An investigation of human antibodies to blood group antigens for the management of haemolytic disease of the fetus and newborn

Abstract

Providing the best possible patient outcomes for haemolytic disease of the fetus and newborn (HDFN) requires quality laboratory testing to guide clinical management. Laboratory testing shows the risk for HDFN by identifying whether the mother: 1) is negative for a paternally inherited blood group antigen on fetal red blood cells (RBCs) and 2) has IgG antibodies to that antigen. When a risk for HDFN is identified, appropriate clinical care is provided. There are, however, challenges in the management of HDFN. This thesis aims to investigate novel approaches to address the challenges which arise in the management of HDFN from two perspectives, one from pregnancy management and the other from the management of a prophylactic, polyclonal antibody supply.In Chapter 2, a challenge in pregnancy management was presented as a case study, where clinical care was necessary in managing HDFN without fully identifying the specificity of the maternal antibody. Extensive serological testing and targeted exome sequencing revealed that the maternal antibody was recognising a new low-frequency antigen in the Augustine (AUG) blood group system. Additional evidence for this AUG antigen discovery was provided by a novel serology-based assay, where enhanced levels of maternal antibody binding to antigen-positive RBCs was found to be similar with antibodies against another antigen in the AUG blood group system. This case study led to the discovery of a new low-frequency antigen in the AUG blood group system (ATML; AUG3) and evidence for its management in future pregnancies.Although maternal antibodies to low-frequency antigens can be clinically significant, one of the most common risks of HDFN arises from maternal antibodies against the RhD blood group antigen. Hence, preventing maternal anti-D alloimmunisation for all RhD-negative women using a prophylactic antibody product, RhD-immunoglobulin (RhIg), is considered a major medical breakthrough. Manufacturing RhIg, however, relies on a limited supply of plasma containing high titres of polyclonal anti-D antibodies from volunteer donors. This brings challenges to the management of HDFN as maintaining such supply raises ethical questions. For this challenge, this thesis aims to investigate novel approaches that can further the development of a prophylactic monoclonal antibody blend, which mimics the safety and efficacy of polyclonal RhIg preparations.The investigation into developing these monoclonal antibodies began with constructing immune human phage display libraries to be a resource for the development of monoclonal antibodies from anti-D donors. Chapter 3 overcomes the logistical difficulties in collecting samples from anti-D donors for phage display with a novel blood collection approach. This led to the successful development of four monoclonal antibodies derived from an anti-D donor in the early stages of RhD immunisation: three were anti-D antibodies and one had unresolved specificity (clone PR3-3).Defining the specificity of the PR3-3 monoclonal antibody was considered important in investigating the potential utility of this antibody. Based on the initial reactivity profile, the hypothesis that the PR3-3 monoclonal antibody was recognising an antigen in the Landsteiner-Weiner (LW) blood group system is reported in Chapter 4. The findings, however, were not consistent with an anti-LW specificity. Further studies were required to fully identify its specificity.The availability of monoclonal anti-D antibodies provided the material required to better understand the mixture of antibodies contributing to polyclonal anti-D preparations of RhIg. This is important for informing the successful development of a monoclonal antibody blend. Chapter 5 investigates the potential operation of the immune network theory in anti-D donors, where anti-D alloimmunisation results in the production of anti-idiotypic antibodies (anti-Ids) against the immunogenic idiotopes on the V-region of anti-D alloantibodies. Furthermore, that these anti-Ids are present in anti-D donor plasma contributing to RhIg preparations. To provide a diversity of anti-D idiotopes for anti-Ids detection, an additional two human monoclonal anti-D antibodies derived from a long-term anti-D donor were developed using phage display. Preliminary evidence suggested there were anti-Ids in anti-D donor plasma and RhIg. Further validation will be required to confirm the specificity of these anti-Ids. The contribution of anti-Ids to the RhIg mechanisms of action remains an open question.In conclusion, this thesis has investigated novel approaches to help address the challenges in managing HDFN in pregnancy and the prophylactic RhIg supply. The investigation has resulted in novel approaches and 6 monoclonal antibodies derived from two anti-D donors. These may prove useful for managing HDFN in future blood group discoveries and in furthering the successful development of a prophylactic monoclonal antibody blend, which mimics the safety and efficacy of polyclonal RhIg preparations. Ultimately, these investigations will help address challenges in the management of HDFN to provide the best possible patient outcomes.