Mimicry-based strategy between human and commensal antigens for the development of a new family of immune therapies for cancer

Abstract

Background: Molecular mimicry between commensal bacterial antigens and tumor-associated antigens (TAAs) has shown potential in enhancing antitumor immune responses. This study leveraged this concept using commensal bacterial antigens, termed OncoMimics, to induce TAA-derived peptide (TAAp)-specific cross-reactive cytotoxic T cells and improve the efficacy of peptide-based immunotherapies.

Methods: The discovery of OncoMimics primarily relied on a bioinformatics approach to identify commensal bacteria-derived peptide sequences mimicking TAAps. Several OncoMimics peptide (OMP) candidates were selected in silico based on multiple key parameters to assess their potential to elicit and ameliorate immune responses against TAAs. Selected OMPs were synthesized and tested for their affinity and stability on the major histocompatibility complex (MHC) in vitro and for their capacity to elicit cross-reactive OMP-specific/TAAp-specific CD8+T cell responses in human leukocyte antigen (HLA)-A2-humanized mice, human peripheral blood mononuclear cells (PBMC) and patients with cancer.

Results: Selected OMPs demonstrated superior HLA-A2 binding affinities and stabilities compared with homologous TAAps. Vaccination of HLA-A2-humanized mice with OMPs led to the expansion of OMP-specific CD8+T cells that recognize both OMPs and homologous TAAps, exhibiting cytotoxic capacities towards tumor antigens and resulting in tumor protection in a prophylactic setting. Using PBMCs from HLA-A2+healthy donors, we confirmed the ability of OMPs to elicit potent cross-reactive OMP-specific/TAAp-specific CD8⁺ T-cell responses. Interestingly, we observed a high prevalence of OMP-specific T cells across donors. Cytotoxicity assays revealed that OMP-stimulated human T cells specifically targeted and killed tumor cells loaded with OMPs or TAAps. Preliminary data from an ongoing clinical trial (NCT04116658) support these findings, indicating that OMPs elicit robust OMP-specific/TAAp-specific CD8+T cell responses in patients. Initial immunomonitoring data revealed sustained T-cell responses over time, with T cells maintaining a polyfunctional, cytotoxic and memory phenotype, which is critical for effective antitumor activity and long-term immune surveillance.

Conclusions: These findings suggest that leveraging naturally occurring commensal-derived antigens through OMPs could significantly remodel the tumor immune landscape, offering guidance for a promising strategy for cancer peptide-based immunotherapies.

Keywords: Adaptive; Human leukocyte antigen - HLA; Immunotherapy; T cell; Vaccine.

Figure 1. Discovery and selection process of OMPs for cancer immunotherapy. (A) OMPs discovery: A two-step process. (1) A data set of HLA-A2 peptides derived from tumor-associated antigens (TAAs) was generated from public human databases according to several parameters. (2) OMP candidates were identified by scanning each TAA-derived peptide against all peptide sequences of the same length from gut microbiome catalogs using different homology criteria (detailed in online supplemental M&M). (B) TAA-derived peptides distribution based on the number of their commensal-derived peptides: the graph shows the distribution of 224 TAA-derived peptides (TAAps) based on the number of their associated CDPs. (C) Repartition of TAA-derived peptides based on the HLA-A2 affinities of their commensal-derived peptides: the graph represents the repartition of TAA-derived peptides based on two categories: TAAp with strong binder (SB) CDPs≥TAAp or TAAp with no SB strong binder CDPs≥TAAp. In other words, the presence (in red) or absence (in gray) of at least one strong binder CDP, is stronger than its TAAp counterparts. In the legends of figure 1C and E, the symbol “≥” indicates “a stronger predicted affinity than”, while “<” signifies “a weaker predicted affinity than”. (D) Commensal-derived peptide distribution based on HLA-A2 predicted binding affinities: the graph shows the distribution of commensal-derived peptides based on their HLA-A2 predicted binding affinity. Commensal-derived peptides with the highest affinities (lower nanomolar values) are shown on the left side of the graph. We classified the CDPs into three categories based on their %rank (according to NetMHCpan3.0 recommendations): strong binders (SB) were defined as having %rank<0.5, weak binders (WB) with %rank<2 and no binder peptides with %rank>2. (E) Commensal-derived peptide repartition based on their HLA-A2 predicted binding affinity compared with their TAAp counterparts: The graph shows how commensal-derived peptides are categorized into five groups: SB commensal-derived peptide with an HLA-A2 affinity equal to or stronger than the associated TAAp; SB commensal-derived peptide with an HLA-A2 affinity equal to or weaker than the associated TAAp; WB commensal-derived peptide with an HLA-A2 affinity equal to or stronger than the associated TAAp; WB commensal-derived peptide with an HLA-A2 affinity equal to or weaker than the associated TAAp; Commensal-derived peptides that are no binders to HLA-A2. (F) Characteristics of the 10 selected HLA-A2 OMPs in this study and their homologous TAAps: gene ID of the targeted TAA, sequence name of TAAp and corresponding OMP. Amino acid (aa) sequences predicted affinity rank and binding affinity (nM) to HLA-A*02:01 molecule of each pair and the numbers of mismatches are shown. Amino acid substitutions are indicated in red and those in dark bold are the five amino acids in the peptide core. Fold increases in affinity between OMP and corresponding TAAp. CDPs, commensal-derived peptide; HLA, human leukocyte antigen; OMP, OncoMimics peptide.

Figure 2. OMPs selected in vitro for their HLA-A2 binding and stability properties and in vivo for their immunogenicity are capable of eliciting potent cross-reactive responses in HLA-A2/DR1 mice. (A, B) Binding and stability of TAA-derived peptides and their OMP counterparts. Comparison of HLA-A2 binding (A) and stability (B) of TAA-derived peptides (red), their OMP counterparts (blue) and the HIV reference peptide (green) in the T2-binding assay. The data represent four to seven independent experiments. Symbols represent the mean and error bars indicate the SEM. (C) Schematic representation of the in vivo experimental setup used to assess OMPs immunogenicity and CD8⁺ T cell-dependent cross-reactive responses against TAAps. (D) In vivo immunogenicity and cross-reactivity. The frequency of peptide-specific T cells that produce IFN-γ was determined by ELISpot analysis of splenocytes from mice vaccinated with the indicated peptides. The negative control, OMP and TAAp are shown in gray, blue and red, respectively. Below each graph, the percentages of cross-reactivity (XR) between OMP and its TAA counterpart are displayed. (E) Schematic representation of the in vivo experimental set-up used to compare the capacity of OMPs and TAA-derived peptides to induce a TAAp-specific response. (F) OMP-induced and TAAp-induced cross-reactive responses. Comparison of OMP efficacy in inducing a TAAp-specific response to that of their TAA counterparts in A2/DR1 humanized mice assessed on splenocytes from mice immunized with the indicated peptides by IFN-γ ELISpot assay. Dark blue, red, light blue and orange indicate OMP vaccination and restimulation conditions, OMP vaccination and TAAp restimulation, TAAp vaccination and OMP restimulation conditions and TAAp vaccination and restimulation conditions, respectively. The data shown in (D) and (F) are from one to five independent experiments; symbols indicate individual mice (n=5–25 mice) and bars represent min and max values. Statistical comparisons were performed using an unpaired non-parametric test (Mann-Whitney). *p<0.05, **p<0.001, ****p<0.0001, ns: non-significant. ELISpot, Enzyme-Linked ImmunoSpot; HLA, human leukocyte antigen; OMP, OncoMimics peptide, TAA, tumor-associated antigen.

Figure 3. OMP-based vaccines elicit functional cytotoxic T cells in mice that are cross-reactive with human TAA-derived peptides in vivo. (A) Schematic representation of the experimental setup employed to assess T cell cytotoxicity elicited in vivo by OMPs. HLA-A2-DR1 mice were vaccinated with OMPs and challenged post-vaccination with syngeneic splenocytes labeled with cell tracking dye. Target T cells (red) were pulsed with a pool of OMPs or TAA-derived peptides and mixed at an equal ratio with unpulsed control cells (black) before being adaptively transferred to immunized mice. (B, C) In vivo cytotoxic activity of OMP-induced T cells against OMP-pulsed or TAAp-pulsed target T cells. Percentages of in vivo specific lysis of splenocytes pulsed with the indicated pool of OMPs (B) or TAA-derived peptide counterparts (C) (challenging peptides) after immunization with the indicated peptide pool or control peptides (immunizing peptides). Data shown in (B) and (C) are from two to four independent experiments, symbols indicate individual mice (n=10–20 mice) and bars represent min and max values. Statistical comparisons were performed using an unpaired non-parametric test (Mann-Whitney). ***p<0.001, ****p<0.0001. (D) Flow cytometry analysis performed on SARC-A2 GFP (left panel) and on SARC-A2-GFP-CD20 (right panel) sarcoma cells showing post-transduction expressions of GFP and hCD20 proteins. (E) Schematic representation of the experimental setup used to evaluate the antitumor effect of OMPs. Mice were immunized with OMP72 using a prime-boost administration regimen. 21 days post-prime immunization, mice were inoculated with hCD20-GFP-expressing or GFP-expressing SARC-A2 sarcoma cells and tumor growth was monitored. (F) Tumor kinetics on individual mice over time for each group. Tumor size in A2/DR1 naïve or OMP vaccinated mice engrafted with 0.5×10⁶ SARC-A2-hCD20-GFP or SARC-A2-GFP tumor cells (n=8 mice) was measured (mm²) over 30 days. (G) hCD20 expression assessment in the vaccinated group. Flow cytometry dot plots showing the expression of human CD20 and GFP in SARC-A2 cells extracted from whole tumors on day 30 in animals still bearing tumors (n=4). (H) Vaccine-specific induced T cell responses. The frequency of peptide-specific T cells producing IFN-γ per million splenic T cells was determined by ELISpot on day 30. ELISpot, Enzyme-Linked ImmunoSpot; HLA, human leukocyte antigen; OMP, OncoMimics peptide; TAAp, tumor-associated antigen-derived peptide.

Figure 4. OMP-specific human T cells recognize TAAs and exert specific cytotoxic activity. (A) Schematic overview for determining antigen-specific CD8⁺ T cell response using healthy volunteer PBMCs and in vitro expansion of CD8⁺ T cells as described in Material and Methods. (B) Cytotoxic activity of the OMP-specific human CD8⁺ T cell. CTL killing activity was assessed against T2 cells loaded with OMP and the TAA-derived peptide (TAAp) counterpart after 24 hours incubation. Each graph displays the representative data from healthy donors. The cytotoxicity percentage (y-axis) and effector-to-target ratio (E:T ratio) (x-axis) are indicated on the graph. EZH2-B2 irrelevant peptide and unpulsed T2 cells were used as negative controls. Error bars represent the mean±SD. Solid lines depict T2 cells loaded with bacterial peptides (OMPs in blue) and tumor-associated derived peptides (TAAp in red) and dashed lines represent controls (unloaded in black, EZH2-loaded in green). The cytotoxicity percentage was calculated as specified in the Materials and Methods section. (C) Prevalence of OMP-specific CD8⁺ T cells in healthy donors (HD). The prevalence of OMP-specific CD8⁺ T cells in HDs was determined by surface staining using pMHC tetramers after expansion of PBMCs using OMPs and is shown as a histogram. The left panel shows the individual detection of OMPs in each HD, with white, blue and black squares representing HDs in which OMPs were not detected, detected, or no analyses were performed, respectively. The right panel shows the general frequency of OMP detection in healthy donors. The number of analyzed HDs is indicated in parentheses for each OMP: OMP10 and OMP11 (n=33), OMP12 (n=27), OMP16 (n=14), OMP17 (n=15), OMP18 (n=14), OMP64, OMP65, OMP66 (n=21) and OMP72 (n=13). CTL, cytotoxic T cells; OMP, OncoMimics peptide; PBMCs, peripheral blood mononuclear cells; pMHC, peptide major histocompatibility complex; TAA, tumor-associated antigen.

Figure 5. Induction of OMP-specific and TAA-cross-reactive T cell responses in patients with glioblastoma post-vaccination Post-IVS T cell responses to OMPs or TAAps from patients are shown in A–D. (A, C) OMP-specific (A) and TAA-specific (C) CD8⁺ cells detected using EO2316, EO2317, EO2318, IL-13RA2, BIRC5 and FOXM1 tetramers on PBMC samples subjected to 12 days in vitro stimulation (IVS) with the OMP peptide pool. Data were obtained from all available samples of patients in Cohort 1 (n=3). Each dot represents the percentage of tetramer-positive CD8⁺ cells in each patient during the defined week. Negative values are plotted at 0.001. (B, D) Immune responses of patients before and after treatment. Total OMP-specific (B) and TAAp-specific (D) CD8⁺ T cells were detected in patient PBMC samples before and after treatment at the indicated time points. Data represent the added percentages (%) of all OMP (B) or TAAp (D), determined by multimer staining, as shown in (A) and (C). A fold increase was calculated and is shown in the graph when OMPs-specific or TAAp-specific T-cells were detected before treatment. (E–F) Generation of functional antigen-specific cells after vaccination. The immune responses of the patients before and after treatment were analyzed using IFN-γ ELISpot post-IVS. IFN-γ ELISpot wells are shown for all three patients before and after vaccination, with positive wells highlighted with colored squares (E) Quantification of the number of spots after vaccination for each patient, as well as the mean±SEM, are shown in (F). The IFN-γ spots were normalized to 10⁶ cells after background subtraction (negative control, DMSO). (G) Polyfunctional T cells are generated by OMP vaccination. ICS quantification of the % of activation marker (IFN-γ, TNF, CD107a, IL-2 and CD154)-positive T-cells on stimulation with the indicated peptides (top of each bar graph). For (F) and (G), Patients #1, #2 and #3 are shown as circles, squares and hexagons, respectively. The symbols filled in green and white represent positive and negative responses, respectively (see Materials and Methods for the positivity criteria). (H–I) (H) OMP-specific CD8⁺ cells detected using EO2316, EO2317 and EO2318 specific tetramers and (I) TAAp-specific CD8⁺ cells detected using IL-13RA2, BIRC5 and FOXM1 tetramers in PBMC samples ex vivo. No TAAp-specific data were available for Patient #3. Each dot represents the percentage of tetramer⁺ CD8⁺ cells in each patient at the indicated week. Negative values are plotted at 0.001. For Patients #1 and #2, only data from the monitoring of the later time points (12–24 weeks) were available. (J) OMP-specific (top) and TAAp-specific (bottom) CD8⁺ cells in PBMC ex vivo or after IVS. DMSO, Dimethyl Sulfoxide; ICS, intracellular cytokine staining; OMP, OncoMimics peptide; PBMCs, peripheral blood mononuclear cells; TAA, tumor-associated antigen; TAAp, TAA-derived peptide.

Figure 6. Long-term memory and polyfunctional T cell responses in patients with post-vaccination glioblastoma. (A–B) Vaccine-specific CD8⁺ T cells are effector memory cells. Representative dot plots (A) for Patient #2 of EO2317-specific and BIRC5-specific CD8⁺ T cells over time (weeks (W) 0, (W24, W32 and W44) based on tetramer staining (left). The memory phenotype (right) was evaluated based on the expression of the CCR7 and CD45RA markers. CCR7 and CD45RA expression profiles of tetramer⁺ cells (orange dots) overlaid on CD14/CD19—cells (gray dots). (B) Differentiation of subsets of tetramers+CD8⁺ T cells. Quantification of the memory phenotype of tetramer⁺ CD8⁺ T cells (central memory (CM), naïve, effector memory (EM) and terminally differentiated effector memory (EMRA)) for Patients #1 and #2 is shown as percentages (%). Only the time points with more than 20 tetramer+CD8+ T cells were used for the analysis. (C–D) Durable and long-term responses of Patient #1 to vaccination. T cells from Patient #1 were analyzed at weeks 88 and 100 after administration of the first vaccine dose. Flow cytometry analysis (C) showing EO2317-specific and BIRC5-specific T cells (left) and the CCR7/CD45RA expression profile (middle) for EO2317-specific and BIRC5-specific T cells (orange) overlaid with the overall CD8⁺ T cell population (gray population). Central memory, effector memory, naïve and terminally differentiated effector memory CD8⁺ subsets were quantified for each indicated tetramer⁺ population (right). T cell polyfunctionality is shown in the pie chart (D). T cells were expanded in culture with the OMP pool and IL-2 for 12 days and restimulated with either EO2317 or BIRC5 peptide for 6 hour before being intracellularly stained for IFN-γ, CD107a and TNF-α. The pie arcs depict the proportion of cells that produce a specific cytokine and the pie slices the proportion of cells co-producing one to three different cytokines. Pie arc overlap represents polyfunctional cells.

Figure 7. Glioblastoma cell killing by OMP-Induced TAAp-specific T cells in an HLA-restricted manner. (A) Experimental workflow for assessing OMP-Induced TAAp-specific T cell cytotoxicity. PBMCs from vaccinated patients (Patient #1 and #2 V15/week28 and Patient #3 V6/week10) were subjected to IVS with OMPs (EO2316, EO2317 and EO2318) followed by FACS analysis to evaluate the presence of TAAp-specific T cells post-IVS. Subsequently, IL13RA2-specific, BIRC5-specific, and FOXM1-specific T cells were isolated using TAAp-tetramers, expanded for several days with αCD3/CD28 stimulation and analyzed again by FACS to determine the frequencies of TAAp-specific T cells in the culture before proceeding to the cytotoxic assay. (B) FACS analysis of TAAp-specific T cells post-IVS using OMPs and post-sorting and αCD3/CD28 amplification. Left panel: TAAp-Tetramer staining showing the percentage of IL13RA2-specific, BIRC5-specific and FOXM1-specific T cells after IVS using OMPs. Right panel: TAAp-Tetramer staining showing the percentage of IL13RA2-specific, BIRC5-specific and FOXM1-specific T cells after tetramer sorting and αCD3/CD28 amplified in the individual cell cultures from the three patients. (C) Cytotoxic activity of sorted TAA-specific T cells against glioblastoma cells. IL13RA2-specific, BIRC5-specific and FOXM-specific T cells were tested for their killing activity against U87 (HLA-A2⁺, IL13RA2⁺, BIRC5⁺ and FOXM1⁺) and U118 (HLA-A2⁻, IL13RA2⁺, BIRC5⁺ and FOXM1⁺) glioblastoma cell lines using various E:T ratios. Cytotoxicity percentages (y-axis) and E:T ratios (x-axis) are shown. The plain lines and closed dark circles indicate U87 cells, while the dotted lines and gray close circles indicate U118 cells. Each point represents the mean of biological duplicates. Cytotoxicity was calculated as specified in the Materials and Methods section. IL13RA2-specific clones were only detected and isolated in patient#1. FACS, Fluorescence-Activated Cell Sorting; HLA, human leukocyte antigen; IVS, in vitro stimulation; OMP, OncoMimics peptide; PBMCs, peripheral blood mononuclear cells; TAAp, tumor-associated antigen -derived peptide.