Impact of target antigen properties on antibody effector mechanisms

Monoclonal antibody (mAb) immunotherapy has proven effective in the treatment of haematological malignancies. Antibodies towards unique epitopes within the same antigen can engage different effector mechanisms to facilitate cell clearance. Understanding what drives the engagement of these mechanisms is important for the development of new therapeutics. This thesis investigated the role of the antigen, in particular the epitope bound by a mAb in defining the effector mechanisms engaged. Specifically, the role of distance between the epitope and the target cell membrane in relation to the engagement of the effector mechanisms; complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) were assessed. A panel of model antigens were generated; incorporating the same epitope for a clinically relevant mAb attached to the N-terminus of various CD137 constructs. Extracellular domains of CD137 were removed or added in order to change the distance of the clinically relevant epitope from the cell membrane. These constructs were transfected into CHO-S and A20 cells and tested in vitro to assess the engagement of the three aforementioned effector mechanisms. It was found that the engagement of CDC and ADCC was diminished when targeting the largest (8-domain) construct (therefore most distal from the membrane), whilst ADCP was impaired with the smallest (membrane-flush) construct and required the presence of at least one extracellular domain for activity. However, ADCP engagement was restored when the membrane-flush epitope was tethered to the membrane via a GPI anchor rather than a transmembrane peptide domain. These findings were confirmed using two separate epitopes targeted by rituximab and CAMPATH-1H antibodies. Finally, the therapeutic response when targeting either the membrane-flush or 8-domain constructs was investigated in vivo. Mice who received A20 cells expressing the membraneflush construct exhibited clearance of tumour in the spleen following antibody therapy, whilst those that received tumours expressing the 8-domain construct did not respond to therapy. Together the work in this thesis demonstrates how the effector mechanisms engaged by a mAb can be altered dependent on the position of the epitope in relation to the cell membrane.<br/

Regulation of monoclonal antibody immunotherapy by FcγRIIB

Monoclonal antibodies (mAb) are revolutionising the treatment of many different diseases. Given their differing mode of action compared to most conventional chemotherapeutics and small molecule inhibitors, they possess the potential to be independent of common modes of treatment resistance and can typically be combined readily with existing treatments without dose-limiting toxicity. However, treatments with mAb rarely result in cure and so a full understanding of how these reagents work and can be optimised is key for their subsequent improvement. Here we review how an understanding of the biology of the inhibitory Fc receptor, Fc?RIIB (CD32B), is leading to the development of improved mAb treatments

Professional Doctorate degrees in nursing and graduate outcomes: A narrative scoping review

Professional Doctorate Degrees in Nursing have existed for many years beginning in the United States in the 1950’s and growing rapidly since; however, the graduate outcomes and impact on the clinical setting have not been explored in depth. This scoping review is being conducted to assess the outcomes of these programmes on doctoral nursing graduates, with the aim of better understanding universal concepts to inform programme development and the impact on clinical and other healthcare settings

Immunological methods to study monoclonal antibody activity

Over recent decades it has become increasingly apparent that malignant cells, including chronic lymphocytic leukemia (CLL) cells, do not exist in isolation. Rather they coalesce with numerous “normal” cells of the body and, in the case of CLL, inhabit key immunological niches within secondary lymphoid organs (SLO), where a plethora of stromal and immune cells mediate their growth and survival. With the advent and approval of targeted immune therapies such as monoclonal antibodies (mAb), which elicit their efficacy by engaging immune-mediated effector mechanisms, it is important to develop accurate methods to measure their activities. Here, we describe a series of reliable assays capable of measuring important antibody-mediated effector functions: antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) that measure these immune activities

Antibody distance from the cell membrane regulates antibody effector mechanisms

Immunotherapy using mAbs, such as rituximab, is an established means of treating hematological malignancies. Abs can elicit a number of mechanisms to delete target cells, including complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, and Ab-dependent cellular phagocytosis. The inherent properties of the target molecule help to define which of these mechanisms are more important for efficacy. However, it is often unclear why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules was generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains to produce a panel of chimeric molecules that held the target epitope at different distances along the protein. It was shown that complement-dependent cytotoxicity and Ab-dependent cellular cytotoxicity favored a membrane-proximal epitope, whereas Ab-dependent cellular phagocytosis favored an epitope positioned further away. These findings were confirmed using reagents targeting the membrane-proximal or -distal domains of CD137 itself before investigating these properties in vivo, where a clear difference in the splenic clearance of transfected tumor cells was observed. Together, this work demonstrates how altering the position of the Ab epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents

Development of a T cell receptor mimic antibody against wild-type p53 for cancer immunotherapy

The tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Due to its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anti-cancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of major histocompatibility class I (MHC I), and can be bound by T cell receptors (TCRs). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201-presented wild-type p53 T cell epitope, p5365-73(RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitro. In vivo, the antibody targets p5365-73 peptide-expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy