Developing a Rat Analogue of a Human Transglutaminase 2 Inhibitory Antibody to Facilitate Preclinical Testing in Treating Kidney Fibrosis

Fibrosis, or scarring, is a frequent feature of a number of relatively common, usually chronic, diseases. These include many types of lung, liver and heart disease and essentially all forms of chronic kidney disease. Studies of the mechanisms underlying the scarring process is an area of intense activity given that currently there is no recognized anti-scarring / anti-fibrotic therapy available for a condition that is ultimately fatal for millions of people each year. Subsequently the potential therapeutic benefits offered by the development of agents able to block or reduce the impact of scarring are considerable. Cumulative data from a range of different groups and experimental designs have highlighted tissue transglutaminase (transglutaminase type 2 ,TG2) as a key effector of the scarring process. Transglutaminases are a family of closely related enzymes that all catalyze the crosslinking of peptide chains via covalent epsilon (gamma-glutamyl) lysine bonds. It is this peptide crosslinking activity of TG2 that acts to produce the increased deposition and stabilization of extracellular matrix observed in chronic scarring.

Knockdown of TG2, either genetically, or through the use of small molecule inhibitors, has been shown to have clear beneficial effects in animal models of chronic kidney disease (CKD). Scarring & fibrosis is reduced and kidney function preserved by up to 80% over the timescale of the experiments. Translation of these results into the patient setting would have profound therapeutic application in the area of CKD, and possibly other diseases that feature chronic scarring. Prolonging kidney function and delaying the requirement for dialysis or renal transplantation would offer a substantial increase in the quality of life of CKD patients and have major cost implications for the health service.

Given the clear case for the development of TG2 inhibitors, there is much interest in this area of research. Development of inhibitors have been hampered by the degree of structural conservation between the 8 different members of the TG family. The catalytic site, or 'core', is highly conserved and all existing small molecule candidates have exhibited prohibitive levels of cross reactivity with other related enzymes. This is particularly important in the case of TG1 and TG3, which are critical to maintaining integrity of the dermis, and factor XIIIa, a major component of the clotting process. Inhibition of these related TG molecules produces serious side effects that would preclude a therapeutic role for existing small molecule inhibitors. A monoclonal antibody approach to this problem offers the potential to overcome these specificity issues due to the very precise nature of antibody targeting.

Using an innovative immunization approach we have successfully generated a series of TG2-specific inhibitory monoclonal antibodies. All are specific for human TG2 and show no cross reactivity to other members of the enzyme family. We have established the inhibitory potential of these mAbs in different in vitro systems and derived IC50 data. The most effective of these antibodies has been humanised, with the affinity and inhibitory activity being preserved. The next stage in the development of this potential therapeutic is to generate preclinical data in FDA approved animal models of chronic fibrosis. The most effective monoclonal, AB1, and the other candidates, are all specific for human TG2, and lack significant inhibitory action against mouse or rat TG2. Accordingly, the aim of this project is to allow us to generate orthologous inhibitory antibodies against rat and mouse TG2, in order that we can gather this crucial preclinical data, attract downstream commercial partners, and accelerate the development of this potentially new class of therapeutic agents.

Background: Transglutaminase 2 (TG2) is central to the development of kidney fibrosis. No specific inhibitor exists. We have therefore developed TG2 inhibitory antibodies targeting 4 defined epitopes in human TG2. These do not inhibit mouse or rat TG2 effectively & are subsequently unsuitable for pre clinical testing.
Purpose: To generate recombinant TG2 antibodies targeting the corresponding inhibitory epitopes in mouse & rat. Undertake preclinical testing of these antibodies as anti-fibrotic agents.

Antibody generation: A scFv phage library will be constructed from the spleens of TG2 KO mice immunised with rat TG2. Phage selection will be used to isolate mAb sequences binding to known inhibitory epitopes. Selected clones will be used to produce inhibitory antibody fragments by recombinant methods, and validated in cell systems.

In vivo application in kidney fibrosis: The best 2 mAbs will be applied to the mouse unilateral ureteric obstruction (UUO) model of kidney fibrosis to provide early proof of principle data on TG2 inhibition, fibrosis & collagen accumulation. The UUO model is rapid, but does not provide functional data & fails to demonstrate typical glomerulosclerosis and tubulointerstitial fibrosis. To address this mAbs will be applied to the rat subtotal nephrectomy model of kidney fibrosis. This is the chronic kidney disease model recognised by the FDA, allowing measurement of function (creatinine clearance, albuminuria) and showing features of human kidney fibrosis (collagen accumulation, fibroblast proliferation, loss of architecture). To achieve this, a series of rat chimeras of the mouse antibodies will need to be generated by framework exchange (MRCT).
Other fibrotic diseases: To demonstrate these therapeutic antibodies have a wider application in the treatment of fibrotic disease, they will be applied to the bleomycin model of pulmonary fibrosis, with lung function (dynamic compliance, blood gasses), collagen & fibrotic index measured.

Our project will potentially impact on a considerable number of other non-academic beneficiaries; within industry, charities, other public bodies, government departments and the pool of patients with CKD, but may also extend to those with lung, liver or heart fibrosis. Our primary route for the dissemination of research data will be publication in scientific journals, presentations at scientific conferences and approaching appropriate charities.

Industry. The Sheffield Group are world leaders in transglutaminase biology in fibrosis having not only initially described changes in TG2 in fibrosis (animals & human), but published 2 papers on TG inhibitors in treating experimental CKD, described the mechanism of TG2 action in ECM expansion and identified the cell export motif on the TG2 beta sandwich. They have clearly demonstrated the disease linkage and validated TG2 as a target for therapeutic intervention. Sheffield also has particular expertise in numerous models of kidney fibrosis and is regarded as one of the premier european centres for models of end stage kidney disease, being contracted on numerous occasions by major phramaceutical companies such as Pfizer and Gherbet. While TG2 is a recognised target for renal fibrosis, the current small molecules have limitations, ie selectivity. A alternative approach which gives exquisite selectivity is a therapeutic antibody approach. This is a growing area with an increasing number of anitbodies now on the market for a wide range of disease indications. This project builds on an already existing collaboration with MRCT which has generated a candidate antibody that inhibits TG2 activity and which is currently being humanised. The MRCT Group have a 20 years track record in humanising antibodies and developing a data packages suitable for partnering with pharma and biotech. The MRCT group have successfully humanised over 45 rodent monoclonals, 12 of which have progressed to the clinic, 8 are currently in active clinical development and 2 antibodies, Tysabri and Actemra have secured marketing approval.

We would engage industry through scientific routes (conferences, presentations, invited seminars) and business routes (preparing a promotional package of our novel antibodies for several big pharmas; attending bio partnering events to engage big and small pharmas).

Funding Bodies. Completion of our program will be attractive to funding bodies too since, it will deliver a tool antibody for POC studies in a disease relevant model of fibrosis. Within the scope of this project, the tool antibodies will be tested in renal fibrosis and lung fibrosis models, but could be made available to other academic groups for testing in other disease models, e.g. liver and heart. TG2 is also implicated in celiacs disease, AD, PD and Huntingdon's disease. These antibodies could be useful as research tools for further investigation of the target, exploring for instance new avenues, thus seeding novel projects.

Patients and Health Service. Development of novel therapeutics to target fibrosis would be beneficial for a large number of patients. The primary indication will be CKD. Between 1 in 10 (over 65) and 1 in 50 (under 30) have CKD. This leads to 108 people per million of population developing kidney failure due to CKD each year in the developed world. In the UK 5600 patients started on dialysis in 2010 with 42 000 patients in total needing RRT. The NHS spent £2.1 Billion (6% of its total budget) on renal care in 2011, 80% of which was in providing RRT. In USA they have a higher incidence of CKD due to susceptibility in Afro-Caribbeans and higher rates of diabetes. More than 380 000 Americans were on dialysis in 2009 costing $39 billion (Medicare $12 billion). Importantly the average survival on dialysis is 5.3 years.

The Sheffield Kidney Group provide integrated clinical, academic and research training into the treatment of kidney disease and directly engage patient groups.