Thomas, Peter; (2023) Modulation of B cell repertoire characteristics during immunisation for improved therapeutic monoclonal antibody generation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Immunisation of transgenic mice containing human immunoglobulin loci (TIg mice) represents the current state-of-the-art for therapeutic monoclonal antibody (mAb) discovery, due to the regulated in vivo affinity maturation that B cells experience within germinal centres (GCs). However, phenomena such as the affinity ceiling and preferential expansion of specific B cell lineages can curtail response affinity and diversity, respectively. This thesis attempted to address these limitations of the GC response in the context of a Human Immunodeficiency Virus (HIV) Envelope gp120 model immunisation regimen within TIg mice to improve generation of therapeutic mAbs. Infusion of high affinity antibody has previously been shown to increase affinity in a model immunisation. In this thesis, this was adapted for therapeutic mAb discovery by sequentially infusing escalating affinity mAbs during an immunisation regimen. These mAbs targeted a consistent epitope, increasing GC selection pressure on specific B cells which was hypothesised to enhance affinity maturation. It was identified that, although affinity maturation of most competing B cells was reduced by mAb infusion, a small but diverse population of B cells that reached greater affinities than both the highest affinity mAb infused and control immunised mice was identified. Plasma cells also exhibited more rapid evolution and more efficient somatic hypermutation than control. To enhance diversity, it was hypothesised that infusion of rapamycin during the immunisation regimen would limit B cell division within GCs and increase B cell diversity compared to control. It was found that rapamycin reduced B cell diversity, hypothesised to be due to limited GC entry. Novel diversity measurements also identified that rapamycin reduced the functional diversity of the plasma cell response, which correlated with weaker serum titres. This thesis therefore presents an assessment of two novel immune modulating strategies for therapeutic mAb discovery and suggests additional strategies and applications for such techniques.

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