RESUMEN
Cancer vaccines aim at generating cytotoxic CD8+ T cells that kill cancer cells and confer durable tumor regression. Hereto, CD8+ peptide epitopes should be presented by antigen presenting cells to CD8+ T cells in lymphoid tissue. Unfortunately, in unformulated soluble form, peptide antigens are poorly taken up by antigen presenting cells and do not efficiently reach lymph nodes. Hence, the lack of efficient delivery remains a major limitation for successful clinical translation of cancer vaccination using peptide antigens. Here we propose a generic peptide nanoformulation strategy by extending the amino acid sequence of the peptide antigen epitope with 10 glutamic acid residues. The resulting overall anionic charge of the peptide allows encapsulation into lipid nanoparticles (peptide-LNP) by electrostatic interaction with an ionizable cationic lipid. We demonstrate that intravenous injection of peptide-LNP efficiently delivers the peptide to immune cells in the spleen. Peptide-LNP that co-encapsulate an imidazoquinoline TLR7/8 agonist (IMDQ) induce robust innate immune activation in a broad range of immune cell subsets in the spleen. Peptide-LNP containing the minimal CD8+ T cell epitope of the HPV type 16 E7 oncoprotein and IMDQ induces high levels of antigen-specific CD8+ T cells in the blood, and can confer protective immunity against E7-expressing tumors in both prophylactic and therapeutic settings.
Asunto(s)
Ratones Endogámicos C57BL , Nanopartículas , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Animales , Nanopartículas/química , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/administración & dosificación , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Lípidos/química , Péptidos/química , Femenino , Proteínas E7 de Papillomavirus/inmunología , Quinolinas/farmacología , Quinolinas/química , Imidazoles/química , Imidazoles/farmacologíaRESUMEN
Poly(I:C) is a synthetic analogue of dsRNA capable of activating both TLR3 and RLRs, such as MDA-5 and RIG-I, as pathogen recognition receptors. While poly(I:C) is known to provoke a robust type I IFN, type III IFN, and Th1 cytokine response, its therapeutic use as a vaccine adjuvant is limited due to its vulnerability to nucleases and poor uptake by immune cells. is encapsulated poly(I:C) into lipid nanoparticles (LNPs) containing an ionizable cationic lipid that can electrostatically interact with poly(I:C). LNP-formulated poly(I:C) triggered both lysosomal TLR3 and cytoplasmic RLRs, in vitro and in vivo, whereas poly(I:C) in an unformulated soluble form only triggered endosomal-localized TLR3. Administration of LNP-formulated poly(I:C) in mouse models led to efficient translocation to lymphoid tissue and concurrent innate immune activation following intramuscular (IM) administration, resulting in a significant increase in innate immune activation compared to unformulated soluble poly(I:C). When used as an adjuvant for recombinant full-length SARS-CoV-2 spike protein, LNP-formulated poly(I:C) elicited potent anti-spike antibody titers, surpassing those of unformulated soluble poly(I:C) by orders of magnitude and offered complete protection against a SARS-CoV-2 viral challenge in vivo, and serum from these mice are capable of significantly reducing viral infection in vitro.
Asunto(s)
Liposomas , Nanopartículas , Poli I-C , Glicoproteína de la Espiga del Coronavirus , Receptor Toll-Like 3 , Animales , Ratones , Humanos , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Adyuvantes Inmunológicos/farmacologíaRESUMEN
Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.
Asunto(s)
Receptor Toll-Like 9 , Vacunas , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/química , Inmunidad Innata , Tejido Linfoide , Oligodesoxirribonucleótidos/farmacología , Oligodesoxirribonucleótidos/químicaRESUMEN
The limited thermostability and need for ultracold storage conditions are the major drawbacks of the currently used nucleoside-modified lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccines, which hamper the distribution of these vaccines in low-resource regions. The LNP core contains, besides mRNA and lipids, a large fraction of water. Therefore, encapsulated mRNA, or at least a part of it, is subjected to hydrolysis mechanisms similar to unformulated mRNA in an aqueous solution. It is likely that the hydrolysis of mRNA and colloidal destabilization are critical factors that decrease the biological activity of mRNA LNPs upon storage under ambient conditions. Hence, lyophilization as a drying technique is a logical and appealing method to improve the thermostability of these vaccines. In this study, we demonstrate that mRNA LNP formulations comprising a reduction-sensitive ionizable lipid can be successfully lyophilized, in the presence of 20% w/v sucrose, both by conventional batch freeze-drying and by an innovative continuous spin lyophilization process. While the chemical structure of the ionizable lipid did not affect the colloidal stability of the LNP after lyophilization and redispersion in an aqueous medium, we found that the ability of LNPs to retain the mRNA payload stably encapsulated, and mediate in vivo and in vitro mRNA translation into protein, post lyophilization strongly depended on the ionizable lipid in the LNP formulation.
Asunto(s)
Lípidos , Nanopartículas , Lípidos/química , ARN Mensajero/genética , Crioprotectores/química , Composición de Medicamentos , Nanopartículas/química , Liofilización , ARN Interferente Pequeño/genéticaRESUMEN
Many anticancer immunotherapeutic agents, including the monoclonal immune checkpoint blocking antibodies, toll-like receptor (TLR) agonists, cytokines and immunostimulatory mRNA are commonly administrated by the intravenous route. Unfortunately, this route is prone to inducing, often life-threatening, side effects through accumulation of these immunotherapeutic agents at off-target tissues. Moreover, additional biological barriers need to be overcome before reaching the tumor microenvironment. By contrast, direct intratumoral injection allows for accomplishing local immune activation and multiple (pre)clinical studies have demonstrated decreased systemic toxicity, improved efficacy as well as abscopal effects. The approval of the oncolytic herpes simplex virus type 1 talimogene laherparepvec (T-VEC) as first approved intratumoral oncolytic virotherapy has fueled the interest to study intensively other immunotherapeutic approaches in preclinical models as well as in clinical context. Moreover, it has been shown that intratumoral administration of immunostimulatory agents successfully synergizes with immune checkpoint inhibitor therapy. Here we review the current state of the art in (pre)clinical intratumoral immunotherapy.