An engineered superantigen SEC2 exhibits promising antitumor activity and low toxicity

Abstract

Recent studies suggested that the histidine residues at 118 and 122 play an important role for the toxicity of staphylococcal enterotoxin C subtype 2 (SEC2), and the substitutions of both histidines with alanine can severely impair the fever activity of SEC2. We hypothesized that promising SEC2 antitumor agent with low toxicity and enhanced superantigen activity can be constructed by introducing related mutations at protein functional sites of SEC2. We showed that the SEC2 mutants H122A and H118A/H122A exhibited improved superantigen activity after introducing the point mutations at Thr20 and Gly22. A resultant mutant, named as SAM-3, has considerable abilities to inhibit the growth of H22 and Hepa1-6 tumor cells in vitro and colon 26 solid tumor in vivo. Furthermore, SAM-3 also exhibits significantly reduced toxicity compared with native SEC2. The study provides a novel strategy for designing promising superantigen immunotherapeutic agent. The constructed SEC2 mutant SAM-3 can be used as a powerful candidate for cancer immunotherapy and could compensate the deficiency caused by toxicity of native SEC2 in clinic.

Fig. 1

Schematic diagram of constructed SEC2 mutants. Shown are the names of SEC2 mutants (left) and the substitutions of amino acid residues at different positions using site-directed mutagenesis. Distinct shading was used to indicate the expressed domains of proteins

Fig. 2

The purified SEC2 and its mutants were confirmed by Western blot. Lane 1 rSEC2; Lane 2 H118A; Lane 3 H122A; Lane 4 H118A/H122A; Lane 5 SAM-1; Lane 6 SAM-2; Lane 7 SAM-3

Fig. 3

Induction of murine lymphocyte proliferation by SEC2 and mutants. SEC2 or mutants had been incubated with murine splenocytes for 72 h before, and the proliferation of splenocytes was determined by MTT assay. Value on the y-axis represents the average proliferation index (PI) ± SD of triplicate values. Each sample was tested in at least three separate assays

Fig. 4

Effects of SEC2 and mutants on inhibition of the growth of Hepa1-6 tumor cells (a) and H22 tumor cells (b) by activating lymphocytes in vitro. PBS was used as negative control. Data shown are the representative of at least three separate experiments, and the values represent the Mean ± SD

Fig. 5

Pyrogenicity of SEC2 and mutants in rabbit model. SEC2 and mutants were separately injected intravenously at dose of 10 μg kg⁻¹ in PBS. The mean rectal temperature rise of the rabbits (3/group) was monitored for 4 h. PBS was used as negative control. The values presented represent the Mean ± SD

Fig. 6

Mean food consumption (MFC) (a) and body weight variety index (BWVI) (b) of mice treated with SEC2 and SAM-3 were investigated. Mice were randomized into five groups: 15 μg/mouse native SEC2 (SEC2 15), 45 μg/mouse native SEC2 (SEC2 45), 15 μg/mouse SAM-3 (SAM-3 15), 45 μg/mouse SAM-3 (SAM-3 45), and PBS negative control (PBS)