A nano-enhanced vaccine for metastatic melanoma immunotherapy

Abstract

Aim: Despite the huge advancements in cancer therapies and treatments over the past decade, most patients with metastasized melanoma still die from the disease. This poor prognosis largely results from resistance to conventional chemotherapies and other cytotoxic drugs. We have previously identified 6 antigenic peptides derived from melanomas that have proven efficacious for activating CD4⁺ T cells in clinical trials for melanoma. Our aim was to improve pharmacodynamics, pharmacokinetic and toxicological parameters by individually encapsulating each of the 6 melanoma helper peptides within their own immunogenic nanoliposomes. Methods: We modified these liposomes as necessary to account for differences in the peptides' chemical properties, resulting in 3 distinct formulations. To further enhance immunogenicity, we also incorporated KDO2, a TLR4 agonist, into the lipid bilayer of all nanoliposome formulations. We then conducted in vivo imaging studies in mice and ex vivo cell studies from 2 patient samples who both strongly expressed one of the identified peptides. Results: We demonstrate that these liposomes, loaded with the different melanoma helper peptides, can be readily mixed together and simultaneously delivered without toxicity in vivo. These liposomes are capable of being diffused to the secondary lymphoid organs very quickly and for at least 6 days. In addition, we show that these immunogenic liposomes enhance immune responses to specific peptides ex vivo. Conclusion: Lipid-based delivery systems, including nanoliposomes and lipid nanoparticles, have now been validated for pharmacological (small molecules, bioactive lipids) and molecular (mRNA, siRNA) therapeutic approaches. However, the utility of these formulations as cancer vaccines, delivering antigenic peptides, has not yet achieved the same degree of commercial success. Here, we describe the novel and successful development of a nanoliposome-based cancer vaccine for melanoma. These vaccines help to circumvent drug resistance by increasing a patient's T cell response, making them more susceptible to checkpoint blockade therapy.

Keywords: Nanoliposomes; cancer vaccines; melanoma drug resistance; metastasized melanoma; nanoscale drug delivery; peptides.

Figure 1

Typical dynamic light scattering (DLS) data attained for each of the 6 melanoma helper peptides. The data shows size distribution by intensity with size, d in nanometers along the X-axis and percentage intensity on the Y axis. (A) shows the DLS profile of the AQN peptide; (B) the DLS profile of the WNR peptide; (C) is that of LLK; (D) FLL; (E) RNG; and (F) is the typical profile for the TSY peptide. The graphs show a homogenous size distribution for all 6 peptides. The Z-average size (d.nm) attained from multiple samples is also provided for each peptide. AQN: AQNILLSNAPLGPQFP; WNR: WNRQLYPEWTEAQRLD; LLK: LLKYRAREPVTKAE; FLL: FLLHHAFVDSIFEQWLQRHRP; RNG: RNGYRALMDKSLHVGTQCALTRR; TSY: TSYVKVLHHMVKISG.

Figure 2

Dynamic light scattering data attained after mixing all 6 peptide nanoliposome formulations together. The data shows size distribution by intensity, with size (d) in nanometers along the X-axis and percentage intensity on the Y axis. The graphs show a homogenous size distribution. The Z-average size (d.nm) attained for this data is 113.5 nm.

Figure 3

Fluorescent imaging of liposome biodistribution in mice on days 0 and 6. Two mice (SQ group) were injected subcutaneously in either flank and 2 mice (IV group) were injected intravenously.

Figure 4

Showing the mean radiant efficiency in the extracted organs of four mice, 2 of which were injected with fluorescent and immunogenic liposomes via intravenous (IV) injection and 2, who were administered the same nanoliposomes via subcutaneous (SQ) injection in either flank.

Figure 5

(A) Graph of lymphocyte viabilities of Sentinel Immunized Nodes (SIN) post-treatment. Thick horizontal bars represent mean viability among treatments for a donor; (B) showing a graph of average proliferation of CD4⁺ cell populations after culture treatments with (i) an empty anionic liposome, (ii) an anionic liposome containing KDO2 but no peptide; (iii) Free TSY (no liposome); (iv) TSY encapsulated in an anionic liposome; and (v) an anionic liposome containing TSY and KDO2; and (C) showing the 2D histograms used to calculate the data in 5B. PBMC: Peripheral blood mononuclear cells.

Figure 6

Showing (A) a schematic diagram of the unilamellar nanoliposome with the peptide dissolved within a stabilizing pH controlled buffer within the aqueous liposome core. Cholesterol embeds within the lipid bilayer; and a sparse PEG brush and immunogenic KDO2 lipid head group are arranged around the outer shell of the nanoliposome. The liposome is stored as a suspension in 1X PBS. It should be noted that additional lipid components are added to create either a positive or negative charge in some of the formulations. A fluorophore may also be added, which depending on the fluorophore used, may embed within the bilayer like cholesterol or be attached to a lipid and incorporate within the bilayer like PEG and KDO2. (B) shows the structure of KDO2-lipid A, which has 6 fatty acid chains and a head group on the surface.