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Vaccines that induce cytotoxic T lymphocyte (CTL)-mediated immune responses constitute an important class of medical tools to fend off diseases like infections and malignancy. Epitope peptides, as a format of CTL vaccines, are being tested preclinically and clinically. To elicit CTL responses, epitope vaccines go through an epitope presentation pathway in dendritic cells (DCs) that has multiple bottleneck steps and hence is inefficient. Here, we report the development of a strategy to overcome one of these barriers, phagolysosomal escape in DCs. First, we furnished a previously established carrier—an immune-tolerant elastin-like polypeptide nanoparticle (iTEP NP)—with the peptides that are derived from the DNA polymerase of herpes simplex virus 1 (Pol peptides). Pol peptides were reported to facilitate phagolysosomal escape. In this study, while we found that Pol peptides promoted the CTL epitope presentation; we also discovered Pol peptides disrupted the formation of the iTEP NP. Thus, we engineered a series of new iTEPs and identified several iTEPs that could accommodate Pol peptides and maintain their NP structure at the same time. We next optimized one of these NPs so that its stability is responsive to its redox environment. This environment-responsive NP further strengthened the CTL epitope presentation and CTL responses. Lastly, we revealed how this NP and Pol peptides utilized biological cues of phagolysosomes to realize phagolysosomal escape and epitope release. In summary, we developed iTEP NP carriers with a new phagolysosomal escape function. These carriers, with their priorly incorporated functions, resolve three bottleneck issues in the CTL epitope presentation pathway: vaccine uptake, phagolysosomal escape, and epitope release.

Targeted suppression of autoimmune diseases without collateral suppression of normal immunity remains an elusive yet clinically important goal. Targeted blockade of the programmed death-1 receptor (PD-1) — an immune checkpoint factor expressed by activated T cells and B cells — is an efficacious therapy for potentiating immune activation against tumours. Here, we show that an immunotoxin consisting of an anti-PD-1 single-chain variable fragment, an albumin-binding domain and Pseudomonas exotoxin targeting PD-1-expressing cells selectively recognizes and induces the killing of the cells. Administration of the immunotoxin to mouse models of autoimmune diabetes delays disease onset, and its administration in mice paralyzed by experimental autoimmune encephalomyelitis ameliorates symptoms. In all mouse models, the immunotoxin reduced the numbers of PD-1-expressing cells, of total T cells and of cells of an autoreactive T cell clone found in inflamed organs, while maintaining active adaptive immunity, as evidenced by full-strength immune responses to vaccinations. The targeted depletion of PD-1-expressing cells contingent to the preservation of adaptive immunity might be effective in the treatment of a wide range of autoimmune diseases.

Rationale: Albumin-binding carriers have been shown to target cytotoxic T lymphocyte (CTL) vaccines to lymph nodes (LNs) and improve the efficacy of the vaccines. However, it was not clear whether the improved efficacy is solely due to the LN targeting, which prompted this study. Methods: First, we generated a fusion protein consisting of an albumin-binding domain (ABD) and an immune-tolerant elastin-like polypeptide (iTEP). Then, we examined the binding between this fusion protein, termed ABD-iTEP, and mouse serum albumin (MSA). Next, we evaluated the accumulation of ABD-iTEP in LNs and dendritic cells (DCs) in the LNs. We also analyzed antigen presentation and in vitro T cell activation of vaccines that were delivered by ABD-iTEP and investigated possible underlying mechanisms of the presentation and activation results. Last, we measured CTL responses induced by ABD-iTEP-delivered vaccines in vivo. Results: ABD-iTEP bound with MSA strongly with an affinity of 1.41 nM. This albumin-binding carrier, ABD-iTEP, accumulated in LNs 3-fold more than iTEP, a control carrier that did not bind with albumin. ABD-iTEP also resulted in 4-fold more accumulation in DCs in the LNs than iTEP. Most importantly, ABD-iTEP drastically enhanced the antigen presentation of its vaccine payloads and the T cell activation induced by its payloads. The enhancement was dependent on the formation of the complex between MSA and ABD-iTEP. Meanwhile, the MSA/ABD-iTEP complex was found to have increased stability in acidic subcellular compartments and increased cytosolic accumulation in DCs, which might explain the enhanced vaccine presentation resulting from the complex. Finally, when ABD-iTEP was used to deliver CTL vaccines derived from both self- and non-self-antigens, it boosted the vaccine-induced responses by 2-fold in either case. Conclusion: ABD-iTEP not only targets vaccines to LNs but also promotes the presentation of the vaccines by DCs. Albumin-binding carriers have more than one mechanism to boost the efficacy of CTL vaccines.

Vaccine carriers have been shown to enhance cytotoxic T lymphocyte (CTL) epitope peptide vaccines by addressing intrinsic limitations of the vaccines. We have previously developed an immune-tolerant elastin-like polypeptide (iTEP)-based nanoparticle (NP) as an effective and unique CTL vaccine carrier. The NP is unique for its humoral immune tolerance, flexible structure, and ability to deliver CTL vaccines as polypeptide fusions. Here, we aimed to improve the NP by increasing its stability since we found it was not stable. We thus generated a more stable iTEP NP (ST-NP) and used it to deliver a CTL peptide vaccine, SIINFEKL. However, we surprisingly found that the ST-NP had a lower efficiency than the previously developed, marginally stable iTEP NP (MS-NP) in terms of promoting vaccine presentation and vaccine-induced CTL responses. On the other hand, dendritic cells (DCs) showed preferential uptake of the ST-NP but not the MS-NP. To develop an iTEP vaccine carrier that outperforms both the MS-NP and the ST-NP, we devised an iTEP NP that has a changeable stability responsive to a cytosolic, reductive environment, termed reductive environment-dependent NP or RED-NP. The RED-NP showed an intermediate ability to promote vaccine presentation and T cell responses in vitro between the MS-NP and the ST-NP. However, the RED-NP induced the strongest CTL responses in vivo among all three NPs. In conclusion, iTEP NPs that have a dynamically changeable stability are most effective to deliver and enhance CTL peptide vaccines. The work also demonstrated the versatile nature of iTEP vaccine carriers.

Dendritic cell (DC)-based cytotoxic T lymphocyte (CTL) epitope vaccines are effective to induce CTL responses but require complex ex vivo DC preparation and epitope-loading. To take advantage of DC-based epitope vaccines without involving the ex vivo procedures, we aimed to develop carriers to directly load CTL epitopes onto DCs in vivo. Here, we first engineered a carrier consisting of a hydrophilic polypeptide, immune-tolerant elastin-like polypeptide (iTEP) and a substrate peptide of matrix metalloproteinases-9 (sMMP). The iTEP was able to solubilize CTL epitopes. CTL epitopes were connected to the carrier, iTEP-sMMP, through sMMP so that the epitopes can be cleaved from the carrier by MMP-9. iTEP-sMMP was found to release its epitope payloads in the DC culture media, which contained MMP-9 released from DCs. iTEP-sMMP allowed for the direct loading of CTL epitopes onto the surface MHC class I complexes of DCs. Importantly, iTEP-sMMP resulted in greater epitope presentation by DCs both in vitro and in vivo than a control carrier that cannot directly load epitopes. iTEP-sMMP also induced 2-fold stronger immune responses than the control carrier. To further enhance the direct epitope-loading strategy, we furnished iTEP-sMMP with an albumin-binding domain (ABD) and found the new carrier, ABD-iTEP-sMMP, had greater lymph node (LN) accumulation than iTEP-sMMP. ABD-iTEP-sMMP also resulted in greater immune responses than iTEP-sMMP by 1.5-fold. Importantly, ABD-iTEP-sMMP-delivered CTL epitope vaccine induced stronger immune responses than free CTL epitope vaccine. Taken together, these carriers utilized two physiological features of DCs to realize direct epitope-loading in vivo: the accumulation of DCs in LNs and MMP-9 released from DCs. These carriers are a potential substitute for DC-based CTL epitope vaccines.

Cytotoxic T lymphocyte (CTL)-mediated immune responses are the primary defense mechanism against cancer and infection. CTL epitope peptides have been used as vaccines to boost CTL responses; however, the efficacy of these peptides is suboptimal. Under current vaccine formulation and delivery strategies, these vaccines are delivered into and processed inside antigen presenting cells such as dendritic cells (DCs). However, the intracellular process is not efficient, which at least partially contributes to the suboptimal efficacy of the vaccines. Thus, we hypothesized that directly loading epitopes onto MHC class I complexes (MHC-Is) on the DC surface would significantly improve the efficacy of the epitopes because the direct loading bypasses inefficient intra-DC vaccine processing. To test the hypothesis, we designed an immune-tolerant elastin-like polypeptide (iTEP)-delivered CTL vaccine containing a metalloproteinase-9 (MMP-9) sensitive peptide and an CTL epitope peptide. We found that the epitope was released from this MMP sensitive vaccine through cleavage by DC-secreted MMP-9 outside of the DCs. The released epitopes were directly loaded onto MHC-Is on the DC surface. Ultimately, the MMP sensitive vaccine strikingly increased epitope presentation by DCs by 7-fold and enhanced the epitope-specific CD8+ T cell response by as high as 9.6-fold compared to the vaccine that was uncleavable by MMP. In summary, this novel direct loading strategy drastically boosted vaccine efficacy. This study offered a new avenue to enhance CTL vaccines.

Cytotoxic T lymphocyte (CTL) epitope peptide-based vaccines are widely used in cancer and infectious disease therapy. We previously generated an immune-tolerant elastin-like polypeptides (iTEPs)-based carrier to deliver a peptide CTL vaccine and enhance the efficiency of the vaccine. To further optimize the vaccine carrier, we intended to potentiate its function by designing an iTEP-based carrier that was able to deliver adjuvant and a vaccine epitope as one molecule. Thus, we fused a 9-mer H100, a peptide derived from the high-mobility group box 1 protein (HMGB1) that could induce activation of dendritic cells (DCs), with an iTEP polymer to generate a new iTEP polymer named H100-iTEP. The H100-iTEP still kept the feature of reversible phase transition of iTEPs and should be able to be used as a polymer carrier to deliver peptide vaccines. The expression levels of CD80/CD86 on DCs were assessed using flow cytometry. The iTEP fusion-stimulated IL-6 secretion by DCs was measured with ELISA. Activation of antigen-specific CD8+ T cells induced by iTEP fusions was examined through a B3Z hybridoma cell activation assay. In vivo CTL activation promoted by iTEP fusions was detected by an IFN-γ-based ELISPOT assay. The iTEP fused with H100 could induce maturation of DCs in vitro as evidenced by increased CD80 and CD86 expression. The iTEP fusion also promoted activation of DCs by increasing secretion of a proinflammatory cytokine IL-6. The N-terminus or C-terminus fusion of H100 to iTEP had a similar effect and a reduced form of cysteine in iTEP fusions was required for DC stimulation. iTEP fusions potentiated a co-administrated CTL vaccine by increasing an antigen-specific CTL response in vitro and in vivo. When the H100-iTEP was fused to a CTL epitope to generate a one-molecule vaccine, this self-adjuvanted vaccine elicited a stronger antigen-specific CTL response than a vaccine adjuvanted by Incomplete Freund's Adjuvant. Thus, we have successfully generated a functional, one-molecule iTEP-based self-adjuvanted vaccine.

Cancer immune checkpoint therapy has achieved remarkable clinical successes in various cancers. However, current immune checkpoint inhibitors block the checkpoint of not only the immune cells that are important to cancer therapy but also the immune cells that are irrelevant to the therapy. Such an indiscriminate blockade limits the efficacy and causes the autoimmune toxicity of the therapy. It might be beneficial to use a carrier to target immune checkpoint inhibitors to cancer-reactive immune cells. Here, we explore a method to load the inhibitors into carriers. We used the anti-programmed death-1 antibody (αPD-1) as a model immune checkpoint inhibitor. First, we generated a recombinant single-chain variable fragment (scFv) of αPD-1. Then, we designed and generated a fusion protein consisting of the scFv and an amphiphilic immune-tolerant elastin-like polypeptide (iTEP). Because of the amphiphilic iTEP, the fusion was able to self-assemble into a nanoparticle (NP). The NP was proved to block the PD-1 immune checkpoint in vitro and in vivo. Particularly, the NP exacerbated diabetes development in non-obese diabetic mice as effectively as natural, intact αPD-1. In summary, we successfully expressed αPD-1 as a recombinant protein and linked αPD-1 to a NP, which lays a foundation to develop a delivery system to target αPD-1 to a subpopulation of immune cells.

Cancer stem cell (CSC) inhibitors are a new category of investigational drugs to treat metastasis. Salinomycin (Sali) is one of most studied CSC inhibitors and has reached clinical tests. Several drug carriers have been developed to improve efficacy of Sali. However, Sali has not been shown to inhibit metastasis from orthotopic tumors, the gold standard for metastasis. To fill this gap, we developed an immune-tolerant, elastin-like polypeptide (iTEP)-based nanoparticle (iTEP-Sali-ABA NP) that released 4-(aminomethyl)benzaldehyde-modified Sali (Sali-ABA) under acidic conditions. We found that the NP increased the area under the curve (AUC) of Sali-ABA by 30-fold and the tumor accumulation by 3.4-fold. Furthermore, no metastasis was detected in any of the mice given the NP. However, all the mice died of primary tumor burdens. To overcome primary tumor growth and improve the overall survival, we applied a combination therapy consisting of the iTEP-Sali-ABA NP and iTEP NP-delivered paclitaxel. This therapy effectively retarded primary tumor growth, and most importantly, improved the overall survival. In conclusion, delivery of Sali-ABA by the NP, alone or in combination with paclitaxel, was more effective than free Sali-ABA in decreasing metastasis and increasing survival. This iTEP-Sali-ABA NP represents a novel and clinically promising therapy to combat metastasis.

Background: Cytotoxic T lymphocyte (CTL) vaccine carriers are known to enhance the efficacy of vaccines, but a search for more effective carriers is warranted. Elastin-like polypeptides (ELPs) have been examined for many medical applications but not as CTL vaccine carriers. Purpose: We aimed to create immune tolerant ELPs using a new polypeptide engineering practice and create CTL vaccine carriers using the ELPs. Results: Four sets of novel ELPs, termed immune-tolerant elastin-like polypeptide (iTEP) were generated according to the principles dictating humoral immunogenicity of polypeptides and phase transition property of ELPs. The iTEPs were non-immunogenic in mice. Their phase transition feature was confirmed through a turbidity assay. An iTEP nanoparticle (NP) was assembled from an amphiphilic iTEP copolymer plus a CTL peptide vaccine, SIINFEKL. The NP facilitated the presentation of the vaccine by dendritic cells (DCs) and enhanced vaccine-induced CTL responses. Discussion: A new ELP design and development practice was established. The non-canonical motif and the immune tolerant nature of the iTEPs broaden our insights about ELPs. ELPs, for the first time, were successfully used as carriers for CTL vaccines. Conclusion: It is feasible to concurrently engineer both immune-tolerant and functional peptide materials. ELPs are a promising type of CTL vaccine carriers.

Intron-containing pre-mRNAs are normally retained and processed in the nucleus but are sometimes exported to the cytoplasm and degraded by the nonsense-mediated mRNA decay (NMD) pathway as a consequence of their inclusion of intronic in-frame termination codons. When shunted to the cytoplasm by autoregulated nuclear export, the intron-containing yeast YRA1 pre-mRNA evades NMD and is targeted by a cytoplasmic decay pathway mediated by the decapping activator Edc3p. Here, we have elucidated this transcript-specific decay mechanism, showing that Edc3p-mediated YRA1 pre-mRNA degradation occurs independently of translation and is controlled through five structurally distinct but functionally interdependent modular elements in the YRA1 intron. Two of these elements target the pre-mRNA as an Edc3p substrate and the other three mediate transcript-specific translational repression. Translational repression of YRA1 pre-mRNA also requires the heterodimeric Mex67p/Mtr2p general mRNA export receptor, but not Edc3p, and serves to enhance Edc3p substrate specificity by inhibiting the susceptibility of this pre-mRNA to NMD. Collectively, our data indicate that YRA1 pre-mRNA degradation is a highly regulated process that proceeds through translational repression, substrate recognition by Edc3p, recruitment of the Dcp1p/Dcp2p decapping enzyme, and activation of decapping.