Anti-tumor antibody isotype response can be modified with locally administered immunoadjuvants

Abstract

In situ vaccination with immunostimulatory compounds is a demonstrated means to treat tumors preclinically. While these therapeutic effects have been attributed to the actions of T cells or innate immune activation, characterisation of the humoral immune response is seldom performed. This study aims to identify whether the injection of immunoadjuvants, Addavax (Adda) and cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG), intratumorally can influence the antibody response. Specifically, whether intratumoral injection of immunoadjuvants can alter the tumor-specific antibody target, titre and isotype. Following this, the study aimed to investigate whether serum obtained from in situ vaccinated mice could neutralise circulating tumor cells. Serum was obtained from mice bearing B16F10-OVA-Luc-GFP tumors treated with immunoadjuvants. Antibody targets' titre and isotype were assessed by indirect ELISA. The ability of serum to neutralise circulating cancer cells was evaluated in a B16F10 pseudo-metastatic model. It was observed that tumor-bearing mice mount a specific anti-tumor antibody response. Antibody titre and target were unaffected by in situ vaccination with immunoadjuvants; however, a higher amount of IgG2c was produced in mice receiving Adda plus CpG. Serum from in situ vaccinated mice was unable to neutralise circulating B16F10 cells. Thus, this study has demonstrated that anti-tumor antibody isotype may be modified using in situ vaccination; however, this alone is not sufficient to neutralise circulating cancer cells.

Keywords: Antibody neutralisation; B16F10; CpG; Immunoadjuvants; In situ vaccination; Isotype.

Fig. 1

Mouse serum was obtained from mice following therapeutic intervention. C57BL/6 mice were implanted with B16F10-OVA-Luc-GFP (N = 6 per group did not receive a tumor (naïve)) (A). On days 5 and 11, 6 mice were injected with PBS, Addavax (25µL) and Addavax (25µL) with CpG (25 µg) respectively. Tumors were measured every 3–4 days and tumor volume is reported in (B). On day 22, mice were sacrificed, and blood was taken for sera analysis. Tumors were excised and weighed (C). Each point represents the mean ± SEM statistical analysis was performed using two-way ANOVA or Student’s T test *p < 0.05 **p < 0.01

Fig. 2

B16F10 cells are the target for antibodies following tumor challenge and antibody titre is unaffected by local administration of the immunoadjuvant. Antibody levels in serum obtained from mice bearing B16F10-OVA were detected using ELISA. ELISA plates were first coated with target antigen followed by blocking using 2% BSA. Following this, serum was added at a range of dilutions ranging from 1:40 to 1:5000. Serum was incubated for 2 h before being washed. Anti-mouse IgG HRP conjugate was added as the secondary antibody and the plate was incubated for 1 h. Following this, the TMB was added and the plate was left to develop (A). The stop solution was added and the absorbance was measured at 450 nm using FLUOstar omega microplate reader. The OD values obtained when either the homologous tumor cell line, B16F10-OVA-Luc-GFP (B); the untransformed cell type, B16F10 (C); or OVA protein (D) was used as target antigen. Points represent the mean of n = 6 mice per group. Error bars represent the standard error of mean

Fig. 3

Antibody isotype is altered following therapeutic intervention. To identify the isotype of the mouse antibodies, ELISAs were carried out as previously described. However, in place on anti-mouse total IgG, either anti-mouse IgM, IgG1 or IgG2c HRP conjugates were used. The OD values obtained for each of these are shown in A, B and C respectively. The points represent the mean of n = 6 mice and the error bars correspond to the standard error of mean. Statistical analysis in graphs was carried out using a two-way ANOVA *p < 0.05, ns non-significant

Fig. 4

Serum of in situ vaccinated mice is unable to neutralise B16F10-OVA-Luc-GFP cells. B16F10-OVA-Luc-GFP cells were mixed with 50% serum comprising of a pool of sera derived from either mice with high IgG1, high IgG2c or (n = 3–4 per group). This was incubated for 30 min at 4 °C. Cells were then implanted IV into the tail vein of C57BL/6 mice. On day 11, the mice were sacrificed and the lungs were taken (A). The lungs were observed using a stereoscopic microscope and the number of B16F10 nodules were counted. The mean number of metastasise (n = 3–4) is shown in B, where the error bars correspond to the standard deviation. The lungs were then frozen and thawed twice at − 80 °C. Following this, the lungs were homogenised in PBS. The mean luciferase extracted was normalised to the amount of protein as calculated by BCA assay is shown in C, where the error bars correspond to the standard deviation. Statistical analysis was carried out using an ordinary one-way ANOVA, ns non-significant