Proiect PN-III-P1-1.2-PCCDI-2017-0769 (Contract 64PCCDI/2018) ONCORAD
The development of radiopharmaceuticals and nuclear techniques in oncology for imaging and personalized treatment at the molecular level
Total Budget: 5287500 RON
Coordinating Institution: Horia Hulubei National Institute for R&D in Physics and Nuclear Engineering (IFIN-HH), Proiect Manager: Dr. Mihai Ciubotaru
Title of project P1: Development of antibodies against myeloid specific markers for radio detection and treatment of myeloid leukemias in incipient stages
Budget of proiect P1: 1595414 RON (Buget INCD-VB: 423.011 RON)
Principal Investigator of P1: Dr. Mihai Ciubotaru
Partners involved in P1:
Horia Hulubei National Institute for R&D in Physics and Nuclear Engineering (IFIN-HH);
Institute of Biochemistry (IBAR);
Cantacuzino National Institute for R&D in Military Medicine (IC);
Victor Babes National Institute of Pathology (IVB)
IVB team in P1: Valeriu Cismasiu (lead person), Sevinci Pop, Gisela Gaina, Cristina Niculite, Stefania Rogozea/Alexandra Popa, Ioana Lambrescu, Victor Ionescu
Abstract :
The ONCORAD project and the associated radiobiology technological platform are built on the foundations of translational medicine and personalized medicine, and have as their main objective the development of targeted radiopharmaceutical products and innovative nuclear medical techniques for imaging diagnosis and improved cancer treatment. The ONCORAD project is based on the unique competence in the field of radioisotopes and radiopharmaceuticals from the coordinating Institute (IFIN-HH), complementing it with the expertise of partner institutions with prodigious research activity in the biomedical field (IVB, IC and IBAR), clinics (Fundeni Clinical Institute, Bucharest), as well as in the field of nanotechnologies/nanomedicine (INCD for Isotopic and Molecular Techniques, Cluj-Napoca). This multidisciplinary mobilization takes shape in four interconnected research projects, with objectives in the field of oncological nuclear medicine. In addition, the ONCORAD project will support the development of the institutions in the consortium by hiring young researchers and training them in top scientific and technological fields, developing the research infrastructure, share of knowledge, technologies, and best practices between the partners in the consortium and to other interested parties. The project creates the framework of interdisciplinary collaboration between the partner institutions, thus constituting the premise for the future participation of the consortium in projects, the diversification of research offers, technological transfer for clinical applications.
The main scientific objective of Project 1 was to obtain antibodies and single-chain Fab fragments immune against evolutionary surface markers both for the early stages of stem cells Sca1 (stem cells antigen 1) and of myeloid differentiation (Ly6C) present on the surface of neoplastic mononuclear cells in the early stages of myeloid leukemia. These single-chain fragments will be labeled with positron-emitting isotopes (Zr-89, Cu-64) to use them as biomarkers. In vitro detection of leukemic cells will be performed in cultures by nuclear techniques, and in vivo by micro PET-CT (positron emission tomography coupled with computerized tomography, with sub-millimeter resolution). The coupling of single-chain immune fragments with therapeutic radionuclides through complementary techniques can lead to antibodies with a therapeutic role, whose effectiveness in stopping tumor growth will be tested through in vitro and in vivo techniques. The therapeutic radionuclides that will be tested within the project are radiation emitters with high energy transfer (high toxic potential), At-211/Ac-225 and/or Cu-64/67.
The IVB team developed the in vivo model for acute myeloid leukemia, analyzed and phenotypically characterized the tumor cells. Later, the research group from INCD-VB tested the new anti-Sca1 and anti-Ly6C antibodies, to select the most efficient clones for radioactive labeling.
Results of the IVB team
The mouse line STOCK Kmt2atm2(MLLT3)Thr/KsyJ mouse (The Jackson Laboratory, # 009079) was used to generate the acute myeloid leukemia model. The transgene that mimics the chromosomal translocation t(9;11)(p22;q23) is inserted into the Kmt2a gene locus. The translocation causes the cancellation of murine MLL protein synthesis, which is replaced by the MLL-AF9 (MA9) fusion protein. The selection of animals carrying the mutation was carried out by PCR genotyping. The mutation causes an epigenetic instability of the hematopoietic cells that ends with the complete oncogenic transformation and the formation of the leukemic stem cell. Thus, both hematopoietic stem cells (HSC) and granulocyte and monocyte precursor cells (GMP), carrying the MA9 mutation, are considered pre-leukemic cells (p-HSC, p-GMP). The appearance and evolution of myeloid leukemia was followed by monitoring the spleen and the general condition of the animals. Figure 1 shows the survival curve of the animals as well as the increased size of the spleen (a sign of tumor cell infiltration).
The average duration of oncogenic transformation is 25 weeks, according to the Kaplan-Meier analysis. Figure 2 shows that the MA9 mutation favors myelopoiesis and the growth of granulocyte and monocyte populations (marked with CD45 and CD11b). The results demonstrate that the oncogenic transformation is accompanied by a reduction of the p-HSC (pre)leukemic stem population (referred to as LSK in figure 2). The second phenomenon associated with the development of leukemia in MA9 mice is the increase in the population of (pre)leukemic p-GMP granulo-monocytic precursors (preGrMo gate, in figure 2). The results are consistent with a published study according to which myeloid differentiation is necessary for tumor transformation and the onset of acute myeloid leukemia (Ye M et al. 2015, Cell Stem Cell 17:611-623).
The results show that tumor evolution is associated with a reduction in the stem population and, simultaneously, with an increase in granulo-monocytic progenitors. Therefore, the identification of specific markers for these two categories of cells will allow in vivo monitoring of the oncogenic transformation. The analysis led to the identification of two suitable markers: Sca-1 and Ly6C. Figure 3 shows the representative results of a set of 4 independent determinations. The reduction of hematopoietic progenitors (cKit+, black rectangle) is observed in MA9 mice starting with age of 5 weeks, which indicates the reduction of stem populations (marked with Sca1). In accordance with this aspect, we can observe the reduction of the positive population for Sca1 (fig 3, blue rectangle, 5 weeks) and the disappearance of these cells in mice with acute myeloid leukemia (MLLAF9, 25 weeks). Instead, population cKit positive Ly6C positive (figure 3, green rectangle) is sustained or increases in frequency as the leukemic development progresses. Therefore, the combined use of these 2 markers indicates tumor progression by reducing the fluorescence signals generated by anti-Sca1 antibodies and increasing the fluorescence signals generated by anti-Ly6C antibodies. Figure 3:
Figura 3:
After two series of immunization, the reactivity of the serum against the hematopoietic cells of the mice was tested by flow cytometry. Immunization was done for the Ly6C antigen, and the tested samples came from three rabbits. Same type of cells were marked with FITC labelled anti-rabbit antibody and with serum (samples) or without serum (background control), and the FACS plots were compared based on the intensity of the fluorescent signal and the percentages of positive populations. In the case of samples treated with serum, the binding of primary antibodies to the cell surface is highlighted by FITC signal. The biological material was represented by mouse cells extracted from the hematogenous marrow of the animals. The results are presented in figure 4. Overall, the data suggest that the post-immunization serum presents polyclonal antibodies with increased affinity in 66% of cases.
Figure 4:
After the completion of the immunization stages of the rabbits and the sorting of antibody-producing lymphocytes, a series of scFV immune fragments reactive to the antigenic markers Sca-1 and Ly6C was received from the partners. FACS analysis (figure 5) indicates that all fragments are reactive against the corresponding antigens.
Figure 5:
In conclusion, all the activities undertaken by IVB team have been fulfilled. Publications and conferences/congresses resulting from this funding
Publicatii si conferinte/congrese rezultate din aceasta finantare
English 
Romanian