Načrtovanje cepljenega superantigena za imunoterapijo raka

Golob Urbanc, Anja (author), Jerala, Roman (mentor)

Terapevtska monoklonska protitelesa se uporabljajo za zdravljenje različnih rakavih obolenj, hkrati pa se njihov proti tumorski učinek lahko še izboljša z dodatkom efektorskih molekul, kot so na primer superantigeni (SAg). Superantigeni so bakterijski ali virusni toksini s sposobnostjo aktivacije velikega števila celic T. Njihova bistvena lastnost je, da se vežejo neposredno na poglavitni kompleks tkivne skladnosti razreda II (MHCII), izražen na antigen predstavitvenih celicah, kar vodi v aktivacijo celic T preko celičnega receptorja T (TCR). Ena izmed pomanjkljivosti terapevtske uporabe superantigenov, spojenih s protitelesi, specifičnimi za tumorske antigene, je sistemska aktivacija T-celičnega odziva, ki je posledica visoke afinitete superantigena do kompleksa MHCII. Naša ideja je bila razdeliti superantigen stafilokokni enterotoksin A (SEA) iz Staphylococcus aureus na dva neaktivna fragmenta, ki se sestavita v biološko aktivno obliko le, ko prideta v neposredno bližino. V študiji smo vzpostavili hitro in enostavno metodo za odkrivanje učinkovitih cepljenih različic superantigena, ki temelji na spojitvi fragmentov SEA s peptidi, ki tvorijo obvite vijačnice. Potrdili smo, da je superantigen SEA mogoče razdeliti na dva neaktivna fragmenta, ki se spontano ne sestavita v biološko aktivno obliko, ampak ju morata peptida, ki tvorita heterodimer, pripeljati v neposredno bližino. Kot dokaz principa uporabe za namene imunoterapije raka smo učinkovito različico cepljenega SEA spojili z enoverižnim variabilnim fragmentom monoklonskega protitelesa proti antigenu CD20 (scFv-CD20). Potrdili smo, da sta fragmenta SEA le ob vezavi na tarčni antigen CD20 preko scFv-CD20, dovolj blizu skupaj, da se sestavita v biološko aktivno obliko, ki aktivira limfocite T. Fuzijski proteini, pripravljeni v doktorskem delu, imajo potencialno terapevtsko vrednost za zdravljenje rakavih obolenj, saj smo potrdili, da je njihovo delovanje usmerjeno le proti tumorskim celicam, brez vpliva na netarčne celice.

• Language: Slovenian
• Keywords: superantigen, cepljeni proteini, protitelesa, aktivacija celic T, imunoterapija raka, aktivacija limfocitov, antigeni novotvorb, proteinski inženiring, novotvorbe, vezava proteinov, T-limfociti, monoklonska protitelesa, biokemija
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2019
• PID: 20.500.12556/RUL-108009
• COBISS.SI-ID: 300696832
• Publication date in RUL: 11.06.2019

Secondary language

• Language: English
• Title: Design of split superantigen for cancer immunotherapy

Abstract

Antibody-based therapeutics that target surface antigens expressed on tumor cells are successfully used for treatment of different types of cancer. Although unconjugated monoclonal antibodies are efficient, conjugating different effector molecules, such as superantigens (SAg) to monoclonal antibodies can enhance their clinical utility. SAg are bacterial or viral toxins and one of the most potent activators of T cells, which directly bind to major histocompatibility complex class II (MHC class II) on antigen presenting cells and activate T cells through T cell receptor (TCR). Strong T cell activation is also one of the main weaknesses of this strategy as it may lead to systemic T cell activation, due to high affinity of superantigens to MHC class II. To overcome the limitation of conventional antibody-superantigen fusion proteins, we have split a staphylococcal enterotoxin A (SEA) from Staphylococcus aureus into two fragments, individually inactive, until both fragments came into close proximity and reassemble into a biologically active form capable of activating T cell response. We developed a fast and simple screening method based on a fusion between SEA and coiled-coil heterodimers that enabled a detection of functional split SEA designs. In this study we showed that it is possible to split SEA into two inactive fragments that do not reassemble spontaneously, but when fused with heterodimerizing domains, which bring fragments into proximity, split SEA can reassemble into a biologically active form. As proof-of-principle of cancer immunotherapeutic the split SEA design that demonstrated efficacy in fusion with coiled-coil dimer forming polypeptides, was fused to a single chain antibody specific for tumor antigen CD20 (scFv-CD20). This design selectively activated T cells by split SEA-scFv fusion binding to target antigen CD20. Prepared fusion proteins have a potential therapeutic value for cancer treatment, as they specifically bind to tumor cells and have no effect on non-target cells.

• Keywords: superantigen, split proteins, antibodies, T cell activation, cancer immunotherapy, Lymphocyte activation, antigens neoplasm, protein engineering, neoplasms, protein binding, T-lymphocytes, antibodies monoclonal, biochemistry