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Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.

Short peptides reflecting major histocompatibility complex (MHC) class I (MHC‐I) epitopes frequently lack sufficient immunogenicity to induce robust antigen (Ag)‐specific CD8+ T cell responses. In the current work, it is demonstrated that position‐scanning peptide libraries themselves can serve as improved immunogens, inducing Ag‐specific CD8+ T cells with greater frequency and function than the wild‐type epitope. The approach involves displaying the entire position‐scanning library onto immunogenic nanoliposomes. Each library contains the MHC‐I epitope with a single randomized position. When a recently identified MHC‐I epitope in the glycoprotein gp70 envelope protein of murine leukemia virus (MuLV) is assessed, only one of the eight positional libraries tested, randomized at amino acid position 5 (Pos5), shows enhanced induction of Ag‐specific CD8+ T cells. A second MHC‐I epitope from gp70 is assessed in the same manner and shows, in contrast, multiple positional libraries (Pos1, Pos3, Pos5, and Pos8) as well as the library mixture give rise to enhanced CD8+ T cell responses. The library mixture Pos1‐3‐5‐8 induces a more diverse epitope‐specific T‐cell repertoire with superior antitumor efficacy compared to an established single mutation mimotope (AH1‐A5). These data show that positional peptide libraries can serve as immunogens for improving CD8+ T‐cell responses against endogenously expressed MHC‐I epitopes. [ABSTRACT FROM AUTHOR]

Tumor-associated self-antigens are potential cancer vaccine targets but suffer from limited immunogenicity. There are examples of mutated, short self-peptides inducing epitope-specific CD8+ T cells more efficiently than the wild-type epitope, but current approaches cannot yet reliably identify such epitopes, which are referred to as enhanced mimotopes ("e-mimotopes"). Here, we present a generalized strategy to develop e-mimotopes, using the tyrosinase-related protein 2 (Trp2) peptide Trp2180-188, which is a murine MHC class I (MHC-I) epitope, as a test case. Using a vaccine adjuvant that induces peptide particle formation and strong cellular responses with nanogram antigen doses, a two-step method systematically identified e-mimotope candidates with murine immunization. First, position-scanning peptide microlibraries were generated in which each position of the wild-type epitope sequence was randomized. Randomization of only one specific residue of the Trp2 epitope increased antitumor immunogenicity. Second, all 20 amino acids were individually substituted and tested at that position, enabling the identification of two e-mimotopes with single amino acid mutations. Despite similar MHC-I affinity compared with the wild-type epitope, e-mimotope immunization elicited improved Trp2-specific cytotoxic T-cell phenotypes and improved T-cell receptor affinity for both the e-mimotopes and the native epitope, resulting in better outcomes in multiple prophylactic and therapeutic tumor models. The screening method was also applied to other targets with other murine MHC-I restriction elements, including epitopes within glycoprotein 70 and Wilms' Tumor Gene 1, to identify additional e-mimotopes with enhanced potency., (©2022 American Association for Cancer Research.)

Background: Induction of CD8 + T cells that recognize immunogenic, mutated protein fragments in the context of major histocompatibility class I (MHC-I) is a pressing challenge for cancer vaccine development., Methods: Using the commonly used murine renal adenocarcinoma RENCA cancer model, MHC-I restricted neoepitopes are predicted following next-generation sequencing. Candidate neoepitopes are screened in mice using a potent cancer vaccine adjuvant system that converts short peptides into immunogenic nanoparticles. An identified functional neoepitope vaccine is then tested in various therapeutic experimental tumor settings., Results: Conversion of 20 short MHC-I restricted neoepitope candidates into immunogenic nanoparticles results in antitumor responses with multivalent vaccination. Only a single neoepitope candidate, Nesprin-2 L4492R (Nes2LR), induced functional responses but still did so when included within 20-plex or 60-plex particles. Immunization with the short Nes2LR neoepitope with the immunogenic particle-inducing vaccine adjuvant prevented tumor growth at doses multiple orders of magnitude less than with other vaccine adjuvants, which were ineffective. Nes2LR vaccination inhibited or eradicated disease in subcutaneous, experimental lung metastasis and orthotopic tumor models, synergizing with immune checkpoint blockade., Conclusion: These findings establish the feasibility of using short, MHC-I-restricted neoepitopes for straightforward immunization with multivalent or validated neoepitopes to induce cytotoxic CD8 + T cells. Furthermore, the Nes2LR neoepitope could be useful for preclinical studies involving renal cell carcinoma immunotherapy., Competing Interests: Competing interests: W-CH and JL hold interest in POP Biotechnologies., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)