Gating strategy for antigen-specific CD8+ T cells in the spleen.

Representative dot plots corresponding to flow cytometry data displayed in Figure 5.

Expansion of nonvaccine antigen-specific CD8+ T cells in response to RNA-LPX and Alb-IL2 RNA-NP. Number of nonvaccine antigen-specific CD8+ T cells per spleen (gp70 (A), OVA (B)) or per 4 x 106 splenocytes (TRP-1 (C)) 7 days after the last of three weekly i.v. vaccinations with gp70 RNA-LPX (A), OVA RNA-LPX (B) or TRP-1 RNA-LPX (C) combined with Alb-IL2 RNA-NP or Alb RNA-NP (gp70: n=6 mice/group for Alb RNA-NP, n=7 mice/group for Alb-IL2 RNA-NP; OVA, TRP-1: n=5 mice/group). Mean + SEM. Two-tailed Student t-test (A). **P≤0.01.

Expansion of Tregs in response to RNA-LPX and Alb-IL2 RNA-NP. Number of regulatory CD4+ T cells per spleen 7 days after the last of three weekly i.v. vaccinations with gp70 RNA-LPX (A) or OVA RNA-LPX (B) combined with Alb-IL2 RNA-NP or Alb RNA-NP (gp70: n=7 mice/group; OVA: n=5 mice/group). Mean + SEM.

Recombinant in vitro-transcribed mRNA is broadly used for vaccination and is evaluated in numerous clinical studies for multiple indications. Typical features of mRNA are the 5' cap, 5' untranslated region, start and stop codons, 3' untranslated region and 3' poly(A) tail. Here, contrary to current dogma, we show that short, chemically synthesized RNA oligonucleotides lacking some or all of these features are efficiently translated when they encode epitopes recognized by CD8+ T cells. In particular, one design that we termed ChemRNA with the structure 5'-OH-AUG-coding sequence-3'-OH strongly stimulates antigen-specific CD8+ T cells both in vitro and in vivo. Our results challenge the current understanding of canonical mRNA structure and introduce the possibility that defective or supposedly non-coding RNA may encode human and murine major histocompatibility complex class I-associated peptides. Moreover, ChemRNA could help overcome challenges associated with the design and purification of individualized anti-cancer vaccines.

Cancer patients typically mount T cell responses to only a small fraction of the neoantigens encoded in their individualized vaccine. Because each patient’s neoantigen set is unique, clinical studies cannot determine whether this restricted breadth reflects limitations of neoantigen prediction algorithms or intrinsic immunological constraints. Here, using a clinically relevant multi-neoantigen RNA–lipoplex vaccine in a preclinical model, we mechanistically dissected CD8+ T-cell breadth. We found that concurrently primed neoantigen-specific CD8+ T cells competed through a peptide-MHC-I-dependent cross-competition, with dominant responses suppressing both the magnitude and differentiation of subdominant responses. Dominant CD8+ T cell responses preferentially acquired a terminally differentiated effector phenotype, while sub-dominant responses adopted memory-precursor and stem-like features. Removing dominant responses enabled sub-dominant responses to expand and adopt terminally differentiated effector phenotypes. These findings provide the first mechanistic explanation for restricted CD8+ T cell breadth in multi-neoantigen vaccination and provide a framework for further optimizing cancer vaccine design.

Gating strategy for antigen-specific CD8+ T cells, Tregs, and NK cells displayed in Figure 6.

Representative dot plots corresponding to flow cytometry data displayed in Figure 6.

Representative dot plots corresponding to flow cytometry data displayed in Figure 4.

Gating strategy for antigen-specific CD8+ T cells and Tregs in the blood.

Representative dot plots and histograms corresponding to flow cytometry data displayed in Figure 3.

Antitumor immunity in response to RNA-LPX and Alb-IL2 RNA-NP. Absolute number of vaccine antigen-specific and nonvaccine antigen-specific CD8+ T cells, Tregs, and NK cells in the tumor (n=6 mice/group; A), tumor-draining lymph node (n=6 mice/group; B), and spleen (Alb RNA-NP: n=5 mice/group; Alb-IL2 RNA-NP: n=6 mice/group; C) of CT26 tumor-bearing BALB/c mice 5 days after i.v. administration of gp70 RNA-LPX combined with Alb-IL2 RNA-NP or Alb RNA-NP on day 12 after tumor cell inoculation. Mean + SEM. Two-tailed Student t-test. *P≤0.05.

Mouse body weight in response to RNA-LPX and Alb-IL2 RNA-NP or rIL-2. Mouse total body weight after each of three weekly i.v. vaccinations with OVA RNA-LPX combined with one (1x) or five (5x) i.p. injections of rIL-2 or one i.v. injection of Alb-IL2 RNA-NP or Alb RNA-NP in C57BL/6 mice (n=7 mice/group) in per cent of initial mouse body weight. Mean + SEM. Vertical lines indicate treatments (dashed: OVA RNA-LPX + Alb RNA-NP, rIL-2 or Alb-IL2 RNA-NP; dotted: repetitive rIL-2).

The therapeutic potential of interleukin-2 (IL-2) in cancer treatment is limited by toxicity challenges, partly due to an unfavorable pharmacokinetic profile and unintended activation of regulatory T (Treg) cells alongside the desired activation of CD8+ effector T cells. To selectively stimulate CD8+ T cells over Treg cells, we engineered an IL-2 variant (IL-2var) with a dual-tuned affinity profile that features reduced binding to IL-2Rα (CD25) and enhanced binding to IL-2Rβ (CD122). To optimize its pharmacokinetics and facilitate tumor enrichment, the variant is fused to albumin and delivered as an mRNA encapsulated in a lipid nanoparticle (Alb-IL-2var RNA-LNP), enabling sustained systemic exposure from hepatic production upon intravenous administration. We show that Alb-IL-2var has a favorable pharmacokinetic profile and is tolerated at biologically active doses in immunocompetent mice and cynomolgus monkeys. Selective enhancement of CD8+ T cell responses over Treg cells is demonstrated in vitro in human peripheral blood mononuclear cells and in vivo in mice and cynomolgus monkeys. When combined with an mRNA cancer vaccine in syngeneic subcutaneous mouse tumor models, Alb-IL-2var RNA-LNP stimulates the expansion of tumor-infiltrating and circulating tumor antigen-specific CD8+ T cells, but not Treg cells. In advanced and cold syngeneic tumor models, it enhances the efficacy of radiotherapy, checkpoint inhibitors, and cancer vaccines. Combination with checkpoint inhibitors and vaccine induces profound proinflammatory conversion of cold tumors. These preclinical results validate a rational design approach to overcome the limitations of IL-2 therapy and support the clinical evaluation of Alb-IL-2var RNA-LNP for solid cancers.

Immunodominance is a universal feature of adaptive immunity that constrains T cell expansion, clonal diversity and breadth resulting in a narrowly focused T cell response. While observed across diverse priming settings and vaccine platforms, the influence of immunodominance on T cell phenotype remains unclear. Using an mRNA lipoplex vaccine encoding multiple antigens to study how immunodominance influences CD8+ T cell fate, we found that dominant CD8+ T cell responses alter the magnitude and phenotype of subdominant responses through peptide-MHC-I stability-mediated T cell cross-competition. Dominant CD8+ T cell responses preferentially acquired markers associated with terminal differentiation and cytotoxic function, while sub-dominant responses adopted memory-precursor and stem-like features. Removal of dominant responses allowed increased expansion of sub-dominant T cell responses and adoption of terminally differentiated effector phenotypes. These findings reveal that immunodominance dynamically shapes the magnitude, breadth and differentiation of CD8+ T cell responses and highlights opportunities to fine-tune T cell responses for therapeutic vaccination. ### Competing Interest Statement MJMC, MH, TDW, AJT, ST, MD, EF, YF, CCB, ID, HL, SL, TN, YO, VJ, AN, YC, AG, SW, CDLC, BH, CB, IM, JS, LD were employees of Genentech, and DE, MV, LK, US were employees of BioNTech at the time of the study.

Abstract Interleukin 2 (IL-2) is a crucial cytokine in T-cell immunity, with a promising potential in cancer vaccines. However, therapeutic application of IL-2 is hampered by its short half-life and substantial toxicity. This study reports preclinical characterization of a mouse serum albumin–IL-2 fusion protein (Alb–IL2) encoded on nucleoside-modified RNA that is delivered via a nanoparticle formulation (Alb–IL2 RNA-NP) mediating prolonged cytokine availability. Alb–IL2 RNA-NP was combined with RNA-lipoplex (RNA-LPX) vaccines to evaluate its effect on the expansion of vaccine-induced antigen specific T-cell immunity. In mice dosed with Alb–IL2 RNA-NP, translated protein was shown to be systemically available up to 2 days, with an albumin-dependent preferred presence in the tumor and tumor-draining lymph node. Alb–IL2 RNA-NP administration prolonged serum availability of the cytokine compared with murine recombinant IL-2. In combination with RNA-LPX vaccines, Alb-IL2 RNA-NP administration highly increased the expansion of RNA-LPX vaccine–induced CD8+ T cells in the spleen and blood. The combination enhanced and sustained the fraction of IL-2 receptor (IL-2R) α-positive antigen-specific CD8+ T cells and ameliorated the functional capacity of the CD8+ T-cell population. Alb–IL2 RNA-NP strongly improved the antitumor activity and survival of concomitant RNA-LPX vaccination and PD-L1 blockade in a subcutaneous mouse tumor model. The favorable pharmacokinetic properties of Alb–IL2 RNA-NP render it an attractive modality for rationally designed combination immunotherapy. RNA vaccines that induce tumor-specific T-cell immunity for Alb–IL2 RNA-NP to further amplify are particularly attractive combination partners.

PDF - 395KB, Supplementary Figure 1: Structure of MO-TES391 and KIAA1864 transcripts.

PDF - 60KB, Supplementary Table 1: Serological reactivity of MO-TES391 assessed by SMARTA.

PDF - 49KB, Supplementary Table 3: Epitope prediction for HLA-A*02 binding peptides derived from KIAA1864 and MO-TES391.

PDF - 61KB, Supplementary Table 2: Primers used in the study.