Therapies based on immune checkpoint inhibitors (ICPI) have yielded promising albeit limited results in patients with hepatocellular carcinoma (HCC). Vaccines have been proposed as combination partners to enhance response rates to ICPI. Thus, we analyzed the combined effect of a vaccine based on the TLR4 ligand cold-inducible RNA binding protein (CIRP) plus ICPI. Mice were immunized with vaccines containing ovalbumin linked to CIRP (OVA-CIRP), with or without ICPI, and antigen-specific responses and therapeutic efficacy were tested in subcutaneous and orthotopic mouse models of liver cancer. OVA-CIRP elicited polyepitopic T-cell responses, which were further enhanced when combined with ICPI (anti-PD-1 and anti-CTLA-4). Combination of OVA-CIRP with ICPI enhanced ICPI-induced therapeutic responses when tested in subcutaneous and intrahepatic B16-OVA tumors, as well as in the orthotopic PM299L HCC model. This effect was associated with higher OVA-specific T-cell responses in the periphery, although many tumor-infiltrating lymphocytes still displayed an exhausted phenotype. Finally, a new vaccine containing human glypican-3 linked to CIRP (GPC3-CIRP) induced clear responses in humanized HLA-A2.01 transgenic mice, which increased upon combination with ICPI. Therefore, CIRP-based vaccines may generate anti-tumor immunity to enhance ICPI efficacy in HCC, although blockade of additional checkpoint molecules and immunosuppressive targets should be also considered.

Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund’s adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 10⁴–3 × 10⁵, depending on the adjuvant) and displaying neutralizing capacity (80–95% inhibition capacity; p < 0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern. Importantly, vaccination with peptide 446-480 or with a cyclic version of peptide 446-488 containing a disulphide bridge between cysteines 480 and 488, protected humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2 (62.5 and 75% of protection; p < 0.01 and p < 0.001, respectively). This region could be the basis for a peptide vaccine or other vaccine platforms against Covid-19.

(1) Background: The ability of cancer cells to evade the immune system is due in part to their capacity to induce and recruit T regulatory cells (Tregs) to the tumor microenvironment. Strategies proposed to improve antitumor immunity by depleting Tregs generally lack specificity and raise the possibility of autoimmunity. Therefore, we propose to control Tregs by their functional inactivation rather than depletion. Tregs are characterized by the expression of the Forkhead box protein 3 (FOXP3) transcription factor, which is considered their “master regulator”. Its interaction with DNA is assisted primarily by its interaction with other proteins in the so-called “Foxp3 interactome”, which elicits much of the characteristic Treg cell transcriptional signature. We speculated that the disruption of such a protein complex by using synthetic peptides able to bind Foxp3 might have an impact on the functionality of Treg cells and thus have a therapeutic potential in cancer treatment. (2) Methods: By using a phage-displayed peptide library, or short synthetic peptides encompassing Foxp3 fragments, or by studying the crystal structure of the Foxp3:NFAT complex, we have identified a series of peptides that are able to bind Foxp3 and inhibit Treg activity. (3) Results: We identified some peptides encompassing fragments of the leuzin zipper or the C terminal domain of Foxp3 with the capacity to inhibit Treg activity in vitro. The acetylation/amidation of linear peptides, head-to-tail cyclization, the incorporation of non-natural aminoacids, or the incorporation of cell-penetrating peptide motifs increased in some cases the Foxp3 binding capacity and Treg inhibitory activity of the identified peptides. Some of them have shown antitumoral activity in vivo. (4) Conclusions: Synthetic peptides constitute an alternative to inhibit Foxp3 protein–protein interactions intracellularly and impair Treg immunosuppressive activity. These peptides might be considered as potential hit compounds on the design of new immunotherapeutic approaches against cancer.

Background and Aims: Glypican-3 (GPC3) is a promising target for T-cell therapy in HCC. While chimeric antigen receptor (CAR) T cells targeting GPC3 have demonstrated therapeutic efficacy, their effectiveness is limited by challenges such as low persistence and shedding of surface GPC3. Natural T-cell receptors (TCRs) may serve as an alternative, though identifying GPC3-specific TCRs within the endogenous repertoire is difficult. Approach and Results: We immunized human leucocyte antigen-A2 (HLA-A2) transgenic mice with an adenovirus expressing human GPC3, identifying a panel of TCRs that recognize the GPC3(522-530) epitope. We cloned 3 murine GPC3-TCRs (TCR-A, TCR-B, and TCR-C) and engineered primary human T cells (TCR-T). TCR-T cells effectively recognized GPC3⁺HLA-A2⁺ human HCC cells, with recognition diminished by GPC3 silencing and HLA-A2 blockade. TCR-B-T and TCR-C-T cells showed the highest reactivity, with TCR-B-T cells exhibiting superior effector functions, proliferative capacity, and therapeutic efficacy in xenograft HCC models. Notable, TCR-B-T cells outperformed second-generation 41BB GPC3-specific CAR-T cells, attributed to lower exhaustion, enhanced proliferation, greater effector function, and improved resilience. Furthermore, mixed dosing of CAR-T and TCR-B-T cells was significantly more effective than staggered dosing of the same cell type, suggesting potential synergistic effects. Conclusions: Transgenic TCRs join forces with CARs, expanding the arsenal of GPC3-targeting receptors for HCC T-cell therapy.

The expression of tumor-specific antigens during cancer progression can trigger an immune response against the tumor. Antigens that have been used as cancer vaccines are those originated by non- synonymous mutations and those derived from cancer/testis antigens. However, the first class is predominantly patient-specific, preventing the development of therapies than can benefit multiple patients, and the second one offers a limited set of actionable targets. A possible alternative is the use of peptides derived from non-canonical ORFs (ncORFs). While many ncORFs have been shown to be translated in cancer cells, their tumor-specificity and patient distribution remains to be determined. Here we analyze RNA sequencing data 117 hepatocellular carcinoma (HCC) tumors and matched healthy tissue, together with ribosome profiling data from an additional 10 HCC tumors, to answer these open questions. Combining HLA-epitope binding predictions and experimental validation experiments we conclude that around 40% of the tumor-specific antigens in HCC are likely to be derived from ncORFs in lncRNAs, including two peptides that can trigger an immune response in mice. We identify a subset of 33 tumor-specific lncRNAs expressing novel cancer antigens shared by more than 10% of the HCC analyzed, which could be combined to target a large proportion of the patients. The results of the study open new avenues for extending the range of anti-cancer vaccines.