Structural studies to advance understanding of IgM and its complement and receptor interactions

The human immune system provides protection against foreign organisms including bacteria, viruses and parasites through various cells such as the white blood cells, and proteins such as antibodies. In man, as in other animals, there are specialized types of antibody, but the type known as IgM is the most evolutionarily conserved and is present in all vertebrates that have antibodies. Not surprisingly, it performs essential functions in immunity, but in fact is present in the blood even before an individual encounters an invading organism or foreign "antigen", thus providing general protection against common pathogens. This circulating IgM also plays a role assisting in the clearance of dead and dying cells, an essential and continuous activity; when it fails, the build up of these cells and their contents can cause dangerous conditions and can lead to autoimmune reactions, in which the immune system turns against the individual's own proteins. IgM also determines the course of a healthy immune response, and once it has recognized an antigen as foreign, it binds and then activates defence mechanisms that will eventually lead to the clearance of the antigen. The initiation of these mechanisms involves interactions with other protein molecules in the blood, one of which is called C1q, or on the surfaces of various cells - these latter molecules are called "receptors" for IgM. One of these receptors was only identified three years ago and appears to enhance the immune response, while another that we will study may play an inhibitory role. Positive and negative feedback in the regulation of an immune respose is critical for health: over-reaction (allergic hypersensitivity for example, although caused by another type of antibody, or an auto-immune reaction initiated by IgM) is just as dangerous as under-reaction. Very little is known about any of these interactions and virtually nothing about the way in which they activate the defence mechanisms; the aim of this project is to advance our understanding of these key steps in the immune response through studying the structures of these molecules.

One remarkable aspect of the structure of the IgM molecule is that it is "polymeric", and much larger than the other types of antibody. Most antibodies are Y-shaped, with two "arms" to detect and bind to antigens, and a "stem" through which they bind to other molecules and to receptors on cells. IgM consists of either five or six of these Y-shaped units linked closely together in a ring. Yet despite their large size, ubiquitous occurrence and fundamental importance to human health, we have no detailed knowledge of the structure of IgM. We don't know how the units fit together, and such knowledge is essential if we are to understand how IgM works: how for example does it prevent binding to C1q until it senses a foreign antigen? Sometimes we would like to control the activities of IgM, such as when it is administered for therapeutic purposes and we want to restrict its ability to activate inflammatory reactions; we will only be able to do this if we know how IgM works and how it binds to C1q and to its receptors.

The work will involve producing the key parts of the IgM molecule, both as the single "monomeric" pieces and as the pentamer and hexamer. These have never been made before in a pure enough form to study their structure. We shall also produce the IgM-binding parts of the C1q and IgM receptor proteins. To determine the detailed three-dimensional structures of these molecules and to see how they interact with each other, we shall employ a technique that involves X-rays to locate the positions of all of the atoms in the structure. With other techniques we will measure the strengths of the binding interactions and understand how we might control them. These studies will thus reveal fundamental mechanisms of immunity and will have longer-term benefits for engineering the activities of IgM when used therapeutically.

IgM is the antibody of the primary immune response, but it also provides protection even before antigen is encountered, and controls the secondary response and the development of autoimmunity, among other roles. Nothing is known in detail about its polymeric structure, nor how this controls its complement-activating and receptor-binding activities. Monomeric IgM also forms part of the B cell receptor for antigen. We shall express, crystallize and carry out binding studies on a range of molecular species in order to provide a sound basis for understanding, and in future engineering, the functional biology of IgM. We shall express IgM-Fc including the Cm2 domains, and without them as Fcm3-4, including various glycoforms both with and without the tailpiece, and also express recombinant pentameric and hexameric IgM-Fc, with and without J-chain respectively. The globular head domain of the first sub-component of complement, gC1q, will be expressed, as well as the IgM-binding Ig-like domains of Fca/mR and FcmR, the two immuno-modulatory Fc receptors. Crystallization and structure determination will be the goal for each of these molecules and their complexes, together with binding studies by surface plasmon resonance (Biacore) and fluorescence anisotropy to determine affinities and mutual compatibility of these interactions, with ITC and/or AUC to determine stoichiometries as appropriate. Ion mobility MS and FRET labeling will be used to determine the shape and flexibility of IgM-Fc, in particular to establish whether the molecule is bent in solution. In advance of the structures of the receptor complexes, mutagenesis of the Fca/mR and FcmR domains, and of IgM-Fc and Fcm3-4 will be used to map the binding sites; the effects of these mutations will be valuable for later studies to modulate the individual activities to investigate functional mechanisms, or for engineering IgM for therapeutic purposes.

This project will provide structural information on IgM-Fc in its monomeric and polymeric forms, and its interactions with complement (C1q), and immune receptors Fca/mR and FcmR. Since there are no high-resolution structural data for any part of IgM-Fc, this will have a substantial impact for all researchers interested in antibody structure and function. The need for such structures is highlighted by numerous modeling studies of IgM used to interpret functional data. Modeling of monomeric IgM as part of the B cell receptor for antigen has also been undertaken in order to understand B cell activation, and conformational change mechanisms proposed but without supporting structural data. The C1q and receptor binding sites in IgM-Fc are not known in detail, nor whether there is steric or allosteric interplay between them; this fundamental information will be valuable to all those interested in the functional roles of IgM or engineering IgM for therapeutic applications, including the pharmaceutical industry. The potential impact of this information may be judged by analogy with the extensive structural data on IgG-Fc and its receptors that has had considerable impact upon engineering IgGs for therapeutic application, and also our work on IgE-Fc and its receptors that has influenced development of anti-IgE and IgE/receptor inhibitors for allergic disease. At a more fundamental level, mutagenesis to remove C1q and receptor activities from IgM can be used to probe functional mechanisms, as has been done extensively for IgG and IgE.

It has only recently been appreciated that IgM plays such a critical role in immune defence, providing early protection against infectious pathogens, directing the secondary response and, through removal of apoptotic cells, restricting atherosclerosis and the development of autoimmunity. Most antibodies isolated from human serum with anti-cancer activity are unmutated IgMs, and their tumour survelliance role is also under investigation. Furthermore, the only IgM isotype-specific receptor, FcmR, was discovered just three years ago. Thus many communities of researchers involved in these diverse disease areas are now looking at IgM. In humans, IgM anti-phosphoryl choline (PC) antibodies constitute a strong protection marker for atherosclerosis, and in SLE patients, higher levels of IgM autoantibodies that recognize epitopes on dead and dying cells, such as PC, correlate with lower susceptibility to cardiovascular events. IgM causes ischaemic reperfusion injury (IRI), and understanding how to control complement activation is important to IgM's therapeutic applications.

There are several ways in which IgM can be used therapeutically. Firstly, it can be administered by i.v. injection, as is IgG; purified IgM from serum is used to prevent sepsis (Pentaglobin). A monoclonal human IgM against GM3 ganglioside on melanoma cells, expressed as both a recombinant pentamer and hexamer in CHO cells, has already undergone a Phase I clinical trial. Three antibodies with in vitro anti-tumor cell activity have been expressed, one as hexamer in a human cell line. Our results may suggest improvements for these therapeutic IgM antibodies in the way that binding sites on IgG for complement and FcgR have been engineered. Secondly, in IRI, where IgM antibodies have a detrimental effect, inhibitory mimetic peptides have been developed which bind to the IgM Fab; one outcome of our project might be mutations in, or peptides/small molecules that bind to, IgM-Fc. Thirdly, it has been suggested that IgM can be used as an adjuvant to boost vaccination against microbes. The alarming rise of bacterial resistance to antibiotics has renewed interest in antibody therapy and vaccination strategies.

There are thus potential benefits for public health and for pharmaceutical industry. We also have a track record of training scientists for industry: two ex-PhD students from the group now lead drug discovery projects in major UK companies.