Cell-penetrating peptides enhance peptide vaccine accumulation and persistence in lymph nodes to drive immunogenicity.

Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action in vivo are poorly understood. We tested eight previously described CPPs conjugated to antigens from multiple syngeneic murine tumor models and found that linkage to CPPs enhanced peptide vaccine potency in vivo by as much as 25-fold. Linkage of antigens to CPPs did not impact dendritic cell activation but did promote uptake of linked antigens by dendritic cells both in vitro and in vivo. However, T cell priming in vivo required Batf3-dependent dendritic cells, suggesting that antigens delivered by CPP peptides were predominantly presented via the process of cross-presentation and not through CPP-mediated cytosolic delivery of peptide to the classical MHC class I antigen processing pathway. Unexpectedly, we observed that many CPPs significantly enhanced antigen accumulation in draining lymph nodes. This effect was associated with the ability of CPPs to bind to lymph-trafficking lipoproteins and protection of CPP-antigens from proteolytic degradation in serum. These two effects resulted in prolonged presentation of CPP-peptides in draining lymph nodes, leading to robust T cell priming and expansion. Thus, CPPs can act through multiple unappreciated mechanisms to enhance T cell priming that can be exploited for cancer vaccines with enhanced potency.

Identification of Highly Cross-Reactive Mimotopes for a Public T Cell Response in Murine Melanoma.

While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.

Neoadjuvant STING Activation, Extended Half-life IL2, and Checkpoint Blockade Promote Metastasis Clearance via Sustained NK-cell Activation.

Combination immunotherapy treatments that recruit both innate and adaptive immunity have the potential to increase cancer response rates by engaging a more complete repertoire of effector mechanisms. Here, we combined intratumoral STimulator of INterferon Genes (STING) agonist therapy with systemically injected extended half-life IL2 and anti-PD-1 checkpoint blockade (hereafter CIP therapy) to drive innate and adaptive antitumor immunity in models of triple-negative breast cancer. Unlike treatment with the individual components, this trivalent immunotherapy halted primary tumor progression and led to long-term remission for a majority of animals in two spontaneously metastasizing orthotopic breast tumor models, though only as a neoadjuvant therapy but not adjuvant therapy. CIP therapy induced antitumor T-cell responses, but protection from metastatic relapse depended on natural killer (NK) cells. The combination of STING agonists with IL2/anti-PD-1 synergized to stimulate sustained granzyme and cytokine expression by lung-infiltrating NK cells. Type I IFNs generated as a result of STING agonism, combined with IL2, acted in a positive-feedback loop by enhancing the expression of IFNAR-1 and CD25 on lung NK cells. These results suggest that NK cells can be therapeutically targeted to effectively eliminate tumor metastases.See related Spotlight by Demaria, p. 3.

An in vivo selection-derived d-peptide for engineering erythrocyte-binding antigens that promote immune tolerance.

When displayed on erythrocytes, peptides and proteins can drive antigen-specific immune tolerance. Here, we investigated a straightforward approach based on erythrocyte binding to promote antigen-specific tolerance to both peptides and proteins. We first identified a robust erythrocyte-binding ligand. A pool of one million fully d-chiral peptides was injected into mice, blood cells were isolated, and ligands enriched on these cells were identified using nano-liquid chromatography-tandem mass spectrometry. One round of selection yielded a murine erythrocyte-binding ligand with an 80 nM apparent dissociation constant, Kd We modified an 83-kDa bacterial protein and a peptide antigen derived from ovalbumin (OVA) with the identified erythrocyte-binding ligand. An administration of the engineered bacterial protein led to decreased protein-specific antibodies in mice. Similarly, mice given the engineered OVA-derived peptide had decreased inflammatory anti-OVA CD8+ T cell responses. These findings suggest that our tolerance-induction strategy is applicable to both peptide and protein antigens and that our in vivo selection strategy can be used for de novo discovery of robust erythrocyte-binding ligands.

Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor.

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.