Allogeneic Antigen Composition for Preparing Universal Cancer Vaccines

Abstract

Recently it was demonstrated that tumors induce specific changes to the surface of human endothelial cells thereby providing the basis for designing endothelial cell-based vaccines that directly target antigens expressed by the tumor endothelium. The present report extends these studies in vitro by investigating the efficacy of allogeneic antigens with regard to their ability to target immune responses against the tumor vasculature since alloantigens simplify vaccine development and implementation in clinical practice. We demonstrated that allogeneic SANTAVAC (Set of All Natural Target Antigens for Vaccination Against Cancer), which presents a specifically prepared composition of cell surface antigens from tumor-stimulated endothelial cells, allows targeting of the tumor vasculature with efficacy of 17, where efficacy represents the killing rate of target cells before normal cells are adversely affected, and efficacy of 60, where efficacy represents the fold decrease in the number of target cells and directly relates to tumor growth arrest. These data suggest that allogeneic SANTAVAC may be considered an antigenic composition that following administration in the presence of respective adjuvants may be clinically tested as a therapeutic or prophylactic universal cancer vaccine without adverse side effects to the normal vasculature.

Figure 1

Primary HMEC cultures. A representative HMEC primary culture from donor 1 (a) and donor 2 (b). Images were obtained using a Leiса DM5000B microscope (scale bar, 50 μm). Flow cytometric analysis of HMEC from donor 1 before activation (c, d) and after activation (e). Cells were stained with PE-conjugated monoclonal mouse anti-hVEGFR-2 or anti-human CD62E antibodies (labeled as “VEGFR-2⁺” or “CD62⁺”). For isotype control, cells were stained with PE-conjugated mouse IgG1 (labeled as “control”).

Figure 2

Dose determination of tumor-conditioned medium used to prepare HMEC with tumor-induced cell surface profiles. HMEC cultures were incubated with 0, 10, 20, 30, 40, or 50% tumor-conditioned medium. After 3 days in culture, cells were counted (red points) in wells using trypan blue exclusion. Cell numbers were approximated using a curve used to determine the concentrations of tumor-conditioned medium that elicited either weak (stimuli just a little higher than in the control), moderate (half of the maximum), or strong (maximum) stimulation of HMEC cultures (green lines). Green lines represent percentage selected for СTA.

Figure 3

Efficacy I (a) and efficacy II (b) of target cell killing by SANTAVAC in CTA. Target HMEC were incubated in the presence of effector CTL at a 1 : 20 ratio. After 3 days, CTL were removed, target cells were carefully washed, and target cell viability was determined. Data is expressed as efficacy I (a) or efficacy II (b) of target cell killing by SANTAVAC. Efficacy I was calculated as a ratio of the number of nonstimulated cells in control wells (i.e., HMEC^0%) to the number of tumor-stimulated cells in experimental wells. Efficacy II was calculated as a ratio of the number of tumor-stimulated cells in control wells (i.e., HMEC^5%, HMEC^15%, or HMEC^25%) to the number of tumor-stimulated cells in experimental wells; that is, the percentage of tumor-conditioned medium in control wells was the same as in the experimental wells. Efficacy I allows in vitro estimation of the SANTAVAC efficacy by demonstrating how many endothelial cells in the tumor vasculature will be destroyed before 1 endothelial cell in normal tissue is destroyed (used to predict vaccine safety). Efficacy II allows in vitro estimation of the SANTAVAC efficacy by demonstrating the degree of HMEC proliferation suppression in the tumor vasculature and is used to establish the degree by which the vaccine can arrest tumor growth (predicted vaccine therapeutic effect). For efficacy calculation, the data representing the mean value of 3 independent measurements was used. “FAA” indicates the data related to the control (■) in CTA where fibroblast-associated antigens were used to simulate CTL. “swap” indicates CTA data where primary cell cultures used to generate antigens and primary cell cultures which were used as target cells were swapped (it was done to demonstrate the reproducibility of the CTA results at defined percentages of the tumor-conditioned medium used to stimulate HMEC). 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 for targeting immune response in CTA.