Antigenic Essence: Upgrade of Cellular Cancer Vaccines

Abstract

The development of anticancer immunotherapy is characterized by several approaches, the most recognized of which include cellular vaccines, tumor-associated antigens (TAAs), neoantigens, and chimeric antigen receptor T cells (CAR-T). This paper presents antigenic essence technology as an effective means for the production of new antigen compositions for anticancer vaccination. This technology is developed via proteomics, cell culture technology, and immunological assays. In terms of vaccine development, it does not fit into any of the above-noted approaches and can be considered a new direction. Here we review the development of this technology, its main characteristics, comparison with existing approaches, and the features that distinguish it as a novel approach to anticancer vaccination. This review will also highlight the benefits of this technology over other approaches, such as the ability to control composition, optimize immunogenicity and similarity to target cells, and evade major histocompatibility complex restriction. The first antigenic essence products, presented under the SANTAVAC brand, are also described.

Keywords: SANTAVAC; antiangiogenic vaccine; antigenic essence; cancer vaccine; endothelial cells; mass spectrometry; preventive vaccine; proteomic footprint.

Figure 1

Anticancer vaccination approaches.

Figure 2

Concept of antigenic essence. Actual antigenic properties of live cells are defined by a pool of antigens presented on the cell surface. Intracellular content is considered noise to be excluded from the essence. After washing away traces of culture medium, cells are treated with a purified protease. Released fragments of the cell surface proteins are collected, analyzed by mass spectrometry, and used for vaccination instead of whole cells. Adapted from [29].

Figure 3

Antigenic equivalence of antigenic essence to whole cell lysate. (a) Cytotoxicity of effector cytotoxic T lymphocytes (CTLs) against human adenocarcinoma (MCF-7) cells. Target MCF- 7 cells were seeded in tissue culture plates, and then effector CTLs were added at CTLs:MCF-7 ratio 4:1. (●) MCF-7 cells incubated with CTLs that had been stimulated with antigenic essence-loaded dendritic cells (DCs). (■) MCF-7 cells incubated with CTLs that had been stimulated with lysate-loaded DCs. (○) MCF-7 cells grown with unstimulated CTLs. Points represent the mean value of three identical measurements. Essence induces 10–40% more cytotoxic activity in CTLs than whole cell lysate, even though the total protein concentration of essence formulation was substantially lower (whole cell lysate: 270 µg/mL vs. essence: 2 µg/mL). Adapted from [30]. (b) Growth of hepatoma H22 on BALB/c mice in the experimental group vaccinated with the essence of H22 cells as compared to unvaccinated controls (mean ± SD). Adapted from [32].

Figure 4

The use of proteomic footprinting in the antigenic essence production. After washing away traces of culture medium, adherent cell culture is treated with a protease. Released fragments of cell surface proteins are collected and analyzed by mass spectrometry. The set of obtained peptide molecular weights represents the essence code. Comparison of this code with mass spectra of the reference cells not only allows for authentication of the essence but also reveals any changes in its composition. This proteomic footprinting method was developed as a part of research and development for antigenic essence products and cell authentication at the subtype level. Adapted from [29].

Figure 5

Stages of design for antigenic essence compositions. This vaccine development routine combines cytotoxicity assays (left plot) and target cell footprinting (right plot). In cytotoxicity assays, the dendritic cells (DCs) present essence antigens to CTLs through major histocompatibility complex (MHC). The target cell killing rate (cytotoxicity) by activated CTLs is reflected in the function describing antigenic essence efficacy from the essence/target similarity (i.e., r value). Selection of the final antigenic essence compositions involved an optimal combination of essence/target similarity and essence immunogenicity influenced by MHC restriction. Antigenic essence compositions should strike an optimal balance between similarity of essence code with the surface of target cells and enrichment of MHC-restricted peptide antigens.

Figure 6

Design of antiangiogenic essence candidates based on comparison of essence code with target cell footprint and cytotoxicity assays. (a) Cytotoxicity is plotted vs. essence/target similarity as determined by correlation of essence composition with cell surface profile of target cells. Legend: 1►2—First letter (1) corresponds to essence, second letter (2) corresponds to the target human microcirculatory endothelial cells (HMECs) used in the cytotoxicity assay. Letters are used to identify the presence in the growth medium of either normal EC growth supplement (N), MCF-7 cell-conditioned medium (M), LNCap cell-conditioned medium (L), or HepG2 cell-conditioned medium (H). All data in the plot were described by a set of linear functions from the essence/target similarity, in which the coefficients were also linearly interdependent and attributed to the essence immunogenicity. Adapted from [74,77]. (b) In vitro efficacy of optimized SANTAVAC compositions for final products. Target HMECs were incubated in the presence of effector CTLs at a 1:20 ratio. After 3 days, CTLs were removed and target cell viability was determined. Efficacy was calculated as a ratio of the number of tumor-stimulated cells in control (i.e., Low: HMEC^5%, Medium: HMEC^15%, or High: HMEC^25%) to the number of tumor-stimulated cells in the experiment. Thus, efficacy predicts the therapeutic effect of the vaccine in vitro. For efficacy calculations, the data representing the mean value of three independent measurements were used. Percentage values indicated in the superscript correspond to the percentage of tumor-conditioned medium used to stimulate target HMEC, or HMEC used to generate SANTAVAC. SANTAVAC¹⁵ corresponds to antigenic essence obtained from HMEC^15%. SANTAVAC²⁵ corresponds antigenic essence obtained from HMEC^25%. Adapted from [75].