Individuals exposed to a foreign and clinically significant red blood cell (RBC) surface structure, known as a blood group antigen, can be alloimmunised and produce an antibody. During pregnancy, mothers who are exposed to a foreign, paternally-inherited, blood group antigen on foetal RBCs can also be alloimmunised. The maternal antibodies destroys foetal RBCs in subsequent pregnancies and, if left untreated, results in foetal death. This is called haemolytic disease of the foetus and newborn (HDFN).

HDFN occurs most frequently in pregnancies where the mother is exposed to the paternally-inherited RhD blood group antigen on foetal RBCs. In 1967, licensing and introduction of a prophylactic antibody product, RhD- immunoglobulin, for all RhD-negative women, was one of the major medical advances of the 20th century. These RhD-immunoglobulin injections prevented the mother from alloimmunisation when exposed to the foetal RhD-positive RBCs. However, there are ongoing problems and challenges associated with RhD- immunoglobulin as it is sourced from blood donors who are deliberately injected with RhD-positive RBCs to produce anti-D antibodies and then, repeatedly injected to maintain “high anti-D levels”. Developing an alternative recombinant antibody source for RhD-immunoglobulin has been difficult as the mechanism of action of RhD-immunoglobulin is unclear. The first component of this study aims to investigate a potentially new mechanism of action and use phage display to capture the complexity of RhD-immunoglobulin. The second component of this study aims to apply phage display for a blood group antigen, other than RhD. Maternal antibodies recognising rare blood group antigens on foetal RBCs are not detected during routine testing and can result in a severe case of HDFN, such as the ATML blood group antigen in the Augustine blood group system. The antisera for typing this antigen is currently sourced from one individual. A recombinant antibody alternative will improve the availability of typing reagents for the ATML blood group antigen to aid in future cases of HDFN.

Typing reagents for hybrid glycophorin blood groups that are rare in Caucasians, but with prevalence of up to 8% in East Asian ethnic groups, are unavailable despite repeated attempts by various groups to use conventional approaches to produce useful mAbs. We will utilise naive human phage libraries to develop reagents that react with RBC with a phenotype defined by the most common hybrid glycophorin but not with pooled human RBC that do not display these antigens. The usefulness of the mAb produced as a typing reagent will be assessed by binding to the target hybrid glycophorin detected by flow cytometry. Subsequently reactivity using the standard immunohaematological agglutination based techniques will be assessed as well as characterising other features required for typing reagents, such as long term stability at room temperature.

Integral membrane proteins are attractive targets for research, diagnostic and therapeutic applications, as they act as biomarkers to define a particular cell type, developmental stage or disease type. As such, mAbs against cell-surface biomarkers are highly sought after as biological therapeutics, laboratory reagents or diagnostic reagents. Based on antibody phage display methodologies.6 AIBN has developed novel whole-cell biopanning techniques to improve the efficiency of screening antibody libraries on whole cells displaying biomarkers. The proposed collaborative project aims to pool the skills of AIBN and CSL researchers to further optimise the whole cell biopanning technique to isolate new mAbs against specific cell surface biomarkers that are of interest to CSL. The outcomes of this project would be further innovations in whole cell panning methodologies, as well as isolation of new antibodies of therapeutic significance.

Using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, ARCBS has identified a unique murine pedigree with a splice-site mutation in a functionally important domain of a gene encoding an acetyltransferase. Red Blood Cells (RBC) from homozygous mutants from this pedigree demonstrate significant modifications of carbohydrates and lack the erythroid lineage marker TER-119. We plan to investigate the extent to which these changes modify the susceptibility to infection for pathogens that target developing and mature cells of the erythroid lineage. We will compare RBC from homozygous and wild type (WT) mice to characterise the RBC surface and identify novel RBC targets to be used as a basis for the development of biopharmaceuticals for treatment of diseases where infectivity is mediated through RBC receptors (e.g. Malaria, Parvovirus (B19)).

Human neutrophil antigens (HNAs) are a group of 5 glycoproteins that are expressed on human neutrophils, and in some cases, also on other cells and tissues. Endogenous antibodies against HNAs have been implicated in cases of alloimmune neonatal neutropenias, autoimmune neutropenias, febrile transfusion reactions and transfusion-related acute lung injury (TRALI). The availability of mAbs to HNAs permits the use of solid phase assays to detect endogenous antibodies specific to these antigens. While mAb reagents against HNA-1, HNA-2, HNA-4 and HNA-5 antigens are available, there are not yet any mAbs available for HNA-3. This project aims to develop mAbs against the two alleles of HNA-3 that can then be used to develop cell-based assays to detect anti-HNA-3a and anti-HNA-3b antibodies in blood donors. Furthermore, this project aims to use the anti-HNA-3 mAbs in existing in vitro transfusion models to help understand the mechanisms by which TRALI develops.

Intracellular processes including protein transport, transcription and signalling present a range of new potential targets that could have application in the treatment of a variety of disease indications. The screening of chemical libraries for binders to intracellular targets has had limited success due to (i) the relative lack of binding clefts and hydrophobic pockets compared to those located on cell surface proteins and (ii) the small “footprint” to which a small molecule can bind on an extensive protein surface interface. As an alternative, antibody fragments are being explored as molecular entities that are capable of disrupting protein-protein interactions within the cell, traditionally thought to be undruggable.5 This project will investigate strategies for intracellular delivery of antibodies and antibody fragments, along with assessing the resulting impact on cellular processes. An antibody against the transcription factor SOX18 (created at AIBN and IMB) will be used as the initial model system for intracellular antibody delivery. A successful outcome of this project will be the establishment of platform technology for the delivery of antibody fragments to intracellular targets.

Project Team