European Review for Medical and Pharmacological Sciences 2021; 25: 1622-1630 Superantigens, superantigen-like proteins and superantigen derivatives for cancer treatment J.-Y. CHEN Xiehe Biology Group, Nobel Institute of Medicine, Shenzhen, Guangdong Province, China Abstract. – OBJECTIVE: Bacterial superanti- gesting a more complex mechanism of immune gens (SAgs) are proteins produced by few types response6. Indeed, the current view is that SAgs of bacteria that have been linked to several hu- bind to multiple coreceptors forming a costimu- man diseases. Due to their potent in vitro and in latory axis between coreceptors critical for T-cell vivo tumoricidal effects, they are extensively in- 7 vestigated for oncological applications either activation . CD28 is a homodimer expressed alone or in combination with classical antican- constitutively on T cells that interacts with its cer drugs. However, the intrinsic toxicity of nat- B7 coligands expressed on antigen-presenting ural SAgs stimulated the development of more cells, transducing the signal essential for T cell effective and less toxic SAg-based immunother- activation. The staphylococcal superantigen-like apy. This review summarizes our current knowl- protein 1 (SSL1) specifically binds to human edge on SAg-based immunotherapy including extracellular signal-regulated kinase 2 (hERK2), SAg-like proteins and SAg derivatives, as well as their potential alone or with other therapeu- an important stress-activated kinase in mito- 8 tic modalities including chemotherapy and ra- gen-activated protein kinase signaling pathways . diotherapy. It is now clearer that SAgs induce the release of cytokines and chemokines through multiple Key Words: pathways as it was recently observed in in vitro Superantigen, Superantigen derivative, Superanti- 9 gen-like, Cancer, Combination therapy experiments . SAgs can cause severe poisoning and several serious human diseases. For instance, Staphylo- coccus aureus enterotoxins have potent super- Introduction antigenic activity associated with frequent food poisoning outbreaks10, toxic shock syndrome11,12, Bacterial Superantigens (SAgs), also common- pneumonia13, Kawasaki disease14-17, nasopha- ly known as erythrogenic toxins or streptococcal ryngeal infections18, atopic dermatitis (AD), and pyrogenic exotoxins1, are the most potent types chronic rhinosinusitis (CRS) and sepsis-related of T cell mitogens. SAgs are produced by only a infections. few bacterial pathogens, including Staphylococ- SAgs can induce the production of cytokines cus aureus2. The majority are produced by the leading to the hypothesis that viral proteins with Gram-positive organisms Staphylococcus aureus a possible superantigen activity may be responsi- and Streptococcus pyogenes3,4. Bacterial SAgs at ble for the systemic shock, acute respiratory syn- a very low concentration (typically < 0.1 pg/ml) drome, multiorgan failure and consequently death are sufficient to stimulate the T lymphocytes5. In observed in patients with COVID-1919. A recent vitro femtomolar concentrations of SAgs are able finding from in silico studies suggests that small to stimulate profound proliferation and cytokine insertions unique to SARS-CoV-2 (SARS-CoV-2 production in up to 20% of all peripheral T cells6. Spike) can display SAg activity and support this It was initially thought that the powerful immune hypothesis20. It is relatively well established that response generated by SAgs was due to its bind- at least one of the mechanisms by which SAgs ing to Major Histocompatibility Complex class II contribute to systemic multiorgan failure is the molecules on antigen-presenting cells and T cell production of a cytokine storm by enhancing receptors on T cells. Later, it was found that the the B7-2/CD28 costimulatory receptor interac- staphylococcal enterotoxin B (SEB) SAg, also tion21,22. The formation of the B7-2/CD28 costim- binds the co-stimulatory molecule CD28 sug- ulatory axis is critical for full T-cell activation21. 1622 Corresponding Author: Juyu Chen, MD; e-mail: [email protected] Superantigens, superantigen-like proteins and superantigen derivatives for cancer treatment Picomolar amounts of staphylococcal entero- at very low dosage but of limited use due to toxin A (SEA) SAg rapidly induced cytotoxic ac- toxic effects27. Natural superantigens have been tivity against K562 and Raji cells, as well as some used for cancer immunotherapy but at the cost of natural-killer (NK)-resistant tumour cell lines23 severe side effects due to their ability to induce which could be of value in therapeutic applica- high systemic levels of a large panel of inflam- tions. Therefore, despite the serious toxic and matory cytokines that may lead to a toxic shock adverse effects, SAgs are actively investigated as syndrome and therefore attempts to translate the therapeutic tools for cancer treatment. The aim of in vitro antitumor effect into clinical trials have this article is to provide a concise review of the been limited. To increase the effect of the SAgs, potential application of SAgs, SAg-like proteins the concept of tumor-targeted SAgs (TTS) was and SAg derivatives for cancer treatment. established with the aim of recruiting a large number of T cells (Figure 1). In addition, be- SAgs for Cancer Treatment cause of the differential species susceptibilities, For their ability to potentially activate T lym- predicting the toxicity of SAgs in humans is phocytes, SAgs have been used clinically as difficult. For instance, mice are not valid human an immunomodifier in the treatment of tumors. disease models because they are significantly The SAg Staphylococcal enterotoxin A (SEA) less sensitive to toxic shock induced by bacterial when co-cultured with human peripheral blood SAgs when compared with primates and rab- mononuclear cells (PBMCs) inhibited the pro- bits28. These limitations triggered the evaluation liferation and induced the death of human lung of other SAg alternatives such as SAg-like pro- carcinoma A549 cells24. The Staphylococcal en- teins and derivatives. terotoxin B (SEB) is an efficient activator of the antitumor immune response that leads to the SAg-Like Proteins eradication of tumor growth and inhibition of The common structure and function of the metastasis25. SEB-Superantigen-activated PBMC SAgs are also shared by another group of staph- (peripheral blood mononuclear cells) significantly ylococcal virulence factors called the superanti- induced apoptosis in transitional cell carcinoma gen-like proteins (SAg-like proteins)4. Staphylo- cells (TCC)26. The Staphylococcal enterotoxin coccal enterotoxin-like toxins (SEls) are proteins C2 (SEC2) is another classical SAg with potent with similar amino acid sequences to those of antitumor activity by activating T lymphocytes classical SAgs but they exhibit low or no emetic Figure 1. Potential immunotherapy using Tumor-Targeted Superantigen (SAg) (Modified from61). A SAg or SAg-based ligand can be linked to a tumor-specific antibody or ligand. The tumor-specific antibody/ligand binds to the tumor antigen whereas the superantigen/ligand crosslink between the major histocompatibility complex class II molecule (MHC-II) and the T cell receptor (TCR) induces T-cell hyperactivation of a T lymphocyte (shown in this figure) and monocytes/macrophages and results in the release of huge amounts of cytokines and chemokines, such as tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), IL-2, interferon γ (IFN-γ). and many others. This leads to T-cell dependent tumor killing likely by apoptosis. APC = Antigen Presenting cell. 1623 J.-Y. Chen Figure 2. Potential mechanism of action of ABR-217620 (Naptumomab estafenatox; 5T4Fab-SEA/E-120). Modified from55. T lymphocyte activation occurs through its T-cell receptor (TCR) upon binding of the fusion protein ABR-217620 to the 5T4 tumor-associated antigen. The T cell kills tumoral cells directly by its cytotoxic T lymphocyte (CTL) activity and indirectly by producing cytokines (tumor necrosis factor [TNFa] – and interferon [IFNg ). activity and have anticancer effects by activat- SAg Derivatives ing lymphocytes particularly CD4+ and CD8+ T SAg derivatives have been created to reduce cells29. Treatment with a low concentration of systemic toxicity while maintaining profound an- Staphylococcal enterotoxin-like Q (SElQ) (30 µg/ titumor effects. SAg side-effects may be caused mouse) could inhibit the growth of tumors by by nonspecific binding to class II positive cells. approximately 30% without significant toxicity29. To overcome this, several authors created mutat- Staphylococcal SAg-like protein 6 (SSL6) inhibit- ed staphylococcal enterotoxin A (SEA). In partic- ed CD47 and promoted Sorafenib-induced apop- ular, Hansson et al35 created a tumor-reactive SAg tosis of hepatocellular carcinoma cells Huh-7 and by engineering a fusion protein composed of a MHCC97H30 that are widely used as liver cancer tumor-reactive mAb (C215Fab) and the bacterial models. Other staphylococcal SAg-like protein SAg staphylococcal enterotoxin A (SEA) (SEA), such as staphylococcal SAg-like protein 7 (SSL7) a well-known SAg. They introduced a point mu- have been shown to inhibit the formation of the tation (D227A) at the major MHC class II binding complement membrane attack complex (MAC) site. The Fab–SEA D227A fusion protein showed and can be useful for treating complement-me- profound antitumor effects with a markedly re- diated hemolysis31. Recombinant staphylococcus duced toxicity as compared with the wild-type aureus SAg-like protein 7 -that inhibits