Rabbitts Group News
The latest news from the Rabbitts Group.
New macromolecules lay the foundations for new cancer therapeutics
Researchers from the MRC WIMM working in collaboration with scientists from AstraZeneca have designed new macromolecules which could potentially be developed as future cancer therapeutics with a greater targeted approach.
For some time, research and development of new cancer medicines have focused on discovering and targeting the genetic drivers and resistance mechanisms of cancer. By identifying the mechanisms through which tumour cells grow and divide (proliferate), we can develop drugs that target these molecules
“By identifying key proteins that are specific to cancer cells, we can then develop tailor-made drugs that impair the function of these specific proteins,” said Professor Terry Rabbitts from the MRC WIMM and MRC MHU, who co-led the study.
A good candidate for such a key protein to target is KRAS, from a protein family that participates in pathway relaying signals from outside the cell to the cell's nucleus. These signals instruct the cell to grow and proliferate: functions which are abnormal in cancer cells.
Previous work has found that the KRAS protein is altered in a quarter of human cancers: while the normal protein switches between active and inactive forms, mutant KRAS is in a permanently active state, telling the affected cell to keep dividing without dying. So this is a good protein to try and block to develop therapies that only target cancer cells.
“The problem is that it is very challenging to specifically target only one member of this protein family, because all the proteins in this family are so similar to each other,” said Dr Nicolas Bery, the first author on the study. “There are small molecules that can block KRAS selectively, but they only work for one particular KRAS mutation that makes up just 12% of cancer-causing KRAS mutations.”
“While earlier work from our lab identified compounds that can block all the members of the RAS family, we needed to find ways to block just the KRAS protein.”
So working with colleagues at AstraZeneca’s BioPharmaceuticals R&D, the Rabbitts lab has been developing a new approach focussed on using fragments of antibodies that normally track invasive proteins or viruses circulating inside cells. By looking for a new antibody-like fragment that can interfere with KRAS inside cells, the research team identified two Designed Ankyrin Repeat Proteins (DARPins, pictured in green) that attach only to KRAS (pictured, in tan) and do not bind the other RAS family members. When the DARPins were artificially expressed in human cancer cells, they only affected mutant KRAS proteins, but not its family members, NRAS or HRAS, even when these were mutated.
“There have been debates in the RAS community for a long time over the relative importance of each RAS isoform, with KRAS generally regarded as the most relevant target for cancer therapy. These DARPins identify a key binding site which enables exquisite KRAS specificity and provide tools to further investigate downstream biology.”, added Ralph Minter, Senior Director, Antibody Discovery & Protein Engineering, BioPharmaceuticals R&D, AstraZeneca.
The team also found that their novel DARPins were blocking the mutant KRAS protein’s function in two different ways, and they attached themselves to the protein at a different place from previous blockers.
“We now need to solve the problem of protein delivery to cells to actually use these molecules as drugs in patients,” said Professor Rabbitts.
The paper is published in Nature Communications.
A route from macrodrugs to small molecular drugs
Intracellular antibody fragments can be used to block protein function in cells by different mechanism including directly interfering with protein-protein interactions, preventing protein-nucleic interactions or causing target protein turnover. These macromolecules (designated as macrodrugs) can therefore be used for initial target validation to ensure that interfering with a target protein is relevant to a clinical indication. However, it has been difficult to implement the use of intracellular antibodies as macrodrugs because of the challenges in delivery into cells.
An alternative to using intracellular antibodies directly as macrodrugs is to exploit the antibody binding site to guide small molecule development and identify small molecule leads (precursors of drugs). This proof of principle has been shown in our recent paper (Nature Communications. doi:10.1038/s41467-018-05707-2) that describes using an anti-mutant RAS inhibitory intracellular antibody to allow the development of a chemical series of drug-like compounds with increasing potency against activated RAS in human cancer cells. These drug-like molecules will be amenable to further improvement to make cancer drugs using a combination of cell biology, medicinal chemistry and structural studies.
The general approach illustrated by our work, starting with an intracellular antibody fragment for target validation and its use to produce drug leads, could be applied to any target protein in a cell, within any clinical indication including cancer, neuropathies, infection and auto-immunity.
Quevedo, C.E., Cruz-Migoni A., Bery N., Miller A., Tanaka T., Petch D., Bataille C.J.R., Lee L.Y.R., Fallon P.S., Tulmin H., Ehebauer M.T., Fernandez-Fuentes N., Russell A.J., Carr S.B., Phillips S.E.V., Rabbitts T.H. (2018) Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment
Nature Communications 9, 3169 doi: 10.1038/s41467-018-05707-2
See also attached Biocentury commentary
Peptide Display Company Orbit Discovery Closes £6.9M Series A Financing
Oxford, UK: 23 May, 2018: Orbit Discovery Ltd, an Oxford, UK-based biotech company developing an industry-leading peptide identification and optimisation platform, today announced the closing of a £6.9M Series A Financing. The round was led by Oxford Sciences Innovation who were joined by new investors RT Ventures, Borealis Ventures, Perivoli Innovations, along with further investment from all existing investors and Oxford University. Additionally, Borealis Managing Partner Phil Ferneau and Oxford Investment Consultants’ Partner James Mallinson join Orbit’s Board of Directors.
Orbit was co-founded in 2015 by Professors Graham Ogg and Terry Rabbitts from Oxford University’s Weatherall Institute of Molecular Medicine. Orbit uses a proprietary technology originally developed to provide a diverse library of peptides to probe the cell surface. The initial scientific objective was to present random peptides to T-cells with unknown specificity as a way to establish their epitopes – something that was challenging with alternative peptide display technologies. As Orbit’s technology also supports the presentation of non-natural amino acids, constrained peptides and peptides modified after translation, the company has also established programs in peptide drug discovery and development, both internally and in partnership with biotechnology and pharmaceutical companies.
“We’ve been very successful in delivering on Orbit’s strategy, leading to growth in both capability development and commercial collaborations,” commented Alex Batchelor, CEO at Orbit. “This Series A funding allows us to greatly expand platform industrialisation whilst achieving the milestones in our existing three pharma partnerships. Moreover, we can support further strategic collaborations that align with development of our platform.”
“Orbit’s unique approach to peptide drug discovery and development, in combination with their well validated business model, has similarities to earlier Borealis investments that have been very successful”, commented Phil Ferneau. He added, “Having sufficient funds to further refine and develop the platform into novel areas such as functional screening of potential peptide drugs in cell-based assays combined with a sophisticated approach to partnering makes Orbit well placed to capitalise on increased interest in peptide therapeutics.”
James Mallinson added, “Since their formation in late 2015, Orbit have delivered on their early commitments and established a skilled and knowledgeable team. We’re delighted to be able to follow our seed investment with increased participation in this Series A round.”
Intracellular Antibody Capture Technology
The intracellular antibody capture technology (IACT) has been updated using improved screening protocols and focussed on single domain antibody fragments (iDAbs). This allows rapid isolation of iDAbs that can be used to block intracellular functions, such as protein-protein interactions, and to use this for target validation in diseases. Thus target validation can be achieved with intracellular antibody fragments prior to drug development programmes (to avoid the latter being undertaken prior to target validation).
Protocol Exchange (2018) DOI: http://dx.doi.org/10.1038/protex.2018.007
The selection of single-domain antibody fragments (iDAbs) by intracellular antibody capture in yeast
The ability to screen for intracellular antibody fragments inside yeast cells has been an important method to obtain single chain Fv (scFv) and intracellular domain antibodies (iDAbs) that express in the reducing environment of cells. The method was originally called Intracellular Antibody Capture (IAC) and, because it is a two hybrid-based method, it does not require immunisation of animals and does not require purified recombinant proteins to elicit an in vivo immune response or for other methods such as phage selection. The IAC method can be used to obtained intracellular antibodies to self-antigens and to conserved antigens. The new Protocol Exchange article sets out the most recent amendments to our screening protocol and tips for best practise.
New book publication on Chromosomal Translocations
A book edited by Janet Rowley, Michelle Le Beau and Terry Rabbitts called “Chromosomal Translocations and Genome Rearrangements in Cancer” has recently been published (Springer).
The multi-author book describes the discovery and characterization chromosomal translocations and aberrations in wide range of cancer types and the aim of the book is to facilitate development of better cancer diagnostics and treatments by informing academic and clinical scientists about the work on aberrant cancer genomes.
The volume was stimulated by work on genome organization from the first cloning of chromosomal translocations in the 1980s to current deep sequencing of cancer genomes. The first chromosomal translocation to be described as somatically acquired in a cancer cell was the Philadelphia chromosome found by Nowell and Hungerford in 1960. Chromosome banding techniques introduced in the 1970s allowed the elucidation of many more recurrent chromosomal abnormalities in cancer cells. The first molecular cloning of chromosomal translocation breakpoints was from the Philadelphia chromosome and from Burkitt’s lymphoma translocations, in the early 1980s. These studies laid the foundation for detection of many gene fusions in cancer as well as oncogene activation in lymphoid tumour chromosomal translocations. Breakpoint cloning and next generation deep sequencing has led to the detection of more than 1,300 different cancer-associated gene fusions.
Cancer genome characterization had reached a zenith by about 2010 that led the book’s Editors to conclude that the details of chromosomal abnormalities in most cancer types was well established and the volume therefore would be timely.
Clotten Foundation Prize 2015
Professor Terry Rabbitts has been awarded the inaugural Clotten Foundation Prize 2015 for discoveries on human antibody genes leading to therapeutic antibodies, and discoveries on chromosomal translocations in cancer leading to novel therapeutic approaches.
The Clotten Foundation was founded in 1994 following the death of Dr Annemarie Clotten, who bequeathed an endowment to establish the organisation. The Clotten Foundation aims to support cancer research that particularly benefits children with malignancies. The Foundation awards a prize every two years to an established scientist who
has contributed in a unique way to the understanding and treatment of cancer with a particular emphasis on children.
LMO2 at 25 years
25 years ago, the LMO2 gene was discovered near areas of chromosome damage in human blood cells, leading to cancer. LMO2 has since become a paradigm of a cancer gene caused by chromosome damage. It was also found to cause cancer in gene therapy trials involving bone marrow transplantation. Normally, LMO2 is an essential protein that performs critical functions in the development of the blood cells and some aspects of blood vessel formation while it has also recently been used as part of a mixture of proteins to create artificial stem cells. LMO2 is both a key-player in stem cell medicine and also a target for anti-cancer therapies. There is potential for therapeutic targeting in diseases where angiogenic processes are involved such as diabetic retinopathy.
A recent review has been published summarizing much of what is known about LMO2.
Intracellular Antibody Capture
Intracellular antibody capture (IAC) was developed to select antibody fragments that function in the reducing environment of cells and recognize native proteins (1, 2). Recent work has enhanced this method to simplify the IAC selection process while retaining the key feature of intracellular screening of diverse single domain libraries (3).
1. Tanaka, T., Rabbitts, T.H. (2010) Nature Protocols 5:1, 67-92
2. Zhang, J., Rabbitts, T.H. (2014) BBA - Proteins and Proteomics 1844, 1970-1976
3. Zeng, J., Li, H.H., Tanaka, T., Rabbitts, T.H. (2015) Selection of human single domain antibodies recognizing the CMYC protein using enhanced intracellular antibody capture
Journal of Immunological Methods
EPSRC Programme Grant to Support New Oxford Centre for Drug Delivery Devices
Our group will be part of the new Oxford Centre for Drug Delivery Devices, starting in July 2014, to develop new methods for drug delivery. The new Centre has been funded by a £10.1 million Programme Grant that includes £6.4 million from the Engineering and Physical Sciences Research Council (EPSRC).
New generation techniques have produced many macromolecules (e.g. antibody fragments) that function in the intracellular environment and these have potential to be drugs but the limitation is in delivering the macromolecules* to their site of action. The new funding will allow us to interact with experts in the Oxford Centre working with new drug delivery techniques to implement clinical delivery of our high affinity antibody fragments for cancer treatment.
* we call macromolecular drugs, macrodrugs to distinguish from conventional small molecule drugs
A group of undergraduate students studying human biosciences at Petroc, a further education college in north Devon, were invited to visit the THR lab & the WIMM on the 19th January 2015.
Students from the FdSc Human Bioscience spent a day at the Weatherall Institute of Molecular Medicine (WIMM), hosted by Dr Peter Canning, visiting Professor Rabbitts’ lab to hear about their research into how antibodies could be used as healthcare tools.
The day activities included meeting with Steve Taylor who gave a talk about Bioinformatics at WIMM , hearing about the role of iron in human immunity studies (by Professor Hal Drakesmith) and learning about research into regenerative medicine using zebrafish. This was followed by a visit to the Wolfson Imaging Centreand a visit to the Flow Cytometry facility.
The students appreciated the opportunity to see equipment that is not available in a classroom, as well as to gain an insight into the work of WIMM scientists. One second year student, Kerry, said: “The day has been inspirational – so many things to think about.”