A lentiviral vector targeting a KRAS neoepitope for cancer immunotherapy

Goloudina, Anastasia; Le Chevalier, Fabien; Authié, Pierre; Ciret, Sylvain; Nemirov, Kirill; Fert, Ingrid; Moncoq, Fanny; Vesin, Benjamin; Noirat, Amandine; Blanc, Catherine; Wei, Yu; Charneau, Pierre; Majlessi, Laleh

doi:10.1038/s41598-025-05134-6

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Published: 02 July 2025

A lentiviral vector targeting a KRAS neoepitope for cancer immunotherapy

Anastasia Goloudina¹,
Fabien Le Chevalier¹,
Pierre Authié¹,
Sylvain Ciret¹,
Kirill Nemirov¹,
Ingrid Fert¹,
Fanny Moncoq¹,
Benjamin Vesin¹,
Amandine Noirat¹,
Catherine Blanc¹,
Yu Wei¹,
Pierre Charneau¹^na1 &
…
Laleh Majlessi¹^na1

Scientific Reports volume 15, Article number: 23171 (2025) Cite this article

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Abstract

Mutated oncogenic Kirsten rat sarcoma virus (KRAS) antigen is expressed in a large variety of cancers, including pancreatic, colorectal, and pulmonary cancers. The oncogenic KRAS mutations cause malignancies and are usually ubiquitously expressed by all cells of a tumor. The KRAS amino acid substitutions at the positions 12 or 13 are among the most frequent mutations in human cancers. Here, we developed immuno-oncotherapeutic non-integrative lentiviral vectors encoding a segment encompassing KRAS^G12D, either alone, or associated with antigen carriers. These carriers can improve the intracellular antigen routing to major histocompatibility complex presentation machineries or provide universal helper CD4⁺ epitopes. Immunotherapy with one of these vectors resulted in significant immune control of tumor growth in colorectal or pulmonary preclinical cancer models, in several murine genetic backgrounds. The antitumor effect was correlated with increased proportions of intra-tumoral hematopoietic cells and notably CD8⁺ T cells. Although this effect was partial, it was robust, reproducible and advantageously combinable with conventional chemotherapies and immunotherapies to improve antitumor protection. Therefore, this approach shows promise as an immuno-oncotherapy against KRAS-mediated malignant transformation.

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Introduction

Cancer is a complex and multistep disease that arises from single genetic modifications that favor 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 attacks¹. 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 expressed by tumor cells and can be presented at their surface in the context of major histocompatibility complex (MHC) molecules^2,3,4. 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 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, head and neck cancers and other solid tumors (NCT05198752)⁵. 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 numerous challenges^6,7.

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, driving tumor initiation and maintenance. These mutations are shared by all cells of the tumor, and harbor neoantigens which can be common to patient cohorts^8,9. 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^3,10. Generation of vaccines targeting this type of common neo-antigens does not need to be initiated after the cancer diagnosis and does not require personalized vaccine design and manufacturing. So far, immunotherapy targeting such shared neoantigens showed encouraging, yet partial, results in pre-clinical and clinical trials (NCT04117087)^5,11.

The small guanosine triphosphatase (GTPase) KRAS switches between its inactive GDP-bound and active GTP-bound forms¹². 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 differentiation¹³. 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^12,13,14 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^15,16—and 10% of endometrial cancers¹⁷. Even though KRAS is altered in only 2% of breast carcinoma patients¹⁸, engagement of RAS functions has been reported, in the progression and metastases of experimental breast cancer studies¹⁹. A single codon substitution at the position 12 or 13 stabilizes KRAS in an active state, amplifying downstream signaling pathways¹⁹. 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 ≈13% of non-small cell lung cancer²⁰, 4% of colorectal cancer²¹ and 1–3% of other solid tumors²². The KRAS^G12A mutation is present in 2.3% of colorectal cancers¹⁸, but only in 0.5% of pancreatic cancers and is associated with poor prognosis²³. The KRAS^G12R mutation is rare in colon and lung cancer but is the third most common KRAS mutation in pancreatic ductal adenocarcinoma²⁴.

Compared to the vaccination strategies based on peptides or proteins, either adjuvanted or packaged in nanoparticles, mRNA-based or various viral vectors^{25,26,27,28,29}, 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 naïve T cells^30,31. Lentiviral vectors induce high quality and long-term 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^4,31,32. Our team has so far largely established the pre-clinical proof of concept for the use of lentiviral vectors in numerous bacterial and viral infectious diseases^{30,31,33,34,35,36,37,38,39,40} and in immuno-oncotherapy^38,41, notably against human papillomavirus (HPV)-induced tumors^42,43, which has recently entered a clinical trial (NCT06319963). We recently cross-sectionally compared the most relevant vaccine strategies so far tested in preclinical anti-HPV immuno-oncotherapies used in murine models and observed that lentiviral vector-based approaches were the most efficient in the control of tumor growth while providing the longest-lasting memory⁴⁴.

Here, we generated several non-integrative lentiviral vectors encoding the KRAS_1-23 N-terminal segment harboring the G12D mutation, either alone or fused to appropriate protein carriers which can improve the intracellular antigen routing to MHC presentation machinery or to provide universal helper CD4⁺ T cell epitopes. Therapeutic vaccination by the generated vectors allowed down-selection of those able to significantly reduce the size of tumors which (over)express the KRAS^G12D mutation in preclinical murine models of lung and colon cancers, in two distinct murine genetic backgrounds. This antitumor effect was correlated with increased intra-tumor infiltration by hematopoietic cells and notably CD8⁺ T cells. Overall, a synergistic antitumor effect of the lentiviral vector-based therapy was observed when this vaccine candidate was used along with one of the compelling first line anti-cancer chemotherapy, cisplatin (cis-diamminedichloroplatine (II))^45,46 and a standard of care immunotherapy against the programmed cell death (PD)1 immune check point. Therefore, lentiviral vector-based vaccination against the common shared KRAS neoantigen, is a promising strategy to be included in combinatory immunotherapy of established tumors expressing mutated KRAS.

Results

Lenti-KRAS vaccine designs

Three modalities of KRAS_1-23^G12D-based antigens were designed to be encoded by non-integrative lentiviral vectors (Fig. 1A). (i) KRAS_1-23^G12D alone (Lenti-KRAS^G12D), (ii) a fusion of the MHC-II light invariant chain (“li”) to the N-terminal part of KRAS_1-23^G12D in order to favor targeting of the encoded antigen to the MHC-II machinery (Lenti-li-KRAS^G12D), in addition to the MHC-I pathway^39,47,48, and (iii) a fusion of the li-KRAS_1–23^G12D to the 202–378 segment of diphtheria toxin (DT) (Accession Number: PRF 1007216B) (Lenti-li-DT-KRAS^G12D), with a view to use in humans and benefiting from the helper functions of memory CD4⁺ T-cell, present in the vast majority of humans vaccinated against diphtheria^27,49 (Fig. 1A). Consequently, delivery of the engineered antigen to the MHC-II machinery by fusion with the li chain will enable presentation of both DT and KRAS epitopes by MHC-II molecules. In fact, in addition to MHC-I restricted KRAS neoepitopes, clinical studies have also identified KRAS neoepitopes restricted by MHC-II molecules and capable of inducing CD4⁺ T cell responses^50,51,52. The ggsg linkers inserted between the between li and DT and between DT and KRAS sequences help avoiding generation of irrelevant junctional T cell epitopes (Fig. 1B). In fact, analysis of the li-DT-KRAS^G12D sequence, which contains two ggsg linkers, by the SYFPEITHI epitope prediction tool (http://www.syfpeithi.de/) did not predict any epitope for H-2^b or-H-2^d molecules, nor for the 30 HLA-I or -II molecules available in this tool.

Sequences encoding each of the designed antigens (Fig. 1B) were inserted into individual lentiviral transfer plasmids under the human β2-microglobulin promoter, whose activity increases spatio-temporally with dendritic cell activation, migration of and MHC-I upregulation³⁸. 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)-293 T cell production system and concentrated by ultracentrifugation with titers ranging from 1.0 to 2.7 × 10¹¹ Transduction Units (TU)/mL.

Antitumor effect of diverse Lenti-KRAS in two colorectal carcinoma preclinical models

The antitumor effect of the generated lentiviral vectors was first explored against the CT26 colorectal cancer murine cell line that endogenously expresses KRAS^G12D²⁵. Syngeneic BALB/c (H-2^d) mice were subcutaneously (s.c.) engrafted with 2 × 10⁵ CT26 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 × 10⁹ TU of an empty lentiviral vector (Ctrl Lenti), Lenti-KRAS^G12D, Lenti-li-KRAS^G12D or Lenti-li-DT-KRAS^G12D (Fig. 2A). At day 18 post tumor engraftment, a tendency to the tumor growth inhibition was observed notably in the Lenti-li-DT-KRAS^G12D-treated mice (Fig. 2B). 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-KRAS^G12D-treated mice was also significantly prolonged (Fig. 2C).

KRAS^G12D mutation generates MHC-I-restricted T cell neoepitope with mild immunogenicity, yet in both H-2^d and H-2^b murine haplotypes²⁵. Consequently, the antitumor effect of the Lenti-KRAS vectors was also evaluated in C57BL/6 (H-2^b) mice, since in this strain several other syngeneic tumor models are available. The C57BL/6-derived murine colon adenocarcinoma MC38 cell line⁵³ was transduced with the full length KRAS^G12D sequence, expressed under the transcriptional control of the ubiquitin promoter^54,55. MC38 clones were screened by qRT-PCR for KRAS^G12D mRNA expression. The clone #1 (Fig. S1) was selected for further experiments after confirmation of the presence of KRAS^G12D mutation in its genomic DNA by sequencing.

C57BL/6 mice were inoculated s.c. with 2 × 10⁵ MC38_KRAS^G12D cells at day 0. Mice were randomized and were treated by a single i.m. injection of 1 × 10⁹ TU of Ctrl Lenti or Lenti-li-DT-KRAS^G12D at day 6 (Fig. 3A). In Lenti-li-DT-KRAS^G12D-vaccinated group a tendency to the tumor growth inhibition was observed at day 14 (Fig. 3B). At day 17, the tumor growth control reached statistical significance in the Lenti-li-DT-KRAS^G12D-treated mice. The efficacy of Lenti-li-DT-KRAS^G12D immunotherapy against MC38_KRAS^G12D tumor was confirmed in > 10 other independent experiments, as exemplified in (Fig. 3C).

To get more insights on the effect of Lenti-li-DT-KRAS^G12D therapy on MC38_KRAS^G12D tumor microenvironment, the tumor immune infiltrates were immunophenotyped at day 11 post vaccine administration. Tumors from the Lenti-li-DT-KRAS^G12D-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 (Fig. 3D, E). However, the CD8⁺—but not CD4⁺—T cell subset was significantly expanded inside the CD45⁺ population within the tumor of Lenti-li-DT-KRAS^G12D-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 MC38_KRAS^G12D tumors, albeit without significant difference between the Lenti-li-DT-KRAS^G12D- and Ctrl Lenti-treated groups (Fig. 3D, E). Lenti-li-DT-KRAS^G12D treatment had no impact on the composition of tumor infiltrating natural killer cells, CD11b^hi CD11c^hi dendritic cells, CD11b⁺ Ly6C⁺ Ly6G^- inflammatory monocytes or CD11b^hi Ly6G⁺ granulocytes/neutrophils, compared to Ctrl Lenti treatment (Fig. S2A, B).

The potential of a lentiviral vector encoding a fusion protein of KRAS_1-23^G12D antigenic segment and the neutral nano-luciferase protein carrier instead of li-DT (Lenti-nLuc-KRAS^G12D) was also explored in immuno-therapy of MC38_KRAS^G12D tumor-bearing mice (Fig. S3A). Lenti-nLuc-KRAS^G12D therapy achieved a significant inhibition of tumor growth (Fig. S3B) and preferential intra-tumoral infiltration/expansion of CD8⁺ T cells (Fig. S3C), reinforcing the robustness of the lentiviral vector platform targeting the mutated KRAS neoantigen in immuno-oncotherapy.

Phenotype of antitumor effector cells

No KRAS^G12D-specific T cell responses were detected in vitro by ELISPOT (Fig. S4A) or intracellular IFN-γ/TNF-α/IL-2 cytokine staining subsequent to peptide stimulation in the spleen of Lenti-li-DT-KRAS^G12D-vaccinated mice (Fig. S4B, C). This probably results from the low affinity of the epitope generated by the KRAS^G12D mutation for murine MHC-I molecules of H-2^b or H-2^d haplotypes (Table S1). However, MC38_KRAS^G12D tumor infiltrating lymphocytes (TILs) in Lenti-li-DT-KRAS^G12D-primed and -boosted C57BL/6 mice contained IFN-γ- and TNF-α-producing CD8⁺ T cells, as evidenced after their co-culture with syngeneic bone-marrow dendritic cells loaded with synthetic KRAS_1-20 peptides (Fig. 4A, B). No IFN-γ- and/or TNF-α-producing CD8⁺ T cells were detected after stimulation of these TILs with an irrelevant negative control peptide. It seems that the whole specific CD8⁺ T cell subset recognized the mutated and WT KRAS epitopes indifferently (Fig. 4B), even though it cannot be excluded that discriminating T clonotypes could be generated. Interestingly, KRAS-specific CD8⁺ TILs were also detected at the same percentages within the CD8⁺ T subset in the tumors of Ctrl Lenti-treated mice, which suggests their tumor-borne origin. Even though the proportions of KRAS-specific CD8⁺ TILs were comparable in mice treated with Ctrl Lenti or Lenti-li-DT-KRAS^G12D, the percentages of total CD8⁺ TILs were higher in Lenti-li-DT-KRAS^G12D-treated mice (Fig. 4C). Although the percentages of KRAS-specific IFN-γ⁺ TNF-α⁺ CD8⁺ T cells were identical in mice treated with Ctrl Lenti or Lenti-li-DT-KRAS^G12D, with the percentages of CD8⁺ cells being significantly higher in the latter group Fig. 4C), the whole tumors in the Lenti-li-DT-KRAS^G12D group contained more KRAS-specific CD8⁺ T cells (Fig. 4D). Furthermore, the total tumor homogenates from Lenti-li-DT-KRAS^G12D-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 Lenti Ctrl-treated mice (Fig. 4E, F).

To identify in vivo the antitumor effector cell subset, MC38_KRAS^G12D-bearing C57BL/6 mice were primed (day 4) and boosted (day 11) with 1 × 10⁹ TU of Lenti-li-DT-KRAS^G12D and received intraperitoneal (i.p) injections of a Ctrl Ig, anti-CD4 or anti-CD8 depleting mAbs on days 2, 4, 7, 10 and 14 (Fig. 5A). In the vaccinated groups, treated with the Ctrl Ig, a significant inhibition of tumor growth was observed, whereas in the Lenti-li-DT-KRAS^G12D-vaccinated group treated with anti-CD8 mAb, the tumor growth was comparable to the Ctrl Lenti-treated mice (Fig. 5B, C). Interestingly, the anti-CD4 depleting mAb treatment had only a slight and 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 (Fig. 5D). Should effector or memory CD4⁺ T cells be able to resist GK1.5 mAb-mediated depletion in vivo, the effects of a KRAS^G12D-specific CD4⁺ response could not be completely ruled out. Therefore, these results indicated that the principal immune effector cells induced by Lenti-li-DT-KRAS^G12D immunization were most likely CD8⁺ T cells.

Advantageous combination of Lenti-li-DT-KRAS^G12D with chemo- and/or immuno-therapies

To enhance the partial antitumor protection provided by Lenti-li-DT-KRAS^G12D, this therapy was first combined with the apoptosis/ferroptosis-inducing agent, ras-selective lethal 3 (RSL3). RSL3 targets and inactivates glutathione peroxidase 4 (GPX4) which leads to reactive oxygen species (ROS) accumulation, lipid peroxidation, and cell membrane disruption^56,57. The antitumor effect of RSL3 was first confirmed in vitro on MC38 tumor cells (Fig. S5A). Then, C57BL/6 mice were grafted s.c. with 1.25 × 10⁵ MC38_KRAS^G12D cells at day 0 and were then: (i) treated i.p. with RSL3 alone, (ii) primed and boosted i.m. with 1 × 10⁹ TU of Lenti-li-DT-KRAS^G12D, or (iii) primed and boosted with Lenti-li-DT-KRAS^G12D and treated with RSL3 (Fig. S5B). The combination with RSL3 showed a tendency to improve the antitumor effect of Lenti-li-DT-KRAS^G12D (Fig. S5C), even though a statistical significance was not reached.

We then evaluated the effect of the combination of Lenti-li-DT-KRAS^G12D with apoptosis/ferroptosis-inducing cisplatin which is one of the compelling first line anti-cancer chemotherapy^45,46 and the standard of care antagonistic anti-PD1 mAb immunotherapy⁵⁸. C57BL/6 mice were engrafted s.c. with 1 × 10⁵ MC38_KRAS^G12D cells and were then: (i) primed and boosted i.m. with 1 × 10⁹ TU of Ctrl Lenti, (ii) primed and boosted i.m. with 1 × 10⁹ TU of Lenti-li-DT-KRAS^G12D, (iii) treated i.p. with a combination of cisplatin + anti-PD1 mAb, or (iv) treated with a triple combination of Lenti-li-DT-KRAS^G12D + cisplatin + anti-PD1 mAb (Fig. 6A). The Lenti-li-DT-KRAS^G12D treatment alone or cisplatin + anti-PD1 bitherapy each provided partial antitumor effect (Fig. 6B) and partial survival increase (Fig. 6C). The Lenti-li-DT-KRAS^G12D + cisplatin + anti-PD1 mAb tritherapy provided the best control of tumor growth and improved the most significantly survival of animals (Fig. 6B, C).

The anti-tumor effect of cisplatin and anti-PD1 in mice treated with Ctrl Lenti certainly resulted from the pro-apoptotic effect of cisplatin, which promotes the presentation of a large number of tumor antigens, including the KRAS^G12D neoantigen and therefore induction of CD8⁺ T cells against them. In mice treated with Lenti-li-DT-KRAS^G12D, the effect of cisplatin and anti-PD1 was amplified, apparently because their tumors contained more KRAS-specific CD8⁺ T cells than those from their Lenti Ctrl-treated counterparts (Fig. 4D).

Antitumor effect of various Lenti-KRAS in a preclinical lung carcinoma model

The LLC1 (Lewis lung carcinoma) tumor cell line is a murine model of pulmonary cancer⁵⁹. The in vivo established LLC1 tumors are characterized by an abundant burden of immunosuppressive myeloid cells⁵⁹. To evaluate the antitumor effect of the developed Lenti-KRAS vectors, LLC1 cells were transduced with the full length KRAS sequence harboring the KRAS^G12D mutation under the control of ubiquitin promoter (LLC1_KRAS^G12D). The resulted LLC1 clones were screened by qRT-PCR for KRAS^G12D mRNA expression (Fig. S6) and the clone #18 was selected for further experiments after confirmation of the presence of KRAS^G12D mutation in its genomic DNA by sequencing.

C57BL/6 mice were engrafted s.c. with 2 × 10⁵ LLC1_KRAS^G12D cells at day 0. Mice were randomized and primed (day 7) and boosted (day 21) i.m. with 1 × 10⁹ TU of Ctrl Lenti or Lenti-KRAS^G12D or Lenti-li-DT-KRAS^G12D (Fig. 7A). The prime-boost immunotherapy with Lenti-li-DT-KRAS^G12D was the most effective in reducing tumor burden in LLC1_KRAS^G12D models (Fig. 7B, C). The survival of tumor bearing mice was prolonged in the group of mice vaccinated Lenti-li-DT-KRAS^G12D (Fig. 7D).

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

Discussion

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^60,61. Based on the high frequencies of mutated KRAS in numerous cancers, neoepitopes resulting from somatic mutations of this oncogene, notably at its position 12, are of the most attractive targets. Constitutively GTP-bound mutated KRAS hyperactivates the MAPK signaling pathway which derives cancer stem cells. Importantly, in contrast to other somatic tumor neoantigens, mutated KRAS neoepitopes can be common to patient cohorts^8,9, which facilitates vaccine design and production in a non-personalized manner. The TCR repertoire, potentially able to recognize somatically mutated KRAS neoantigens, has not been subjected to central T cell tolerance induction, since the mutated KRAS neoantigen is not expressed by healthy tissues^10,62. For the same reason, T cell immunity against mutated KRAS neoantigens will not represent any risk of autoimmunity induction.

In the present study, we took advantage of the high efficiency of the non-integrative lentiviral vector platform to generate vaccine candidates encoding different modalities of KRAS neoantigens for immuno-oncotherapy. We first down selected Lenti-li-DT-KRAS^G12D vector, encoding a fusion protein composed of: (i) li in order to favor targeting of the encoded antigen to the MHC-II machinery, in addition to the MHC-I, (ii) the DT:202–378 segment enabling to take benefit of possible memory CD4⁺ T helper cell functions, present in a human vaccinated against diphtheria, and (iii) the KRAS_1-23 segment encompassing the G12D mutation. Immunotherapy by Lenti-li-DT-KRAS^G12D resulted in significant immune control of the tumor growth, but not tumor regression, in two colorectal and one pulmonary preclinical cancer models, in distinct murine genetic backgrounds. As studied on day 11 post tumor challenge, 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. We compared TIL status at a time when mean tumor size was not excessively different between the two experimental groups, in order to minimize quantitative differences related to this parameter. However, it would also have been informative to add a kinetic point at day 18 post tumor challenge to check whether these differences were persistent.

We emphasize that the immunogenicity of KRAS^G12D-resulted epitope in H-2^b and H-2^d mice is low, which explains why we were unable to visualize KRAS-specific T splenocyte responses in mice immunized with Lenti-li-DT-KRAS^G12D, while lentiviral vectors are particularly effective in inducing T-cell responses³⁸. This observation is in line with the very rare findings of T-cell immunogenicity of KRAS in murine models in the literature^25,28. However, some HLA class I alleles can be particularly efficient in presenting KRAS neoepitopes, resulting in the induction of T-cell responses with excellent antitumor effects⁶³. Consequently, the transposition of the partial antitumor results observed with Lenti-li-DT-KRAS^G12D in mice presages in no way weak immunotherapeutic effects of this vector in humans. On the contrary, KRAS antigen has been shown to induce outperforming antitumor effects in patients carrying certain HLA alleles⁶⁴.

The Lenti-li-DT-KRAS^G12D antitumor effect in the studied preclinical models was partial, even statistically significant. However, a tritherapy combination of Lenti-li-DT-KRAS^G12D + cisplatin + antagonistic anti-PD1 mAb improved the antitumor efficacy and increased the survival of tumor bearing mice. These data are in line with the previous preclinical observations that highlighted the significant synergistic antitumor efficacy of anti-HPV antitumor vaccine candidates particularly when combined with cisplatin among other chemotherapies^65,66. The mechanism involved relies on the ability of cisplatin to cause immunogenic tumor cell death. In fact, by crosslinking the purine bases, cisplatin impacts DNA repair which causes DNA damage, apoptosis and/or ferroptosis⁶⁷. Such tumor cell death favors tumor antigen uptake by dendritic cells and antigen cross-presentation of MHC-I-bound peptides to antitumor CD8⁺ T cells^68,69,70. Addition of the antagonistic anti-PD1 mAb certainly reinvigorates CD8⁺ T cells triggered by the tumor itself and/or by the Lenti-li-DT-KRAS^G12D immunotherapy.

There are numerous clinical evidences of the recognition of mutated KRAS neoantigens in the context of MHC-I by CD8⁺ T cells with antitumor cytotoxic activity^63,64. Additional clinical data suggest that not only cancer patients but also healthy donors develop T cell clones specific to mutated KRAS epitopes, detectable in both peripheral blood mononuclear cells and TILs^71,72. Recently, the presentation of neoepitopes containing KRAS^G12C, KRAS^G12D, KRAS^G12R and KRAS^G12V mutations, in the context of 9 individual HLA-I alleles, has been demonstrated by immunoprecipitation of HLA molecules from tumor cells prior to mass spectrometric analysis of the peptides naturally loaded on their presentation groove⁷³. In addition, peptides containing KRAS^G12A, KRAS^G12C, KRAS^G12D, KRAS^G12R, KRAS^G12S and KRAS^G12V motifs can bind to HLA-A*02–01 and HLA-A*03:01 and induce specific CD8⁺ T-cell responses⁷⁴. A polyclonal, HLA-C*08:02-restricted CD8⁺ T-cell response against KRAS^G12D was also observed in TILs from a patient with metastatic colorectal cancer⁶³. Naturally occurring T cell responses against KRAS mutations were also found in unvaccinated patients with pancreatic cancer, both in TILs and in peripheral blood lymphocytes^75,76. In a recent follow-up study to the clinical trial NCT03953235, the efficacy of KRAS-specific CD8⁺ T cells increased proportionally with the number of repetitions of KRAS neoepitopes in the vaccine design⁷⁷. In addition, there is clinical evidence that KRAS neoepitopes can also be presented by MHC-II molecules and induce CD4⁺ T cells^50,51,52, highlighting the value of the antigenic design used in our study, namely the addition of the li chain to the N-terminus of the antigen and its routing to the MHC-II machinery.

An adjuvanted peptide-based vaccination strategy has been reported to expand these responses to mutated KRAS or to induce de novo priming of specific T cells⁷⁶. In line with these clinical observations, our results also demonstrated that KRAS-specific murine T cells were present in MC38_KRAS^G12D tumor infiltrates, and that Lenti-li-DT-KRAS^G12D vaccination led to an increase in the number of such CD8⁺ TILs. Therefore, immune targeting of the somatic gain-of-function KRAS mutations can be an attractive tumor-specific therapeutic strategy.

Additional clinical data suggest that not only cancer patients but also healthy donors develop T cell clones specific to mutated KRAS epitopes, detectable in both peripheral blood mononuclear cells and TILs^71,72. A case study describes a colorectal cancer patient harboring HLA-C*08:02 that received T cell transfer (TCR T) therapy targeting KRAS^G12D mutation⁶³. After the treatment, all metastases were cleared except for one lesion. Whole-exome and transcriptome sequencing revealed that tumor cells of that single lesion still expressed the KRAS^G12D mutation, yet lost HLA expression and thus evaded the immune surveillance. Another HLA-C*08:02 patient with metastatic pancreatic cancer expressing KRAS^G12D received a leukapheresis of autologous T cells that had been genetically modified to express allogeneic T-cell receptors targeting KRAS^G12D. This patient showed regression of metastases with overall partial response of 72% that lasted for at least 6 months⁶⁴. There are currently several ongoing clinical trials that use anti-KRAS^G12D immunotherapy, mainly based on adjuvanted peptides or mRNA approaches, alone or in combination with anti-immune check point inhibitors (NCT04117087, NCT05254184, NCT03948763)^76,77.

In contrast to personal neoantigens, characterized by their mosaic expression within tumors, the great majority of shared neoantigens, including KRAS neoantigens, result from driver mutations, arise in the early stages of carcinogenesis, are cancer causing and essential to malignancy. These properties of the shared neoantigens minimize the possibility of their mosaic or loss of expression, which could lead to tumor escaping from neoantigen-specific immunity. Therefore, negative selection of tumor cells that, under the pressure of an immune response, might lose expression of driving neoantigens such as KRAS neoepitopes is rather unlikely⁷⁸. However, the loss of HLA molecules capable of presenting the corresponding neoepitopes by tumor cells is possible and has been clinically proven⁶⁴.

In a recent review, we compared the most relevant preclinical strategies used in tumor immunotherapy in terms of antitumor efficacy and persistence of T-cell memory in the HPV-induced murine TC-1 tumor model⁴⁴. Comparing adjuvanted proteins, bacterial or viral vectors, including adenoviral, arenaviral, lentiviral and poxviral vectors, as well as DNA- or mRNA-based approaches, we concluded that lentiviral vectors induced the strongest antitumor effect and the longest-lasting antitumor T-cell immune memory⁴⁴. On the basis of these comprehensive comparisons and the observations reported in the present paper, lentiviral vector-based anti-KRAS immunotherapy may be particularly promising if used in combination with appropriate chemotherapies and immune checkpoint inhibitors, especially in cancer patients expressing mutated KRAS and presenting favorable HLA MHC-I alleles including HLA-C*08:02. In the case of low immunogenicity of KRAS G12 mutated neoepitopes in certain HLA haplotypes and difficulty of recruiting specific T cells into the primary inaccessible solid tumors, the use of KRAS-based immunotherapies will remain still promising in reducing metastases and post-surgery residual malignant cells.

Methods

Mice

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.

Sex as a biological variable

Our study exclusively examined female mice because the pertinent experimental tumor models were only available in females.

Declarations of conformity

All methods were carried out in accordance with ARRIVE guidelines and regulations (https://arriveguidelines.org) and with the European and French guidelines (Directive 86/609/CEE and Decree 87–848 of 19 October 1987).

Study approval statements

The experiments on animals were performed in accordance with ARRIVE guidelines (https://arriveguidelines.org). Euthanasia was performed by cervical dislocation. All experimental 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#43914-2023062210343762v2).

Tumor cell lines

CT26 mouse colon carcinoma cell line (CRL-2638), derived from BALB/c mouse strain, was purchased from ATCC (cat # 59227052) and maintained in RPMI media (GIBCO) media supplemented with 100 U/mL penicillin/streptomycin (Gibco) and 10% heat-inactivated fetal bovine serum (Serana).

MC38 mouse colon carcinoma cell line, derived from C57BL/6 murine colon adenocarcinoma⁵³, 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 lentiviral vector coding full-length murine Kras gene with G12D amino acid substitution, under the ubiquitin promoter³⁸. Transduced populations of MC38 or LLC1 cells were cloned and the resulted clones were analyzed for KRAS^G12D mRNA expression by RT-PCR with following primers: KRAS F: 3’-ggcctgctgaaaatgactga-5’, KRAS R: 3’-ggctgccgtcctttacaag-5’. Clones with highest KRAS^G12D mRNA expression were used in the experiments. MC38 and MC38-_KRAS^G12D (#1) 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 LLC1_KRAS^G12D cells were cultured in DMEM media containing Glutamax (Gibco) supplemented with 100 U/mL penicillin/streptomycin (Gibco) and 10% heat-inactivated fetal bovine serum (Serana).

All the tumor clones used in this study expressed surface MHC-I molecules (Fig. S7A-C).

Construction and production of diverse Lenti-KRAS G12D

Codon-optimized sequences of the designed KRAS G12D antigens were synthesized and inserted into the pFlap lentiviral plasmid between the BamHI and XhoI sites, located between the human β2-microglobulin promoter and the mutated atg starting codon of the woodchuck post-transcriptional regulatory element (WPRE) sequence, as detailed elsewhere (Fig. S8A-D). 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^31,38.

Immunotherapy of tumor-bearing mice

Mice were shaved with electric shaver device (ChroMini Tcut, WAHL) 2 days before tumor implantation. For s.c. tumor cell inoculation, mice were anesthetized with 3% isoflurane for anesthesia induction and then 1–2% isoflurane for anesthesia maintenance. Mice were engrafted s.c. with 2 × 10⁵ or 1 × 10⁵ tumor 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 × 10⁹ TU of lentiviral vector-based vaccine, contained in 50 µl of PBS. For the MC38_KRAS^G12D model, mice were randomized and primed at day 3 post tumor challenge.

Tumors were measured 2-to-3 times a week using a digital caliper. The tumor volume was calculated as V = L × 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 mm³ in volume or when the mice became moribund.

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 μm-pore filters and washed in PBS. The recovered cells were stained as follows.

i.
To detect T cells and their activation profile, Near IR Live/Dead (Invitrogen), FcγII/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-KLRG1 (clone 2F1, Invitrogen), and FITC-anti-PD1 (clone RMP1-30, Invitrogen) were used. Samples were incubated with the mAb mixtures for 30 min at 4 °C, washed with PBS + 3% fetal bovine serum and fixed with Cytofix (BD Biosciences) for 20 min at 4 °C.
ii.
To study intra-tumoral innate immune cells, tumor suspensions were stained by a mixture of Near IR Live/Dead (Invitrogen), FcγII/III receptor blocking anti-CD16/CD32 (clone 2.4G2, BD Biosciences), BV605-anti-CD45 (clone 30-F11, BD Biosciences), PE-anti-CD11b (clone M1/70, Invitrogen), PE-Cy7-anti-CD11c (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.
iii.
To detect intra-tumoral anti-KRAS T cells, following enzymatic tumor dissociation, cell suspensions were enriched in lymphocytes by centrifugation for 20 min 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 × 10⁶/well overnight with 5 × 10⁵/well of syngeneic bone-marrow-derived dendritic cells, loaded with 10 µg/mL of appropriate peptides, in the presence of 1 µg/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 1X buffer from the Cytofix/Cytoperm kit and incubated with a mixture of FITC-anti-TNFα (clone MP6-XT22, BD Pharmigen), and APC-anti-IFN- γ (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.
iv.
Expression of MHC-I molecules and PDL1 on tumor cells were checked by use of PE-anti-H-2 Kb (REA1198, Miltenyi Biotec), PerCP/Cyanine5.5-anti-H-2D^b (KH95, BioLegend), APC-anti-PDL1 (10F.9G2, BioLegend) mAbs used, in presence of Near IR Live/Dead (Invitrogen) and FcγII/III receptor blocking anti-CD16/CD32 (clone 2.4G2, BD Biosciences).

Samples were acquired in an Attune NxT cytometer (Invitrogen) and were analyzed using FlowJo software (Treestar, OR, USA).

ELISPOT

IFNγ ELISPOT assay on splenocytes were performed as detailed elsewhere⁷⁹.

Statistics

Statistical significance was determined using the two‐tailed unpaired t‐test or two-way ANOVA test. The statistical significance of the percent survival was determined using the Log‐rank Mantel‐Cox test. All the statistical analyses were performed using Prism GraphPad v10.

Data availability

The article includes all datasets generated and analyzed during this study. All plasmids and LV generated in this study will be available under an MTA for research use, given a pending patent directed to Lenti‐KRAS immunotherapy vectors. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author Laleh Majlessi (laleh.majlessi@pasteur.fr).

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Acknowledgements

This work was supported by Institut Pasteur and TheraVectys.

Author information

Pierre Charneau and Laleh Majlessi: Co-senior authors

Authors and Affiliations

Pasteur-TheraVectys Joint Laboratory, Institut Pasteur, Virology Department, Université de Paris, 28 Rue du Dr. Roux, 75015, Paris, France
Anastasia Goloudina, Fabien Le Chevalier, Pierre Authié, Sylvain Ciret, Kirill Nemirov, Ingrid Fert, Fanny Moncoq, Benjamin Vesin, Amandine Noirat, Catherine Blanc, Yu Wei, Pierre Charneau & Laleh Majlessi

Authors

Anastasia Goloudina
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Fabien Le Chevalier
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Pierre Authié
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Sylvain Ciret
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Kirill Nemirov
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Ingrid Fert
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Fanny Moncoq
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Benjamin Vesin
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Amandine Noirat
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Catherine Blanc
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Yu Wei
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Pierre Charneau
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Laleh Majlessi
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Contributions

AG, FLC, PC and LM conceptualized the project. AG, FLC, PA, SC, KN, BV, MB and YW realized experiments and acquired data. IF performed cytometry, FM, AN and CB constructed the plasmids and produced the lentiviral vectors, AG and LM wrote the paper. All authors reviewed the manuscript.

Corresponding author

Correspondence to Laleh Majlessi.

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PC is the founder and CSO of TheraVectys. AG, FLC, PA, KN, IF, FM, BV and AN are employees of TheraVectys. LM has a consultancy activity for TheraVectys. AG, FLC, IF, AN, CB, PC and LM are inventors of a pending patent directed to the potential of lentiviral vectors encoding KRAS mutations in immuno-oncotherapy. Other authors declare no competing interests.

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Goloudina, A., Le Chevalier, F., Authié, P. et al. A lentiviral vector targeting a KRAS neoepitope for cancer immunotherapy. Sci Rep 15, 23171 (2025). https://doi.org/10.1038/s41598-025-05134-6

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Received: 17 March 2025
Accepted: 30 May 2025
Published: 02 July 2025
DOI: https://doi.org/10.1038/s41598-025-05134-6