Development of Staphylococcal immune evasion protein Sbi as a potential therapeutic agent

Student thesis: Doctoral ThesisPhD

Abstract

Monoclonal antibodies (mAbs) have been clinically utilised as a monotherapy for cancer treatment, demonstrating mixed evidence of effectiveness as patients developed acquired resistance to their activity. Research has shown that mAbs biological effector function can be enhanced, by using antibodies as a targeting mechanism to deliver cytotoxic drugs as seen in antibody drug conjugates (ADCs). It has been shown, Sbi (Staphylococcus binder of IgG) enables \textit{Staphylococcus aureus} to evade the host immune response by futile fluid-phase consumption of the complement protein C3 through Alternative pathway (AP) activation.

The aim of this study was to conjugate Sbi to Trastuzumab (Tmab) and target it on the surface of HER2 positive cancer cells to induce AP complement cascade activation. This could cause complement-dependent cytotoxicity (CDC), cell recognition-opsonisation and cell lysis by the formation of the MAC (membrane attack complex) leading to cancer reduction or clearance. Sbi was recombinantly made for subsequent conjugation reactions with previously produced Fabs (antigen-binding fragment) or Trastuzumab for the production of the novel Fab-Sbi and Tmab-Sbi drug immunoconjugates. By site-directed mutagenesis (SDM) different mutants were developed and evaluated with variable immunostimulatory capabilities and stability. Terminal cysteines were introduced for subsequent conjugation reactions for the production of the final Sbi novel immunoconjugates. Novel Sbi-Tmab immunoconjugates were produced using previously characterised novel disulfide-rebridging agents. The factors affecting the construction of novel immunoconjugates with selected Sbi mutants were evaluated by two strategies (antibody functionalisation vs Sbi functionalisation). Strategy two (Sbi functionalisation) proved successful for the production of an anti-HER2 antibody Tmab-Sbi immunoconjugate at a good yield. The same strategy was then applied for the production of novel anti-HER2 and anti-MUC1 Fab-Sbi products. A novel eGFP-Sbi protein was explored as a diagnostic for the detection of C3 fragment deposition. The protein was used for C3 fragment deposition detection on HER2+ cancer cell lines surfaces using a combination of cell culturing techniques, confocal microscopy and by utilising the AP activation capabilities of the produced anti-HER2 Tmab-Sbi immunoconjugate. C3 deposition was detected under various conditions using two HER2+ human expressing cancer cell lines but also in a aHUS animal model.

Preliminary findings imply the applicability of the tested conjugation method to construct multiple mAbs and Fabs immunoconjugates with variable Sbi mutants, allowing the production of specific targeted treatments as novel interventions in the field of oncology. Furthermore it was demonstrated the applicability of the eGFP-Sbi fusion protein to detect C3 fragment deposition that could be further explored in animal studies and human trials in multiple complement mediated diseases.
Date of Award20 Jan 2020
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorAndrew Watts (Supervisor) & Jean Van Den Elsen (Supervisor)

Keywords

  • Antibodies
  • immunotherapy
  • Conjugation
  • Cancer
  • cancer cells
  • fluorescence probes
  • microscopy
  • recombinant expression
  • Papain
  • enzymatic digestion

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