Final Report Summary - EUCAAD (European Consortium for Anticancer Antibody Development)
Executive summary:
Angiogenesis, the formation of new blood vessels to supply growing tissue with oxygen and nutrients, was early recognised as a process that could be targeted to inhibit tumour growth. The idea was compelling, and was based on the realisation that endothelial cells lining the capillaries are normal and stable cells that are less prone to genetic instability and hence less likely to develop escape mechanisms when put under the pressure of a drug. However, the biology and the molecular targets to capitalise on this concept remained unknown for a long period of time, and it was not until the factors and pathways driving angiogenesis were discovered and defined that focused research and drug development could commence. At the same time as the discovery of the potential of targeting the angiogenic processes, antibodies were also realised to have a potential in therapy. Antibodies, being natural compounds and specific for their target, were considered potentially valuable for specific treatment of disease, particularly for those caused by infectious agents. The appealing property of antibodies is their specificity. They can single out and bind to a particular molecular structure in the midst of an ocean of other structures. This means that the effect of the antibody can be targeted to cells either expressing the antigen or that are dependent on the antigen, for example, a growth factor.
Research in the laboratories of beneficiary 1 has led to the identification of a unique protein called angiomotin (AMOT) which has so far only been detected in endothelial cells of angiogenic tissues as well as cytotrophoblasts of the human placenta. Several lines of investigations have also revealed that targeting AMOT may impede neovascularisation and inhibit the growth of tumours. A recent report from the laboratories of beneficiary 1 demonstrated that a Deoxyribonucleic acid (DNA) vaccine targeting AMOT inhibited angiogenesis and tumour growth in a mouse model. The effect of the vaccine was predominantly mediated through the induction of antibodies since the antitumour effects were abrogated in B cell knockout mice. Subsequently, two other related proteins AMOT-like molecules 1 and 2 (AMOTL1 and AMOTL2) were also identified. These data provide the rationale for generating therapeutic antibodies to target AMOT and related molecules during tumour angiogenesis.
EUCAAD will identify and validate new target molecules expressed by tumour cells or associated with tumour vascular-matrix interactions, develop new antibody-based therapeutic strategies and examine such strategies in vitro and in animal leading to clinical testing in humans. The project represents a virtual research institution in Europe and consists of 5 highly competent academic centres together with three leading Small and medium-sized enterprises (SMEs) and one non-SME commercial partner within the field of oncology research. Antibodies against these proteins will be tested for affinity and specificity for tumour or stromal components following which, the efficacy of the antibodies in animal models of tumourigenesis will be examined. Following validation the antibodies will be produced in a clinical scale paralleled with pharmacological and toxicity testing prior to a clinical trial. In this project, basic science, translational research and clinical activities are strongly integrated, in order to validate defined targets and to develop new therapeutic entities and principles for clinical implementation.
Project context and objectives:
The EUCAAD project has the overall objective of defining novel target structures and generating new antibodies that may be used for therapy of cancers. The two fundamental types of cancer-associated molecules to be targeted in the course of the EUCAAD project molecules related to tumour angiogenesis and tumour-associated antigens.
Angiogenesis, the formation of new blood vessels to supply growing tissue with oxygen and nutrients, was early recognised as a process that could be targeted to inhibit tumour growth. The idea was compelling, and was based on the realisation that endothelial cells lining the capillaries are normal and stable cells that are less prone to genetic instability and hence less likely to develop escape mechanisms when put under the pressure of a drug. However, the biology and the molecular targets to capitalise on this concept remained unknown for a long period of time, and it was not until the factors and pathways driving angiogenesis were discovered and defined that focused research and drug development could commence. At the same time as the discovery of the potential of targeting the angiogenic processes, antibodies were also realised to have a potential in therapy. Antibodies, being natural compounds and specific for their target, were considered potentially valuable for specific treatment of disease, particularly for those caused by infectious agents. The appealing property of antibodies is their specificity. They can single out and bind to a particular molecular structure in the midst of an ocean of other structures. This means that the effect of the antibody can be targeted to cells either expressing the antigen or that are dependent on the antigen, for example, a growth factor.
Research has led to the identification of a unique protein, called AMOT, which has so far only been detected in endothelial cells of angiogenic tissues as well as cytotrophoblasts of the human placenta. Several lines of investigations have also revealed that targeting AMOT may impede neovascularisation and inhibit the growth of tumours. A recent report from the laboratories of beneficiary 1 demonstrated that a DNA vaccine targeting AMOT inhibited angiogenesis and tumour growth in a mouse model. The effect of the vaccine was predominantly mediated through the induction of antibodies since the anti-tumour effects were abrogated in B cell knockout mice. Subsequently, two other related proteins AMOT-like molecules 1 and 2 (AMOTL1 and AMOTL2) were also identified. These data provide the rationale for generating therapeutic antibodies to target AMOT and related molecules during tumour angiogenesis.
Another target that is being developed by beneficiary 1 for antibody-based therapy of Chronic lymphocytic leukemia (CLL) is a molecule called ROR1. ROR1 is located on the chromosomal region 1p31.3 (http://www.ensembl.org) and encodes a 105 kDa protein with potential tyrosine kinase function. ROR1 messenger Ribonucleic acid (mRNA) is normally expressed in heart, lung and kidneys in adult humans and to a lesser extent in the placenta, pancreas and skeletal muscles and a truncated ROR1 is known to be expressed in human leukemia and lymphoma cell lines. Recent studies have demonstrated that ROR1 can bind Wnt5a in CLL cells and is currently being actively explored in vitro by several research groups as a potential target for therapy of CLL.
EUCAAD represents a virtual research institution in Europe and consists of five highly competent academic centres together with three leading SMEs and one non-SME commercial partner within the field of oncology research. The project will identify and validate new target molecules expressed by tumour cells or associated with tumour vascular-matrix interactions, develop new antibody-based therapeutic strategies and examine such strategies in vitro and in animal leading to clinical testing in humans. Antibodies against these proteins will be tested for affinity and specificity for tumour or stromal components following which, the efficacy of the antibodies in animal models of tumour genesis will be examined. Following validation the antibodies will be produced in a clinical scale paralleled with pharmacological and toxicity testing prior to a clinical trial. In this project, basic science, translational research and clinical activities are strongly integrated, in order to validate defined targets and to develop new therapeutic entities and principles for clinical implementation.
In the first 24 months, there has been considerable progress in the tasks intended for the project and overall the implementation of EUCAAD is on schedule. In WP1, collection of tissue and serum samples is ongoing and a set of standard protocols have been developed in the first 12 months. In WP2 several proteins have been identified co-localising with AMOT. These tasks in WP2 have been achieved before schedule and a list of candidate partners for AMOT, AMOTL1 and AMOTL2 have been partially described. These include Syx, Patj, Pals1, Lin7, Mupp1. Several monoclonal antibodies have been developed against the Ror1 molecules and are currently being tested for their ability to biologically antagonise and facilitate antibody-mediated cellular cytotoxicity of CLL cells. Studies have demonstrated that Ror1 is specifically upregulated in CLL in comparison to normal peripheral blood cells and silencing of Ror1 can lead to apoptosis of CLL cells. This contributes to one of the tasks in WP3. Expression of AMOT, AMOTL1 and AMOTL2 has been studied extensively in zebrafish, mouse and human tissue under WP4. Functional effects of these molecules have been studied by using siRNA mediated silencing. There is a strict direct relationship between tumour grade and level of ROR1 positivity with poorly differentiated carcinomas more often expressing high levels of ROR1.
AMOT knockout mice have been created. The effect of vaccination against AMOT in the cure of neu+ established tumours. New insights into the mechanism of this inhibition demonstrating that vessel alterations were due to an NK mediated ADCC. In WP6, a panel of antibodies against different regions of the Ror1 molecule has been tested for their ability to induce apoptosis in CLL cells. Further investigations are under way to characterise the leading candidates of ROR1 antibodies that have demonstrated promise in initial experiments. At least two antibodies have been validated as benchmarks for imaging studies in WP7 and two separate methods for radioisotopic or non-radioistopic labelling of antibodies for imaging have been established.
Project management activities of EUCAAD have been implemented in parallel to the scientific activities. The last period review of the EUCAAD project was held at Frankfurt / Main, Germany on 20 April 2012. The suggestions of the reviewer have been taken into consideration and duly the implementation activities of the EUCAAD consortium has been adjusted to emphasise on the development of antibodies against Ror1.
Project results:
WP1 - Acquisition of clinical samples and relevant clinical data
According to the plan, we have established a bio bank consisting of samples from breast cancer and renal cancer. These samples have been used to map target gene expression as well as detection of marker antibodies in blood.
WP2 - Identification of AMOT associated proteins
This work package has revealed a novel signalling pathway that controls the migration of blood vessels during angiogenesis (the term for blood vessel formation from pre-existing vessels). We show that AMOT forms a protein complex that regulates cytoskeletal turnover in the migrating cell and this controls directional movement. This is the biological effect that we aim to target with anti-AMOT antibodies.
WP3 - Discovery of potential target structures expressed by tumour and endothelial cells
MALDI imaging was developed in this WP. This technique combines the advances in proteomics technology to histological levels. We have analysed the profile of proteins in biopsies from renal cancer patients. The technique allowed differentiation of expression of protein profiles in different tumour regions. such as stroma, normal tissue and cancer cells. WP4 - Target validation in vitro
ROR1 antibodies induce tumour cell death in vitro
The receptor tyrosine kinase ROR1 is shown to be expressed in CLL and not in samples from normal donors. Furthermore, the receptor is highly phosphorylated which is indicative of receptor activation. Thus, ROR1 may play an essential role in expansion of CLL tumour cells. Inactivation of ROR1 signalling using either siRNA or mouse monoclonal antibodies induced cell death in CLL patient samples in vitro. Taken together, these data shows that CLL tumour cells depend on ROR1 signalling and when this pathway is inhibited they undergo programmed cell death.
AMOT controls endothelial cell migration. Here we used either genetic ablation of the AMOT gene or overexpression of AMOT in an endothelial cell-line. Both approaches indicated a role of AMOT in directional cell migration.
WP5 - Target validation in vivo
Roles of AMOT, AMOTL1 and AMOTL2 in normal vascular development: We have used two powerful genetic approaches to analyse the role of the AMOT protein family during blood vessel formation in vivo. These include studies of developmental angiogenesis in zebrafish and mouse.
We have silenced all AMOT family members during zebrafish vascular development. The effects of this gene silencing approach have been evaluated in transgenic zebrafish where blood vessels are highlighted by expression of fluorescent markers. In mouse we have used homologous recombination to genetically silence AMOT, AMOTL1 or AMOT2. The chosen approach has allowed us to specifically silence the studied gene in the blood vessel endothelial cells at any time point during development. The data show that each individual member of this family has a distinct function in blood vessel formation. AMOT controls the directional migration of capillaries; AMOTL1 is affecting cellular contacts and AmotL2 controls lumen formation. These are three vital processes during the establishment of a functional blood circulation and opens up the possibility to target these distinct events during pathological blood vessel formation in disease.
DNA vaccination: Targeting AMOT and AMOTL1 in a mouse breast cancer model. The mouse mmtv-neuT transgenic model mimics human breast cancer in that tumours are driven by the rodent counterpart of the HER2 oncogene. These mice invariably develop tumours and succumb to disease 22 weeks after birth. Single vaccination with an AMOT DNA increases survival with over two months. Perhaps even more exciting are the findings two vaccinations using a combination of Amot, AmotL1 and HER2 completely protect the mice from dying from dying of breast cancer. This effect is antibody dependent and thus provides the rationale for generating recombinant therapeutic antibodies targeting AMOT and AMOTL1.
Taken together genetic inactivation and DNA vaccination data provide the rationale for targeting both AMOT and AMOTL1 to efficiently inhibit tumour angiogenesis.
WPs 6,7 - Antibody selection and in vitro characterisation
Generation of a CLL model in mouse. We have selected human recombinant antibodies on their capacity to induce apoptosis of human primary CLL cells from patients at the Karolinska Hospital. Prior to our studies, no animal model of CLL existed. A mouse model was established by grafting CLL cells from patients into immune deficient mice. This is to our knowledge the first model system of CLL and it was then used to analyse antibody efficacy. AMOT antibodies inhibit endothelial cell migration in vitro.
We have isolated human single chain antibodies reactive against AMOT. These antibodies have been tested for anti-endothelial migration activity in vitro. The best candidates have been selected and tested in mice for anti-angiogenic and anti-tumour activities. However, an unforeseen problem arose when it proved impossible to maintain biological activity when the single chain antibodies where converted into immunoglobulin format. This conversion increases half-life in circulation and is presently the standard forma when going into clinical trials. We have solved this problem by fusing the single chain antibody to an albumin-binding domain. Once injected, the single chain antibody binds to albumin in circulation and thus has increased half-life. Our in vivo data show that this antibody specifically targets migrating endothelial cells in developing vessels of the new born retina. The specificity is unprecedented and opens up the possibility to remove the migrating tip cells during angiogenesis but leaving the rest of the vasculature intact.
WPs 8,9
A number of ROR1 antibody candidates have been generated in the consortium. These have been tested in the novel mouse model system described in WPs 6-7. Clinical trials with the lead candidate are planned within 1-2 years from the ending of the EUCAAD project.
Potential impact:
Cancer is the second leading cause of death in the European Union after heart disease and one of the most urgent health problems in the developed world. 3.2 million Europeans are diagnosed with cancer every year, according to EU figures, and the disease is responsible for 29 per cent of deaths among men and 23 per cent amongst women.
While survival rates are improving all the time, the huge number of people still losing their battle with cancer highlights the urgent need for further research. Indeed, the in three men and one in four women are expected to be directly affected by the disease by the age of 75, and these figures are predicted to rise as Europe's population ages further. Such high levels of incidence have led the EU to set a goal of cutting cancer rates by 15 % by 2020. To this end, it is channelling hundreds of millions of euros into research for innovative cancer treatments every year. That includes investing in a ?virtual research institute drawn from some of Europe's best known centres of excellence to develop targeted new therapies aimed specifically at cancer cells or cells of the stroma the surrounding mass of tissue including blood vessels that support tumour growth.
The consortiums main focus has been to develop antibodies against new target structures on tumour cells, and on the blood vessels supplying tumours, which are responsible for the cancer?s growth, progression and spread. The goal of a targeted therapy is to increase anti-tumour efficacy with lowest possible side effects.
Unpleasant side effects are one of the problems associated with conventional treatments such as chemotherapy and radiotherapy. Cancer cells share many common features with normal host cells from which they derive, and traditional cancer therapy often fails to discriminate between normal and malignant tissues.
Also, patients with advanced cancers which have spread to other parts of the body frequently relapse and succumb to their disease after developing resistance to such conventional treatments. That is why there has been such interest in developing more targeted forms of therapy, with antibodies having become the designer cancer treatment of choice in recent years.
The advantage of antibodies is that they are extremely specific; each one will bind to and attack one particular antigen. Antibodies have been developed to inhibit cell growth in tumours, induce apoptosis sometimes dubbed as cell suicide and deliver radiation or other potent anti-cancer drugs directly to malignant cells.
The scale of the cancer challenge is such that new strategies for targeting widespread metastases are urgently needed. To this end, EUCAAD has seven key objectives, one of the most important of which has been to identify new structures in cancer cells and in the tissue and blood vessels supporting tumours that can be targeted using antibody therapy. The EUCAAD consortium has identified a list of candidate drug targets expressed either by the tumour or in the supporting normal tissues. From this list, the consortium has gone on to test the capacity of DNA vaccination against selected target proteins in breast and lymphoma models. Three of the tested molecules provided exciting anti-cancer activities.
Moving new discoveries from the bench to the bedside requires the kind of interdisciplinary, collaborative, team-oriented approach taken by the consortium and is the fastest way to achieve the kind of major breakthroughs in cancer treatment we all want to see. The joint goal for the consortium has been to generate human antibodies to treat cancer patients. EUCAAD has proceeded to produce human monoclonal antibodies against two of the target molecules. These have been validated in terms of tissue specificity, binding affinity and functional properties; and examining the ability of selected antibodies against the genes ROR1 and AMOT inhibit tumour progression in animal models. These tests have shown great promise and EUCAAD intends to begin clinical trials to assess efficacy in cancer patients in the near future.
The activities and results generated in the EUCAAD have been disseminated to society in several ways. The project has been presented at the EUCAAD web site where progress and meetings are advertised and open to the public. Data from the collaboration has been presented in a number of oral and poster presentations at international conferences as well as publications in internationally high ranking journals.
In order to promote exchange of ideas in the field of cancer therapy, EUCAAD has organised and sponsored a workshop on anti-angiogenesis therapy in Marseille 2009 with participation of invited leading scientists. The importance of the interaction between academia and small medium enterprise has been acknowledged among European decision makers. EUCAAD has actively participated in the symposia, such as 'Biotech SMEs in health and disease' organised by the State of North Rhine-Westphalia representation in Brussels 2010. These events provided the opportunity for dialogue amongst Europe's political leaders, innovative business operators and researchers. Here EUCAAD has been highlighted as a positive example on how SMEs can benefit from the interaction with European research institutes.
Taken together, EUCAAD has linked education, research and biotech business to answer to the urgent demand of new modern cancer therapies. After clinical trials, the ultimate dissemination is to provide an efficient treatment to cancer patients.
List of websites: www.eucaad.net
Angiogenesis, the formation of new blood vessels to supply growing tissue with oxygen and nutrients, was early recognised as a process that could be targeted to inhibit tumour growth. The idea was compelling, and was based on the realisation that endothelial cells lining the capillaries are normal and stable cells that are less prone to genetic instability and hence less likely to develop escape mechanisms when put under the pressure of a drug. However, the biology and the molecular targets to capitalise on this concept remained unknown for a long period of time, and it was not until the factors and pathways driving angiogenesis were discovered and defined that focused research and drug development could commence. At the same time as the discovery of the potential of targeting the angiogenic processes, antibodies were also realised to have a potential in therapy. Antibodies, being natural compounds and specific for their target, were considered potentially valuable for specific treatment of disease, particularly for those caused by infectious agents. The appealing property of antibodies is their specificity. They can single out and bind to a particular molecular structure in the midst of an ocean of other structures. This means that the effect of the antibody can be targeted to cells either expressing the antigen or that are dependent on the antigen, for example, a growth factor.
Research in the laboratories of beneficiary 1 has led to the identification of a unique protein called angiomotin (AMOT) which has so far only been detected in endothelial cells of angiogenic tissues as well as cytotrophoblasts of the human placenta. Several lines of investigations have also revealed that targeting AMOT may impede neovascularisation and inhibit the growth of tumours. A recent report from the laboratories of beneficiary 1 demonstrated that a Deoxyribonucleic acid (DNA) vaccine targeting AMOT inhibited angiogenesis and tumour growth in a mouse model. The effect of the vaccine was predominantly mediated through the induction of antibodies since the antitumour effects were abrogated in B cell knockout mice. Subsequently, two other related proteins AMOT-like molecules 1 and 2 (AMOTL1 and AMOTL2) were also identified. These data provide the rationale for generating therapeutic antibodies to target AMOT and related molecules during tumour angiogenesis.
EUCAAD will identify and validate new target molecules expressed by tumour cells or associated with tumour vascular-matrix interactions, develop new antibody-based therapeutic strategies and examine such strategies in vitro and in animal leading to clinical testing in humans. The project represents a virtual research institution in Europe and consists of 5 highly competent academic centres together with three leading Small and medium-sized enterprises (SMEs) and one non-SME commercial partner within the field of oncology research. Antibodies against these proteins will be tested for affinity and specificity for tumour or stromal components following which, the efficacy of the antibodies in animal models of tumourigenesis will be examined. Following validation the antibodies will be produced in a clinical scale paralleled with pharmacological and toxicity testing prior to a clinical trial. In this project, basic science, translational research and clinical activities are strongly integrated, in order to validate defined targets and to develop new therapeutic entities and principles for clinical implementation.
Project context and objectives:
The EUCAAD project has the overall objective of defining novel target structures and generating new antibodies that may be used for therapy of cancers. The two fundamental types of cancer-associated molecules to be targeted in the course of the EUCAAD project molecules related to tumour angiogenesis and tumour-associated antigens.
Angiogenesis, the formation of new blood vessels to supply growing tissue with oxygen and nutrients, was early recognised as a process that could be targeted to inhibit tumour growth. The idea was compelling, and was based on the realisation that endothelial cells lining the capillaries are normal and stable cells that are less prone to genetic instability and hence less likely to develop escape mechanisms when put under the pressure of a drug. However, the biology and the molecular targets to capitalise on this concept remained unknown for a long period of time, and it was not until the factors and pathways driving angiogenesis were discovered and defined that focused research and drug development could commence. At the same time as the discovery of the potential of targeting the angiogenic processes, antibodies were also realised to have a potential in therapy. Antibodies, being natural compounds and specific for their target, were considered potentially valuable for specific treatment of disease, particularly for those caused by infectious agents. The appealing property of antibodies is their specificity. They can single out and bind to a particular molecular structure in the midst of an ocean of other structures. This means that the effect of the antibody can be targeted to cells either expressing the antigen or that are dependent on the antigen, for example, a growth factor.
Research has led to the identification of a unique protein, called AMOT, which has so far only been detected in endothelial cells of angiogenic tissues as well as cytotrophoblasts of the human placenta. Several lines of investigations have also revealed that targeting AMOT may impede neovascularisation and inhibit the growth of tumours. A recent report from the laboratories of beneficiary 1 demonstrated that a DNA vaccine targeting AMOT inhibited angiogenesis and tumour growth in a mouse model. The effect of the vaccine was predominantly mediated through the induction of antibodies since the anti-tumour effects were abrogated in B cell knockout mice. Subsequently, two other related proteins AMOT-like molecules 1 and 2 (AMOTL1 and AMOTL2) were also identified. These data provide the rationale for generating therapeutic antibodies to target AMOT and related molecules during tumour angiogenesis.
Another target that is being developed by beneficiary 1 for antibody-based therapy of Chronic lymphocytic leukemia (CLL) is a molecule called ROR1. ROR1 is located on the chromosomal region 1p31.3 (http://www.ensembl.org) and encodes a 105 kDa protein with potential tyrosine kinase function. ROR1 messenger Ribonucleic acid (mRNA) is normally expressed in heart, lung and kidneys in adult humans and to a lesser extent in the placenta, pancreas and skeletal muscles and a truncated ROR1 is known to be expressed in human leukemia and lymphoma cell lines. Recent studies have demonstrated that ROR1 can bind Wnt5a in CLL cells and is currently being actively explored in vitro by several research groups as a potential target for therapy of CLL.
EUCAAD represents a virtual research institution in Europe and consists of five highly competent academic centres together with three leading SMEs and one non-SME commercial partner within the field of oncology research. The project will identify and validate new target molecules expressed by tumour cells or associated with tumour vascular-matrix interactions, develop new antibody-based therapeutic strategies and examine such strategies in vitro and in animal leading to clinical testing in humans. Antibodies against these proteins will be tested for affinity and specificity for tumour or stromal components following which, the efficacy of the antibodies in animal models of tumour genesis will be examined. Following validation the antibodies will be produced in a clinical scale paralleled with pharmacological and toxicity testing prior to a clinical trial. In this project, basic science, translational research and clinical activities are strongly integrated, in order to validate defined targets and to develop new therapeutic entities and principles for clinical implementation.
In the first 24 months, there has been considerable progress in the tasks intended for the project and overall the implementation of EUCAAD is on schedule. In WP1, collection of tissue and serum samples is ongoing and a set of standard protocols have been developed in the first 12 months. In WP2 several proteins have been identified co-localising with AMOT. These tasks in WP2 have been achieved before schedule and a list of candidate partners for AMOT, AMOTL1 and AMOTL2 have been partially described. These include Syx, Patj, Pals1, Lin7, Mupp1. Several monoclonal antibodies have been developed against the Ror1 molecules and are currently being tested for their ability to biologically antagonise and facilitate antibody-mediated cellular cytotoxicity of CLL cells. Studies have demonstrated that Ror1 is specifically upregulated in CLL in comparison to normal peripheral blood cells and silencing of Ror1 can lead to apoptosis of CLL cells. This contributes to one of the tasks in WP3. Expression of AMOT, AMOTL1 and AMOTL2 has been studied extensively in zebrafish, mouse and human tissue under WP4. Functional effects of these molecules have been studied by using siRNA mediated silencing. There is a strict direct relationship between tumour grade and level of ROR1 positivity with poorly differentiated carcinomas more often expressing high levels of ROR1.
AMOT knockout mice have been created. The effect of vaccination against AMOT in the cure of neu+ established tumours. New insights into the mechanism of this inhibition demonstrating that vessel alterations were due to an NK mediated ADCC. In WP6, a panel of antibodies against different regions of the Ror1 molecule has been tested for their ability to induce apoptosis in CLL cells. Further investigations are under way to characterise the leading candidates of ROR1 antibodies that have demonstrated promise in initial experiments. At least two antibodies have been validated as benchmarks for imaging studies in WP7 and two separate methods for radioisotopic or non-radioistopic labelling of antibodies for imaging have been established.
Project management activities of EUCAAD have been implemented in parallel to the scientific activities. The last period review of the EUCAAD project was held at Frankfurt / Main, Germany on 20 April 2012. The suggestions of the reviewer have been taken into consideration and duly the implementation activities of the EUCAAD consortium has been adjusted to emphasise on the development of antibodies against Ror1.
Project results:
WP1 - Acquisition of clinical samples and relevant clinical data
According to the plan, we have established a bio bank consisting of samples from breast cancer and renal cancer. These samples have been used to map target gene expression as well as detection of marker antibodies in blood.
WP2 - Identification of AMOT associated proteins
This work package has revealed a novel signalling pathway that controls the migration of blood vessels during angiogenesis (the term for blood vessel formation from pre-existing vessels). We show that AMOT forms a protein complex that regulates cytoskeletal turnover in the migrating cell and this controls directional movement. This is the biological effect that we aim to target with anti-AMOT antibodies.
WP3 - Discovery of potential target structures expressed by tumour and endothelial cells
MALDI imaging was developed in this WP. This technique combines the advances in proteomics technology to histological levels. We have analysed the profile of proteins in biopsies from renal cancer patients. The technique allowed differentiation of expression of protein profiles in different tumour regions. such as stroma, normal tissue and cancer cells. WP4 - Target validation in vitro
ROR1 antibodies induce tumour cell death in vitro
The receptor tyrosine kinase ROR1 is shown to be expressed in CLL and not in samples from normal donors. Furthermore, the receptor is highly phosphorylated which is indicative of receptor activation. Thus, ROR1 may play an essential role in expansion of CLL tumour cells. Inactivation of ROR1 signalling using either siRNA or mouse monoclonal antibodies induced cell death in CLL patient samples in vitro. Taken together, these data shows that CLL tumour cells depend on ROR1 signalling and when this pathway is inhibited they undergo programmed cell death.
AMOT controls endothelial cell migration. Here we used either genetic ablation of the AMOT gene or overexpression of AMOT in an endothelial cell-line. Both approaches indicated a role of AMOT in directional cell migration.
WP5 - Target validation in vivo
Roles of AMOT, AMOTL1 and AMOTL2 in normal vascular development: We have used two powerful genetic approaches to analyse the role of the AMOT protein family during blood vessel formation in vivo. These include studies of developmental angiogenesis in zebrafish and mouse.
We have silenced all AMOT family members during zebrafish vascular development. The effects of this gene silencing approach have been evaluated in transgenic zebrafish where blood vessels are highlighted by expression of fluorescent markers. In mouse we have used homologous recombination to genetically silence AMOT, AMOTL1 or AMOT2. The chosen approach has allowed us to specifically silence the studied gene in the blood vessel endothelial cells at any time point during development. The data show that each individual member of this family has a distinct function in blood vessel formation. AMOT controls the directional migration of capillaries; AMOTL1 is affecting cellular contacts and AmotL2 controls lumen formation. These are three vital processes during the establishment of a functional blood circulation and opens up the possibility to target these distinct events during pathological blood vessel formation in disease.
DNA vaccination: Targeting AMOT and AMOTL1 in a mouse breast cancer model. The mouse mmtv-neuT transgenic model mimics human breast cancer in that tumours are driven by the rodent counterpart of the HER2 oncogene. These mice invariably develop tumours and succumb to disease 22 weeks after birth. Single vaccination with an AMOT DNA increases survival with over two months. Perhaps even more exciting are the findings two vaccinations using a combination of Amot, AmotL1 and HER2 completely protect the mice from dying from dying of breast cancer. This effect is antibody dependent and thus provides the rationale for generating recombinant therapeutic antibodies targeting AMOT and AMOTL1.
Taken together genetic inactivation and DNA vaccination data provide the rationale for targeting both AMOT and AMOTL1 to efficiently inhibit tumour angiogenesis.
WPs 6,7 - Antibody selection and in vitro characterisation
Generation of a CLL model in mouse. We have selected human recombinant antibodies on their capacity to induce apoptosis of human primary CLL cells from patients at the Karolinska Hospital. Prior to our studies, no animal model of CLL existed. A mouse model was established by grafting CLL cells from patients into immune deficient mice. This is to our knowledge the first model system of CLL and it was then used to analyse antibody efficacy. AMOT antibodies inhibit endothelial cell migration in vitro.
We have isolated human single chain antibodies reactive against AMOT. These antibodies have been tested for anti-endothelial migration activity in vitro. The best candidates have been selected and tested in mice for anti-angiogenic and anti-tumour activities. However, an unforeseen problem arose when it proved impossible to maintain biological activity when the single chain antibodies where converted into immunoglobulin format. This conversion increases half-life in circulation and is presently the standard forma when going into clinical trials. We have solved this problem by fusing the single chain antibody to an albumin-binding domain. Once injected, the single chain antibody binds to albumin in circulation and thus has increased half-life. Our in vivo data show that this antibody specifically targets migrating endothelial cells in developing vessels of the new born retina. The specificity is unprecedented and opens up the possibility to remove the migrating tip cells during angiogenesis but leaving the rest of the vasculature intact.
WPs 8,9
A number of ROR1 antibody candidates have been generated in the consortium. These have been tested in the novel mouse model system described in WPs 6-7. Clinical trials with the lead candidate are planned within 1-2 years from the ending of the EUCAAD project.
Potential impact:
Cancer is the second leading cause of death in the European Union after heart disease and one of the most urgent health problems in the developed world. 3.2 million Europeans are diagnosed with cancer every year, according to EU figures, and the disease is responsible for 29 per cent of deaths among men and 23 per cent amongst women.
While survival rates are improving all the time, the huge number of people still losing their battle with cancer highlights the urgent need for further research. Indeed, the in three men and one in four women are expected to be directly affected by the disease by the age of 75, and these figures are predicted to rise as Europe's population ages further. Such high levels of incidence have led the EU to set a goal of cutting cancer rates by 15 % by 2020. To this end, it is channelling hundreds of millions of euros into research for innovative cancer treatments every year. That includes investing in a ?virtual research institute drawn from some of Europe's best known centres of excellence to develop targeted new therapies aimed specifically at cancer cells or cells of the stroma the surrounding mass of tissue including blood vessels that support tumour growth.
The consortiums main focus has been to develop antibodies against new target structures on tumour cells, and on the blood vessels supplying tumours, which are responsible for the cancer?s growth, progression and spread. The goal of a targeted therapy is to increase anti-tumour efficacy with lowest possible side effects.
Unpleasant side effects are one of the problems associated with conventional treatments such as chemotherapy and radiotherapy. Cancer cells share many common features with normal host cells from which they derive, and traditional cancer therapy often fails to discriminate between normal and malignant tissues.
Also, patients with advanced cancers which have spread to other parts of the body frequently relapse and succumb to their disease after developing resistance to such conventional treatments. That is why there has been such interest in developing more targeted forms of therapy, with antibodies having become the designer cancer treatment of choice in recent years.
The advantage of antibodies is that they are extremely specific; each one will bind to and attack one particular antigen. Antibodies have been developed to inhibit cell growth in tumours, induce apoptosis sometimes dubbed as cell suicide and deliver radiation or other potent anti-cancer drugs directly to malignant cells.
The scale of the cancer challenge is such that new strategies for targeting widespread metastases are urgently needed. To this end, EUCAAD has seven key objectives, one of the most important of which has been to identify new structures in cancer cells and in the tissue and blood vessels supporting tumours that can be targeted using antibody therapy. The EUCAAD consortium has identified a list of candidate drug targets expressed either by the tumour or in the supporting normal tissues. From this list, the consortium has gone on to test the capacity of DNA vaccination against selected target proteins in breast and lymphoma models. Three of the tested molecules provided exciting anti-cancer activities.
Moving new discoveries from the bench to the bedside requires the kind of interdisciplinary, collaborative, team-oriented approach taken by the consortium and is the fastest way to achieve the kind of major breakthroughs in cancer treatment we all want to see. The joint goal for the consortium has been to generate human antibodies to treat cancer patients. EUCAAD has proceeded to produce human monoclonal antibodies against two of the target molecules. These have been validated in terms of tissue specificity, binding affinity and functional properties; and examining the ability of selected antibodies against the genes ROR1 and AMOT inhibit tumour progression in animal models. These tests have shown great promise and EUCAAD intends to begin clinical trials to assess efficacy in cancer patients in the near future.
The activities and results generated in the EUCAAD have been disseminated to society in several ways. The project has been presented at the EUCAAD web site where progress and meetings are advertised and open to the public. Data from the collaboration has been presented in a number of oral and poster presentations at international conferences as well as publications in internationally high ranking journals.
In order to promote exchange of ideas in the field of cancer therapy, EUCAAD has organised and sponsored a workshop on anti-angiogenesis therapy in Marseille 2009 with participation of invited leading scientists. The importance of the interaction between academia and small medium enterprise has been acknowledged among European decision makers. EUCAAD has actively participated in the symposia, such as 'Biotech SMEs in health and disease' organised by the State of North Rhine-Westphalia representation in Brussels 2010. These events provided the opportunity for dialogue amongst Europe's political leaders, innovative business operators and researchers. Here EUCAAD has been highlighted as a positive example on how SMEs can benefit from the interaction with European research institutes.
Taken together, EUCAAD has linked education, research and biotech business to answer to the urgent demand of new modern cancer therapies. After clinical trials, the ultimate dissemination is to provide an efficient treatment to cancer patients.
List of websites: www.eucaad.net