brain crossprotection against SARS-CoV-2 variants by a lentiviral vaccine in new transgenic mice. EMBO Mol Med 13: el4459), 2020 (A Single Dose of NIL V-Based Vaccine Provides Rapid and Durable Protection against Zika Virus. Mol Ther J Am Soc Gene Ther 28: 1772-1782); Iglesias et al., 2006, A single immunization with a minute dose of a lentiviral vector-based vaccine is highly effective at eliciting protective humoral immunity against West Nile virus. J Gene Med 8: 265-274; de Wispelaere et al. , 2015, A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs. PLoS Negl Trap Dis 9: e0004081; Nemirov et al., 2023) and in immuno-oncotherapy (Adotevi et al., 2010, Targeting human telomerase reverse transcriptase with recombinant lentivector is highly effective to stimulate antitumor CD8 T-cell immunity in vivo. Blood 115: 3025-3032; Ku et al., 2021c), notably against human papillomavirus (HPV)-induced tumors (Douguet et al. , 2023, Full eradication of pre-clinical human papilloma virus-induced tumors by a lentiviral vaccine. EMBO Mol Med'. el7723), which has recently entered a clinical trial.

[0013] Recently, the most relevant vaccine strategies so far tested in preclinical anti- HPV immuno-oncotherapies used in murine models were cross-sectionally compared and it was observed that lentiviral vector-based approaches were the most efficient in the control of tumor growth while providing the longest-lasting memory (Demidova et al., 2024, Comparison of preclinical efficacy of immunotherapies against HPV-induced cancers. Expert Review of Vaccines, 23(1), 674-687).

[0014] Therefore, there remains a need in the art for an efficient and long-lasting treatment against cancers, in particular against cancer expressing a KRAS mutation.

[0015] There also remains a need for a vaccine that can improve the intracellular antigen routing to MHC presentation machineries or to provide helper CD4+ T cell epitopes In particular, there remains a need for a therapeutic vaccine which is efficient and can optimize the anti-cancerous effects when used as a combinatory treatment with first line anti-cancer chemotherapy and / or a standard of care immunotherapy.

[0016] The present invention has for purpose to meet at least one of the above- mentioned needs.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention accordingly relates to the following items:

[0019] Item 1: A lentiviral vector encoding a KRAS 1-23 N-terminal segment, said KRAS 1-23 N-terminal segment comprising at least one amino acid substitution in position 12 or 13, in particular in position 12, compared to the amino acid sequence set forth as SEQ ID NO: 1.

[0020] As illustrated in the examples, lentiviral vectors of the invention allow for a strong therapeutic and prophylactic activity against HPV-induced tumors.

[0021] Item 2: The lentiviral vector according to item 1, further encoding at least one support protein. Item 3 The lentiviral vector according to item 1 or 2, wherein the KRAS 1-23 N-terminal segment comprises, and in particular consists in, an amino acid sequence selected from the group consisting of amino acid sequences set forth as SEQ ID NO: 2 (KRASI-23G12A), SEQ ID NO: 3 (KRASI-23G12C), SEQ ID NO: 4 (KRASi-23G12D), SEQ ID NO: 5 (KRASI-23G12R), SEQ ID NO: 6 (KRASi-23G12v), SEQ ID NO: 7 (KRASi-23G13D) and variants of these sequences having at least 70% sequence identity thereof, in particular the amino acid sequence set forth as SEQ ID NO: 4 or a variant thereof.

[0022] Item 4: The lentiviral vector according to any one of the preceding items, encoding at least two KRAS 1-23 N-terminal segments comprising at least one amino acid substitution in position 12 or 13, in particular in position 12, compared to the amino acid sequence set forth as SEQ ID NO: 1, more particularly encoding at least two KRAS 1-23 N-terminal segments having an amino acid sequence independently selected from the group consisting of the amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and variants of these sequences having at least 70% sequence identity thereof.

[0023] Item 5: The lentiviral vector according to any one of the preceding items, encoding:

[0024] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0025] 2, or a variant thereof having at least 70% sequence identity thereof ; and

[0026] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0027] 3, or a variant thereof having at least 70% sequence identity thereof; and

[0028] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0029] 4, or a variant thereof having at least 70% sequence identity thereof; and

[0030] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0031] 5, or a variant thereof having at least 70% sequence identity thereof; and

[0032] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0033] 6, or a variant thereof having at least 70% sequence identity thereof; and

[0034] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0035] 7, or a variant thereof having at least 70% sequence identity thereof.

[0036] Item 6: The lentiviral vector according to item 4 or 5 wherein the at least two

[0037] KRASi -23 N-terminal segments are fused together with or without a linker. Item 7: The lentiviral vector according to any one of items 4 to 6, further encoding an amino acid segment 1-77 of the mutated human ubiquitin CG77V, in particular said amino acid segment 1-77 of the mutated human ubiquitin CG77Vbeing located at the N- terminal end of the KRAS 1-23 N-terminal segments.

[0038] Item 8: The lentiviral vector according to any one of items 2 to 7, wherein the at least one support protein is selected from the group consisting of:

[0039] (i) proteins that improve the intracellular antigen routing to major histocompatibility complex presentation machineries; and

[0040] (ii) proteins that provide helper CD4+ T-cell epitopes.

[0041] Item 9: The lentiviral vector according to any one of items 2 to 8, wherein the at least one support protein is selected from the group consisting of the MHC-II light invariant chain (“li”); diphteria toxins, or fragments thereof, in particular the 203-379 fragment of diphteria toxin (“DT”); ubiquitin proteins, or fragments thereof, in particular the fragment 1-77 of the mutated human ubiquitin CG77V; tetanus toxoids (TT) or fragments thereof; and mixtures thereof.

[0042] Item 10: The lentiviral vector according to any one of items 2 to 9, wherein the at least one support protein is selected from the group consisting of:

[0043] (i) the MHC-II light invariant chain (“li”), in particular having the amino acid sequence set forth as SEQ ID NO: 8,

[0044] (ii) the 203-379 segment of diphteria toxin (“DT”), in particular having the amino acid sequence set forth as SEQ ID NO: 9 and

[0045] (iii) the MHC-II light invariant chain (“li”) and the 203-379 segment of diphteria toxin (“DT”), in particular having the amino acid sequences set forth as SEQ ID NO: 8 and SEQ ID NO: 9.

[0046] Item 11: The lentiviral vector according to any one of items 1 to 10, encoding an amino acid sequence set forth as SEQ ID NO: 12 or SEQ ID NO: 13.

[0047] Item 12: The lentiviral vector according to any one of items 2 to 9, wherein the support protein is located at the N-terminal end of the at least one KRAS 1-23 N-terminal segment, in particular is fused together at the N-terminal end of the at least one KRAS 1-23 N-terminal segment. Item 13: The lenti viral vector according to any one of the preceding items, wherein the lenti viral vector comprises an MHC Class I promoter, and in particular a human |32-microglobulin promoter.

[0048] Item 14: The lenti viral vector according to any one of the preceding items, wherein the lentiviral vector comprises a cPPT / CTS sequence, in particular the cPPT / CTS sequence set forth as sequence SEQ ID NO: 10.

[0049] Item 15: The lentiviral vector according to any one of the preceding items, wherein the lentiviral vector comprises a 3’ long terminal repeat (LTR) which is devoid of its U3 promoter sequence.

[0050] Item 16: The lentiviral vector according to any one of the preceding items, wherein the lentiviral vector comprises a mutant form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), and in particular having the sequence set forth as sequence SEQ ID NO: 11.

[0051] Item 17: A lentiviral vector particle comprising at least a lentiviral vector as defined in any one of items 1 to 16.

[0052] Item 18: The lentiviral vector particle according to item 17, further comprising a functional lentiviral integrase protein.

[0053] Item 19: The lentiviral vector particle according to item 17 or 18, further comprising a vesicular stomatitis virus glycoprotein (VSVG), in particular a VSV-G Indiana serotype or a VSV-G New Jersey serotype.

[0054] Item 20: The lentiviral vector particle according to any one of items 17 to 19, wherein the lentiviral vector particle comprises HIV-1 subtype D Gag and Pol proteins.

[0055] Item 21: An isolated cell comprising a lentiviral vector according to any one of items 1 to 17 or a lentiviral vector particle according to any one of items 17 to 20.

[0056] Item 22: A pharmaceutical composition, and in particular a vaccine composition, comprising, in a pharmaceutically acceptable medium, a lentiviral vector according to any one of items 1 to 16 or a lentiviral vector particle according to any one of items 17 to 20 or an isolated cell according to item 21.

[0057] Item 23: A combinatory treatment comprising:

[0058] (i) at least one lentiviral vector according to any one of items 1 to 16 or lentiviral vector particle according to according to any one of items 17 to 20, or an isolated cell according to item 21 or a pharmaceutical composition according to item 22, and (ii) at least one chemotherapy agent or treatment and / or at least one immunotherapy agent or treatment.

[0059] Item 24: The combinatory treatment according to item 23, wherein the at least one chemotherapy agent or treatment is selected from the group consisting of, cisplatin, carboplatin, oxaliplatin, pemetrexed, 5-fluoracil (5-FU), taxane, and mixtures thereof.

[0060] Item 25: The combinatory treatment according to item 23 or 24, wherein the at least one immunotherapy agent or treatment is selected from the group consisting of immune checkpoint inhibitors, in particular anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti-TIM-3, anti- LAG3, anti-TIGIT and anti-NKG2A antagonist antibodies; and mixtures thereof.

[0061] Item 26: A lenti viral vector according to any one of items 1 to 16 or a lenti viral vector particle according to any one of items 17 to 20, or an isolated cell according to item 21, or a pharmaceutical composition according to item 22, or a combinatory treatment according to any one of items 23 to 25, for their use in the treatment and / or prevention of cancers, in particular of cancers wherein the KRAS1-23 N-terminal segment is mutated, more particularly of pancreatic, colorectal, and / or pulmonary cancers.

[0062] The details, examples and preferences provided in relation to any particular one or more of the stated aspects of the present invention will be further described herein and apply equally to all aspects of the present invention. Any combination of the embodiments, examples and preferences described herein in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein, or otherwise clearly contradicted by context.

[0063] BRIEF DESCRIPTION OF THE FIGURES

[0064] Figure 1 shows schematic representations of KRAS 1-23 segment designs.

[0065] Figure 1A is a schematic representation of KRASi-23G12Dprotein segment: alone (top design), fused at its N-terminal end with full length li to facilitate its routing through the MHC-II pathway (middle design), or fused at its N-terminal end with li-DT (bottom design). Figure IB is a schematic representation of a fusion of human ubiquitin CG77Vlinked to N- terminal end of a poly-antigen composed of KRASG12A, KRASG12C, KRASG12D, KRASG12R, KRASG12Vand KRASG13Dneoepitopes, and linked at its C-terminal part with the well-known immunogenic ETTDPDRAHYNIVTF peptides derived from the Early E7 oncoprotein of human papilloma virus (HPV)16 which contains an H-2Db-restricted T cell epitope, as an immunological tag.

[0066] Figure 2 shows the therapeutic vaccination of mice bearing CT26 colorectal tumors with lentiviral vectors coding various designs of KRASG12Dantigen.

[0067] Figure 2A represents the variation of tumor size in individual mice (in mm3) over time (in days post tumor transplantation) from left to right: for the control group (Ctrl Lenti) (empty circle), for the Lenti-KRASG12Dgroup (black triangle), for the Lenti-li-KRASG12Dgroup (black square) and for the Lenti-li-DT-KRASG12Dgroup (black circle), 7 days, 10 days, 12 days, 15 days, 18 days and 20 days after tumor transplantation. Statistical significance was determined by two-way ANOVA test by Log-rank Mantel-Cox test (*p < 0.05).

[0068] Figure 2B represents the survival curve of the animals for the Ctrl Lenti group (dotted line) and the Lenti-li-DT-KRASG12Dgroup (full line). Statistical significance was determined by Log-rank Mantel-Cox tests (*p < 0.05). Mice were sacrificed when the tumor volume reached 1500 mm3, defined as humane endpoints. The experiment shown is representative of two independent experiments.

[0069] Figure 3 shows the therapeutic vaccination of mice bearing MC38KRASG12Dtumors with Lenti-li-DT-KRASG12D.

[0070] Figures 3A and 3B show the variation of tumor size in individual mice (in mm3) over time (in days post tumor transplantation) from left to right: in the Ctrl Lenti group (empty circle) and in the Lenti-li-DT- KRASG12Dgroup (black circle). Statistical significance was determined by two-way ANOVA test by Log-rank Mantel-Cox tests (*p < 0.05). Mice were sacrificed when the tumor volume reached 1500 mm3, defined as humane endpoints.

[0071] Figures 3C and 3D show the tumor infiltrating T cells in Ctrl Lenti- or Lenti-li-DT- KRASG12D-treated mice. C57BL / 6 mice were engrafted with 2 x 105MC38KRASG12Dcells as detailed in B and injected on day 6 with 1 x 109TU Lenti Ctrl or Lenti-li-DT-KRASG12D(n = 4-5 / group). Tumors were studied on day 11 post- vaccination. Figure 3C shows the gating strategy and representative blots of tumor infiltrating T cells, studied by cytometry. Figure 3D shows the percentage of each subset compared between the two groups and statistical significance determined using two-tailed unpaired t tests (*p < 0.05). The top line of figure 3D shows, from left to right, the results for %CD45+ vs total cells, %CD4+ vs CD45+ cells, %CD8+ vs CD45+ cells, CD8 / CD4 cells ratio and the bottom line shows, from left to right, the results for %CD44+ CD69+ vs CD8+ cells, % Trm vs CD8+ cells and the %PD1+ KLRG1+ vs CD8+ cells.

[0072] Figure 4 shows the therapeutic vaccination of MC38KRASG12Dtumor-bearing mice with Lenti-nLuc-KRASG12D

[0073] Figure 4A shows the variation of tumor size in individual mice (in mm3) over time (in days post tumor transplantation) in the Ctrl lenti group (on the left - empty circle) and in the Lenti-nLuc- KRASG12Dgroup (on the right - black circle). Statistical significance was determined by 2-way ANOVA (**p < 0.01). Mice were sacrificed when the tumor volume reached 1500 mm3, defined as humane endpoints.

[0074] Figure 4B shows T cell infiltrates inside the MC38KRASG12Dtumors in mice treated with Ctrl Lenti or with Lenti-nLuc-KRASG12D, as studied at day 20 post tumor inoculation. From left to right, the results are shown for %CD45+ vs total cells, %CD4+ vs CD45+ cells, %CD8+ vs CD45+ cells and %PD1+ vs CD8+ cells. The percentage of each subset was compared between the two groups and statistical significance determined using two-tailed unpaired t tests (*p < 0.05).

[0075] Figure 5 shows the presence of anti-KRAS CD8+T cell effectors within tumor infiltrates. C57BL / 6 mice were engrafted with 1 x 105MC38KRASG12Dcells and were primed (day 2) and boosted (day 9) i.m. with 1 x 109TU / mouse of Ctrl Lenti or Lenti-li-DT-KRASG12D. The tumor infiltrates were studied at day 20 after enzymatical digestion of tumors, enrichment of cell suspensions in lymphocytes on Ficoll, and overnight co-culture with syngeneic bone-marrow-derived DCs loaded with KRAS I-20G12Dor KRASI-2OWTor a negative control peptide.

[0076] The Figure shows the cytometric analysis of the tumor infiltrates after enzymatical digestion without enrichment on Ficoll. Part (A) represents the cytometric gating strategy. Part (B) shows the typical results of intracellular IFN-gamma and TNF-alpha detection in CD8+ T cell infiltrates. Part (C) shows percentages of CD4+ and CD8+ within the CD45+ cells in the tumor infiltrates of Ctrl Lenti- or Lenti-li-DT-KRASG12D-treated mice. Part (D) shows cytometric analysis of the tumor infiltrates after enzymatical digestion without enrichment on Ficoll. Part (E) shows the percentages of CD45+ or tumor cells, identified as large CD45- cells, within the whole cells in Ctrl Lenti- or Lenti-li-DT-KRASG12D-treated mice. The percentage of each subset was compared between the two groups and statistical significance determined using two-tailed unpaired t tests (*p < 0.05).

[0077] Figure 6 shows the phenotype of antitumor effector T cells generated by Lenti-li-DT- KRASG12Dtherapy.

[0078] Figure 6A shows the variation of tumor size (in mm3) in individual mice over time (in days post tumor transplantation) for each of the following groups, from left to right at each time point: Ctrl Lenti group (empty circle), Lenti-li-DT- KRASG12D+ Ctrl Ig group (black circle), Lenti-li-DT- KRASG12D+ anti-CD4 group (asterisk) and Lenti-li-DT- KRASG12D+ anti-CD8 group (black square). From left to right, the tumor was measured 7, 10, 12, 14, 17 and 19 days after tumor transplantation.

[0079] Figure 6B shows the variation of the tumor size (in mm3) over time (in days post tumor transplantation) in each group, from highest to lowest tumor volume at day 19: Lenti-li-DT- KRASG12D+ anti-CD8 group (black square), Lenti-li-DT- KRASG12D+ Ctrl Ig group (black circle), Ctrl Lenti group (empty circle) and Lenti-li-DT- KRASG12D+anti-CD4 group (asterisk).

[0080] Statistical significance was determined by two-way ANOVA test by Log-rank Mantel-Cox tests (*p < 0.05).

[0081] Figure 6C shows the efficacy of T subset depletion in anti-CD4 or anti-CD8 mAb-treated mice, assessed at day 6 on the peripheral blood leukocytes from one representative mouse / group, by anti-CD3, anti-CD4 and anti-CD8 mAb staining and cytometry study.

[0082] Figure 7 shows the beneficial anti-tumor effect of a combination of Lenti-li-DT-KRASG12Dwith cisplatin and anti-PDl treatment.

[0083] Figure 7 A shows the evolution of tumor size (in mm3) in individual mice over time (in days post tumor transplantation) in the following different groups, from left to right at each time point: Ctrl Lenti group (empty circle), Ctrl Lenti + cisplatin + anti-PDl group (asterisk), Lenti-li-DT- KRASG12Dgroup (black circle) and Lenti-li-DT- KRASG12D+ cisplatin + anti- PDl (black square). From left to right, the tumor size was measured 9, 11, 14 and 16 days after tumor transplantation. Significance was determined by two-way ANOVA test by Logrank Mantel-Cox tests (*p < 0.05). Figure 7B shows the survival curve of the animals (% survival) over time (in days post tumor transplantation) for the following groups, from shortest to longest survival time: Ctrl Lenti, Lenti-li-DT-KRASG12D, Ctrl Lenti + cisplatin + anti-PDl and Lenti-li-DT-KRASG12D+ cisplatin + anti-PDl. Statistical significance was determined by Log-rank Mantel-Cox tests (*p < 0.01, ***p < 0.0001). Mice were sacrificed when the tumor volume reached 1500 mm3, defined as humane endpoints.

[0084] Figure 8 shows the therapeutic vaccination of mice bearing LLC1KRASG12Dtumors with lentiviral vectors coding various designs of KRASG12Dantigen

[0085] Figure 8A shows the variation of tumor size (in mm3) in individual mice over time (in days post tumor transplantation) in the following groups, from left to right at each time point: Ctrl Lenti, Lenti-KRASG12Dand Lenti-li-DT-KRASG12D. Mice were sacrificed when the tumor volume reached 1500 mm3, defined as humane endpoints. Statistical significance was determined by 2-way ANOVA (**p < 0.01).

[0086] Figure 8B shows the mean tumor size (or volume) (in mm3) in each group over time (days post tumor engraftment) in the following groups, from the highest to the lowest tumor volume at day 22: Ctrl Lenti and Lenti-li-DT-KRASG12D. Statistical significance was determined by two-way ANOVA test by Log-rank Mantel-Cox tests (**p < 0.01).

[0087] DETAILED DESCRIPTION OF THE INVENTION

[0088] The inventors have discovered that T cell-based immunotherapy against completely tumor- specific KRAS GTPase mutations represents a promising approach to treat a large variety of cancers, because of their ubiquitous expression by all cells of the tumors and the narrow diversity of their mutated variants. In particular, the inventors showed that immunotherapy by lentiviral vectors according to the invention resulted in significant immune control of the tumor growth in two colorectal and one pulmonary preclinical cancer models, in distinct murine genetic backgrounds.

[0089] The antitumor effect was correlated with increased proportions of intra-tumor hematopoietic cells and notably CD8+ T cells, resulting from their preferential infiltration or intra-tumor expansion. It was further demonstrated that a tri-therapy combination of a lentiviral vector of the invention with cisplatin and antagonistic anti-PDl mAb improved the anti-tumor efficacy of these first line anti-cancer chemotherapy and standard of care.

[0090] Definitions

[0091] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

[0092] As used herein, “transgene” means a polynucleotide that can be expressed, via recombinant techniques, in a non-native environment or heterologous cell under appropriate conditions.

[0093] 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 an endogenous and / or heterologous nucleic acid or protein into the cell or the alteration of a native cell or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes or nucleic acid that are not found within the native (non-recombinant) form of the cell or express native (eg endogenous) genes at a different level than their native level or express additional or supplementary copies of native (eg endogenous) at a different level than their native level. An isolated cell according to the invention is recombinant in that it comprises at least one lentiviral vector according to the invention and / or at least one lentiviral vector particle according to the invention.

[0094] As used herein, the term “recombinant”, when used in reference to a vector, are sequences formed / obtained by technics of genetic engineering well known to the man skilled in the art.

[0095] As used herein, the term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and containing more than five amino acid residues. The amino acids are identified by either the single-letter or three-letter designations. The term "protein" as used herein 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, famesylated, prenylated, sulfonated, and the like) to add functionality. Polypeptides exhibiting activity may be referred to as enzymes. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given polypeptide may be produced.

[0096] The term "operably linked" as used herein refers to two or more nucleic acid sequence or amino acid sequence elements that are physically linked and are in a functional relationship with each other. For instance, in a lentiviral vector according to the invention, a promoter is operably linked to a coding sequence, also termed herein “antigen construct” as the promoter is able to initiate or regulate the transcription or expression of the antigen construct, in which 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 will be in the same orientation and usually also in the same reading frame. They usually will be essentially contiguous, although this may not be required.

[0097] The terms "encoding" or "coding for" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an aminoacid sequence.

[0098] For each or the amino acid sequences of interest, reference sequences are described herein. The present description also encompasses amino acid sequences having specific percentages of amino acid identity with a reference amino acid sequence.

[0099] For obvious reasons, in all the present description, a specific nucleic acid sequence or a specific amino acid sequence which complies with, respectively, the considered nucleotide or amino acid identity, should further lead to obtaining a protein (or antigen) which displays the desired biological activity. As used herein, the "percentage of identity" between two nucleic acid sequences or between two amino acid sequences is determined by comparing both optimally aligned sequences through a comparison window.

[0100] The portion of the nucleotide or amino-acid sequence in the comparison window may thus include additions or deletions (for example "gaps") as compared to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment between both sequences.

[0101] The terms "sequence homology" or "sequence identity" or "homology" or "identity" are used interchangeably herein. For the purpose of the invention, it is defined here that in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids / based or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

[0102] A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).

[0103] The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE.

[0104] For the purpose of the 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. LongdenJ. and Bleasby,A. Trends in Genetics 16, (6) pp276 — 277, http: / / emboss.bioinformatics.nl / ). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is 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, Yes has been indicated in response to the question “Brief identity and similarity” and “SRS pairwise” indicated as Output alignment format.

[0105] After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest- identity".

[0106] The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments using several other art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on https: / / www.ebi.ac.uk / Tools / msa / clustalo / or the GAP program (mathematical algorithm of the University of Iowa) or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17) or Clone Manager 9. Preferred parameters used are the default parameters as they are set on https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0107] The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of 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 that are homologous to those nucleic acids which encode the relevant protein.

[0108] 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 comparative 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 may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: 154-162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

[0109] In particular embodiments, % identity between two sequences is determined using CLUSTAL O (version 1.2.4). The terms non-oncogenic used herein are used in their traditional meaning, i.e. it relates to an element, in the present case to antigens, unable to cause the formation of tumors. As detailed elsewhere, antigens implemented in the present invention have been genetically amended in order to become nononcogenic. It means, according to the usual meaning of these terms, that the nucleic acid sequences encoding the antigens implemented herein are not found in nature and are modified either by introduction or by deletion or by modification of their nucleic acid sequences, leading to encoded amino acid sequences that also do not naturally exist in nature.

[0110] The man skilled in the art has known for a long time a variety of means to perform a deletion, substitution or introduction in a nucleic acid sequence.

[0111] As will be understood by those of skill in the art, it can moreover be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called “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 (www.idtdna.com / CodonOptfrom) from Integrated DNA Technologies. 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 rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a nonoptimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al, 1996, Nucl Acids Res. 24: 216- 8).

[0112] A “non-integrative” lentiviral vector means that, when this lentiviral vector is in a cell, it does not integrate into the host cell genome. A non-integrative lentiviral vector particle relates to a lentiviral vector particle that comprises a non-integrative lentiviral vector. It can also be termed integration-defective lentiviral vectors or non-integrating lenti viral vectors.

[0113] As used herein, “treating” refers to causing a detectable improvement in one or more symptoms associated with cancers, in particular cancers wherein the KRAS 1-23 N- terminal segment is mutated, more particularly pancreatic, colorectal, and / or pulmonary cancers or causing a biological effect (e.g., a decrease in the level of a particular biomarker) that is correlated with the underlying pathologic mechanism(s) giving rise to the condition or symptom(s).

[0114] “Prevention” or “preventing” means any treatment of a disease or condition that causes the clinical symptoms of the disease or condition not to develop. Compounds may, in some embodiments, be administered to a subject (including a human) or an individual in need thereof who is at risk or has a family history of the disease or condition, in particular of the cancer as described elsewhere.

[0115] “Subject” or “individual in need thereof’ refers to an animal, such as a mammal (including a human), that has been or will be the object of treatment, observation, or experiment. The methods described herein may be useful in human therapy and / or veterinary applications. In some embodiments, the subject or individual is a mammal. In one embodiment, the subject or individual is a human.

[0116] As used herein, a “fragment” generally refers to a fragment of a given sequence, in particular of an amino acid sequence, that has the same biological activity as the original peptide from which it originates that has a given number of consecutive amino acids from the original peptide. In a particular embodiment, the fragment is a biologically active fragment. A biologically active fragment may be an immunologically active fragment, which refers to a fragment that has the same immunological activity as the peptide from which it originates.

[0117] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a segment,” is understood to represent one or more segments. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0118] Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements. The term “consisting of’ implies the inclusion of the stated element(s), to the exclusion of any additional elements. The term “consisting essentially of’ implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the basic characteristic(s) of the disclosure. It is understood that the different embodiments of the disclosure using the term “comprising” or equivalent cover the embodiments where this term is replaced with “comprising only”, “consisting of’ or “consisting essentially of’.

[0119] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0120] Lentiviral vector according to the invention

[0121] The inventors have conceived novel therapeutic and prophylactic lentiviral vectors-based vaccines against cancers wherein the KRAS 1-23 N-terminal segment is mutated. The non mutated KRAS 1-23 N-terminal segment, that does not contain any amino acid substitutions, and that serves as a reference segment, consists of the sequence set forth as SEQ ID NO: 1. The amino acid sequence of the full wild type KRAS protein from which the KRAS 1 -23 N-terminal segment is from is set forth as SEQ ID NO: 47.

[0122] In particular, the present invention relates to a lentiviral vector encoding a mutated KRAS 1-23 N-terminal segment.

[0123] Indeed, the lentiviral vector encodes a KRAS 1-23 N-terminal segment, said KRASi -23 N-terminal segment comprising at least one amino acid substitution in position 12 or 13, in particular in position 12, compared to the amino acid sequence set forth as SEQ ID NO: 1.

[0124] The amino acid sequence set forth as SEQ ID NO: 1 is the the amino acid sequence of KRAS 1-23 N-terminal segment, which has not been mutated, i.e. that does not contain any amino acid substitutions. It corresponds to the segment from the 1stto the 23rdamino acid of the Kirsten rat sarcoma viral oncogene. This amino acid sequence serves as a reference to the KRAS1-23 N-terminal segments. In a particular embodiment, the KRAS 1-23 N-terminal segment comprises an amino acid sequence selected from the group consisting of amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and variants of these sequences having at least 70%, and in particular the amino acid sequence set forth as SEQ ID NO: 4.

[0125] In a particular embodiment, the KRAS 1-23 N-terminal segment comprises a variant sequence, that is to say it comprises an amino acid sequence having at least 70%, in particular 80%, more particularly 90% sequence identity with an amino acid sequence selected from the group consisting of amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.

[0126] Such variant sequences differ from amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 by the fact that they contain at least a mutation in position 12 or 13.

[0127] Further, such variant sequences are different from SEQ ID NO: 1

[0128] The variant sequences have the same biological activity as the KRAS 1-23 N- terminal segments that comprise an amino acid sequence selected from the group consisting of amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.

[0129] As described herein, an amino acid sequence having at least 70% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence.

[0130] In a particular embodiment, the variant sequences comprise an amino acid sequence having from 70% to 95% amino acid identity with the said reference amino acid sequence, in particular have from 75% to 70%, more particularly have from 80% to 85 % amino acid identity with the said reference amino acid sequence.

[0131] In a particular embodiment, the variant sequences are seventeen-mers of the reference amino acid sequence, that is to say that they are fragments of the reference amino acid sequences consisting in seventeen consecutive amino acid sequences of the reference amino acid sequences. In a particular embodiment, the variant sequences are 5-21 segments of the reference amino acid sequences. In other words, the variant sequences comprise, in particular consist in, the amino acid segment including the consecutive amino acids from the 5thto the 21stamino acids from the reference amino acid sequences.

[0132] In a particular embodiment, the variant sequences comprise an amino acid sequence selected from the group consisting of the amino acid sequences set forth as SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38.

[0133] In a particular embodiment the variant sequences comprise a nucleic acid sequence having at least 70%, in particular 80%, more particularly 90% sequence identity with an nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

[0134] In a particular embodiment, the variant sequences consist in a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth as SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

[0135] In a particular embodiment, the KRAS 1-23 N-terminal segment consists of an amino acid sequence selected from the group consisting of amino acid sequences set forth as SEQ ID NO: 2 (KRASI-23G12A), SEQ ID NO: 3 (KRASi-23G12C), SEQ ID NO: 4 (KRASi-23G12D), SEQ ID NO: 5 (KRASI-23G12R), SEQ ID NO: 6 (KRASi-23G12v), SEQ ID NO: 7 (KRASi-23G13D).

[0136] In a particular embodiment, the KRASI-23N-terminal segment comprises an amino acid sequence set forth as SEQ ID NO: 4. In particular, the KRASI-23N-terminal segment consists of an amino acid sequence set forth as SEQ ID NO: 4.

[0137] In a particular embodiment, the KRASI-23N-terminal segment comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences set forth as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and variants of these sequences having at least 70%sequence identity thereof. In a particular embodiment, the KRAS 1-23 N-terminal segment consists of a nucleic acid sequence selected from the group consisting of nucleic acid sequences set forth as SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and variants of these sequences having at least 70% sequence identity thereof.

[0138] In a particular embodiment, the KRAS 1-23 N-terminal segment comprises a nucleic acid sequence set forth as SEQ ID NO: 17. In particular, the KRAS1-23 N-terminal segment consists of a nucleic acid sequence set forth as SEQ ID NO: 17.

[0139] In a particular embodiment, the lentiviral vector encodes at least two KRASi- 23 N-terminal segments comprising at least one amino acid substitution in position 12 or 13, in particular in position 12, compared to the amino acid sequence set forth as SEQ ID NO:

[0140] 1.

[0141] In particular, the lentiviral vector encodes at least two KRAS 1-23 N-terminal segments having an amino acid sequence independently selected from the group consisting of the amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and variants of these sequences having at least 70% sequence identity thereof.

[0142] In a particular embodiment, the lentiviral vector encodes:

[0143] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0144] 2, or a variant thereof having at least 70%sequence identity thereof ; and

[0145] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0146] 3, or a variant thereof having at least 70%sequence identity thereof ; and

[0147] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0148] 4, or a variant thereof having at least 70%sequence identity thereof ; and

[0149] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0150] 5, or a variant thereof having at least 70%sequence identity thereof ; and

[0151] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0152] 6, or a variant thereof having at least 70%sequence identity thereof ; and

[0153] - the KRAS 1 -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0154] 7, or a variant thereof having at least 70%sequence identity thereof. In a particular embodiment, the lentiviral vector encodes, from N-terminus to C-terminus, the KRAS 1-23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO: 2, the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO: 3, the KRAS 1-23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO: 4, the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO: 5, the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO: 6, and the KRAS1-23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO: 7.

[0155] In a particular embodiment, the lentiviral vector encodes, from N-terminus to C-terminus, the KRAS 1-23 N-terminal segment having the nucleic acid sequence set forth as SEQ ID NO: 15, the KRASi -23 N-terminal segment having the nucleic acid sequence set forth as SEQ ID NO: 16, the KRASi -23 N-terminal segment having the nucleic acid sequence set forth as SEQ ID NO: 17, the KRASi -23 N-terminal segment having the nucleic acid sequence set forth as SEQ ID NO: 18, the KRAS1-23 N-terminal segment having the nucleic acid sequence set forth as SEQ ID NO: 19, and the KRAS1-23 N-terminal segment having the nucleic acid sequence set forth as SEQ ID NO: 20.

[0156] In a particular embodiment, the at least two KRAS 1-23 N-terminal segments are fused together with or without a linker.

[0157] A linker, or linking sequence, is to be understood herein as an amino acid sequence or a nucleic acid sequence that serves to link two other amino acid or nucleic acid sequences together. In particular, the linker’ s sole role is to link two amino acid or nucleic acid sequences together. In particular, the linker does not provide any additional biological activity to the amino acid or nucleic acid sequences it links.

[0158] In a particular embodiment, the linker is located between two KRAS 1-23 N- terminal segments, in particular between the C-terminal end of a first KRAS 1-23 N-terminal segment and the N-terminal end of a second KRAS 1-23 N-terminal segment.

[0159] In a particular embodiment, the KRAS 1-23 N-terminal segments are linked without a linker. In a particular embodiment, the KRAS 1-23 N-terminal segments are directly linked to one another.

[0160] In a particulier embodiment, the lentiviral vector further encodes an amino acid segment 1-77 of the mutated human ubiquitin CG77V. In particular said amino acid segment 1-77 of the mutated human ubiquitin CG77Vis located at the N-terminal end of the KRAS 1-23 N-terminal segments. In particular, the amino acid segment 1-77 is a segment from the full amino acid sequence of the human ubiquitin C set forth as SEQ ID NO: 45. The amino acid segment 1-77 further comprises the addition of an alanine in position 2 compared with the CG76Vversjon. The corresponding wild type 1-76 segment for the human ubiquitin is set forth as SEQ ID NO: 46.

[0161] The amino acid segment 1-77 of the mutated human ubiquitin CG77V(wherein the CG77Vmutation corresponds to the CG76Vmutation described in Liu el al, 2011 but wherein the mutated human ubiquitin further comprises the addition of an alanine in position 2), may serve to favor the recognition and processing of the protein antigens by the proteasome and MHC-I presentation (Loureiro & Ploegh, 2006, Antigen presentation and the ubiquitin-proteasome system in host-pathogen interactions. Adv Immunol 92: 225-305).

[0162] In a particular embodiment, the segment 1-77 of the mutated human ubiquitin CG77Vcomprises an amino acid sequence set forth as SEQ ID NO: 25. In a particular embodiment, the segment 1-77 of the mutated human ubiquitin CG77Vconsists of an amino acid sequence set forth as SEQ ID NO: 25.

[0163] In a particular embodiment, the segment 1-77 of the mutated human ubiquitin CG77Vcomprises a nucleic acid sequence set forth as SEQ ID NO: 26. In a particular embodiment, the segment 1-77 of the mutated human ubiquitin CG77Vconsists of a nucleic acid sequence set forth as SEQ ID NO: 26.

[0164] In a particular embodiment, the lentiviral vector further encodes an immunogenic peptide derived from the Early E7 oncoprotein of human papilloma virus (HPV)16 containing an H-2Db-restricted T cell epitope. In particular the E7 immunogenic peptide is located at the C-terminal end of the KRAS1-23 N-terminal segments.

[0165] The E7 immunogenic peptide may serve as an immunological tag, in particular in preclinical murine models.

[0166] In a particular embodiment, the E7 immunogenic peptide comprises an amino acid sequence set forth as SEQ ID NO: 27. In a particular embodiment, the E7 immunogenic peptide consists of an amino acid sequence set forth as SEQ ID NO: 27. In a particular embodiment, the E7 immunogenic peptide comprises a nucleic acid sequence set forth as SEQ ID NO: 28. In a particular embodiment, the E7 immunogenic peptide consists of a nucleic acid sequence set forth as SEQ ID NO: 28.

[0167] In a particular embodiment, the lentiviral vector encodes at least two KRASi- 23 N-terminal segments having an amino acid sequence independently selected from the group consisting of the amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and variants of these sequences having at least 70% sequence identity thereof, wherein the lentiviral vector further encodes an amino acid segment 1-77 of the mutated human ubiquitin CG77V, in particular located at the N-terminal end of the KRASi- 23 N-terminal segments.

[0168] In a particular embodiment, the lentiviral vector encodes:

[0169] - the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO: 2, or a variant thereof having at least 70% sequence identity thereof ; and

[0170] - the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0171] 3, or a variant thereof having at least 70%sequence identity thereof ; and

[0172] - the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0173] 4, or a variant thereof having at least 70%sequence identity thereof ; and

[0174] - the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0175] 5, or a variant thereof having at least 70%sequence identity thereof ; and

[0176] - the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0177] 6, or a variant thereof having at least 70%sequence identity thereof ; and

[0178] - the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:

[0179] 7, or a variant thereof having at least 70%sequence identity thereof, and

[0180] - an amino acid segment 1-77 of the mutated human ubiquitin CG77V, in particular located at the N-terminal end of the KRAS1-23 N-terminal segments.

[0181] In a particular embodiment, the lentiviral vector encodes an amino acid sequence comprising the sequence set forth as SEQ ID NO: 29, or a variant having at least 70%, in particular 80%, more particularly 90% sequence identity thereof. In a particular embodiment, the lentiviral vector comprises a nucleic acid sequence having the sequence set forth as SEQ ID NO: 30, or a variant having at least 70%, in particular 80%, more particularly 90% sequence identity thereof.

[0182] A lentiviral vector according to the invention may further encode at least one support protein.

[0183] In particular, the at least one support protein may be selected from the group consisting of:

[0184] (i) proteins that improve the intracellular antigen routing to major histocompatibility complex presentation machineries;

[0185] (ii) proteins that provide helper CD4+ T-cell epitopes; and

[0186] (iii) proteins that direct the antigen to the proteasome and presentation machinery.

[0187] In particular, the at least one support protein may be selected from the group consisting of the MHC-II light invariant chain (“li”); diphteria toxins, or fragments thereof, in particular the 203-379 fragment of diphteria toxin (“DT”); ubiquitin proteins, or fragments thereof, in particular the fragment 1-77 of the mutated human ubiquitin CG77V; tetanus toxoids (TT) or fragments thereof; and mixtures thereof. The reference amino acid sequence of the full length diphteria toxin is the amino acid sequence set forth as SEQ ID NO: 48. The fragment 203-379 of DT is defined by reference to the amino acid sequence of full length DT as set forth in SEQ ID NO:48.

[0188] In a particular embodiment, the at least one support protein is selected from the group consisting of:

[0189] (i) the MHC-II light invariant chain (“li”), in particular having the amino acid sequence set forth as SEQ ID NO: 8,

[0190] (ii) the 203-379 segment of diphteria toxin (“DT”), in particular having the amino acid sequence set forth as SEQ ID NO: 9 and

[0191] (iii) the MHC-II light invariant chain (“li”) and the 203-379 segment of diphteria toxin (“DT”), in particular having the amino acid sequences set forth as SEQ ID NO: 8 and SEQ ID NO: 9.

[0192] The MHC-II light invariant chain (“li”) allows to favor targeting of the encoded KRASi -23 N-terminal segment(s), the 203-379 segment of diphteria toxin (“DT”), and / or tetanus toxoids (TT) to the MHC-II machinery in addition to the MHC-I pathway (Lopez et al, 2022; Diebold et al, 2001, MHC class II presentation of endogenously expressed antigens by transfected dendritic cells. Gene Ther 8: 487-493; Rowe et al, 2006, Immunization with a lentiviral vector stimulates both CD4 and CD8 T cell responses to an ovalbumin transgene. Mol Ther J Am Soc Gene Ther 13: 310-319).

[0193] The 203-379 segment of diphtheria toxin (DT) or tetanus toxoids (TT) allows to benefit from the helper functions of memory CD4+T-cell, present in the vast majority of humans vaccinated against diphtheria (Diethelm- Okita et al, 2000, Universal epitopes for human CD4+ cells on tetanus and diphtheria toxins . J Infect Dis 181: 1001-1009; Morse et al, 2013).

[0194] In a particular embodiment, the at least one support protein is located at the N- terminal end of the KRAS 1-23 N-terminal segment(s).

[0195] In a particular embodiment, the support protein is fused together at the N- terminal end of the at least one KRAS1-23 N-terminal segment(s).

[0196] In a particular embodiment, wherein the support protein is the MHC-II light invariant chain (“li”), in particular having the amino acid sequence set forth as SEQ ID NO: 8, it is located at the N-terminal end of the KRAS1-23 N-terminal segment(s).

[0197] In a particular embodiment, wherein the support protein is the 203-379 segment of diphteria toxin (“DT”), in particular having the amino acid sequence set forth as SEQ ID NO: 9, it is located at the N-terminal end of the KRAS1-23 N-terminal segment(s).

[0198] In a particular embodiment, wherein the support protein is the MHC-II light invariant chain (“li”), in particular having the amino acid sequence set forth as SEQ ID NO: 8, and the 203-379 segment of diphteria toxin (“DT”), in particular having the amino acid sequence set fort as SEQ ID NO: 9 the 203-379 segment of diphteria toxin (“DT”) is located at the C-terminal end of the MHC-II light invariant chain (“li”), and the 203-379 segment of diphteria toxin (“DT”) is located at the N-terminal end of the KRASi -23 N-terminal segment(s).

[0199] In a particular embodiment, the MHC-II light invariant chain (“li”) comprises a nucleic acid sequence set forth as SEQ ID NO: 21. In a particular embodiment, the MHC- II light invariant chain (“li”) consists of a nucleic acid sequence set forth as SEQ ID NO: 21. In a particular embodiment, the 203-379 segment of diphteria toxin (“DT”) comprises a nucleic acid sequence set forth as SEQ ID NO: 22. In a particular embodiment, the 203-379 segment of diphteria toxin (“DT”) consists of a nucleic acid sequence set forth as SEQ ID NO: 22.

[0200] In a particular embodiment, the lentiviral vector according to the invention encodes an amino acid sequence set forth as SEQ ID NO: 12 or SEQ ID NO: 13.

[0201] A lentiviral vector according to the invention may be single- stranded or doublestranded. A lentiviral vector according to the invention may be an RNA or DNA molecule.

[0202] In the context of the present invention, a “lentiviral vector” means a nonreplicating vector for the transduction of a host cell with a transgene comprising cis-acting lentiviral RNA or DNA sequences, and requiring essential lentiviral proteins (e.g., Gag, Pol, and / or Env) and accessory proteins (e.g, Tat, Rev) that are provided in trans. The lentiviral vector lacks expression of all functional HIV proteins. The lentiviral vector genome may be present in the form of an RNA or DNA molecule, depending on the stage of production or development of said retroviral vectors.

[0203] In a particular embodiment, the lentiviral vector is an integrative lentiviral vector. As used herein, “integrative” refers to a lentiviral vector that is able to integrate the delivered genetic material permanently into the host cell's DNA, in particular without any exogeneous tools.

[0204] In a particular embodiment, the lentiviral vector is a non-integrative lentiviral vector.

[0205] Non-integrating lentiviral vectors have been designed to mitigate the risks of potential oncogenesis linked to eventual insertional mutagenesis events, particularly for vaccination and immunotherapy purposes. Examples of non-integrating lentiviral vectors are provided in Coutant et al., PLOS ONE 7(l l):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). Consequently, it has been reported that a nonintegrating lentiviral vector system can mitigate the potential risk of insertional mutagenesis as compared to an integrating system (Hu et al., Vaccine 28:6675-6683 (2010)). It has been further reported that in some functional analysis, both the magnitude and quality of the immune responses elicited by DC-directed integration-defective lentiviral vectors (IDLVs) are comparable to that of its integrating counterpart. Thus, integration-defective lentiviral vectors (IDLVs) have been considered safer vectors than integrating vectors for human administration, with comparable effectiveness.

[0206] In addition, a lentiviral vector according to the invention may comprise long terminal repeats (LTRs) sequences in cis as known in the art and in particular comprise a 3’ long terminal repeat (LTR) which is devoid of its U3 promoter sequence (Miyoshi H et al, 1998, J Virol. 72(1 0):81 50-7; Zufferey et al., 1998, J V / ro / 72(12):9873-80).

[0207] In a particular embodiment, the lentiviral vector comprises a 3’ long terminal repeat (LTR) which is devoid of its U3 promoter sequence.

[0208] Enhancers are cis-acting sequences, which can act as transcriptional activators at a distance. They have been widely employed in viral derived vectors because they appear to be the most efficient for obtaining transgene strong expression in a variety of cell types, in particular dendritic cells (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, given the safety issue of insertional mutagenesis, such transcriptional enhancer sequences should be deleted from the lentiviral vector constructs to abolish the risk of insertional mutagenesis by enhancer proximity effect. This enhancer proximity effect is by far the most frequent mechanism of insertional mutagenesis and is the only effect described in human or animal cases of tumorigenic events after gene transfer.

[0209] Accordingly, in a particular embodiment, the lentiviral vector may not comprise a constitutive enhancer sequence.

[0210] Previous studies have reported on the replacement of viral promoters by DC- specific promoters deriving from major histocompatibility complex class II genes (MHC class II) (Kimura et al., 2007, Mol Ther 15(7): 1390-9) and dectin-2 genes (Lopes et al., 2008, J Virol 82(l):86-95). The dectin-2 gene promoter used in Lopes et al. contains a putative enhancer and an adenoviral conserved sequence (inverted terminal repeats in adenovirus promoter) (Bonkabara et al., 2001, J. Immunology, 167:6893-6900). The MHC class II gene promoter used by Kimura et al. does not contain any known enhancer. Yet, without an enhancer, the MHC class II promoter was found not to provide sufficient transgene expression in DCs, when administered intravenously. In particular, lentiviral vectors including MHC class II promoters did not provoke an immune reaction in immunocompetent C57BL / 6 mice, in contrast to the immune responses observed with CMV promo ters / enhancers. Although integration and persistent transgene expression were observed after injection in mice, the lentiviral vectors transcribed through MHC class II promoters failed to stimulate an antigen- specific CD8+ cytotoxic T-lymphocyte response, even after vaccination boost. The authors of these studies therefore concluded that the use of MHC class II promoters was of interest only for applications where persistence of expression is sought as in gene replacement therapy, but not in the context of immunotherapy. Of note, MHC class II promoters are expressed poorly in most cell types.

[0211] Thus, the MHC class II promoter is not an adequate promoter for lentiviral vectors for induction of an immune response against an antigen via IV injection. Moreover, the dectin-2 promoter is expressed poorly in most cell types and appears to contain an enhancer. Thus, the dectin-2 promoter is not a good promoter for lentiviral vectors for safety reasons.

[0212] Accordingly, in a particular embodiment, the lentiviral may comprise an MHC class I promoter, i.e. the nucleic acid sequences encoding antigens of a lentiviral vector according to the invention may be under the control of an MHC class I promoter.

[0213] An appropriate MHC Class I promoter may be selected from the group consisting of a p2-microglobulin promoter, a HLA-A2 promoter, a HLA-B7 promoter, a HLA-Cw5 promoter, a HLA-E promoter or a HLA-F promoter and is more particularly a P2-microglobulin promoter.

[0214] In a particular embodiment, the lentiviral may comprise a p2-microglobulin promoter.

[0215] MHC Class I promoters are particularly active in antigen presenting cells like dendritic cells in that expression of the promoter in BDCA+ dendritic cells is higher than the expression in kidney, smooth muscle, liver, and heart cells. They also have relatively high expression in other transduced cell types, for example, expression of the promoter in BDCA+ dendritic cells is only 12-100 times the expression of that promoter in skeletal muscle cells, in contrast to 900 times with the MHCII HLA-DRa promoter. This promoter drives in particular the transcription of the nucleic acid sequences encoding HPV antigens in a lentiviral vector of the invention.

[0216] Said promoter can be a naturally occurring or a synthetic MHC Class I promoter, obtained using well known molecular biological techniques.

[0217] In a particular embodiment, the lentiviral vector may comprise a cPPT / CTS sequence, such as described in EP2169073. This cPPT / CTS sequence may in particular be the sequence set forth as sequence SEQ ID NO: 10.

[0218] Indeed, efficient integration and replication in non-dividing cells generally requires the presence of two cis-acting sequences at 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 uncoating of the pre-integration complex at the nuclear pore and efficient importation of the expression cassette into the nucleus of non-dividing cells, such as dendritic cells.

[0219] In a particular embodiment, the lentiviral vector may comprise a Woodchuck hepatitis B virus (WHV) Post-Transcriptional Regulatory Element (WPRE), which allows a more stable expression of the transgene in vivo, and in particular a mutant form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE).

[0220] The mutated Woodchuck Posttranscriptional Regulatory Element (mWPRE) is characterized in that point mutations are introduced to avoid expression of the X protein contained in the WPRE region as said X protein may have oncogenic properties (Kingsman et al., Gene Ther. 2005 Jan;12(l):3-4).

[0221] The mutant form of the woodchuck hepatitis B virus (WHV) posttranscriptional regulatory element (WPRE) comprised in a lentiviral vector of the invention may in particular have the nucleic acid sequence set forth as sequence SEQ ID NO: 11.

[0222] In a particular embodiment, the lentiviral vector comprises a mutant form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), and in particular having the sequence set forth as sequence SEQ ID NO: 11.

[0223] In a particular embodiment, the lentiviral vector comprises an MHC Class I promoter, and in particular a human |32-microglobulin promoter and / or comprises a cPPT / CTS sequence, in particular the cPPT / CTS sequence set forth as sequence SEQ ID NO: 10, and / or comprises a 3’ long terminal repeat (LTR) which is devoid of its U3 promoter sequence and / or does not comprise a constitutive enhancer sequence and / or comprises a mutant form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), and in particular having the sequence set forth as sequence SEQ ID NO: 11.

[0224] In a particular embodiment, a lentiviral vector according to the invention:

[0225] (i) encodes a KRAS 1-23 N-terminal segment, said KRAS 1-23 N-terminal segment comprising at least one amino acid substitution in position 12 or 13, in particular in position 12, compared to the amino acid sequence set forth as SEQ ID NO: 1;

[0226] (ii) comprises a 3’ long terminal repeat (LTR) which is devoid of its U3 promoter sequence;

[0227] (iii) does not comprise a constitutive enhancer sequence;

[0228] (iv) comprises an MHC Class I promoter, and in particular a p2-microglobulin promoter;

[0229] (v) comprises a cPPT / CTS sequence, having in particular the sequence set forth as sequence SEQ ID NO: 10; and

[0230] (vi) comprises a mutant form of the woodchuck hepatitis B virus (WHV) post- transcriptional regulatory element (WPRE), having in particular the nucleic acid sequence set forth as sequence SEQ ID NO: 11.

[0231] Lentiviral vector particles according to the invention

[0232] Another object of the present invention relates to a lentiviral vector particle comprising at least one lentiviral vector according to the invention, and in particular at least one lentiviral vector as defined above.

[0233] A lentiviral vector particle according to the invention, which contains a lentiviral vector according to the invention, can be produced by recombinant technology known in the art upon transient transfection of cells, for example HEK 293T human cultured cells, by different DNA plasmids:

[0234] (i) a packaging plasmid, which expresses at least the Gag, Pol, Rev, Tat and, in some cases, structural and enzymatic proteins necessary for the packaging of the transfer construct; (ii) a lentiviral vector according to the invention, containing an expression cassette (antigens) and HIV cis-acting factors necessary for packaging, reverse transcription, and integration; and

[0235] (iii) an envelope-encoding plasmid, in most cases the glycoprotein of vesicular stomatitis virus (VSV.G), a protein that allows the formation of mixed particles (pseudotypes) that can target a wide variety of cells, especially major histocompatibility (MHC) antigen-presenting cells (APCs), including DCs.

[0236] Such a method allows producing a recombinant vector particle according to the invention, comprising the following steps of: i) transfecting a suitable host cell with a lentiviral vector according to the invention; ii) transfecting said host cell with a packaging plasmid vector, containing viral DNA sequences encoding at least structural and polymerase activities of a retrovirus (preferably lentivirus); Such packaging plasmids are for example described 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 said transfected host cell in order to obtain expression and packaging of said lentiviral vector into lentiviral vector particles; and iv) harvesting the lentiviral vector particles resulting from the expression and packaging of step iii) in said cultured host cells.

[0237] In order to pseudotype the retroviral particles of the invention, the host cell can be further transfected with one or several envelope DNA plasmid(s) encoding viral envelope protein(s), preferably a VSV-G envelope protein.

[0238] This procedure allows obtaining transient production of lentiviral particle vectors by the transfected cells. However, the lentiviral particle vectors may also be continuously produced by cells by stably inserting the packaging genes, the proviral coding DNA, and the envelope gene into the cellular genome. This allows the 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, with some of the DNAs / plasmids integrated into the cellular genome and others provided by transient transfection.

[0239] A lentiviral vector particle may be a non-integrating lentiviral vector particle. Non-integrating vector particles have one or more mutations that eliminate most or all of the integrating capacity of the lentiviral vector particles. For, example, a non-integrating vector particle can contain mutation(s) in the integrase encoded by the lentiviral pol gene that cause a reduction in integrating capacity.

[0240] A lentiviral vector particle according to the invention in particular comprises a non-integrating lentiviral vector of the invention.

[0241] According to a particular embodiment, the lentiviral vector particle further comprises a functional lentiviral integrase protein.

[0242] A lentiviral vector particle according to the invention may further comprise a vesicular stomatitis virus glycoprotein (VSVG), in particular a VSV-G Indiana serotype or a VSV-G New Jersey serotype.

[0243] In matter of vaccination strategy, VSV-G-pseudotyped lentiviral vector particles are not target of pre-existing anti- vector immunity because human population have been barely exposed to VSV.

[0244] The lentiviral vector particle may comprise HIV-1 Gag and Pol proteins, and in particular HIV-1 subtype D Gag and Pol proteins.

[0245] A further object of the present invention relates to an isolated cell comprising (i.e. transformed with) a lentiviral vector according to the invention or a lentiviral vector particle of the invention.

[0246] A cell according to the invention is preferably a mammalian cell, particularly a human cell. Particularly preferred are human non-dividing cells.

[0247] Another object of the present invention relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable medium, a lentiviral vector according to the invention, a lentiviral vector particle according to the invention or a cell according to the invention.

[0248] In a particular embodiment, the invention relates to a vaccine composition comprising, in a pharmaceutically acceptable medium, a lentiviral vector according to the invention, a lentiviral vector particle according to the invention or a cell according to the invention.

[0249] A pharmaceutical composition or a vaccine composition according to the invention comprises a pharmaceutically acceptable medium. By ''pharmaceutically acceptable medium” is meant any solution used to solubilize and deliver a lentiviral vector, a lentiviral vector particle or a cell according to the invention to an individual. A desirable pharmaceutically acceptable carrier is saline. In desirable embodiments, a pharmaceutically acceptable medium includes an adjuvant.

[0250] Appropriate pharmaceutically acceptable mediums and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (20th edition), ed. A. Gennaro, 2003, Lippincott Williams & Wilkins.

[0251] Another object of the present invention relates to a combinatory treatment.

[0252] A combinatory treatment refers to the use of two or more different therapeutic approaches simultaneously or sequentially to treat a disease, such as cancer. The rationale behind combinatory treatment is to enhance the effectiveness of therapy by targeting the disease in multiple ways, which can lead to better outcomes and / or fewer undesirable side effects compared to using a single treatment modality alone. Using multiple treatments simultaneously may render the tumor cells more susceptible to the action of vaccine-induced effector T cells, and may also enhance the effector functions of vaccine-induced T cells.

[0253] The invention therefore relates to a combinatory treatment comprising:

[0254] (i) at least one lentiviral vector according to the invention or lentiviral vector particle according to the invention or an isolated cell according to the invention or a pharmaceutical composition according to the invention, and

[0255] (ii) at least one chemotherapy agent or treatment and / or at least one immunotherapy agent or treatment.

[0256] In particular, a combinatory treatment is a combination of one or more products, in particular a combination of one or more vectors, particles, agents, compositions, and / or treatments.

[0257] Chemotherapy agents and treatments, in particular chemotherapy agents and treatments against pancreatic, colorectal, and / or pulmonary cancers are well known in the art.

[0258] Similarly, immunotherapy agents and treatments against pancreatic, colorectal, and / or pulmonary cancers are well known in the art.

[0259] In a particular embodiment, the at least one chemotherapy agent or treatment is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, pemetrexed, 5- fluoracil (5-FU), taxane, and mixtures thereof. In a particular embodiment, the chemotherapy agent or treatment is cisplatin.

[0260] In a particular embodiment, the at least one immunotherapy agent or treatment is selected from the group consisting of immune checkpoint inhibitors, in particular anti-PD- 1, anti-PD-Ll (PD-1 Ligand), anti-CTLA-4 (Cytotoxic T-Lymphocyte- Associated protein 4), an anti-NKG2A, anti-TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), anti-TIGIT (T cell immunoreceptor with Ig and ITIM domains) or anti-LAG-3 (Lymphocyte- activation gene 3) antagonist antibodies; and mixtures thereof. In a particular embodiment, the immunotherapy agent or treatment is an anti-PDl antibody, in particular an anti-PDl monoclonal antibody.

[0261] Further immune checkpoints according to the invention are described below.

[0262] Further characteristics describing the administration of the combinatory treatment are provided below.

[0263] In a particular embodiment, the combinatory treatment comprises:

[0264] (i) at least one lentiviral vector according to the invention or lentiviral vector particle according to according to the invention or an isolated cell according to the invention or a pharmaceutical composition according to the invention, and

[0265] (ii) cisplatin and an anti-PDl antibody, in particular an anti-PDl monoclonal antibody.

[0266] Implementations according to the invention

[0267] An 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 or a pharmaceutical composition of the invention for use as a medicament or vaccine.

[0268] It is further described the use of a lentiviral vector of the invention, a lentiviral vector particle of the invention or an isolated cell of the invention or a pharmaceutical composition of the invention for use in the manufacture of a medicament, in particular a medicament for the treatment of a cancers.

[0269] In particular, an object of the present invention relates to a lentiviral vector of the invention, a lentiviral vector particle of the invention, an isolated cell of the invention, or a pharmaceutical composition of the invention, in particular in the form of a vaccine composition according to the invention, for use in the treatment or prevention of cancers. In a particular embodiment, the cancers are cancers wherein the KRAS 1-23 N- terminal segment is mutated.

[0270] In a particular embodiment, the cancers are pancreatic, colorectal, and / or pulmonary cancers.

[0271] Such prevention and / or treatment implies the administration of the considered active, in particular a vaccine composition of the invention as defined above, to an individual in need thereof.

[0272] An individual in need thereof is an animal, in particular a mammal, and may more particularly be a human being.

[0273] Lentiviral vectors, lentiviral vector particles, isolated cells and pharmaceutical compositions according to the invention are administered to an individual in need thereof by conventional methods, in dosages which are sufficient to elicit an immunological response, which can be easily determined by those skilled in the art.

[0274] Lentiviral vectors, lentiviral vector particles, isolated cells and pharmaceutical compositions according to the invention may accordingly be administered intravenously or intramuscularly as indicted below.

[0275] Lentiviral vectors, lentiviral vector particles, isolated cells and pharmaceutical compositions according to the invention may alternatively be administered intranasally. This route of administration is particularly useful in the treatment or prevention of oropharyngeal cancers and / or pulmonary metastases.

[0276] Lentiviral vectors, lentiviral vector particles, isolated cells and pharmaceutical compositions according to the invention are administered in a therapeutically effective amount, and may in particular be administered in a dose corresponding to at least 1 x 106, 2 x 106, 5 x 106, 107, 2 x 107, 5 x 107, 1 x 108, 2 x 108, 5 x 108, or at least 1 x 109TU (Transduction units) of lentiviral vectors according to the invention, in particular in a dose corresponding to at least 1 x 107, 2 x 107, 5 x 107, 1 x 108TU or at least 1 x 109TU of lentiviral vectors according to the invention. In a preferred embodiment, the lentiviral vectors, lentiviral vector particles, cells and pharmaceutical compositions according to the invention are administered in a dose corresponding to at least 1 x 107TU of lentiviral vectors according to the invention, more particularly at least 1 x 108TU of lentiviral vectors according to the invention and in particular at least 1 x 109TU of lentiviral vectors according to the invention. By a “therapeutically effective amount” is for example meant the amount of a lentiviral vector or lentiviral vector particle, cell or pharmaceutical composition according to the invention required to generate in a subject one or more of the following effects: an immune response against the KRAS expressing tumor; an inhibition of the tumor growth and / or a decrease in the size of the induced tumor, i.e. a reduction of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or of 100% of the tumor size in weeks following the start of the immunotherapy compared to the size of the tumor at the time of administration); an increase in the intratumoral CD45+ hematopoietic cells, notably CD8+ T cells, and an engagement of CD8+T cells toward either CD44+CD69+CD103+resident memory T cells (Trm) or KLRG1+(killer cell lectin like receptor Gl) and PD1+(programmed death 1) activated and terminally differentiated phenotype.

[0277] Administration can be performed using well known routes including, for example, intravenous, intramuscular, intradermal, intranasal, intraperitoneal or subcutaneous injection, and in particular intravenous, intranasal or intramuscular, and may be intravenous or intramuscular.

[0278] The appropriate dose and regimen will obviously vary between species and individuals depending on many factors.

[0279] Lentiviral vectors, lentiviral vector particles, cells and pharmaceutical compositions according to the invention may for example be administered in a single dose, as illustrated in the examples, or in two or more administrations. Practitioners will determine, in each case, the appropriate regimen and dosage for the administration of actives according to the invention.

[0280] Lentiviral vectors, lentiviral vector particles, cells and pharmaceutical compositions according to the invention may advantageously be administered in combination with at least one immune checkpoint inhibitor (ICI). In a particular embodiment, the lentiviral vectors, lentiviral vector particles, cells and pharmaceutical compositions according to the invention may advantageously be administered in combination with at least one immune checkpoint inhibitor (ICI) in the form of a combinatory treatment, in particular as described above.

[0281] An immune checkpoint inhibitor (ICI) according to the invention may in particular be an antibody, in particular an anti-PD-1, an anti-PD-Ll (PD-1 Ligand), an anti- CTLA-4 (Cytotoxic T-Lymphocyte-Associated protein 4), an anti-NKG2A, an anti-TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), an anti-TIGIT (T cell immunoreceptor with Ig and ITIM domains) or an anti-LAG-3 (Lymphocyte-activation gene 3) antibody. More particularly, the at least one immune checkpoint inhibitor according to the invention may be a monoclonal antibody selected from the group consisting of anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti-NKG2A, anti-TIM-3, anti-TIGIT and anti-LAG-3 antagonistic monoclonal antibodies. Even more particularly, the at least one immune checkpoint inhibitor according to the invention may be a monoclonal antibody selected from the group consisting of anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti-NKG2A, anti-TIM-3, anti-LAG-3 and anti-TIGIT monoclonal antibodies.

[0282] An anti-PD-1 monoclonal antibody may for example be selected from the group consisting of Nivolumab, Pembrolizumab and Cemiplimab.

[0283] An anti-PD-Ll monoclonal antibody may for example be selected from the group consisting of Atezolizumab, Avelumab and Durvalumab.

[0284] An anti-CTLA-4 monoclonal antibody may for example be selected from the group consisting of ipilimumab, tremelimumab and quavonlimab.

[0285] NKG2A is an ITIM (intracytoplasmic tyrosine-based inhibitory motifs)- bearing receptor expressed at the surface of 50% of peripheral blood NK cells and 5% of human peripheral blood CD8+ T cells. This cell surface molecule is expressed as a heterodimer with CD94 and interacts with the Major Histocompatibility Complex class I (MHC-I) non-classical molecules, i.e., human leukocyte antigen (HLA)-E in and murine Qa- 1b. This interaction inhibits both T and NK effector anti-tumor functions (Andre et al, Cell. 2018 Dec 13; 175(7): 1731- 1743.el3). It has been described that an anti-NKG2A mAb can be used as a checkpoint inhibitor and can promote anti-tumor cellular immunity by unleashing not only NK, but also CD8+ T cells in mice (Andre et al, Cell. 2018 Dec 13;175(7): 1731- 1743.el3). An anti-NKG2A monoclonal antibody may for example be monalizumab.

[0286] T-cell immunoglobulin and mucin domain-3 (TIM-3) is a negative regulatory immune checkpoint. TIM-3 is expressed by various immune cells, notably T cells. TIM-3 has four ligands, including galectin-9 (Gal-9), carcinoembryonic antigen cell adhesion molecule 1 (CEACAM-1), high-mobility group protein Bl (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 displays an important role in T-cell exhaustion. In cancer immunotherapy anti-TIM-3 mAh treatment displays beneficial effects comparable to those of anti-PD-1 mAb therapy. An anti-TIM-3 monoclonal antibody may for example be selected from the group consisting of Sym023 and sabatolimab.

[0287] An anti-TIGIT monoclonal antibody may for example be tiragolumab.

[0288] An anti-LAG-3 monoclonal antibody may for example be relatlimab.

[0289] In particular, the ICI may be an anti-PD-Ll or an anti-PD-1 monoclonal antibody, and in particular be an anti-PD-1 monoclonal antibody.

[0290] In particular, the ICI may be selected in the group consisting of an anti-PD-Ll an anti-NKG2A, an anti-TIM-3 and an anti-PD-1 monoclonal antibody, and more particularly in the group consisting of an anti-NKG2A, an anti-TIM-3 and an anti-PD-1 monoclonal antibody.

[0291] The vaccine composition, lentiviral vector, lentiviral vector particle or cell for use according to the invention and the immune checkpoint inhibitor may be administered simultaneously or separately.

[0292] Considering the synergistic advantageous properties obtained when combining a lentiviral vector according to the invention with an immune checkpoint inhibitor as demonstrated in the examples, it can be anticipated that vaccination with a lentiviral vector according to the invention may increase the number of patients eligible for immune checkpoint inhibitor therapy, especially anti-PD-1, an anti-NKG2A or an anti-TIM-3.

[0293] By simultaneously, it is understood that (i) the vaccine composition, lentiviral vector, lentiviral vector particle or cell and (ii) the immune checkpoint inhibitor, may be administered at the same moment or up to the same day or couple of days. In this case, they can be administered in the same composition or in separate compositions.

[0294] By separately, it is understood that (i) the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the invention and (ii) the immune checkpoint inhibitor may be administered with at least several days, for example at least two days of difference.

[0295] 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. The same characteristics may be applied to the combinatory therapy according to the invention and described above.

[0296] Advantageously, the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the invention may be administered at least 2 and in particular at least 4 days before the administration of the immune checkpoint inhibitor. Accordingly, the immune checkpoint inhibitor may advantageously be administered at least 2 and in particular at least 4 days after the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the invention. The immune checkpoint inhibitor may more particularly be administered 4 days to 1 month after the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the invention, in particular 4 days to 15 days, and more particularly 4 days to 10 days after the vaccine composition, lentiviral vector, lentiviral vector particle or cell according to the invention.

[0297] The vaccine composition, lentiviral vector, lentiviral vector particle, cell or pharmaceutical composition for use according to the invention and the immune checkpoint inhibitor may be administered by the same route or through different routes.

[0298] The at least one immune checkpoint inhibitor herein is administered in a therapeutically effective dose, i.e. a dose that produces the effects for which it is administered. The exact dose of immune checkpoint inhibitor will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques.

[0299] The characteristics described above for the use of a lentiviral vector, lentiviral vector particle, cell or pharmaceutical composition in combination with a check point inhibitor may be applied to the combinatory treatment according to the invention and described elsewhere in the text.

[0300] The invention further relates to a method for the treatment and / or prevention of cancers in an individual in need thereof, comprising the administration to said individual of at least one lentiviral vector of the invention, lentiviral vector particle of the invention or isolated cell of the invention, in particular in the form of a vaccine composition according to the invention.

[0301] The invention further relates to the use of at least one lentiviral vector of the invention, lentiviral vector particle of the invention, isolated cell of the invention, or pharmaceutical composition of the invention, in particular in the form of a vaccine composition according to the invention for the treatment and / or prevention of cancers in an individual in need thereof.

[0302] In particular, the cancers are cancers wherein the KRAS 1-23 N-terminal segment is mutated, more particularly the cancers are pancreatic, colorectal, and / or pulmonary cancers wherein the KRAS 1-23 N-terminal segment is mutated.

[0303] The examples and figures which follow are presented by way of illustration and without implied limitation of the invention.

[0304] EXAMPLES

[0305] Materials and methods

[0306] Mice

[0307] Six- to 8-week-old female BALB / c or C57BL / 6JRj mice (Janvier, Le Genest Saint Isle, France) were housed in ventilated cages under specific pathogen-free conditions at the Institut Pasteur animal facilities. All procedures were performed in accordance with the European and French guidelines (Directive 86 / 609 / CEE and Decree 87-848 of 19 October 1987) after approval of the protocol by the Institut Pasteur Safety, Animal Care and Use Committee delivered by the local ethics committee (CETEA #DAP190130 and CETEA #DAP220103) and Ministry of High Education and Research (APAFIS#43914- 2023062210343762 v2).

[0308] Tumor cell lines

[0309] CT26 mouse colon carcinoma cell line (CRL-2638), derived from BALB / c mouse strain, was purchased from ATCC and maintained in RPMI media (GIBCO) media supplemented with 100 U / mL penicillin / streptomycin (Gibco) and 10 % heat-inactivated fetal bovine serum (Serana).

[0310] MC38 mouse colon carcinoma cell line, derived from C57BL / 6 murine colon adenocarcinoma (Corbett et al, 1975, Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays, with a note on carcinogen structure. Cancer Res 35: 2434-2439), was purchased from Kerafast (Boston MA, USA). LLC1 mouse lung carcinoma cell line (CRL-1462), derived from C57BL / 6 strain, was purchased from ATCC. MC38 and LLC1 cells were transduced with an integrative lenti viral vector coding full-length murine Kras gene with G12D amino acid substitution, under the ubiquitin promoter (Ku et al, 2021b, Lentiviral vector induces high- quality memory T cells via dendritic cells transduction. Commun Biol 4: 713). Transduced populations of MC38 or LLC1 cells were cloned and clones were analyzed for KRASG12DmRNA expression by RT-PCR with following primers: KRAS F: 3’ -ggcctgctgaaaatgactga- 5’ (SEQ ID NO: 31), KRAS R: 3’ -ggctgccgtcctttacaag-5’ (SEQ ID NO: 32). Clones with highest KRASG12DmRNA expression were used in the experiments. MC38 and MC38- KRASG12D(#9S) cell lines were cultured in DMEM medium containing Glutamax (Gibco) and supplemented with 100 U / mL penicillin / streptomycin (Gibco), 10 % heat- inactivated fetal bovine serum (Serana), 0.1 mM nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco) and 10 mM Hepes (Gibco). LLC1 and LLC1KRASG12Dcells were cultured in DMEM media containing Glutamax (Gibco) supplemented with 100 U / mL penicillin / streptomycin (Gibco) and 10 % heat-inactivated fetal bovine serum (Serana).

[0311] All the tumor clones used in this study expressed surface MHC-I molecules.

[0312] Construction and production of diverse Lenti-KRAS G12D

[0313] Codon-optimized sequences of the designed KRAS G12D antigens were synthesized and inserted into the pFlap lentiviral plasmid between the BamHI and Xhol sites, located between the human [32-microglobulin promoter and the mutated atg starting codon of the woodchuck post-transcriptional regulatory element (WPRE) sequence, as detailed elsewhere. The envelope plasmid encodes Vesicular Stomatitis Virus glycoprotein (VSV-G) under the transcriptional control of cytomegalovirus promoter, and the packaging plasmid contains gag, pol, tat, and rev genes. The integrase resulting from the packaging plasmid carries a missense amino acid in its catalytic triad, i.e., the D64V mutation, which prevents the integration of viral DNA into the host chromosome (Ku et al, 2021c), (Nemirov et al, 2023).

[0314] Immunotherapy of tumor-bearing mice

[0315] Mice were shaved with electric shaver device (ChroMini Tcut, WAHL) 2 days before tumor implantation and were engrafted s.c. with 2 x 105or 1 x 105tumor cells on the right flank at day 0. When the tumors became palpable, i.e., usually at day 6 post tumor engraftment, mice were randomized and injected i.m. with 1 x 109TU of lentiviral vectorbased vaccine, contained in 50 pl of PBS. For the MC38KRASG12Dmodel, mice were randomized and primed at day 3 post tumor challenge.

[0316] Tumors were measured 2-to-3 times a week using a digital caliper. The tumor volume was calculated as V = L x W2 / 2, where V is the volume, L the length (the longest diameter), and W the width (the shortest diameter). Due to ethical reasons, mice were sacrificed if tumors became ulcerated or when they reached 1500 mm3in volume or when the mice became moribund.

[0317] Cytometric study of tumor immune infiltrates Tumors were harvested and individually treated with the Mouse Tumor Dissociation kit (Miltenyi). Cell suspensions were then filtered through 70 pm-pore filters and washed in PBS. The recovered cells were stained as follows.

[0318] (i) To detect T cells and their activation profile, Near IR Live / Dead (Invitrogen), Fcyll / III receptor blocking anti-CD16 / CD32 (clone 2.4G2, BD Biosciences), BV605-anti-CD45 (clone 30-F11, BD Biosciences), APC-anti-CD8 (clone 53-6.7, Invitrogen), eF450-anti-CD4 (clone RM4-5, eBioscience), BV711-anti-CD103 (clone 2E7, Biolegend), PE-Cy7-anti-CD69 (clone H1.2F3, BD Biosciences), FITC-anti-CD44 (clone IM7, Biolegend), and PerCP-eF710-anti-KLRGl (clone 2F1, Invitrogen), and FITC-anti- PD1 (clone RMP1-30, Invitrogen) were used. Samples were incubated with the mAb mixtures for 30 minutes at 4°C, washed with PBS + 3% fetal bovine serum and fixed with Cytofix (BD Biosciences) for 20 min at 4°C.

[0319] (ii) To study intra- tumoral innate immune cells, tumor suspensions were stained by a mixture of Near IR Live / Dead (Invitrogen), Fey 11 / 111 receptor blocking anti- CD16 / CD32 (clone 2.4G2, BD Biosciences), BV605-anti-CD45 (clone 30-F11, BD Biosciences), PE-anti-CDl lb (clone MI / 70, Invitrogen), PE-Cy7-anti-CDl lc (clone N418, eBioscience), PE-anti-Ly6G (clone 1A8, Biolegend), BV421-anti-NKp46 (clone 29A1.4, Biolegend), and PerCP-Cy5.5-anti-Ly6C (clone HK1.4, eBioscience) were used.

[0320] (iii) To detect intra-tumoral anti-KRAS T cells, following enzymatic tumor dissociation, cell suspensions were enriched in lymphocytes by centrifugation for 20 minutes at 3000 rpm at RT without brake on Ficoll medium (Lympholyte M, Cedarlane Laboratories). Recovered cells were co-cultured in 24-well plates at 1 x 106 / well overnight with 5 x 105 / well of syngeneic bone-marrow-derived dendritic cells, loaded with 10 pg / mL of appropriate peptides, in the presence of 1 pg / mL final of anti-CD28 (clone 37.51) and anti-CD49d (clone 9C10-MFR4.B) mAbs (BD Biosciences). During the last 3 h of incubation, cell cultures were treated with a Golgi Plug and Golgi Stop (BD Biosciences) mixture. Cells were then washed with PBS containing 3% fetal bovine serum and incubated for 25 min at 4°C with a mixture of Near IR Live / Dead (Invitrogen), FcgII / III receptor blocking anti-CD16 / CD32 (clone 2.4G2), BV605-anti-CD45 (clone 30-F11, BD Biosciences), eF450-anti-CD4 (clone RM4-5, eBioscience), and BV711-anti-CD8 (clone 53-6.7, BD Horizon) mAbs. Cells were washed twice and permeabilized with Cytofix / Cytoperm kit (BD Bioscience). Cells were then washed twice with PermWash IX buffer from the Cytofix / Cytoperm kit and incubated with a mixture of FITC-anti-TNFcx (clone MP6-XT22, BD Pharmigen), and APC-anti-IFN- y (clone XMG1.2, BD Pharmigen) mAbs during 30 min at 4C. Cells were then washed twice in PermWash and once in PBS containing 3% fetal bovine serum, then fixed with Cytofix (BD Biosciences) overnight at 4°C.

[0321] (iv) Expression of MHC-I molecules and PDL1 on tumor cells were checked by use of PE-anti-H-2Kb(REA1198, Miltenyi Biotec), PerCP / Cyanine5.5-anti-H-2Db(KH95, BioLegend), APC-anti-PDLl (10F.9G2, BioLegend) mAbs used, in presence of Near IR Live / Dead (Invitrogen) and Fcyll / III receptor blocking anti-CD16 / CD32 (clone 2.4G2, BD Biosciences).

[0322] Samples were acquired in an Attune NxT cytometer (Invitrogen) and were analyzed using Flow Jo software (Treestar, OR, USA).

[0323] Example 1: Lenti-KRAS vaccine designs

[0324] Three modalities of KRASi-23G12D-based antigens were designed to be encoded by non-integrative lentiviral vectors (see Figure 1A). (i) KRASi-23G12Dalone (Lenti- KRASG12D), (ii) a fusion of the MHC-II light invariant chain (“li”) to the N-terminal end of KRASI-23G12D, and (iii) a fusion of the li-KRASi-23G12Dto the 203-379 segment of diphtheria toxin (DT) (Lenti-li-DT-KRASG12D), present in the vast majority of humans vaccinated against diphtheria

[0325] The inventors further generated an additional lentiviral vector encoding a polyantigen composed of sequences containing KRASG12A, KRASG12C, KRASG12D, KRASG12R, KRASG12Vand KRASG13Dmutations (Hobbs et al, 2016). This poly-antigen was fused, at its N-terminal end, to the amino acid segment 1-77 of the mutated human ubiquitin CG77V(Liu et al, 2011, EGF signalling activates the ubiquitin proteasome system to modulate C. elegans lifespan. EMBO J 30: 2990-3003). (see Figure IB).

[0326] Sequences encoding each of the designed antigens were inserted into individual lentiviral transfer plasmids under the human |32-microglobulin promoter, with increasing spatio-temporal activity with activation and migration of dendritic cells and upregulation of MHC-I molecules (Ku et al, 2021b). Recombinant lentiviral vector particles, pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) from Indiana serotype, were successfully produced in the human embryonic kidney (HEK)-293T cell production system and concentrated by ultracentrifugation with titers ranging from 1.0 to 2.7 x 1011Transduction Units (TU) / mL.

[0327] Example 2: Antitumor effect of diverse Lenti-KRAS in two colorectal carcinoma preclinical models

[0328] The antitumor effect of the generated lentiviral vectors was first explored against the CT26 colorectal cancer murine cell line that endogenously expresses KRASG12D(Arbelaez et al, 2020). BALB / c (H-2d) mice were subcutaneously (s.c.) engrafted with 2 x 105CT26 cells at day 0. At day 7 post tumor challenge, when tumors became palpable, mice were randomized and vaccinated with a single intramuscular (i.m.) injection of 1 x 109TU of an empty lentiviral vector (Ctrl Lenti), Lenti-KRASG12D, Lenti-li-KRASG12Dor Lenti-li- DT-KRASG12D.

[0329] At day 18 post tumor engraftment, a tendency to the tumor growth inhibition was observed notably in the Lenti-li-DT-KRASG12D-treated mice (see Figure 2A). At day 20, the tumor growth inhibition was statistically significant in this group, compared to the Ctrl Lenti-treated group. The mean survival of Lenti-li-DT-KRASG12D-treated mice was also significantly prolonged (see Figure 2B).

[0330] KRASG12Dmutation generates MHC-I-restricted T cell neoepitope with mild immunogenicity, yet in both H-2dand H-2bmurine haplotypes (Arbelaez et al, 2020). Therefore, the antitumor effect of the Lenti-KRAS vectors was also evaluated in C57BL / 6 (H-2b) mice, in which several other tumor cell lines are available. The C57BL / 6-derived murine colon adenocarcinoma MC38 cell line (Corbett et al, 1975) was transduced by the full length KRASG12Dprotein, expressed under the transcriptional control of the ubiquitin promoter (Wiborg et al, 1985, The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences. EMBO J 4: 755-759; Christensen & Quail, 1996, Ubiquitin promoter-based vectors for high-level expression of selectable and / or screenable marker genes in monocotyledonous plants. Transgenic Res 5: 213-218). The resulted clones were screened for KRAS mRNA. The clone #9S1 of MC38KRASG12D, in which the presence of KRASG12Dmutation was confirmed by sequencing, was selected for further experiments. C57BL / 6 mice were inoculated s.c. with 2 x 105MC38KRASG12Dcells at day 0. Mice were randomized and were treated by an i.m. mono-injection of 1 x 109TU of Ctrl Lenti or Lenti-li-DT-KRASG12Dat day 6.

[0331] In Lenti-li-DT-KRASG12D-vaccinated group a tendency to the tumor growth inhibition was observed at day 14 (see Figure 3A). At day 17, the tumor growth control reached statistical significance in the Lenti-li-DT-KRASG12D-treated mice. The efficacy of Lenti-li-DT-KRASG12Dimmunotherapy against MC38KRASG12Dtumor was confirmed in numerous other independent experiments, as exemplified in (see Figure 3B).

[0332] To get more insights on the effect of Lenti-li-DT-KRASG12Dtherapy on MC38KRASG12Dtumor microenvironment, the tumor immune infiltrates were immunophenotyped at day 14 post tumor challenge, which corresponded to day 11 post vaccine administration. Tumors from the Lenti-li-DT-KRASG12D-treated mice contained a higher density of CD45+hematopoietic cells than those from their Ctrl Lenti-treated counterparts, even if the difference did not reach statistical significance (see Figures 3C and 3D). However, the CD8+- but not CD4+- T cell subset was significantly expanded inside the CD45+population within the tumor microenvironment of Lenti-li-DT-KRASG12D-treated mice, with a net increase in the intra-tumoral CD8+ / CD4+ratio. Engagement of CD8+T cells toward either CD44+CD69+CD103+resident memory T cells (Trm) or killer cell lectin like receptor G1 KLRG1+and programmed death 1 PD1+exhausted phenotype was detectable in the MC38KRASG12Dtumors, albeit without significant difference between the Lenti-li-DT- KRASG12D- and Ctrl Lenti-treated groups (see Figures 3C and 3D).

[0333] The potential of a lentiviral vector encoding a fusion protein of KRASi-23G12Dantigenic segment and the neutral nano-luciferase protein instead of li-DT (Lenti-nLuc- KRASG12D) was also explored in immuno-therapy of MC38KRASG12Dtumor-bearing mice. Lenti-nLuc-KRASG12Dtherapy achieved a significant inhibition of tumor growth (see Figure 4A) and preferential intra-tumoral infiltration / expansion of CD8+T cells (see Figure 4B), reinforcing the robustness of the lentiviral vector platform targeting the mutated KRAS neoantigen in immuno-oncotherapy.

[0334] Example 3: Phenotype of antitumor effector cells

[0335] Probably due to the low affinity of the epitope generated by the KRASG12Dmutation for murine MHC-I molecules of haplotypes H-2bor H-2d, no KRASG12D-specific T cell responses were detected in vitro by ELISPOT or intracellular IFN-y / TNF-oc / IE-2 cytokine staining subsequent to peptide stimulation in the spleen of Eenti-li-DT-KRASG12D- vaccinated mice.

[0336] However, MC38KRASG12Dtumor infiltrating lymphocytes (TIFs) in Fenti-li- DT-KRASG12D-primed and -boosted C57BF / 6 mice contained IFN-y- and TNF-oc-producing CD8+T cells, as evidenced after their co-culture with syngeneic bone-marrow dendritic cells loaded with synthetic KRAS i-io pcptidcs. No IFN-y- and / or TNF-oc-producing CD8+T cells were detected after stimulation of these TIFs with an irrelevant negative control peptide.

[0337] KRAS-specific CD8+TIFs were also detected at the same percentages within the CD8+T subset in the tumors of Ctrl Fenti-treated mice, which suggests their tumor-bom origin. Even though the proportions of KRAS-specific CD8+TIFs were comparable in mice treated with Ctrl Eenti or Eenti-li-DT-KRASG12D, the percentages of total CD8+TIFs were higher in Eenti-li-DT-KRASG12D-treated mice. Consequently, the total absolute number of KRAS-specific CD8+TIES can only be higher in the tumors from Eenti-li-DT-KRASG12D- vaccinated mice (see Figure 5). Furthermore, the total tumor homogenates from Eenti-li-DT- KRASG12D-treated mice contained significantly reduced percentages of tumor cells and displayed a clear upward trend in the percentages of CD45+hematopoietic cells, compared to the tumor homogenates from Eenti Ctrl-treated mice (see Figure 5).

[0338] To identify in vivo the antitumor effector cell subset, MC38KRASG12D-bearing C57BE / 6 mice were primed (day 4) and boosted (day 11) with 1 x 109TU of Eenti-li-DT- KRASG12Dand received intraperitoneal (i.p) injections of a Ctrl Ig, anti-CD4 or anti-CD8 depleting mAbs on days 2, 4, 7, 10 and 14. In the vaccinated groups, treated with the Ctrl Ig, a significant inhibition of tumor growth was observed, whereas in the Eenti-li-DT- KRASG12D-vaccinated group treated with anti-CD8 mAb, the tumor growth was comparable to the Ctrl Fenti-treated mice (see Figures 6A and 6B). Interestingly, the anti-CD4 depleting mAb treatment had only a slight and statistically unsignificant negative effect on the vaccine efficacy at the early time points, i.e., until day 14, possibly suggesting a role for an initial CD4+T helper function. The anti-CD4- or anti-CD8 treatments were efficient in T subset depletion, as determined by the drastically reduced percentages of CD4+or CD8+T cells, respectively in the peripheral blood leukocytes of treated mice (see Figure 6C).

[0339] Therefore, these results established that the principal immune effector cells induced by Eenti-li-DT-KRASG12Dimmunization were CD8+T cells. Example 4: Advantageous combination of Lenti-li-DT-KRASG12D with chemo- and immuno-therapies

[0340] The inventors evaluated the effect of the combination of Lenti-li-DT- KRASG12Dwith apoptosis / ferroptosis-inducing cisplatin which is one of the compelling first line anti-cancer chemotherapy and the standard of care antagonistic anti-PDl mAb immunotherapy. C57BL / 6 mice (n = 6-7 / group) were engrafted s.c. with 1 x 105MC38KRASG12Dcells and were then: (i) primed and boosted i.m. with 1 x 109TU of Ctrl Lenti, (ii) primed and boosted i.m. with 1 x 109TU of Lenti-li-DT-KRASG12D, (iii) treated i.p. with a combination of cisplatin + anti-PDl mAb, or (iv) treated with a triple combination of Lenti- li-DT-KRASG12D+ cisplatin + anti-PDA mAb.

[0341] The Lenti-li-DT-KRASG12Dtreatment alone or cisplatin + anti-PDl bitherapy each provided partial anti-tumor effect (see Figure 7A) and partial survival increase (see Figure 7B). The Lenti-li-DT-KRASG12D+ cisplatin + anti-PDl mAb tri-therapy provided the best control of tumor growth and improved the most significantly the survival of animals.

[0342] Example 5: Antitumor effect of various Lenti-KRAS in a preclinical lung carcinoma model

[0343] The LLC1 (Lewis lung carcinoma) tumor cell line is a murine model of pulmonary cancer (Sun et al, 2021, Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1. Cancers 13: 5508). The in vivo established LLC1 tumors are characterized by an abundant burden of immunosuppressive myeloid cells (Sun et al, 2021). To evaluate the antitumor effect of the developed Lenti- KRAS vectors, LLC1 were transduced by the full length KRAS protein harboring the KRASG12Dmutation under the control of ubiquitin promoter (LLC1KRASG12D). The resulted clones were screened for KRAS mRNA expression and the clone LLC1KRASG12D#18, in which the presence of KRASG12Dmutation was confirmed by sequencing, was selected for further experiments.

[0344] C57BL / 6 mice were engrafted s.c. with 2 x 105LLC 1KRASG12Dcells at day 0. Mice were randomized and primed (day 7) and boosted (day 21) i.m. with 1 x 109TU of Ctrl Lenti or Lenti-KRASG12Dor Lenti-li-DT-KRASG12D. The prime-boost immunotherapy with Lenti-li-DT-KRASG12Dwas the most effective in lowering tumor burden in LLC1KRASG12Dmodels (see Figure 8 A). The survival of these tumor bearing mice was prolonged in all groups vaccinated against KRASG12D, without the difference reaching a statistical significance (survival data not shown for this group of mice inoculated with LLC1KRASG12D).

[0345] Therefore, these results provided the proof of concept of an effective lentiviral vector-based immunotherapy of a preclinical lung carcinoma model.

[0346] SEQUENCES

[0347] SEO ID NO: 1 is the amino acid sequence of KRAS 1-23 N-terminal segment MTEYKLVVVGAGGVGKSALTIQL

[0348] SEO ID NO : 2 is the amino acid sequence of KRASI-23G12A

[0349] MTEYKLVVVGAAGVGKSALTIQL

[0350] SEO ID NO : 3 is the amino acid sequence of KRASI-23G12C

[0351] MTEYKLVVVGACGVGKSALTIQL

[0352] SEO ID NO : 4 is the amino acid sequence of KRASi-23G12DMTEYKLVVVGADGVGKSALTIQL

[0353] SEO ID NO : 5 is the amino acid sequence of KRASI-23G12RMTEYKLVVVGARGVGKSALTIQL

[0354] SEO ID NO : 6 is the amino acid sequence of KRASi-23G12vMTEYKLVVVGAVGVGKSALTIQL

[0355] SEO ID NO : 7 is the amino acid sequence of KRASi-23G13DMTEYKLVVVGAGDVGKSALTIQL

[0356] SEO ID NO : 8 is the amino acid sequence of MHC-II light invariant chain (“li”)

[0357] MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVALLLAGQATTAYFLY

[0358] QQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLMRPMSMDNMLLG

[0359] PVKNVTKYGNMTQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVNWKIF

[0360] ESWMKQWLLFEMSKNSLEEKKPTEAPPKEPLDMEDLSSGLGVTRQELGQVTL

[0361] SEO ID NO : 9 is the amino acid sequence of the 203-379 segment of DT

[0362] INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEH

[0363] PELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSV MGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQV

[0364] VHNSYNRP is the nucleic acid sequence of the cPPT / CTS sequence aattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaa gaattacaaaaacaaattacaaaaattcaaaatttt is the nucleic acid sequence of woodchuck hepatitis B virus (WHY) post- transcriptional regulatory element (WPRE) ttcccgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctg ctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgt ggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcag ctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctc ggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccgcggctgctcgcctgtgttgccacctggattct gcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctct tccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgc is the amino acid sequence of li KRASG12D

[0365] MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVALLLAGQATTAYFLY

[0366] QQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLMRPMSMDNMLLG

[0367] PVKNVTKYGNMTQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVNWKIF

[0368] ESWMKQWLLFEMSKNSLEEKKPTEAPPKEPLDMEDLSSGLGVTRQELGQVTLGGS

[0369] GMTEYKLVVVGADGVGKSALTIQLIQ* is the amino acid sequence of li DT-KRASG12D

[0370] MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVALLLAGQATTAYFLY

[0371] QQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLMRPMSMDNMLLG

[0372] PVKNVTKYGNMTQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVNWKIF

[0373] ESWMKQWLLFEMSKNSLEEKKPTEAPPKEPLDMEDLSSGLGVTRQELGQVTLGGS

[0374] GINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALE

[0375] HPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGS VMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQ

[0376] VVHNSYNRPGGSGMTEYKLVVVGADGVGKSALTIQLIQ* is the nucleic acid sequence of KRAS 1-23 N terminal segment atgacggaatataagctggtggtggtgggcgccggcggtgtgggcaagagtgcgctgaccatccagctg is the nucleic acid sequence of KRASI-23G12Aatgacggaatataagctggtggtggtgggcgccgccggtgtgggcaagagtgcgctgaccatccagctg is the nucleic acid sequence of KRASI-23G12Catgacggaatataagctggtggtggtgggcgcctgcggtgtgggcaagagtgcgctgaccatccagctg is the nucleic acid sequence of KRASi-23G12Datgacggaatataagctggtggtggtgggcgccgacggtgtgggcaagagtgcgctgaccatccagctg is the nucleic acid sequence of KRASI-23G12Ratgacggaatataagctggtggtggtgggcgccagaggtgtgggcaagagtgcgctgaccatccagctg is the nucleic acid sequence of KRASi-23G12vatgacggaatataagctggtggtggtgggcgccgtgggtgtgggcaagagtgcgctgaccatccagctg is the nucleic acid sequence of KRASi-23G13Datgacggaatataagctggtggtggtgggcgccggcgacgtgggcaagagtgcgctgaccatccagctg is the nucleic acid sequence of MHC II light invariant chain (“li”) atggatgaccagagagacctgataagcaaccatgaacaactccctatcctgggcaacagaccccgggagccagagaggtgcag ccgaggggcactgtatactggcgtgtcggtactggtagcattattgctggctggccaagccacgacggcatacttcctgtaccagc agcaaggccgcttggacaagttgactatcaccagccagaatcttcagttggaaagcctgcgcatgaaactgcccaagtctgcaaa gccagtttcccagatgagaatggccacacccctactgatgaggcccatgtcaatggacaacatgctgttggggccagtgaaaaat gttaccaaatatgggaatatgacccaggaccacgtcatgcatctactcacgcggagtggcccgcttgagtatccacagcttaaagg gaccttccctgagaacctcaagcacctgaaaaatagcatggatggtgtcaactggaagatatttgaatcctggatgaagcagtggct gctatttgagatgagcaagaattctctggaggagaagaagcctacagaggcccctccaaaggagcctctggacatggaggacct gagctctggtttgggagtaacacggcaggagctgggacaagtcacatta is the nucleic acid sequence of the 203 379 segment of DT atcaatcttgactgggatgtaattcgtgataaaaccaagacaaaaattgaatcgctaaaagaacatggtcccatcaagaacaagatg tctgagtcccccaacaaaactgtgtctgaagagaaagccaaacagtacctggaggagttccaccagacagcccttgagcatccag aactctcagagctcaagactgttactggcaccaaccctgtgtttgctggggccaactatgctgcctgggctgtgaatgtggcgcaag tcattgacagtgaaacggctgataatctagaaaagaccactgctgctctctccatattaccaggcattggatcagtgatggggatagc agatggagctgtccaccacaacaccgaagaaattgtggcgcagagcattgccctgagttccctcatggtggcacaggccatccct ctagttggagaactggtggacattggctttgcagcttacaacttcgtggagtccatcatcaacctcttccaggtggtgcacaatagtta caataggccg is the nucleic acid sequence of li KRASG12Datggatgaccagagagacctgataagcaaccatgaacaactccctatcctgggcaacagaccccgggagccagagaggtgcag ccgaggggcactgtatactggcgtgtcggtactggtagcattattgctggctggccaagccacgacggcatacttcctgtaccagc agcaaggccgcttggacaagttgactatcaccagccagaatcttcagttggaaagcctgcgcatgaaactgcccaagtctgcaaa gccagtttcccagatgagaatggccacacccctactgatgaggcccatgtcaatggacaacatgctgttggggccagtgaaaaat gttaccaaatatgggaatatgacccaggaccacgtcatgcatctactcacgcggagtggcccgcttgagtatccacagcttaaagg gaccttccctgagaacctcaagcacctgaaaaatagcatggatggtgtcaactggaagatatttgaatcctggatgaagcagtggct gctatttgagatgagcaagaattctctggaggagaagaagcctacagaggcccctccaaaggagcctctggacatggaggacct gagctctggtttgggagtaacacggcaggagctgggacaagtcacattaggtggtccggaatgacagaatacaagcttgttgttgtcg gtgccgatggagtgggcaaaagtgcgctgaccatccagctcatccaatga is the nucleic acid sequence of li DT-KRASG12Datggatgaccagagagacctgataagcaaccatgaacaactccctatcctgggcaacagaccccgggagccagagaggtgcag ccgaggggcactgtatactggcgtgtcggtactggtagcattattgctggctggccaagccacgacggcatacttcctgtaccagc agcaaggccgcttggacaagttgactatcaccagccagaatcttcagttggaaagcctgcgcatgaaactgcccaagtctgcaaa gccagtttcccagatgagaatggccacacccctactgatgaggcccatgtcaatggacaacatgctgttggggccagtgaaaaat gttaccaaatatgggaatatgacccaggaccacgtcatgcatctactcacgcggagtggcccgcttgagtatccacagcttaaagg gaccttccctgagaacctcaagcacctgaaaaatagcatggatggtgtcaactggaagatatttgaatcctggatgaagcagtggct gctatttgagatgagcaagaattctctggaggagaagaagcctacagaggcccctccaaaggagcctctggacatggaggacct gagctctggtttgggagtaacacggcaggagctgggacaagtcacattaggtggtccggaatcaatcttgactgggatgtaattcgtg ataaaaccaagacaaaaattgaatcgctaaaagaacatggtcccatcaagaacaagatgtctgagtcccccaacaaaactgtgtct gaagagaaagccaaacagtacctggaggagttccaccagacagcccttgagcatccagaactctcagagctcaagactgttactg gcaccaaccctgtgtttgctggggccaactatgctgcctgggctgtgaatgtggcgcaagtcattgacagtgaaacggctgataatc tagaaaagaccactgctgctctctccatattaccaggcattggatcagtgatggggatagcagatggagctgtccaccacaacacc gaagaaattgtggcgcagagcattgccctgagttccctcatggtggcacaggccatccctctagttggagaactggtggacattgg ctttgcagcttacaacttcgtggagtccatcatcaacctcttccaggtggtgcacaatagttacaataggccgggaggctocggaatga cagaatacaagcttgttgttgtcggtgccgatggagtgggcaaaagtgcgctgaccatccagctcatccaatga is the amino acid sequence of segment 1 77 of the mutated human ubiquitin G77V

[0377] MAQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTL

[0378] SDYNIQKESTLHLVLRLRGV is the nucleic sequence of segment 1-77 of the mutated human ubiquitin G77V atggcccagatcttcgtgaagaccctgacaggaaaaactatcacactggaggtggagccgagtgacaccattgagaatgtcaaag ccaagattcaagacaaggagggcatccccccagaccagcagcgactcatctttgctggtaagcaactggaagatggccgcacttt gtctgattataacatccagaaggaatccactctgcacctggtgctgaggcttagaggcgtg

[0379] ID NO 27: amino acid sequence of E7 HPV 16

[0380] ETTDPDRAHYNIVTF the nucleic sequence of E7 HPV 16 gaaacaacagaccctgaccgggcccattacaacattgtgaccttctga is the amino acid sequence of fusion protein + CG77Vsegment

[0381] MAQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTL

[0382] SDYNIQKESTLHLVLRLRGV AAAAKLVVVGADGVGKSALTIAAAAKLVVVGACG

[0383] VGKSALTIAAAAKLVVVGAVGVGKSALTIAAAAKLVVVGAAGVGKSALTIAAAA

[0384] KLVVVGAGDVGKSALTIAAAAKLVVVGARGVGKSALTIAAAA is the nucleic acid sequence of fusion protein + CG77Vsegment atggcccagatcttcgtgaagaccctgacaggaaaaactatcacactggaggtggagccgagtgacaccattgagaatgtcaaag ccaagattcaagacaaggagggcatccccccagaccagcagcgactcatctttgctggtaagcaactggaagatggccgcacttt gtctgatataacatccagaaggaatccactctgcacctggtgctgaggctagaggcgtggcagctgctgcaaaactagtcgtggttg gagcagatggtgtggggaagtcagctcttactatcgccgcagcigccaaacttgtggtcgtcggagcctgcggggttggcaagtccgc tttgacaatcgcagcagcagctaagttggtagttgttggagctgtgggtgtcgggaaaagcgctctcaccatagcagcigccgcgaaactcgt ggtcgtaggtgcggctggagtggggaaaagtgccttaacgattgcigccgcagcgaagctggtagtggtaggagctggcgatgtggg caaatetgccctgaccattgccgccgcagccaagctagttgtcgtgggggccagaggcgttgggaagagcgccctcacgatagcggca gcagcc

[0385] G12D the KRAS F primer ggcctgctgaaaatgactga

[0386] G12D the KRAS R primer ggctgccgtcctttacaag is the amino acid sequence of KRAS5-21GI2A

[0387] KLVVVGAAGVGKSALTI is the amino acid sequence of KRAS5-21GI2C

[0388] KLVVVGACGVGKSALTI is the amino acid sequence of KRAS5-21GI2D

[0389] KLVVVGADGVGKSALTI is the amino acid sequence of KRAS5-21GI2R

[0390] KLVVVGARGVGKSALTI is the amino acid sequence of KRAS5-21GI2V

[0391] KLVVVGAVGVGKSALTI the amino acid sequence of KRASs-2iG13D

[0392] KLVVVGAGDVGKSALTI is the nucleic acid sequence of KRAS5-21GI2A aaactcgtggtcgtaggtgcggctggagtggggaaaagtgccttaacgatt is the nucleic acid sequence of KRAS5-21GI2C aaacttgtggtcgtcggagcctgcggggttggcaagtccgctttgacaatc is the nucleic acid sequence of KRAS5-21GI2D aaactagtcgtggttggagcagatggtgtggggaagtcagctcttactatc is the nucleic acid sequence of KRAS5-21GI2R aagctagttgtcgtgggggccagaggcgttgggaagagcgccctcacgata is the nucleic acid sequence of KRAS5-21GI2V aagttggtagttgttggagctgtgggtgtcgggaaaagcgctctcaccata is the nucleic acid sequence of KRASs-2iG13D aagctggtagtggtaggagctggcgatgtgggcaaatctgccctgaccatt is the full amino acid sequence of the wild type human ubiquitin C

[0393] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLS

[0394] DYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPP

[0395] DQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTL is the sequence of 1 76 amino acids of wild type human ubiquitin C

[0396] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLS

[0397] DYNIQKESTLHLVLRLRGG is the amino acid sequence of the full length wild type KRAS protein

[0398] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDIL

[0399] DTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPM

[0400] VLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYR

[0401] LKKISKEEKTPGCVKIKKCIIM SEO ID NO: 48 is the amino acid sequence of the diphteria toxin

[0402] MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGF

[0403] YSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELG LSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVEL

[0404] EINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIE

[0405] SLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFA

[0406] GANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIV

[0407] AQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKT QPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVN

[0408] KSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIH

[0409] SNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

Claims

CLAIMS1. A lentiviral vector encoding a KRAS 1-23 N-terminal segment, said KRAS 1-23 N-terminal segment comprising at least one amino acid substitution in position 12 or 13, in particular in position 12, compared to the amino acid sequence set forth as SEQ ID NO: 1.

2. The lentiviral vector according to claim 1, further encoding at least one support protein.

3. The lentiviral vector according to claim 1 or 2, wherein the KRAS 1-23 N-terminal segment comprises, and in particular consists in, an amino acid sequence selected from the group consisting of amino acid sequences set forth as SEQ ID NO: 2 (KRASI-23G12A), SEQ ID NO: 3 (KRASI-23G12C), SEQ ID NO: 4 (KRASi-23G12D), SEQ ID NO: 5 (KRASI-23G12R), SEQ ID NO: 6 (KRASI-23G12V), SEQ ID NO: 7 (KRASI-23G13D) and variants of these sequences having at least 70% sequence identity thereof, in particular the amino acid sequence set forth as SEQ ID NO: 4 or a variant thereof.

4. The lentiviral vector according to any one of the preceding claims, encoding at least two KRASi -23 N-terminal segments comprising at least one amino acid substitution in position 12 or 13, in particular in position 12, compared to the amino acid sequence set forth as SEQ ID NO: 1, more particularly encoding at least two KRAS 1-23 N-terminal segments having an amino acid sequence independently selected from the group consisting of the amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and variants of these sequences having at least 70% sequence identity thereof.

5. The lentiviral vector according to any one of the preceding claims, encoding:- the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:2, or a variant thereof having at least 70% sequence identity thereof ; and- the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:3, or a variant thereof having at least 70% sequence identity thereof ; and- the KRASi -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:4, or a variant thereof having at least 70% sequence identity thereof ; and- the KRAS i -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:5, or a variant thereof having at least 70% sequence identity thereof ; and- the KRAS i -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:6, or a variant thereof having at least 70% sequence identity thereof ; and- the KRAS i -23 N-terminal segment having the amino acid sequence set forth as SEQ ID NO:7, or a variant thereof having at least 70% sequence identity thereof.

6. The lentiviral vector according to claim 4 or 5 wherein the at least two KRAS1-23 N- terminal segments are fused together with or without a linker.

7. The lentiviral vector according to any one of claims 4 to 6, further encoding an amino acid segment 1-77 of the mutated human ubiquitin CG77V, in particular said amino acid segment 1-77 of the mutated human ubiquitin CG77Vbeing located at the N-terminal end of the KRASi -23 N-terminal segments.

8. The lentiviral vector according to any one of claims 2 to 7, wherein the at least one support protein is selected from the group consisting of:(i) proteins that improve the intracellular antigen routing to major histocompatibility complex presentation machineries; and(ii) proteins that provide helper CD4+ T-cell epitopes.

9. The lentiviral vector according to any one of claims 2 to 8, wherein the at least one support protein is selected from the group consisting of the MHC-II light invariant chain (“li”); diphteria toxins, or fragments thereof, in particular the 203-379 fragment of diphteria toxin (“DT”); ubiquitin proteins, or fragments thereof, in particular the fragment 1-77 of the mutated human ubiquitin CG77V; tetanus toxoids (TT) or fragments thereof; and mixtures thereof.

10. The lentiviral vector according to any one of claims 2 to 9, wherein the at least one support protein is selected from the group consisting of:(i) the MHC-II light invariant chain (“li”), in particular having the amino acid sequence set forth as SEQ ID NO: 8,(ii) the 203-379 segment of diphteria toxin (“DT”), in particular having the amino acid sequence set forth as SEQ ID NO: 9; and(iii) the MHC-II light invariant chain (“li”) and the 203-379 segment of diphteria toxin (“DT”), in particular having the amino acid sequences set forth as SEQ ID NO: 8 and SEQ ID NO: 9.

11. The lenti viral vector according to any one of claims 1 to 10, encoding an amino acid sequence set forth as SEQ ID NO: 12 or SEQ ID NO: 13.

12. The lentiviral vector according to any one of claims 2 to 10, wherein the support protein is located at the N-terminal end of the at least one KRAS 1-23 N-terminal segment, in particular is fused together at the N-terminal end of the at least one KRAS 1-23 N-terminal segment.

13. The lentiviral vector according to any one of the preceding claims, wherein the lentiviral vector comprises an MHC Class I promoter, and in particular a human |32-microglobulin promoter and / or comprises a cPPT / CTS sequence, in particular the cPPT / CTS sequence set forth as sequence SEQ ID NO: 10, and / or comprises a 3’ long terminal repeat (LTR) which is devoid of its U3 promoter sequence and / or does not comprise a constitutive enhancer sequence and / or comprises a mutant form of the woodchuck hepatitis B virus (WHV) post-transcriptional regulatory element (WPRE), and in particular having the sequence set forth as sequence SEQ ID NO: 11.

14. A lentiviral vector particle comprising at least a lentiviral vector as defined in any one of claims 1 to 13.

15. The lentiviral vector particle according to claim 14, further comprising a functional lentiviral integrase protein and / or a vesicular stomatitis virus glycoprotein (VSVG), inparticular a V S V-G Indiana serotype or a V SV -G New Jersey serotype and / or HIV - 1 subtype D Gag and Pol proteins.

16. A pharmaceutical composition, and in particular a vaccine composition, comprising, in a pharmaceutically acceptable medium, a lentiviral vector according to any one of claims 1 to 13, or a lentiviral vector particle according to any one of claims 14 or 15.

17. A combinatory treatment comprising:(i) at least one lentiviral vector according to any one of claims 1 to 13 or lentiviral vector particle according to according to any one of claims 14 or 15, or a pharmaceutical composition according to claim 16, and(ii) at least one chemotherapy agent or treatment and / or at least one immunotherapy agent or treatment.

18. The combinatory treatment according to claim 17, wherein the at least one chemotherapy agent or treatment is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, pemetrexed, 5-fluoracil (5-FU), taxane, and mixtures thereof and / or wherein the at least one immunotherapy agent or treatment is selected from the group consisting of immune checkpoint inhibitors, in particular anti-PD-1, anti-PD-Ll, anti- CTLA-4, anti-TIM-3, anti-LAG3, anti-TIGIT and anti-NKG2A antagonist antibodies; and mixtures thereof.

19. A lentiviral vector according to any one of claims 1 to 13 or a lentiviral vector particle according to any one of claims 14 or 15, or a pharmaceutical composition according to claim 16, or a combinatory treatment according to any one of claims 17 or 18, for their use in the treatment and / or prevention of cancers, in particular of cancers wherein the KRAS1-23 N- terminal segment is mutated, more particularly of pancreatic, colorectal, and / or pulmonary cancers.

Images