Folate-Targeted Nanoliposomal Chemophototherapy.

Light-responsive liposomes have been developed for the on-demand release of drugs. However, efficient delivery of chemotherapeutic drugs to tumor for cancer theranostics remains a challenge. Herein, folic acid (FA), an established ligand for targeted drug delivery, was used to decorate light-sensitive porphyrin-phospholipid (PoP) liposomes, which were assessed for FA-targeted chemophototherapy (CPT). PoP liposomes and FA-conjugated PoP liposomes were loaded with Doxorubicin (Dox), and physical properties were characterized. In vitro, FA-PoP liposomes that were incubated with FA receptor-overexpressing human KB cancer cells showed increased uptake compared to non-targeted PoP liposomes. Dox and PoP contributed towards chemophototherapy (CPT) in vitro, and PoP and FA-PoP liposomes induced cell killing. In vivo, mice bearing subcutaneous KB tumors treated with PoP or FA-PoP liposomes loaded with Dox, followed by 665 nm laser treatment, had delayed tumor growth and improved survival. Dox delivery to tumors increased following laser irradiation for both PoP and FA-PoP liposomes. Thus, while Dox-FA-PoP liposomes were effective following systemic administration and local light irradiation in this tumor model, the FA targeting moiety did not appear essential for anti-tumor responses.

Antibody induction in mice by liposome-displayed recombinant enterotoxigenic Escherichia coli (ETEC) colonization antigens.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) strains cause infectious diarrhea and colonize host intestine epithelia via surface-expressed colonization factors. Colonization factor antigen I (CFA/I), a prevalent ETEC colonization factor, is a vaccine target since antibodies directed to this fimbria can block ETEC adherence and prevent diarrhea. METHODS: Two recombinant antigens derived from CFA/I were investigated with a vaccine adjuvant system that displays soluble antigens on the surface of immunogenic liposomes. The first antigen, CfaEB, is a chimeric fusion protein comprising the minor (CfaE) and major (CfaB) subunits of CFA/I. The second, CfaEad, is the adhesin domain of CfaE. RESULTS: Owing to their His-tag, recombinant CfaEB and CfaEad, spontaneously bound upon admixture with nanoliposomes containing cobalt-porphyrin phospholipid (CoPoP), as well as a synthetic monophosphoryl lipid A (PHAD) adjuvant. Intramuscular immunization of mice with sub-microgram doses CfaEB or CfaEad admixed with CoPoP/PHAD liposomes elicited serum IgG and intestinal IgA antibodies. The smaller CfaEad antigen benefitted more from liposome display. Serum and intestine antibodies from mice immunized with liposome-displayed CfaEB or CfaEad recognized native CFA/I fimbria as evidenced by immunofluorescence and hemagglutination inhibition assays using the CFA/I-expressing H10407 ETEC strain. CONCLUSION: These data show that colonization factor-derived recombinant ETEC antigens exhibit immunogenicity when delivered in immunogenic particle-based formulations.

Immune checkpoint blockade enhances chemophototherapy in a syngeneic pancreatic tumor model.

Pancreatic cancer (PaCa) suffers from poor treatment options for locally advanced cases. Chemophototherapy (CPT) is an emerging anti-tumor modality, and porphyrin-phospholipid liposomes have been shown to be versatile drug carriers for CPT in preclinical rodent models. Here we show that in the syngeneic subcutaneous KPC PaCa tumor model, exhausted CD8+ T cells are localized in the tumor, and that CPT is enhanced in combination with immune checkpoint blockade (ICB). Addition of ICB using anti-programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies resulted in ablation of medium-sized, established KPC tumors (∼200 mm3) without recurrence for over 100 days. Mice rejected subsequent tumor re-challenge. Flow cytometry and tumor slice analysis following injection of a fluorescently labeled anti-PD-1 antibody showed that CPT improved antibody delivery to the tumor microenvironment. Treatment of large established tumors (∼400 mm3) using with CPT and ICB induced appreciable tumor regression and delay in regrowth. Taken together, these data demonstrate the utility of combining CPT with immunotherapies.

An In Vivo Screen to Identify Short Peptide Mimotopes with Enhanced Antitumor Immunogenicity.

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.

Immunization with short peptide particles reveals a functional CD8+ T-cell neoepitope in a murine renal carcinoma model.

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.

Position-Scanning Peptide Libraries as Particle Immunogens for Improving CD8+ T-Cell Responses.

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.

Labeling of Erythrocytes by Porphyrin-Phospholipid.

A method is developed for membrane labeling of erythrocytes with porphyrin-phospholipid (PoP). To generate a concentrated PoP solution for labeling human red blood cells (RBCs), various surfactants and solvents are screened to identify conditions that avoid hemolysis, while minimizing non-specific PoP co-precipitation with RBCs in the pellet during centrifugation washes. Cholate, Tween 80 and Tween 40 are identified as useful surfactants for this purpose. When labeled RBCs are mixed with unlabeled ones, substantial non-specific PoP exchange is observed. Egg-yolk lecithin is included in a washing buffer to remove loosely bound PoP and reduce PoP exchange with unlabeled erythrocytes, based on flow cytometry and photodynamic hemolysis assays. Murine RBCs that are labeled with 64Cu-chelated PoP displayed altered biodistribution with longer blood circulation relative to directly administered 64Cu-chelated PoP.

A liposome-displayed hemagglutinin vaccine platform protects mice and ferrets from heterologous influenza virus challenge.

Recombinant influenza virus vaccines based on hemagglutinin (HA) hold the potential to accelerate production timelines and improve efficacy relative to traditional egg-based platforms. Here, we assess a vaccine adjuvant system comprised of immunogenic liposomes that spontaneously convert soluble antigens into a particle format, displayed on the bilayer surface. When trimeric H3 HA was presented on liposomes, antigen delivery to macrophages was improved in vitro, and strong functional antibody responses were induced following intramuscular immunization of mice. Protection was conferred against challenge with a heterologous strain of H3N2 virus, and naive mice were also protected following passive serum transfer. When admixed with the particle-forming liposomes, immunization reduced viral infection severity at vaccine doses as low as 2 ng HA, highlighting dose-sparing potential. In ferrets, immunization induced neutralizing antibodies that reduced the upper respiratory viral load upon challenge with a more modern, heterologous H3N2 viral strain. To demonstrate the flexibility and modular nature of the liposome system, 10 recombinant surface antigens representing distinct influenza virus strains were bound simultaneously to generate a highly multivalent protein particle that with 5 ng individual antigen dosing induced antibodies in mice that specifically recognized the constituent immunogens and conferred protection against heterologous H5N1 influenza virus challenge. Taken together, these results show that stable presentation of recombinant HA on immunogenic liposome surfaces in an arrayed fashion enhances functional immune responses and warrants further attention for the development of broadly protective influenza virus vaccines.

HPV-Associated Tumor Eradication by Vaccination with Synthetic Short Peptides and Particle-Forming Liposomes.

Human papilloma virus (HPV)-16 is associated with cervical cancers and induces expression of the E6 and E7 oncogenes. Using a murine cell line that expresses these, the genes are sequenced, and six predicted major histocompatibility complex (MHC) class I (MHC-I) epitopes are identified. A liposomal vaccine adjuvant based on cobalt-porphyrin-phospholipid (CoPoP) is admixed with synthetic 9-mer epitopes appended with three histidine residues, resulting in rapid formation of peptide-liposome particles. Immunization with multivalent peptides leads to protection from tumor challenge. Of the peptides screened, only the previously identified E749-57 epitope is functional. The peptide-liposome particles that form upon mixing E7HHH49-57 with CoPoP liposomes are stable in serum and are avidly taken up by immune cells in vitro. Immunization results in robust protection from tumor challenge and re-challenge. A 100 ng peptide dose protects mice in a therapeutic tumor challenge when admixed with CoPoP liposomes, whereas 200-fold higher peptide doses are ineffective with the polyinosinic-polycytidylic (poly(I:C)) adjuvant. CoPoP induces a strong infiltrating CD8+ T-cell response within the tumor microenvironment with an improved functional profile. Vaccine monotherapy using nanogram dosing of the E7HHH49-57 peptide admixed with CoPoP reverses the growth of large established tumors, eradicating subcutaneous tumors upwards of 100 mm3 . Immunization also eradicates lung tumors in a metastasis model.

A Potent Cancer Vaccine Adjuvant System for Particleization of Short, Synthetic CD8+ T Cell Epitopes.

Short major histocompatibility complex (MHC) class I (MHC-I)-restricted peptides contain the minimal biochemical information to induce antigen (Ag)-specific CD8+ cytotoxic T cell responses but are generally ineffective in doing so. To address this, we developed a cobalt-porphyrin (CoPoP) liposome vaccine adjuvant system that induces rapid particleization of conventional, short synthetic MHC-I epitopes, leading to strong cellular immune responses at nanogram dosing. Along with CoPoP (to induce particle formation of peptides), synthetic monophosphoryl lipid A (PHAD) and QS-21 immunostimulatory molecules were included in the liposome bilayer to generate the "CPQ" adjuvant system. In mice, immunization with a short MHC-I-restricted peptide, derived from glycoprotein 70 (gp70), admixed with CPQ safely generated functional, Ag-specific CD8+ T cells, resulting in the rejection of multiple tumor cell lines, with durable immunity. When cobalt was omitted, the otherwise identical peptide and adjuvant components did not result in peptide binding and were incapable of inducing immune responses, demonstrating the importance of stable particle formation. Immunization with the liposomal vaccine was well-tolerated and could control local and metastatic disease in a therapeutic setting. Mechanistic studies showed that particle-based peptides were better taken up by antigen-presenting cells, where they were putatively released within endosomes and phagosomes for display on MHC-I surfaces. On the basis of the potency of the approach, the platform was demonstrated as a tool for in vivo epitope screening of peptide microlibraries comprising a hundred peptides.

Experimental and Computational Observations of Immunogenic Cobalt Porphyrin Lipid Bilayers: Nanodomain-Enhanced Antigen Association.

Cobalt porphyrin phospholipid (CoPoP) can incorporate within bilayers to enable non-covalent surface-display of antigens on liposomes by mixing with proteins bearing a polyhistidine tag (his-tag); however, the mechanisms for how this occurs are poorly understood. These were investigated using the his-tagged model antigen Pfs25, a protein antigen candidate for malaria transmission-blocking vaccines. Pfs25 was found to associate with the small molecule aquocobalamin, a form of vitamin B12 and a cobalt-containing corrin macrocycle, but without particle formation, enabling comparative assessment. Relative to CoPoP liposomes, binding and serum stability studies indicated a weaker association of Pfs25 to aquocobalamin or cobalt nitrilotriacetic acid (Co-NTA) liposomes, which have cobalt displayed in the aqueous phase on lipid headgroups. Antigen internalization by macrophages was enhanced with Pfs25 bound to CoPoP liposomes. Immunization in mice with Pfs25 bound to CoPoP liposomes elicited antibodies that recognized ookinetes and showed transmission-reducing activity. To explore the physical mechanisms involved, we employed molecular dynamics (MD) simulations of bilayers containing phospholipid, cholesterol, as well as either CoPoP or NTA-functionalized lipids. The results show that the CoPoP-containing bilayer creates nanodomains that allow access for a limited but sufficient amount of water molecules that could be replaced by his-tags due to their favorable free energy properties allowing for stabilization. The position of the metal center within the NTA liposomes was much more exposed to the aqueous environment, which could explain its limited capacity for stabilizing Pfs25. This study illustrates the impact of CoPoP-induced antigen particleization in enhancing vaccine efficacy, and provides molecular insights into the CoPoP bilayer properties that enable this.

SARS-CoV-2 RBD Neutralizing Antibody Induction is Enhanced by Particulate Vaccination.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a candidate vaccine antigen that binds angiotensin-converting enzyme 2 (ACE2), leading to virus entry. Here, it is shown that rapid conversion of recombinant RBD into particulate form via admixing with liposomes containing cobalt-porphyrin-phospholipid (CoPoP) potently enhances the functional antibody response. Antigen binding via His-tag insertion into the CoPoP bilayer results in a serum-stable and conformationally intact display of the RBD on the liposome surface. Compared to other vaccine formulations, immunization using CoPoP liposomes admixed with recombinant RBD induces multiple orders of magnitude higher levels of antibody titers in mice that neutralize pseudovirus cell entry, block RBD interaction with ACE2, and inhibit live virus replication. Enhanced immunogenicity can be accounted for by greater RBD uptake into antigen-presenting cells in particulate form and improved immune cell infiltration in draining lymph nodes. QS-21 inclusion in the liposomes results in an enhanced antigen-specific polyfunctional T cell response. In mice, high dose immunization results in minimal local reactogenicity, is well-tolerated, and does not elevate serum cobalt levels. Taken together, these results confirm that particulate presentation strategies for the RBD immunogen should be considered for inducing strongly neutralizing antibody responses against SARS-CoV-2.

Antibody response of a particle-inducing, liposome vaccine adjuvant admixed with a Pfs230 fragment.

Pfs230 is a malaria transmission-blocking antigen candidate, expressed on the surface of Plasmodium falciparum gametocytes. A recombinant, his-tagged Pfs230 fragment (Pfs230C1; amino acids 443-731) formed serum-stable particles upon incubation with liposomes containing cobalt-porphyrin-phospholipid (CoPoP). In mice, immunization with Pfs230C1, admixed with the adjuvants Alum, Montanide ISA720 or CoPoP liposomes (also containing synthetic monophosphoryl lipid A; PHAD), resulted in elicitation of IgG antibodies, but only those induced with CoPoP/PHAD or ISA720 strongly reduced parasite transmission. Immunization with micrograms of Pfs230C1 adjuvanted with identical liposomes lacking cobalt (that did not induce particle formation) or Alum was less effective than immunization with nanograms of Pfs230C1 with CoPoP/PHAD. CoPoP/PHAD and ISA720 adjuvants induced antibodies with similar Pfs230C1 avidity but higher IgG2-to-IgG1 ratios than Alum, which likely contributed to enhanced functional activity. Unlike prior work with another transmission-blocking antigen (Pfs25), Pfs230C1 was found to be effectively taken up by antigen-presenting cells without particle formation. The anti-Pfs230C1 IgG response was durable in mice for 250 days following immunization with CoPoP/PHAD, as were antibody avidity and elevated IgG2-to-IgG1 ratios. Immunization of rabbits with 20 µg Pfs230C1 admixed with CoPoP/PHAD elicited antibodies that inhibited parasite transmission. Taken together, these results show that liposomes containing CoPoP and PHAD are an effective vaccine adjuvant platform for recombinant malaria transmission blocking antigens.

Immunogenicity of the Lyme disease antigen OspA, particleized by cobalt porphyrin-phospholipid liposomes.

Outer surface protein A (OspA) is a Borrelia lipoprotein and an established Lyme disease vaccine target. Admixing non-lipidated, recombinant B. burgdorferi OspA with liposomes containing cobalt porphyrin-phospholipid (CoPoP) resulted in rapid, particulate surface display of the conformationally intact antigen. Particleization was serum-stable and led to enhanced antigen uptake in murine macrophages in vitro. Mouse immunization using CoPoP liposomes that also contained a synthetic monophosphoryl lipid A (PHAD) elicited a Th1-biased OspA antibody response with higher IgG production compared to other vaccine adjuvants. Antibodies were reactive with intact B. burgdorferi spirochetes and Borrelia lysates, and induced complement-mediated borreliacidal activity in vitro. One year after initial immunization, mice maintained high levels of circulating borreliacidal antibodies capable of blocking B. burgdorferi transmission from infected ticks to human blood in a feeding chamber.

A malaria vaccine adjuvant based on recombinant antigen binding to liposomes.

Pfs25 is a malaria transmission-blocking vaccine antigen candidate, but its apparently limited immunogenicity in humans has hindered clinical development. Here, we show that recombinant, polyhistidine-tagged (his-tagged) Pfs25 can be mixed at the time of immunization with pre-formed liposomes containing cobalt porphyrin-phospholipid, resulting in spontaneous nanoliposome antigen particleization (SNAP). Antigens are stably presented in uniformly orientated display via his-tag insertion in the cobalt porphyrin-phospholipid bilayer, without covalent modification or disruption of antigen conformation. SNAP immunization of mice and rabbits is well tolerated with minimal local reactogenicity, and results in orders-of-magnitude higher functional antibody generation compared with other 'mix-and-inject' adjuvants. Serum-stable antigen binding during transit to draining lymph nodes leads to enhanced antigen uptake by phagocytic antigen-presenting cells, with subsequent generation of long-lived, antigen-specific plasma cells. Seamless multiplexing with four additional his-tagged Plasmodium falciparum polypeptides induces strong and balanced antibody production, illustrating the simplicity of developing multistage particulate vaccines with SNAP immunization.

Short Drug-Light Intervals Improve Liposomal Chemophototherapy in Mice Bearing MIA PaCa-2 Xenografts.

Chemophototherapy (CPT) is an emerging tumor treatment that combines phototherapy and chemotherapy. Long-circulating (LC) liposomes can stably incorporate 2 mol % porphyrin-phospholipid (PoP) in the bilayer and load doxorubicin (Dox) to generate LC-Dox-PoP liposomes, for single-agent CPT. Following intravenous administration to mice, LC-Dox-PoP liposomes (2 mg/kg Dox) circulated with similar blood concentration ranges produced by a typical human clinical dose of DOXIL (50 mg/m2 Dox). This dosing approach aims to achieve physiologically relevant Dox and PoP concentrations as well as CPT vascular responses in mice bearing subcutaneous human pancreatic MIA PaCa-2 xenografts. Phototreatment with 2 mg/kg LC-Dox-PoP induced vascular permeabilization, leading to a 12.5-fold increase in Dox tumor influx estimated by a pharmacokinetic model, based on experimental data. Shorter drug-light intervals (0.5-3 h) led to greater tumoral drug deposition and improved treatment outcomes, compared to longer drug-light intervals. At 2 mg/kg Dox, CPT with LC-Dox-PoP liposomes induced tumor regression and growth inhibition, whereas chemotherapy using several other formulations of Dox did not. LC-Dox-PoP liposomes were well tolerated at the 2 mg/kg dose.

RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells.

Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvß3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvß3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvß3 integrin-overexpressing cancer cells.