Final Report Summary - ANTICARB (Monoclonal ANTIbody-targeted CARBon nanobues against cancer)
ANTICARB attempts to exploit the advantages offered by a novel nanotechnology platform - carbon nanotubes - and apply them to a clinically established therapeutic modality - targeted antibody therapy - for the creation of hybrid nanotechnology-based monoclonal antibody targeted cancer therapeutics. ANTICARB combines two emerging technologies, antibody and nanotube technology, in a way that will allow safe development of antibody-nanotube conjugates and explore their swift translation into a clinical oncology setting. By combining proven, clinically used, anti-cancer agents - antibodies - with a novel nanotechnology-based platform made of advanced nanomaterials, ANTICARB aims at enhancing the therapeutic potency of the antibody and establish a new paradigm for oncology therapeutics. The ability of carbon nanotube technology to transport antibodies into the tumour cell cytoplasm may lead to validation of specific intracellular targets for oncology. This objective will be reached by adopting a multidisciplinary approach and by bringing together expertise from the fields of drug delivery, molecular biology, chemistry, engineering, pharmacology and toxicology. The proposal capitalises on this industry-academia multidisciplinary and perfectly integrated team, whose expertise spans from advanced biotechnology to sophisticated nanotechnology.
Project context and objectives:
Background
Cancer is a serious and costly threat to human health today. According to the Public Health Portal of the European Union (EU), 3.2 million Europeans annually are diagnosed with cancer, mostly breast, colorectal or lung cancers. While there are many different types of cancer, the underlying problem is the same: cells divide and grow in an uncontrolled manner, most commonly resulting in solid primary tumours that may spread to other organs, leading to metastasis.
One of the challenges with cancer treatment is the non-selective nature of therapy, i.e. both healthy and malignant tissues are affected. In addition, most treatments also inhibit the production of erythrocytes and white blood cells, causing patients to become anaemic and neutropenic, leading to enhanced susceptibilities to secondary infections. Cancer is projected to become more widespread in the next few decades due to ageing of the population and increased chemo- and radio-resistance.
Project aim
The aim of the ANTICARB project is the improvement of oncology treatment options using systemic administration of nanotechnology-mediated antibody therapeutics. Antibodies were selected as they allow for therapeutic and active targeting to specific cell receptors. Carbon nanotubes were chosen because their large surface area offers a template for conjugation with a variety of targeting and therapeutic monoclonal antibodies.
The CNT-Ab constructs are tagged with fluorescent probes and radioisotopes to help determine their cell biology and pharmacology. Fluorescent probes allow for in vitro imaging to understand the mechanism of the interaction between the CNT-Ab constructs and cancer cells. Radioisoprobes offer capabilities for in vivo diagnosis and quantitative tissue distribution, pharmacokinetics and toxicokinetics. The ANTICARB project also attempted to determine the safety profile of the novel CNT-Ab constructs using an array of toxicokinetic, immunological and cytotoxicity assays.
Project objectives
The ANTICARB project seeks to create new technologies that may offer more treatment and diagnostic options to clinicians and patients. The main objective of ANTICARB is the design and development of carbon nanotube-antibody (CNT-Ab) constructs. They are investigated as novel platforms for cancer treatment with the purpose to act as combinatory therapeutic/diagnostic agents.
The specific ANTICARB objectives are:
- improve and expand oncology treatment options based on systemic administration of nanotechnology-mediated antibody therapeutics;
- determine the safety profile of the novel CNT-Ab constructs based on pharmacokinetic, immunological and toxicological assays;
- develop CNT-Ab constructs that can be rapidly translated into larger animal and clinical pre-Phase I trials;
- determine the pharmacology and cell biology of functionalised carbon nanotubes with and without antibody conjugation;
- increase the competitiveness of the European industry (pharmaceutical and nanotechnology sector).
Project workplan
The ANTICARB workplan is divided in three almost distinct stages. Even though stages I-III follow a scientifically logical sequence, our intention was to engage in all three Stages simultaneously very early on in the project in order to allow for optimisation of the CNT-Ab constructs through multiple feedback loops: lab-to-cells-to-animals.
Stage I: CNT-Ab engineering carbon nanotube platform material [contribution from work packages (WPs) 1 and 2]
A very important problem related to the use of carbon nanotubes is their extraordinary aggregation due to very strong intertube forces, which causes insolubility in any type of organic solvent or aqueous solution. For biomedical applications, it is therefore mandatory to modify the pristine material in order to disrupt the bundles and make the manipulation of CNTs easier. To render the CNTs soluble or, better, biocompatible, we followed several strategies. Nanocyl provided CVD-synthesised carbon nanotubes: single-walled carbon nanotubes (SWNT), double-walled carbon nanotubes (DWNT) and multi-walled carbon nanotubes (MWNT). The purity of the raw MWNTs was always above 90 % and always further purified. All purified new products of DWNTs and MWNTs CNT were shortened and functionalised at the same time and available at a laboratory scale too.
Functionalisation of CNTs was performed in order to have different functional groups at the CNT surface that influenced reactivity towards antibodies. NH2 functional groups were introduced on the surface of CNTs. Optimisation steps included purification, functionalisation and shortening of CNTs to achieve as narrow as possible the size distribution of CNTs. The objective is to increase the reactivity of CNTs towards antibodies conjugation, a higher yield of simple functional group such as NH2 can be envisaged througha plasma functionalisation process. Based on the reactivity of such modified CNT with selected antibodies, the choice of the ideal number of functional groups were estimated.
Alternatively, the CNT were treated with strong oxidising acids to generate carboxylic functions at their tips and side-walls at much higher concentrations. After oxidation, the nanotubes were also chemically shortened and compared with those obtained by the ball milling process (vide supra). The material so produced was much more easily dispersable than the pristine nanotubes, and can be further functionalised with many relevant biomolecules. Another approach in the modification will consist on the use of organic addition reactions, including the 1,3-dipolar cycloaddition of azomethine ylides. This process, optimised in the last few years by the UTr and CNRS groups of this Consortium, will allow the direct introduction of the functionalities at the side-walls and tips of the unmodified tubes. The functional groups will be again modified with the suitable bio fragments (vide infra). These two types of functionalisation strategies were applied to double- and multi-walled CNTs provided by Nanocyl. The two functionalisation processes were applied contemporarily, so that we can produce CNTs derivatised with two different molecular entities, such as an antibody and/or a drug, or a labelling unit. In addition, the functionalisation allowed the complete removal of the catalytic particles that may not be totally eliminated during the manufacturing process.
Antibody construct [contribution from WP3 and WP4]
UCB has over twenty years history of taking antibodies into the clinic, indeed it was the pioneer in evaluating chimeric antibodies. Over the decades it has acquired a large library of antibodies - human and murine - different IgG sub-classes and whole IgG, as well as fragments such as Fabs. This library of therapeutic antibodies has been mainly produced in E.Coli and has in general a clinical specification of 99.9 % purity. For the purposes of ANTICARB, a series of antibodies were used that were clinically developed to Phase II clinical trials.
Carbon nanotube - antibody conjugate [contribution from WP2 & WP3]
Following the first step, the f-CNTs will contain carboxylic and/or amino groups at their tips and side walls. These f-CNTs, derived from the oxidation reaction and the organic cycloaddition reactions, will be used as starting materials for the conjugation to the cell specific targeting monoclonal antibodies and to the therapeutic monoclonal antibodies (IgG or fragments). To generate the different conjugates, the f-CNTs will possess amino functions, fundamental for the introduction of suitable linkers for the monoclonal antibody binding. The amino groups around the nanotubes will be modified with a maleimide function. Indeed, the maleimide will allow a direct chemo-selective reaction with the thiol-free cysteine amino acid residues within the antibody sequence. All these types of CNT-Ab conjugates are of covalent nature.
Radiation and optical probes [contribution from WP2, WP4, WP5]
The f-CNTs, derived from the oxidation reaction and the organic cycloaddition reactions, will be further functionalised with chelating agents for radioisotopes emitting and Auger radiation to perform in vivo diagnosis and intracellular radiotherapy. To generate the different radiolabelled conjugates, f-CNTs will be modified with amino functions for the introduction of suitable ligands including DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and DTPA (diethylentriaminepentaacetic dianhydride) for the complexation of the different radionuclei. In parallel, a similar approach will be exploited to link fluorescent probes to f-CNTs for in vitro imaging. Alternatively, the radioisotopes and the fluorescent molecules will be grafted to the monoclonal antibodies before or after the conjugation to the nanotubes.
Stage II: CNT-Ab biology - CNT-Ab construct cell biology [contribution from WP2, WP4, WP5]
All CNT-Ab conjugates, including the fluorescent derivatives, will be assessed for their cellular uptake behaviour. A wide panel of human and murine cells lines, murine T and B lymphocytes, and murine macrophages will be used to study the cell penetration, the intracellular trafficking and the mechanisms of internalisation. Fluorescent spectroscopy (epifluorescence and/or confocal laser scanning microscopy) will be used to observe the internalisation process. Flow cytometry (FACS) will be helpful not only for the uptake analysis, but also for the determination of the kinetic, the time and concentration dependence of the process. In addition, FACS will be fundamental for the assessment of the cytotoxic effects. The lymphocytes and macrophages will be directly isolated from the animal models available in our laboratories (vide infra). Surface plasmon resonance analysis (Biacore technology) will be used to measure the parameters of binding between the CNT-Ab constructs and the receptor expressed at the cell surface. 3D human tumor spheroids are routinely cultured in our laboratories and will be used as an in vitro model for metastatic nodules and the avascular interstitial space of solid tumors. Penetration into the tumor volume will be assessed by optical slicing of the spheroid using confocal laser scanning microsocpy tracking the optical signal from fluorescently labelled CNT-Ab constructs. Image analysis packages will allow modelling of the capacity of different CNT-Ab conjugates to penetrate deeper into the tumor mass and also simulate the predicted radiation dose deposition and tumor cell kill.
CNT-Ab pharmacology [contribution from WP2, WP4, WP5]
The pharmacological properties of the CNT-Ab conjugates will be highly dependent on the tissue distribution, pharmacokinetics, blood residence time and body excretion kinetics following their in vivo administration. A variety of critical parameters will be assessed:
a) route of administration - three main routes relevant to current clinical application of antibodies and proteins will be explored: intravenous, subcutaneous; pulmonary;
b) dose escalation - our starting point will be the currently administered therapeutic antibody doses (approximately 3mg/kg in a single administration) and escalate from there;
c) in vivo model upscaling - we will carry out all of the routine tissue biodistribution and pharmacokinetic analysis using mice and dynamic whole-body imaging (microSPECT/CT - requested for ULSOP) deemed essential to allow rapid, accurate, ethical (minimisation of animal numbers) and feasible (minimisation of labour intensity required) determination of the in vivo transport kinetics and organ distribution of the CNT-Ab. In that way, fundamental understanding of the CNT-Ab pharmacology will be obtained.
Stage III: CNT-Ab safety and therapeutic efficacy - CNT-Ab toxicology, immunocompatibility and radioprotection [contribution from WP2, WP4, WP5]
Toxicology - To achieve a long-term risk assessment of the CNT-Ab constructs, dose escalation studies will be carried out first to determine doses to the various organs and tissues of relevance. In a second step we will study the toxicological effects in those organs. We will carry out long-term biokinetics after intravenous injection (two, four weeks and three months) of stably radiolabeled Ab-CNT in rats. Once we know which secondary target organ is going to be dosed we will perform toxicological studies on some of these organs at selected time points (determined by the dose escalation studies) by pro-inflammatory cytokine assays and limited induction tests. These tests will include gene array analyses. We are expecting to be able to determine which organs are affected and the lower dose limit, under which toxicity will not be expected to act as an adverse effect (toxicity threshold).
Immunocompatibility - The rationale behind studying complement activation in vitro and complement-mediated pseudoallergic reactions in vivo, as predictors of short-term immune toxicity of CNT-Ab, lies in the fact that complement activation might cause, or play a major role in those unusual hypersensitivity reactions (HSRs) wherein prior exposure to the reactogen agent, and, hence, a causal role of IgE, cannot be established. These reactions have been referred to as 'complement activation-related pseudoallergy' (CARPA), and include widely known and feared 'infusion' reactions to anticancer drugs solubilised by micellar solvents, such as Cremophor EL and polysorbate-80 (in Taxol and Taxotere), liposomal anticancer and antifungal drugs (Doxil and Ambisome), radiocontrast media, plasma expanders and blood substitutes. The pathomechanism of CARPA involves complement activation through both the classical and the alternative pathways, giving rise to C3a and C5a anaphylatoxins that trigger mast cells and basophils for secretory response that underlies the clinical symptoms of HSRs. Considering their cylindrical structure with extended length, CNTs have a substantial surface area for binding plasma and complement proteins, particularly if, after functionalisation and targeted binding, they deposit on cell surfaces in a tandem or aligned manner. CNT-Ab may therefore activate complement, posing realistic danger for CARPA. In addition, unexpected cross reactions with immune cell surface antigens might trigger the release of cytokines or allergomedins, as observed with TGN1412. The proposed measurement of complement and basophil activation by CNT-Ab in human serum and blood in vitro and reactogenicity studies in animals in vivo will serve as essential predictors of biocompatibility or toxic effects in the clinic.
Radioprotection - Gamma (or X) ray emitting radionuclides suitable for imaging emit photons which are energetic enough to escape the body and, thus, in addition to the self-dose in the residing tissue are also capable of cross-irradiating adjacent organs. Given their relatively uniform mode of irradiation, a quantitative estimate of the energy deposition to the various organs (radiation absorbed dose) will be obtained by the MIRD mathematical formalism using experimental biokinetic data for the radionuclides, its characteristic emission properties (type and energy of emitting photons/electrons, and half life) as well as its source-to-target specific absorption values. For radionuclides that also (or solely) emit Auger electrons which deposit their energy in a highly non-uniform and localised manner (usually within less than 100 nm from their origin), the MIRD scheme is not suitable, therefore we will perform detailed, nanoscale Monte-Carlo simulations to assess the energy deposition by the Auger electrons in radiobiologically sensitive subcellular domains (e.g. mitochondrium, cell nucleus, DNA). Appropriate weighting factors will then be used for the irradiation component that resulted from the Auger electrons, to properly account for their increased radiobiological effectiveness in comparison to the gamma and X rays.
CNT-Ab preclinical efficacy [contribution from WP2, WP4, WP5]
The therapeutic efficacy of the CNT-Ab conjugates were performed using human tumour xenograft models in nude mice. The main objective is to use antibody nanotubes bearing no isotope or cytotoxin but with antibodies that are biologically active against an intracellular target. The changes in tissue biodistribution and pharmacokinetics of radiolabelled CNT-Ab in mice were initially established by microSPECT/CT, followed by tumor xenograft therapeutic studies using a dose regime previously established to be lower than the toxicity threshold. The intratumoral route was primarily explored to investigate the therapeutic activity of the CNT-Ab.
Project results:
The main aim of ANTICARB has been the design and development of novel, safe and effective carbon nanotube -antibody (CNT-Ab) constructs for combinatory therapeutic / diagnostic (theranostic) oncology indications. The individual components employed to achieve this technology are the following:
a) Antibodies - monoclonal antibodies - full (approximately size 8 nm) or fragments (5 nm) will be used for therapeutic and active targeting to specific cell receptors
b) Carbon nanotubes - single- (1 nm diameter) and multi-walled (20-30 nm in diameter) functionalised carbon nanotubes will be used as platforms for controlled conjugation of the antibodies. Also, antibodies with the capacity for intracellular activity
c) Radionuclides - radioisotopes emitting and Auger radiation will be grafted either on the carbon nanotube or the antibody to offer capability for in vivo diagnosis (SPECT/CT imaging) and synergistic intracellular radiotherapy.
d) Optical probes - fluorescent probe molecules will be grafted on the carbon nanotubes or antibodies to offer capability for in vitro imaging (microscopy)
ANTICARB had four major scientific objectives:
ANTICARB has achieved the following science and technology (S&T) foregrounds in each of its objectives:
1. Fabrication and synthesis of CNT-Ab
Two types of MWNT material, morphologically different, were compared throughout the project (Nanocyl and NanoAmorph). The Nanocyl material was thinner (5-10 nm) than the NanoAmorphous (20-30 nm) material. It was also found that the oxidation of the nanotubes in strong acids shortens the length of both Nanocyl and NanoAmorph materials but also creates defects in their sidewalls.
One of the interesting findings in ANTICARB, is that oxidation renders MWNT prone to enzymatic degradation when the oxidised MWNT were incubated in the phagolysosomal stimulating fluid (PSF) and horseradish peroxidase (HRP).
A series of Phase II clinically developed antibodies were used in ANTICARB. This include the full IgG; a humanised monoclonal antibody (hCTM01) , the antigen binding fragment (Fab') and the single chain variable fragment (scFV). These antibodies were targeting the polymorphic epithelial mucins (MUC-1) which is over-expressed in many tumours.
Therefore, different CNT-Ab constructs were synthesised, characterised and studied in ANTICARB. These constructs had dual functional groups which allowed their conjugation to the antibodies (IgG, Fab', scFV) at one end and to a fluorescent probe (FITC) or a chelating molecule (DTPA) to the other end.
2. Intracellular delivery of CNT-Ab
The CNT-Fab' construct internalised in both MUC-1 positive cells (MCF7) and MUC-1 negative cells (Calu6) as shown by the presence of the green signal (FITC conjugated to CNT) and red signal (secondary antibody against Fab's) in both cell lines.
3. Tumor targeting of CNT-Ab
In ANTICARB, we used multicellular tumour spheroids as an vitro 3D model to study the tumour targeting capacity of the CNT-Ab constructs. Both MWNT-IgG and MWNT-Fab' constructs showed enhanced affinity for MUC-1 positive tumour spheroids (MCF7). However, MWNT-Fab' exhibited a deeper localisation within the tumour avascular interstitium which was even observed with the MUC-1 negative (HCT116) tumour spheroids as shown in the cryosections of the spheroids).This indicates that MWNT were driving the uptake when the antibody was a smaller fragment (Fab').
4. Pharmacokinetics of CNT-Ab
The biodistribution profile of the CNT-Ab constructs was studied after intravenous administration. As seen in the SPECT/CT images, there was no change in the biodistribution profile when the antibodies were conjugated onto the nanotubes. However, the NanoAmorph CNT-Ab constructs accumulated mainly in the liver and spleen; while the Nanocyl CNT-Ab constructs were mainly detected in the lungs and liver. This indicates that the starting material has a drastic effect on the biological profile of materials in vivo.
5. Safety and toxicity of CNT-Ab
The toxicity of CNT-Ab constructs was assessed after intra-tracheal administration. It was found that the MWNT-Fab' construct caused the most significant acute and chronic inflammation followed by the Fab' fragment alone and then the MWNT alone. In addition, acute macrophage depletion was observed with the same construct after 24 hrs post-injection but was also observed to a lesser degree with the Fab' alone and MWNT.
Moreover, the cardiovascular changes were assessed using a porcine model which was intra-arterially administrated with repeated doses of CNT. While the in vitro data indicated complement activation with the oxidised MWNT, the most severe cardiovascular changes were also observed with the same oxidised MWNT. Interestingly, amino functionalisation and Fab' conjugation to MWNT alleviated those risks.
S&T ANTICARB achievements
- Fabrication, Characterisation and Upscaling of complex MWNT-Ab
- Confirmation of the cellular internalisation of MWNT-Ab
- Elucidation of mechanisms leading to cellular internalisation of MWNT-Ab
- Determination of pharmacokinetic and pharmacodynamic profile of MWNT-Ab
- Revelation that surface carboxylation increases risk of unwanted reactivity and chemical ways to alleviate this
- Development of antibodies with biological activity against intracellular targets
- Biologically active MWNT-Ab conjugates.
S&T ANTICARB reflections
- Conjugation of proteins on MWNT is not easy, straightforward and cannot avoid physisorption (contrary to what literature indicates).
- Batch variation in terms of starting material and functionalisation still persist and affects biological interactions.
- Size of MWNT-Ab constructs (depending on the Ab size: IgG, Fab', scFv) determines localisation within tumor interstitium.
- The kinetics of free probes (in our case radionuclides Gd-153 and In-111) can affect exact determination of biokinetic profiles.
- Fibre-related toxicity is not really relevant to short, chemically functionalised carbon nanotubes.
- It is exceedingly challenging for CNT and other novel nanomaterials to match the short-term scope of pharmaceutical industry, maintain novelty (i.e. attractive patentability) and avoid hype (positive and negative) to keep public funding (that requires industry participation / support) for the medium / long term.
Potential impact:
The ANTICARB consortium brought together five of the most qualified research centres in Europe, one multinational pharmaceutical company that leads the global biopharmaceutical market segment (UCB) and two young and dynamic SMEs (NANOCYL and SeroScience) with a major scientific aim: to generate a new class of nanomedicine constructs as anticancer therapeutics by joining the carbon nanotube and the monoclonal antibody technologies. The proposal was based on a large and well-coordinated effort from seven different countries (United Kingdom, Belgium, France, Germany, Hungary, Italy and Greece).
Training a new generation of European researchers
The development of nanopharmaceuticals is a new emerging multidisciplinary area and research within a European-wide network is mandatory for progress in the field. The ANTICARB consortium brought together the three sides of the 'knowledge triangle': industrial innovation, cutting-edge academic research and education within an inter-sectorial approach. One of the key impacts that were addressed is the improvement of technical skills, training and the level of innovation of the new generation of European researchers, so as to improve European competitiveness within the global nanotechnology and pharmaceutical market. The participation of researchers from different European countries is in line with the spirit of EU cohesion and joint policymaking for research. The sharing of knowledge between the members of ANTICARB led to research activities that could not be achieved at the national scale. The ANTICARB network contributed to a strong interdisciplinary and inter-sectoral training of top-researchers that will be the workforce of the future.
Further the understanding the hurdles of clinical translation
The interaction between cancer physiology and the nanotechnology-based innovative strategy proposed (the CNT-Ab hybrids) has been of fundamental importance throughout the development of ANTICARB. ANTICARB has been seeking to contribute to the creation of fundamental knowledge at the European level and at the same time, through the central role of the pharmaceutical industry partner (UCB), maximised the possibilities of translation into the clinical setting.
Exploit basic knowledge to develop novel drugs and treatment strategies
ANTICARB created necessary knowledge that was lacking on how to transform a novel nanomaterial of known fascinating physical properties, such as carbon nanotubes, in the biomedical arena and explore the possibility to translate it clinically into an effective cancer therapeutic. One of the key conclusions from ANTICARB is that the particular nanotechnology has been shown to have the capacity to transform otherwise ineffective known entities into therapeutically active and effective anti-cancer drugs. ANTICARB offers evidence that a new market in therapeutic oncology can be opened - that of using antibodies and their fragments against intracellular targets.
European economical and social advantages ANTICARB provided a variety of social and economical advantages to the European Community. It will reinforce the collaboration among European scientists and laboratories and will contribute to establish a European scientific identity. US and Asian Universities and companies represent the major competitors in genome science and nano- and biotechnology. The quality of life for European citizens depends on Europe's ability to compete and, where possible, to take the lead. In addition, shrinking or closing the gaps in sectors where Europe currently lags behind is of vital importance. If successful, this policy will create opportunities and increased prosperity within Europe. To this end no individual country within Europe is in the position to either assemble a consortium of this calibre or to fund it to the extent necessary to either compete with or gain a competitive advantage over the US and Asia. Finally, the multifaceted nature of the nanobiotechnology research area requires a multidisciplinary approach, which is best achieved through coordinated and integrated European wide research.
Main dissemination activities
1. Project coordination activities
Although the project coordination mainly involved the consortium, in several cases they involved dissemination of project information beyond the consortium, via each partner's collaborations network.
During the whole project period the partners held 33 coordination activities:
- 7 project meetings with 93 % participation
- 14 teleconferences with 92 % participation
- 12 visits, exchanges and joint work involving:
- 4 meetings involving 2 or more partners
- 1 sabbatical visit
- 7 visits in order to perform jointly experiments
2. Publications
The ANTICARB consortium published in all 53 publications in wich ANTICARB project is acknowledged.
- 47 articles in journals and
- 6 book chapters.
Regarding the authorship of the above publications:
- 32 were submitted by 1 partner
- 10 by two partners
- 11 were submitted by three or more partners.
Regarding the area the publishing houses are situated:
- 42 of them were published in journals and books published in Europe and
- 11 in journals and books published outside EU, mainly in US.
3.Presentations
The ANTICARB consortium presented their work in 85 activities of which:
- 50 were conferences
- 15 were summer schools - seminars
- 20 were workshops - meetings.
The impact level of the above activities was for:
- 16 national
- 22 European
- 47 international.
Regarding the activity audience for:
- 61 involved mainly the research community
- 24 involved the industry community (together with the research community in some cases)
- 14 involved mainly policy makers and media (together with the research and the industry community in some cases).
4. PhD studentships
The project employed in all 13 PhD students, 3 of which worked entirely on ANTICARB and acknowledged the project in their thesis. The thesis were submitted, examined and published while the ANTICARB project was still running.
5. Events organised
Four major events were organised under or supported by ANTICARB project:
Second Carbon Nanotube Biology, Medicine and Toxicology Satellite Symposium
20 June 2009, Tsinghua University Campus, Beijing, China
The ANTICARB project sponsored the organisation and implementation of the above symposium. For that reason the ANTICARB logo features until today in the symposium website. This one-day symposium took place one day prior to the official opening and within the content of the 'Tenth International Conference on the Science and Application of Nanotubes - NT09'.
The symposium involved participation of about 120 scientists presenting their work and discussing the interaction of nanotubes with cells and tissues in vitro and in vivo, and exploring specific applications of nanotubes in the biomedical field.
For more information please see the symposium website at http://www.cntbiology.org/CBTN09/
Third Carbon Nanotube Biology, Medicine and Toxicology Satellite Symposium
27-28 June 2010, Hilton Bonaventure, Montréal, Canada
The ANTICARB project sponsored the organisation and implementation of the above symposium. For that reason the ANTICARB logo features until today in the symposium website. This two-days symposium took place during the first two days and within the content of the 'Eleventh International Conference on the Science and Application of Nanotubes - NT10'.
The symposium involved participation of about 90 scientists presenting their work and discussing the correlation between the specific characteristics of carbon nanomaterials (method of synthesis and manufacturing, purity, type of surface functionalisation) and their biological effect in various model systems (immortalised or primary cells, in vivo models).
For more information please see the symposium website at http://www.cntbiology.org/CBTN10/
Fourth Carbon Nanotube Biology, Medicine and Toxicology Satellite Symposium
15-16 July 2011, University of Cambridge, United Kingdom
The ANTICARB project sponsored the organisation and implementation of the above symposium. For that reason the ANTICARB logo features until today in the symposium website. This two-days symposium took place during the last two days and within the content of the 'Twelfth International Conference on the Science and Application of Nanotubes - NT11'.
The symposium involved participation of about 78 scientists presenting their work and discussing the determining the therapeutic and diagnostic potential of carbon nanomaterials and the challenges ahead. Moreover, particular attention was placed on attempts to understand the toxicological and environmental risks posed by use of carbon nanotubes.
For more information please see the symposium website at http://www.cntbiology.org/CBTN11/ and http://www-g.eng.cam.ac.uk/nms/cnbmt/
Carbon Nanotubes: From Safety Assessment to Biomedical Applications
One-day workshop, 29 November 2011, Brussels, Belgium
The ANTICARB project organised in synergy with another FP7 STREP project NANOMMUNE, an one day workshop. In this workshop it was attempted to present the state-of-the-art in the field of carbon nanotechnology in medicine, by bringing together success stories and lessons learnt from the research work carried out by the two FP7 projects that have worked in parallel in this domain for the last three years.
Distinguished speakers were invited beyond the two projects' consortia, like independent experts, Nano - Fora and industrial associations' representatives, EU policy makers and EC Project Officers. The workshop was advertised also in the Nanosafety Cluster and the Nanotechnology Industries Association news' webpages.
Web dissemination
Project website
The project website was created within the first six months of the project and since then it provide information to the project partners and the wider public. In its current status the public website is fully updated with latest information regarding publications and dissemination activities of the consortium. The private (only for partners) webpage is also fully updated with all information and presentations for all partners coordination activities. The website is planned to remain online for at least the next 12 months.
For more information please see the project website at http://www.anticarb.org
Coordinator's website
Project information can also be found in the coordinator's website.
For more information please see the Nanomedicine lab website at http://www.nanomedicinelab.com/collaborations.html
Other websites
Here are some other websites relevant to the ANTICARB partners or subject providing information about the project:
IST WORLD
http://www.ist-world.org/ProjectDetails.aspx?ProjectId=cc03235c40904005884360b8a4d57bba&SourceDatabaseId=018774364ea94468b3f4dec24aa1ee53
NANOCYL
http://www.nanocyl.com/CNT-Expertise-Centre/R-D-Projects-Alliances/Nanocyl-can-Quote-10-European-Projects-and-4-Belgian-Ones
HELMHOLTZ INSTITUTE
http://www.helmholtz-muenchen.de/ilbd/about-ilbd/projects-and-collaborations/index.html
Exploitation of results
1. Research era
The project results will be exploited in the following way in order to contribute to the EU research era.
- New knowledge and know-how acquired during the project will serve to set up a new research field around antibody therapeutics and drug delivery.
- The results of the project will be used to explore the carbon nanotube-based drug delivery concept for several applications as cancer and neurological disorders.
- The results obtained will be used to design new protocols for the exploration of gene involvement in various diseases (e.g. Parkinson's).
- Research and evaluation of the results by upscaling to large animal studies for eventual use in the design of clinical studies.
- New techniques to be introduced into small animal imaging courses and newly developed therapy options can be evaluated in order to transfer them into clinical trials.
2. Training and education
The project results will be exploited in the following way in order to contribute to the training and education of new scientists.
- New knowledge and expertise acquired during the project will be utilised in postgraduate course curriculum at the University partners (ULSOP, UTr and UoI).
- Novel technologies on the development of carbon nanomaerials will be part of the curriculum for a nanomedicine -based approach to cancer treatment and will be incorporated in our existing teaching modules in drug delivery.
- Some of the topics can feed into master's curricula of biomedical engineering faculties as teaching material.
3. Industry
The project results will be exploited in the following way in order to contribute to the EU nanotechnology and medicine industry.
- Improve our knowledge in nanoparticles design. Extend nanoparticles applications to innovative imaging and therapy applications.
- Extending the application of carbon nanomaterials as key components in new sophisticated drug delivery systems and further development of the market of anticancer therapeutics in the long term.
- The investigated methods will be used in the further development for intracellularly-targeted antibody therapeutics. The tools developed for the preclinical work in this project may in itself become a commercially viable option for the research oriented gene and protein delivery research market (e.g. nanotube-based transfection agents).
- The new therapy options and methods for delivery of antibodies directly adds to the industrial partners (UCB, Nanocyl and SeroScience) production and exploration of new products programme.
Policy making
The project results will be exploited in the following way in order to influence EU policies regarding advancements in nanotechnology and nanomedicine.
- New activities are already initiated using the Nanosafety Cluster (see http://www.nanosafetycluster.eu online) and other technologies in collaboration with other FP7 consortia (see http://www.nanommune.eu online).
- Attempts to bridge the gap between regulation of nanomaterials in the context of public health safety and nanotoxicology and nanomedicine through the EMA.
Project website address:
http://www.anticarb.org
Contact details:
Prof. Kostas Kostarelos
Centre for Drug Delivery Research
The School of Pharmacy
Tel: +44 (0)20 7753 5861
Fax: +44 (0)20 7753 5942
E-mail: kostas.kostarelos@pharmacy.ac.uk
Project beneficiaries:
P1. ULSOP Core Scientific Team (UK)
Prof. Kostas Kostarelos
Dr Chang Guo
Dr Khuloud Al-Jamal
Dr Hanene Ali-Boucetta
Dr Tzu-Wen Wang
P2. UCB Core Scientific Team (Belgium/UK)
Dr Mike Eaton
Dr Martyn Robinson
Mrs Laura Newham
P3. NANOCYL Core Scientific Team (Belgium)
Dr Julien Aamadou
P4. CNRS Core Scientific Team (France)
Dr Alberto Bianco
Dr Cécilia Menard-Moyon
Dr Enrica Venturelli
Dr Julie Russier
P5. UTr Core Scientific Team (Italy)
Prof. Maurizio PRATO
Dr Tatiana Da Ros
Dr Chiara Fabbro
P6. HelmZ Core Scientific Team (Germany)
Dr Wolfgang G. Kreyling
Dr Tobias Stoeger
Dr Stephanie Hirn
P7. UoI Core Scientific Team (Greece)
Dr Dimitris Emfietzoglou
Dr Ioanna Kyriakou
P8. SeroS Core Scientific Team (Hungary)
Prof. Janos Szebeni
Dr Rudolf Urbanics
Dr Zoltan Rozsnyay
Project context and objectives:
Background
Cancer is a serious and costly threat to human health today. According to the Public Health Portal of the European Union (EU), 3.2 million Europeans annually are diagnosed with cancer, mostly breast, colorectal or lung cancers. While there are many different types of cancer, the underlying problem is the same: cells divide and grow in an uncontrolled manner, most commonly resulting in solid primary tumours that may spread to other organs, leading to metastasis.
One of the challenges with cancer treatment is the non-selective nature of therapy, i.e. both healthy and malignant tissues are affected. In addition, most treatments also inhibit the production of erythrocytes and white blood cells, causing patients to become anaemic and neutropenic, leading to enhanced susceptibilities to secondary infections. Cancer is projected to become more widespread in the next few decades due to ageing of the population and increased chemo- and radio-resistance.
Project aim
The aim of the ANTICARB project is the improvement of oncology treatment options using systemic administration of nanotechnology-mediated antibody therapeutics. Antibodies were selected as they allow for therapeutic and active targeting to specific cell receptors. Carbon nanotubes were chosen because their large surface area offers a template for conjugation with a variety of targeting and therapeutic monoclonal antibodies.
The CNT-Ab constructs are tagged with fluorescent probes and radioisotopes to help determine their cell biology and pharmacology. Fluorescent probes allow for in vitro imaging to understand the mechanism of the interaction between the CNT-Ab constructs and cancer cells. Radioisoprobes offer capabilities for in vivo diagnosis and quantitative tissue distribution, pharmacokinetics and toxicokinetics. The ANTICARB project also attempted to determine the safety profile of the novel CNT-Ab constructs using an array of toxicokinetic, immunological and cytotoxicity assays.
Project objectives
The ANTICARB project seeks to create new technologies that may offer more treatment and diagnostic options to clinicians and patients. The main objective of ANTICARB is the design and development of carbon nanotube-antibody (CNT-Ab) constructs. They are investigated as novel platforms for cancer treatment with the purpose to act as combinatory therapeutic/diagnostic agents.
The specific ANTICARB objectives are:
- improve and expand oncology treatment options based on systemic administration of nanotechnology-mediated antibody therapeutics;
- determine the safety profile of the novel CNT-Ab constructs based on pharmacokinetic, immunological and toxicological assays;
- develop CNT-Ab constructs that can be rapidly translated into larger animal and clinical pre-Phase I trials;
- determine the pharmacology and cell biology of functionalised carbon nanotubes with and without antibody conjugation;
- increase the competitiveness of the European industry (pharmaceutical and nanotechnology sector).
Project workplan
The ANTICARB workplan is divided in three almost distinct stages. Even though stages I-III follow a scientifically logical sequence, our intention was to engage in all three Stages simultaneously very early on in the project in order to allow for optimisation of the CNT-Ab constructs through multiple feedback loops: lab-to-cells-to-animals.
Stage I: CNT-Ab engineering carbon nanotube platform material [contribution from work packages (WPs) 1 and 2]
A very important problem related to the use of carbon nanotubes is their extraordinary aggregation due to very strong intertube forces, which causes insolubility in any type of organic solvent or aqueous solution. For biomedical applications, it is therefore mandatory to modify the pristine material in order to disrupt the bundles and make the manipulation of CNTs easier. To render the CNTs soluble or, better, biocompatible, we followed several strategies. Nanocyl provided CVD-synthesised carbon nanotubes: single-walled carbon nanotubes (SWNT), double-walled carbon nanotubes (DWNT) and multi-walled carbon nanotubes (MWNT). The purity of the raw MWNTs was always above 90 % and always further purified. All purified new products of DWNTs and MWNTs CNT were shortened and functionalised at the same time and available at a laboratory scale too.
Functionalisation of CNTs was performed in order to have different functional groups at the CNT surface that influenced reactivity towards antibodies. NH2 functional groups were introduced on the surface of CNTs. Optimisation steps included purification, functionalisation and shortening of CNTs to achieve as narrow as possible the size distribution of CNTs. The objective is to increase the reactivity of CNTs towards antibodies conjugation, a higher yield of simple functional group such as NH2 can be envisaged througha plasma functionalisation process. Based on the reactivity of such modified CNT with selected antibodies, the choice of the ideal number of functional groups were estimated.
Alternatively, the CNT were treated with strong oxidising acids to generate carboxylic functions at their tips and side-walls at much higher concentrations. After oxidation, the nanotubes were also chemically shortened and compared with those obtained by the ball milling process (vide supra). The material so produced was much more easily dispersable than the pristine nanotubes, and can be further functionalised with many relevant biomolecules. Another approach in the modification will consist on the use of organic addition reactions, including the 1,3-dipolar cycloaddition of azomethine ylides. This process, optimised in the last few years by the UTr and CNRS groups of this Consortium, will allow the direct introduction of the functionalities at the side-walls and tips of the unmodified tubes. The functional groups will be again modified with the suitable bio fragments (vide infra). These two types of functionalisation strategies were applied to double- and multi-walled CNTs provided by Nanocyl. The two functionalisation processes were applied contemporarily, so that we can produce CNTs derivatised with two different molecular entities, such as an antibody and/or a drug, or a labelling unit. In addition, the functionalisation allowed the complete removal of the catalytic particles that may not be totally eliminated during the manufacturing process.
Antibody construct [contribution from WP3 and WP4]
UCB has over twenty years history of taking antibodies into the clinic, indeed it was the pioneer in evaluating chimeric antibodies. Over the decades it has acquired a large library of antibodies - human and murine - different IgG sub-classes and whole IgG, as well as fragments such as Fabs. This library of therapeutic antibodies has been mainly produced in E.Coli and has in general a clinical specification of 99.9 % purity. For the purposes of ANTICARB, a series of antibodies were used that were clinically developed to Phase II clinical trials.
Carbon nanotube - antibody conjugate [contribution from WP2 & WP3]
Following the first step, the f-CNTs will contain carboxylic and/or amino groups at their tips and side walls. These f-CNTs, derived from the oxidation reaction and the organic cycloaddition reactions, will be used as starting materials for the conjugation to the cell specific targeting monoclonal antibodies and to the therapeutic monoclonal antibodies (IgG or fragments). To generate the different conjugates, the f-CNTs will possess amino functions, fundamental for the introduction of suitable linkers for the monoclonal antibody binding. The amino groups around the nanotubes will be modified with a maleimide function. Indeed, the maleimide will allow a direct chemo-selective reaction with the thiol-free cysteine amino acid residues within the antibody sequence. All these types of CNT-Ab conjugates are of covalent nature.
Radiation and optical probes [contribution from WP2, WP4, WP5]
The f-CNTs, derived from the oxidation reaction and the organic cycloaddition reactions, will be further functionalised with chelating agents for radioisotopes emitting and Auger radiation to perform in vivo diagnosis and intracellular radiotherapy. To generate the different radiolabelled conjugates, f-CNTs will be modified with amino functions for the introduction of suitable ligands including DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and DTPA (diethylentriaminepentaacetic dianhydride) for the complexation of the different radionuclei. In parallel, a similar approach will be exploited to link fluorescent probes to f-CNTs for in vitro imaging. Alternatively, the radioisotopes and the fluorescent molecules will be grafted to the monoclonal antibodies before or after the conjugation to the nanotubes.
Stage II: CNT-Ab biology - CNT-Ab construct cell biology [contribution from WP2, WP4, WP5]
All CNT-Ab conjugates, including the fluorescent derivatives, will be assessed for their cellular uptake behaviour. A wide panel of human and murine cells lines, murine T and B lymphocytes, and murine macrophages will be used to study the cell penetration, the intracellular trafficking and the mechanisms of internalisation. Fluorescent spectroscopy (epifluorescence and/or confocal laser scanning microscopy) will be used to observe the internalisation process. Flow cytometry (FACS) will be helpful not only for the uptake analysis, but also for the determination of the kinetic, the time and concentration dependence of the process. In addition, FACS will be fundamental for the assessment of the cytotoxic effects. The lymphocytes and macrophages will be directly isolated from the animal models available in our laboratories (vide infra). Surface plasmon resonance analysis (Biacore technology) will be used to measure the parameters of binding between the CNT-Ab constructs and the receptor expressed at the cell surface. 3D human tumor spheroids are routinely cultured in our laboratories and will be used as an in vitro model for metastatic nodules and the avascular interstitial space of solid tumors. Penetration into the tumor volume will be assessed by optical slicing of the spheroid using confocal laser scanning microsocpy tracking the optical signal from fluorescently labelled CNT-Ab constructs. Image analysis packages will allow modelling of the capacity of different CNT-Ab conjugates to penetrate deeper into the tumor mass and also simulate the predicted radiation dose deposition and tumor cell kill.
CNT-Ab pharmacology [contribution from WP2, WP4, WP5]
The pharmacological properties of the CNT-Ab conjugates will be highly dependent on the tissue distribution, pharmacokinetics, blood residence time and body excretion kinetics following their in vivo administration. A variety of critical parameters will be assessed:
a) route of administration - three main routes relevant to current clinical application of antibodies and proteins will be explored: intravenous, subcutaneous; pulmonary;
b) dose escalation - our starting point will be the currently administered therapeutic antibody doses (approximately 3mg/kg in a single administration) and escalate from there;
c) in vivo model upscaling - we will carry out all of the routine tissue biodistribution and pharmacokinetic analysis using mice and dynamic whole-body imaging (microSPECT/CT - requested for ULSOP) deemed essential to allow rapid, accurate, ethical (minimisation of animal numbers) and feasible (minimisation of labour intensity required) determination of the in vivo transport kinetics and organ distribution of the CNT-Ab. In that way, fundamental understanding of the CNT-Ab pharmacology will be obtained.
Stage III: CNT-Ab safety and therapeutic efficacy - CNT-Ab toxicology, immunocompatibility and radioprotection [contribution from WP2, WP4, WP5]
Toxicology - To achieve a long-term risk assessment of the CNT-Ab constructs, dose escalation studies will be carried out first to determine doses to the various organs and tissues of relevance. In a second step we will study the toxicological effects in those organs. We will carry out long-term biokinetics after intravenous injection (two, four weeks and three months) of stably radiolabeled Ab-CNT in rats. Once we know which secondary target organ is going to be dosed we will perform toxicological studies on some of these organs at selected time points (determined by the dose escalation studies) by pro-inflammatory cytokine assays and limited induction tests. These tests will include gene array analyses. We are expecting to be able to determine which organs are affected and the lower dose limit, under which toxicity will not be expected to act as an adverse effect (toxicity threshold).
Immunocompatibility - The rationale behind studying complement activation in vitro and complement-mediated pseudoallergic reactions in vivo, as predictors of short-term immune toxicity of CNT-Ab, lies in the fact that complement activation might cause, or play a major role in those unusual hypersensitivity reactions (HSRs) wherein prior exposure to the reactogen agent, and, hence, a causal role of IgE, cannot be established. These reactions have been referred to as 'complement activation-related pseudoallergy' (CARPA), and include widely known and feared 'infusion' reactions to anticancer drugs solubilised by micellar solvents, such as Cremophor EL and polysorbate-80 (in Taxol and Taxotere), liposomal anticancer and antifungal drugs (Doxil and Ambisome), radiocontrast media, plasma expanders and blood substitutes. The pathomechanism of CARPA involves complement activation through both the classical and the alternative pathways, giving rise to C3a and C5a anaphylatoxins that trigger mast cells and basophils for secretory response that underlies the clinical symptoms of HSRs. Considering their cylindrical structure with extended length, CNTs have a substantial surface area for binding plasma and complement proteins, particularly if, after functionalisation and targeted binding, they deposit on cell surfaces in a tandem or aligned manner. CNT-Ab may therefore activate complement, posing realistic danger for CARPA. In addition, unexpected cross reactions with immune cell surface antigens might trigger the release of cytokines or allergomedins, as observed with TGN1412. The proposed measurement of complement and basophil activation by CNT-Ab in human serum and blood in vitro and reactogenicity studies in animals in vivo will serve as essential predictors of biocompatibility or toxic effects in the clinic.
Radioprotection - Gamma (or X) ray emitting radionuclides suitable for imaging emit photons which are energetic enough to escape the body and, thus, in addition to the self-dose in the residing tissue are also capable of cross-irradiating adjacent organs. Given their relatively uniform mode of irradiation, a quantitative estimate of the energy deposition to the various organs (radiation absorbed dose) will be obtained by the MIRD mathematical formalism using experimental biokinetic data for the radionuclides, its characteristic emission properties (type and energy of emitting photons/electrons, and half life) as well as its source-to-target specific absorption values. For radionuclides that also (or solely) emit Auger electrons which deposit their energy in a highly non-uniform and localised manner (usually within less than 100 nm from their origin), the MIRD scheme is not suitable, therefore we will perform detailed, nanoscale Monte-Carlo simulations to assess the energy deposition by the Auger electrons in radiobiologically sensitive subcellular domains (e.g. mitochondrium, cell nucleus, DNA). Appropriate weighting factors will then be used for the irradiation component that resulted from the Auger electrons, to properly account for their increased radiobiological effectiveness in comparison to the gamma and X rays.
CNT-Ab preclinical efficacy [contribution from WP2, WP4, WP5]
The therapeutic efficacy of the CNT-Ab conjugates were performed using human tumour xenograft models in nude mice. The main objective is to use antibody nanotubes bearing no isotope or cytotoxin but with antibodies that are biologically active against an intracellular target. The changes in tissue biodistribution and pharmacokinetics of radiolabelled CNT-Ab in mice were initially established by microSPECT/CT, followed by tumor xenograft therapeutic studies using a dose regime previously established to be lower than the toxicity threshold. The intratumoral route was primarily explored to investigate the therapeutic activity of the CNT-Ab.
Project results:
The main aim of ANTICARB has been the design and development of novel, safe and effective carbon nanotube -antibody (CNT-Ab) constructs for combinatory therapeutic / diagnostic (theranostic) oncology indications. The individual components employed to achieve this technology are the following:
a) Antibodies - monoclonal antibodies - full (approximately size 8 nm) or fragments (5 nm) will be used for therapeutic and active targeting to specific cell receptors
b) Carbon nanotubes - single- (1 nm diameter) and multi-walled (20-30 nm in diameter) functionalised carbon nanotubes will be used as platforms for controlled conjugation of the antibodies. Also, antibodies with the capacity for intracellular activity
c) Radionuclides - radioisotopes emitting and Auger radiation will be grafted either on the carbon nanotube or the antibody to offer capability for in vivo diagnosis (SPECT/CT imaging) and synergistic intracellular radiotherapy.
d) Optical probes - fluorescent probe molecules will be grafted on the carbon nanotubes or antibodies to offer capability for in vitro imaging (microscopy)
ANTICARB had four major scientific objectives:
ANTICARB has achieved the following science and technology (S&T) foregrounds in each of its objectives:
1. Fabrication and synthesis of CNT-Ab
Two types of MWNT material, morphologically different, were compared throughout the project (Nanocyl and NanoAmorph). The Nanocyl material was thinner (5-10 nm) than the NanoAmorphous (20-30 nm) material. It was also found that the oxidation of the nanotubes in strong acids shortens the length of both Nanocyl and NanoAmorph materials but also creates defects in their sidewalls.
One of the interesting findings in ANTICARB, is that oxidation renders MWNT prone to enzymatic degradation when the oxidised MWNT were incubated in the phagolysosomal stimulating fluid (PSF) and horseradish peroxidase (HRP).
A series of Phase II clinically developed antibodies were used in ANTICARB. This include the full IgG; a humanised monoclonal antibody (hCTM01) , the antigen binding fragment (Fab') and the single chain variable fragment (scFV). These antibodies were targeting the polymorphic epithelial mucins (MUC-1) which is over-expressed in many tumours.
Therefore, different CNT-Ab constructs were synthesised, characterised and studied in ANTICARB. These constructs had dual functional groups which allowed their conjugation to the antibodies (IgG, Fab', scFV) at one end and to a fluorescent probe (FITC) or a chelating molecule (DTPA) to the other end.
2. Intracellular delivery of CNT-Ab
The CNT-Fab' construct internalised in both MUC-1 positive cells (MCF7) and MUC-1 negative cells (Calu6) as shown by the presence of the green signal (FITC conjugated to CNT) and red signal (secondary antibody against Fab's) in both cell lines.
3. Tumor targeting of CNT-Ab
In ANTICARB, we used multicellular tumour spheroids as an vitro 3D model to study the tumour targeting capacity of the CNT-Ab constructs. Both MWNT-IgG and MWNT-Fab' constructs showed enhanced affinity for MUC-1 positive tumour spheroids (MCF7). However, MWNT-Fab' exhibited a deeper localisation within the tumour avascular interstitium which was even observed with the MUC-1 negative (HCT116) tumour spheroids as shown in the cryosections of the spheroids).This indicates that MWNT were driving the uptake when the antibody was a smaller fragment (Fab').
4. Pharmacokinetics of CNT-Ab
The biodistribution profile of the CNT-Ab constructs was studied after intravenous administration. As seen in the SPECT/CT images, there was no change in the biodistribution profile when the antibodies were conjugated onto the nanotubes. However, the NanoAmorph CNT-Ab constructs accumulated mainly in the liver and spleen; while the Nanocyl CNT-Ab constructs were mainly detected in the lungs and liver. This indicates that the starting material has a drastic effect on the biological profile of materials in vivo.
5. Safety and toxicity of CNT-Ab
The toxicity of CNT-Ab constructs was assessed after intra-tracheal administration. It was found that the MWNT-Fab' construct caused the most significant acute and chronic inflammation followed by the Fab' fragment alone and then the MWNT alone. In addition, acute macrophage depletion was observed with the same construct after 24 hrs post-injection but was also observed to a lesser degree with the Fab' alone and MWNT.
Moreover, the cardiovascular changes were assessed using a porcine model which was intra-arterially administrated with repeated doses of CNT. While the in vitro data indicated complement activation with the oxidised MWNT, the most severe cardiovascular changes were also observed with the same oxidised MWNT. Interestingly, amino functionalisation and Fab' conjugation to MWNT alleviated those risks.
S&T ANTICARB achievements
- Fabrication, Characterisation and Upscaling of complex MWNT-Ab
- Confirmation of the cellular internalisation of MWNT-Ab
- Elucidation of mechanisms leading to cellular internalisation of MWNT-Ab
- Determination of pharmacokinetic and pharmacodynamic profile of MWNT-Ab
- Revelation that surface carboxylation increases risk of unwanted reactivity and chemical ways to alleviate this
- Development of antibodies with biological activity against intracellular targets
- Biologically active MWNT-Ab conjugates.
S&T ANTICARB reflections
- Conjugation of proteins on MWNT is not easy, straightforward and cannot avoid physisorption (contrary to what literature indicates).
- Batch variation in terms of starting material and functionalisation still persist and affects biological interactions.
- Size of MWNT-Ab constructs (depending on the Ab size: IgG, Fab', scFv) determines localisation within tumor interstitium.
- The kinetics of free probes (in our case radionuclides Gd-153 and In-111) can affect exact determination of biokinetic profiles.
- Fibre-related toxicity is not really relevant to short, chemically functionalised carbon nanotubes.
- It is exceedingly challenging for CNT and other novel nanomaterials to match the short-term scope of pharmaceutical industry, maintain novelty (i.e. attractive patentability) and avoid hype (positive and negative) to keep public funding (that requires industry participation / support) for the medium / long term.
Potential impact:
The ANTICARB consortium brought together five of the most qualified research centres in Europe, one multinational pharmaceutical company that leads the global biopharmaceutical market segment (UCB) and two young and dynamic SMEs (NANOCYL and SeroScience) with a major scientific aim: to generate a new class of nanomedicine constructs as anticancer therapeutics by joining the carbon nanotube and the monoclonal antibody technologies. The proposal was based on a large and well-coordinated effort from seven different countries (United Kingdom, Belgium, France, Germany, Hungary, Italy and Greece).
Training a new generation of European researchers
The development of nanopharmaceuticals is a new emerging multidisciplinary area and research within a European-wide network is mandatory for progress in the field. The ANTICARB consortium brought together the three sides of the 'knowledge triangle': industrial innovation, cutting-edge academic research and education within an inter-sectorial approach. One of the key impacts that were addressed is the improvement of technical skills, training and the level of innovation of the new generation of European researchers, so as to improve European competitiveness within the global nanotechnology and pharmaceutical market. The participation of researchers from different European countries is in line with the spirit of EU cohesion and joint policymaking for research. The sharing of knowledge between the members of ANTICARB led to research activities that could not be achieved at the national scale. The ANTICARB network contributed to a strong interdisciplinary and inter-sectoral training of top-researchers that will be the workforce of the future.
Further the understanding the hurdles of clinical translation
The interaction between cancer physiology and the nanotechnology-based innovative strategy proposed (the CNT-Ab hybrids) has been of fundamental importance throughout the development of ANTICARB. ANTICARB has been seeking to contribute to the creation of fundamental knowledge at the European level and at the same time, through the central role of the pharmaceutical industry partner (UCB), maximised the possibilities of translation into the clinical setting.
Exploit basic knowledge to develop novel drugs and treatment strategies
ANTICARB created necessary knowledge that was lacking on how to transform a novel nanomaterial of known fascinating physical properties, such as carbon nanotubes, in the biomedical arena and explore the possibility to translate it clinically into an effective cancer therapeutic. One of the key conclusions from ANTICARB is that the particular nanotechnology has been shown to have the capacity to transform otherwise ineffective known entities into therapeutically active and effective anti-cancer drugs. ANTICARB offers evidence that a new market in therapeutic oncology can be opened - that of using antibodies and their fragments against intracellular targets.
European economical and social advantages ANTICARB provided a variety of social and economical advantages to the European Community. It will reinforce the collaboration among European scientists and laboratories and will contribute to establish a European scientific identity. US and Asian Universities and companies represent the major competitors in genome science and nano- and biotechnology. The quality of life for European citizens depends on Europe's ability to compete and, where possible, to take the lead. In addition, shrinking or closing the gaps in sectors where Europe currently lags behind is of vital importance. If successful, this policy will create opportunities and increased prosperity within Europe. To this end no individual country within Europe is in the position to either assemble a consortium of this calibre or to fund it to the extent necessary to either compete with or gain a competitive advantage over the US and Asia. Finally, the multifaceted nature of the nanobiotechnology research area requires a multidisciplinary approach, which is best achieved through coordinated and integrated European wide research.
Main dissemination activities
1. Project coordination activities
Although the project coordination mainly involved the consortium, in several cases they involved dissemination of project information beyond the consortium, via each partner's collaborations network.
During the whole project period the partners held 33 coordination activities:
- 7 project meetings with 93 % participation
- 14 teleconferences with 92 % participation
- 12 visits, exchanges and joint work involving:
- 4 meetings involving 2 or more partners
- 1 sabbatical visit
- 7 visits in order to perform jointly experiments
2. Publications
The ANTICARB consortium published in all 53 publications in wich ANTICARB project is acknowledged.
- 47 articles in journals and
- 6 book chapters.
Regarding the authorship of the above publications:
- 32 were submitted by 1 partner
- 10 by two partners
- 11 were submitted by three or more partners.
Regarding the area the publishing houses are situated:
- 42 of them were published in journals and books published in Europe and
- 11 in journals and books published outside EU, mainly in US.
3.Presentations
The ANTICARB consortium presented their work in 85 activities of which:
- 50 were conferences
- 15 were summer schools - seminars
- 20 were workshops - meetings.
The impact level of the above activities was for:
- 16 national
- 22 European
- 47 international.
Regarding the activity audience for:
- 61 involved mainly the research community
- 24 involved the industry community (together with the research community in some cases)
- 14 involved mainly policy makers and media (together with the research and the industry community in some cases).
4. PhD studentships
The project employed in all 13 PhD students, 3 of which worked entirely on ANTICARB and acknowledged the project in their thesis. The thesis were submitted, examined and published while the ANTICARB project was still running.
5. Events organised
Four major events were organised under or supported by ANTICARB project:
Second Carbon Nanotube Biology, Medicine and Toxicology Satellite Symposium
20 June 2009, Tsinghua University Campus, Beijing, China
The ANTICARB project sponsored the organisation and implementation of the above symposium. For that reason the ANTICARB logo features until today in the symposium website. This one-day symposium took place one day prior to the official opening and within the content of the 'Tenth International Conference on the Science and Application of Nanotubes - NT09'.
The symposium involved participation of about 120 scientists presenting their work and discussing the interaction of nanotubes with cells and tissues in vitro and in vivo, and exploring specific applications of nanotubes in the biomedical field.
For more information please see the symposium website at http://www.cntbiology.org/CBTN09/
Third Carbon Nanotube Biology, Medicine and Toxicology Satellite Symposium
27-28 June 2010, Hilton Bonaventure, Montréal, Canada
The ANTICARB project sponsored the organisation and implementation of the above symposium. For that reason the ANTICARB logo features until today in the symposium website. This two-days symposium took place during the first two days and within the content of the 'Eleventh International Conference on the Science and Application of Nanotubes - NT10'.
The symposium involved participation of about 90 scientists presenting their work and discussing the correlation between the specific characteristics of carbon nanomaterials (method of synthesis and manufacturing, purity, type of surface functionalisation) and their biological effect in various model systems (immortalised or primary cells, in vivo models).
For more information please see the symposium website at http://www.cntbiology.org/CBTN10/
Fourth Carbon Nanotube Biology, Medicine and Toxicology Satellite Symposium
15-16 July 2011, University of Cambridge, United Kingdom
The ANTICARB project sponsored the organisation and implementation of the above symposium. For that reason the ANTICARB logo features until today in the symposium website. This two-days symposium took place during the last two days and within the content of the 'Twelfth International Conference on the Science and Application of Nanotubes - NT11'.
The symposium involved participation of about 78 scientists presenting their work and discussing the determining the therapeutic and diagnostic potential of carbon nanomaterials and the challenges ahead. Moreover, particular attention was placed on attempts to understand the toxicological and environmental risks posed by use of carbon nanotubes.
For more information please see the symposium website at http://www.cntbiology.org/CBTN11/ and http://www-g.eng.cam.ac.uk/nms/cnbmt/
Carbon Nanotubes: From Safety Assessment to Biomedical Applications
One-day workshop, 29 November 2011, Brussels, Belgium
The ANTICARB project organised in synergy with another FP7 STREP project NANOMMUNE, an one day workshop. In this workshop it was attempted to present the state-of-the-art in the field of carbon nanotechnology in medicine, by bringing together success stories and lessons learnt from the research work carried out by the two FP7 projects that have worked in parallel in this domain for the last three years.
Distinguished speakers were invited beyond the two projects' consortia, like independent experts, Nano - Fora and industrial associations' representatives, EU policy makers and EC Project Officers. The workshop was advertised also in the Nanosafety Cluster and the Nanotechnology Industries Association news' webpages.
Web dissemination
Project website
The project website was created within the first six months of the project and since then it provide information to the project partners and the wider public. In its current status the public website is fully updated with latest information regarding publications and dissemination activities of the consortium. The private (only for partners) webpage is also fully updated with all information and presentations for all partners coordination activities. The website is planned to remain online for at least the next 12 months.
For more information please see the project website at http://www.anticarb.org
Coordinator's website
Project information can also be found in the coordinator's website.
For more information please see the Nanomedicine lab website at http://www.nanomedicinelab.com/collaborations.html
Other websites
Here are some other websites relevant to the ANTICARB partners or subject providing information about the project:
IST WORLD
http://www.ist-world.org/ProjectDetails.aspx?ProjectId=cc03235c40904005884360b8a4d57bba&SourceDatabaseId=018774364ea94468b3f4dec24aa1ee53
NANOCYL
http://www.nanocyl.com/CNT-Expertise-Centre/R-D-Projects-Alliances/Nanocyl-can-Quote-10-European-Projects-and-4-Belgian-Ones
HELMHOLTZ INSTITUTE
http://www.helmholtz-muenchen.de/ilbd/about-ilbd/projects-and-collaborations/index.html
Exploitation of results
1. Research era
The project results will be exploited in the following way in order to contribute to the EU research era.
- New knowledge and know-how acquired during the project will serve to set up a new research field around antibody therapeutics and drug delivery.
- The results of the project will be used to explore the carbon nanotube-based drug delivery concept for several applications as cancer and neurological disorders.
- The results obtained will be used to design new protocols for the exploration of gene involvement in various diseases (e.g. Parkinson's).
- Research and evaluation of the results by upscaling to large animal studies for eventual use in the design of clinical studies.
- New techniques to be introduced into small animal imaging courses and newly developed therapy options can be evaluated in order to transfer them into clinical trials.
2. Training and education
The project results will be exploited in the following way in order to contribute to the training and education of new scientists.
- New knowledge and expertise acquired during the project will be utilised in postgraduate course curriculum at the University partners (ULSOP, UTr and UoI).
- Novel technologies on the development of carbon nanomaerials will be part of the curriculum for a nanomedicine -based approach to cancer treatment and will be incorporated in our existing teaching modules in drug delivery.
- Some of the topics can feed into master's curricula of biomedical engineering faculties as teaching material.
3. Industry
The project results will be exploited in the following way in order to contribute to the EU nanotechnology and medicine industry.
- Improve our knowledge in nanoparticles design. Extend nanoparticles applications to innovative imaging and therapy applications.
- Extending the application of carbon nanomaterials as key components in new sophisticated drug delivery systems and further development of the market of anticancer therapeutics in the long term.
- The investigated methods will be used in the further development for intracellularly-targeted antibody therapeutics. The tools developed for the preclinical work in this project may in itself become a commercially viable option for the research oriented gene and protein delivery research market (e.g. nanotube-based transfection agents).
- The new therapy options and methods for delivery of antibodies directly adds to the industrial partners (UCB, Nanocyl and SeroScience) production and exploration of new products programme.
Policy making
The project results will be exploited in the following way in order to influence EU policies regarding advancements in nanotechnology and nanomedicine.
- New activities are already initiated using the Nanosafety Cluster (see http://www.nanosafetycluster.eu online) and other technologies in collaboration with other FP7 consortia (see http://www.nanommune.eu online).
- Attempts to bridge the gap between regulation of nanomaterials in the context of public health safety and nanotoxicology and nanomedicine through the EMA.
Project website address:
http://www.anticarb.org
Contact details:
Prof. Kostas Kostarelos
Centre for Drug Delivery Research
The School of Pharmacy
Tel: +44 (0)20 7753 5861
Fax: +44 (0)20 7753 5942
E-mail: kostas.kostarelos@pharmacy.ac.uk
Project beneficiaries:
P1. ULSOP Core Scientific Team (UK)
Prof. Kostas Kostarelos
Dr Chang Guo
Dr Khuloud Al-Jamal
Dr Hanene Ali-Boucetta
Dr Tzu-Wen Wang
P2. UCB Core Scientific Team (Belgium/UK)
Dr Mike Eaton
Dr Martyn Robinson
Mrs Laura Newham
P3. NANOCYL Core Scientific Team (Belgium)
Dr Julien Aamadou
P4. CNRS Core Scientific Team (France)
Dr Alberto Bianco
Dr Cécilia Menard-Moyon
Dr Enrica Venturelli
Dr Julie Russier
P5. UTr Core Scientific Team (Italy)
Prof. Maurizio PRATO
Dr Tatiana Da Ros
Dr Chiara Fabbro
P6. HelmZ Core Scientific Team (Germany)
Dr Wolfgang G. Kreyling
Dr Tobias Stoeger
Dr Stephanie Hirn
P7. UoI Core Scientific Team (Greece)
Dr Dimitris Emfietzoglou
Dr Ioanna Kyriakou
P8. SeroS Core Scientific Team (Hungary)
Prof. Janos Szebeni
Dr Rudolf Urbanics
Dr Zoltan Rozsnyay
