Targeted Liposome Based Delivery System to Present HLA Class I Antigens to Tumor Cells: Two papers
Reporter: Stephen J. Williams, Ph.D.
Modular Three-component Delivery System Facilitates HLA Class I Antigen Presentation and CD8+ T-cell Activation Against Tumors
Cell-mediated immunotherapies have potential as stand-alone and adjuvant therapies for cancer. However, most current protocols suffer from one or more of three major issues: cost, safety, or efficacy. Here we present a nanoparticle delivery system that facilitates presentation of an immunogenic measles antigen specifically in cancer cells. The delivery system does not contain viral particles, toxins, or biologically derived material. Treatment with this system facilitates activation of a secondary immune response against cancer cells, bypassing the need to identify tumor-associated antigens or educate the immune system through a primary immune response. The delivery system consists of a stealth liposome displaying a cancer-specific targeting peptide, named H1299.3, on its exterior surface and encapsulating H250, an immunogenic human leukocyte antigen class 1 restricted peptide. This targeted-nanoparticle facilitates presentation of the H250 peptide in major histocompatibility complex class I molecules. Activation is dependent on the targeting peptide, previous antigen exposure, and utilizes a novel autophagy-mediated mechanism to facilitate presentation. Treatment with this liposome results in a significant reduction of tumor growth using an aggressive LLC1 model in vaccinated C57BL/6 mice. These data provide proof-of-principle for a novel cell-mediated immunotherapy that is scalable, contains no biologically derived material, and is an efficacious cancer therapy.
Cell-mediated (CM) immunotherapies for cancer treatment are designed to activate the body’s adaptive immune responses against a malignant growth.1,2 Generally, the goal of a CM response is to activate a cytotoxic T-cell response against a tumor to eliminate cancer cells. The principle of these treatments is straightforward, yet current work studying the complexity of the tumor micro-environment2,3 as well as methods that attempt to directly activate T cells against tumor antigens4,5,6 demonstrate the difficulty associated generating an immune response against a tumor.
Several CM cancer immunotherapies exist today. Major examples include PD-1 inhibitors, injection of live virus or viral particles into tumors, and adoptive T-cell therapies.1,6,7,8 However, concerns regarding efficacy, safety, and/or cost have limited the use of many of these treatments. To address these concerns, we sought to develop a novel treatment based on developing a fully synthetic, minimal delivery system that facilitates presentation of human leukocyte antigen (HLA) class I restricted immunogenic peptides specifically on cancer cells without using live virus, viral subunits, or biologically derived material.
Based on these requirements, we developed a liposomal based agent consisting of a neutral, stealth liposome that encapsulates a synthetically manufactured immunogenic HLA class I restricted peptide derived from measles virus.1,2,9 In addition, the liposome has a targeting peptide on the external surface that both specifically accumulates in cancer cells and facilitates presentation of the immunogenic peptide in HLA class I molecules (Figure 1a). Thus, this treatment is designed to generate a secondary CM immune response specifically against the tumor if the patient was previously vaccinated against or infected with measles.
In this proof-of-concept study, we synthesized a liposome that encapsulates H250,1 an immunogenic HLA class 1 restricted peptide identified from measles hemagglutinin protein. The liposome is designed to specifically internalize in cancer cells by displaying the recently identified targeting peptide H1299.3 on the exterior surface (Figure 1b).10 H1299.3 is a 20mer, cancer-specific targeting peptide that was recently identified by our group. The peptide was identified using a novel phage display technique that allows for selection of cancer-specific targeting peptides that preferentially internalize in cancer cells via a defined mechanism of endocytosis. This peptide was dimerized on a lysine core and is fully functional outside the context of the phage particle. The H1299.3 peptide accumulates specifically in a panel of non-small cell lung cancer (NSCLC) cell lines compared to a normal bronchial epithelial cell control cell line via a clathrin-dependent mechanism of endocytosis. In this study, we demonstrate that H1299.3 facilitates functional presentation of an immunogenic antigen in both major histocompatibility complex (MHC) and HLA class I molecules as indicated by CD8+-specific interferon (IFN)γ secretion. In addition, H1299.3 facilitated presentation utilizes an autophagy-dependent mechanism. Finally, treatment with H1299.3 targeted liposomes containing H250 substantially reduces the growth rate of subcutaneous LLC1 tumors implanted in vaccinated C57BL/6 mice compared to treatment with vehicle control.
- The H1299.3 targeting ligand specifically accumulates in cancer and facilitates HLA class I presentation: H250 is an immunogenic peptide identified from sequencing peptides present in HLA A*0201 molecules following measles infection. identified two donors that were HLA A*02 positive and had previously been vaccinated against measles virus (the human NSCLC cell line, H1993, which we determined to be HLA A*02 positive)
- identified three different cancer-specific targeting peptides that internalize into H1993 that have been previously published: H1299.2, H2009.1, and H1299.3. Each of these peptides specifically internalize in NSCLC cell lines compared to normal bronchial epithelial cells
- H1299.3 facilitated HLA class I presentation requires autophagy. H1299.3 peptide colocalizes with Lamp-1 which is a marker of both lysosomes and autolysosomes, therefore it was possible autophagy involved and shown that H1299.3 colocalizes with autophagosomes. Chlorpromazine, which inhibits clathrin coated mediatated endocytosis, decreased the HLA1 presentation of H250.
- H1299.3-targeted liposomes encapsulating H250 reduce tumor burden in vivo. Mice were first vaccinated against H250. The J1299.3 targeted liposome encapsulation H250 reduced tumor growth of LLC1 s.c. xenograpfts by 50%.
Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy.
Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge.
To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro.
We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses.
Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.
- [PubMed – indexed for MEDLINE]