DNA-delivered monoclonal antibodies targeting the p53 R175H mutant epitope inhibit tumor development in mice

Abstract

The tumor suppressor p53 is the most common mutated gene in cancer, with the R175H as the most frequent p53 missense mutant. However, there are currently no approved targeted therapies or immunotherapies against mutant p53. Here, we characterized and investigated a monoclonal antibody (mAb) that recognizes the mutant p53-R175H for its affinity, specificity, and activity against tumor cells in vitro. We then delivered DNA plasmids expressing the anti-R175H mAb or a bispecific antibody (BsAb) into mice to evaluate their therapeutic effects. Our results showed that the anti-R175H mAb specifically bound to the p53-R175H antigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells, with no cross-reactivity with wild-type p53. In cultured cells, the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity. We made a recombinant MC38 mouse cell line (MC38-p53-R175H) that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles. In vivo, administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice. The administration of the anti-R175H BsAb plasmid showed no therapeutic effects, yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody. These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.

Keywords: BsAb; Cytotoxicity; Mutant p53; PD-1; R175H; mAb.

Figure 1

Characterization of p53-R175H mAb. (A) The purified anti-R175H protein was separated on a 4%–20% SDS-PAGE gel and stained with Coomassie solution. Lanes 1 and 4 were loaded with reducing anti-R175H mAb; Lanes 2 and 5 were loaded with non-reducing protein; Lane 3 indicates the protein ladder (250, 130, 100, 70, 55, 35, 25, 15, 10 kDa, Thermo Fisher Scientific). (B) BLI kinetics of R175H mAb association (t = 0 s–120 s) and dissociation (t > 120 s) with R175H antigen. (C) Forty-8 h after transfection of 293T cells with WT p53, p53-R175H, or the vector control, a Western blot was used to evaluate the expression of WT p53 or p53-R175H. (D) The protein expression levels of p53-R175H were detected using Western blot in MC38 cells without p53 or with human p53-R175H. (E) The mutp53 protein expression in CT26-p53-R172H cells was detected by Western blot with the R175H mAb. (F) Knocking the R172H mutation into mouse p53 gene in CT26 cells.

Figure 2

The cytotoxicity of R175H mAb in cultured cells. MC38-p53-R175H or H1299-p53-R175H cells were added into a 96-well tissue culture plate at a density of 1 × 10⁴ cells/well in the presence of various concentrations of R175H mAb or isotype control. PBMCs (1 × 10⁵ cells/well) were co-cultured with tumor cells in a humidified incubator with 5% CO₂ at 37 °C for three days. (A) At the mAb concentration of 10, 1, 0.1, and 0.01 μg/mL, the cytotoxicity of MC38-p53-R175H cell supernatant was measured using the lactate dehydrogenase (LDH) assay. (B) Cytotoxicity was determined using the LDH assay with 10 μg/mL mAb. (C, D) The cytotoxicity of the H1299-R175H cell supernatant was measured using the LDH assay after cells were incubated with 10, 1, 0.1, and 0.01 μg/mL mAb. (E, F) H1299-p53-R175H cells (1 × 10⁴ cells) harboring a luciferase reporter gene were co-cultured with CAR-T cells at various concentrations in 96-well plates at 37 °C overnight. Cytotoxicity was determined by measuring the amount of luciferase in lysed target cells. The data shown are representative of three experiments. The data are expressed as mean ± SD. Statistical significance was set at ^∗P < 0.05. ns, not significant.

Figure 3

The therapeutic effect of pR175H-mAb in mouse MC38 cell tumor model. (A) Schema of the pR175H-mAb all-in-one construct. (B) The expression of R175H was detected using a Western blot with anti-p53-R175H mAb in 293T cells transfected with WT p53 or p53-R175H. (C) Purified protein from pR175H-mAb-transfected 293 cells was separated on an SDS-PAGE gel and stained with Coomassie solution. (D) Schema of MC38-R175H tumor model establishment and experiment. The subcutaneous MC38-R175H cell tumor model was intramuscularly inoculated with pR175H-mAb, followed by electroporation on days 5 and 12 after tumor inoculation. Meanwhile, αPD-1 was administrated intraperitoneally on days 8 and 15. (E) The serum levels of anti-R175H Ab were detected using ELISA. (F) Tumor volume was measured at different times after inoculation. The data are expressed as mean ± SEM. (G) Tumor volume was statistically analyzed on day 25 post-tumor inoculation. (H) Tumor diameter of individual mice from the groups in (F) as a function of time. Six mice in each group were tested. The data shown are representative of three experiments. The data are expressed as mean ± SD. ^∗∗P < 0.01 and ^∗∗∗∗P < 0.0001. ns, not significant.

Figure 4

Anti-tumor response induced by pR175H-mAb on CT26 tumor model. (A) Schema of CT26-R172H tumor model establishment and experiment. The subcutaneous tumor model was established and intramuscularly inoculated with pR175H-mAb on days 1 and 8 after tumor inoculation. Meanwhile, αPD-1 was administrated intraperitoneally on days 4 and 11. Each group included six mice. (B) Tumor growth was measured and compared on the indicated days after initial inoculation. The data were expressed as mean ± SEM. (C) Tumor volume was statistically analyzed on day 20 post-tumor inoculation. (D) Tumor volume of individual mice from the groups in (B) as a function of time. The data are shown as mean ± SD. ^∗∗P < 0.01. ns, not significant.

Figure 5

The therapeutic effect of pR175H-BsAb was evaluated in vivo. (A) Schema of the pR175H-BsAb construct using the knob-into-hole technology. (B) The purified protein from pR175H-BsAb transfection in 293 cells was separated on an SDS-PAGE gel and stained with Coomassie solution. (C) The expression of p53-WT or R175H was detected using a Western blot with anti-R175H BsAb in 293T cells transfected with an empty vector or that expressing p53-WT or p53-R175H. (D) BLI kinetics of R175H/mCD3 mAb association (t = 0 s–120 s) and dissociation (t > 120 s) with R175H antigen. (E) Schema of MC38-R175H tumor model establishment and experiment. MC38-R175H subcutaneous tumor model was intramuscularly inoculated with pR175H-BsAb on days 4 and 3 before tumor inoculation. Meanwhile, three doses of αPD-1 were administrated intraperitoneally on days 7, 10, and 14. (F) The serum levels of anti-R175H/mCD3 BsAb were detected using an ELISA. (G) Tumor volume was measured at different times after inoculation. The data are expressed as mean ± SEM. (H) Statistical analysis of mean tumor volume was performed on days 16–22 post-tumor inoculation. (I) Tumor diameter of individual mice from the groups in (G) as a function of time. The experiments were performed with five mice per group. The data are expressed as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗∗P < 0.0001.

Figure 6

Target antigen recognized by pR175H-mAb or BsAb in vivo. The subcutaneous tumors (MC38-p53-R175H on C57BL/6J and CT26-p53-R175H on BALB/c) were established before mice were intramuscularly inoculated with pSEAP, pR175H-mAb, or pR175H-BsAb. Tumor tissues were collected 7 days after the last treatment, and single-cell suspension was subjected to flow cytometry using antibodies against human IgG Fc and mouse Cd45. The experiments were performed with three mice per group. (A) The frequency of human Fc-positive Cd45⁺ and Cd45⁻ cells from tumors. Representative images were shown. (B) Statistical analysis of cells that were stained positive by the anti-Fc antibody. The data are shown as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.