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Research

Our major scientific interest is focused on B lymphocyte development and the tumoral lymphoproliferative disorders of this cell type. In this context we chose a leukemic model as Chronic Lymphocytic Leukemia (CLL) and we directed on different target proteins-pathways in order to understand the basis of tumoral development in B-cells. Our work lies on the interface between biochemistry, molecular and cellular biology in combination with crystallography and biophysics protein constituting the core of our experimental methods.

Three major lines of research are being pursued::

  • Functional role of the mutagenic B-cell specific enzyme, Activation-Induced Cytidine Deaminase (AID) in the tumoral process
  • Genomic and proteomic characterization of a proliferating CLL B-cells subset over-expressing AID enzyme with a typically profile to an aggressive tumoral disease. In these two items special interest is given to understand the molecular mechanisms underlying the tumoral process in those lymphoproliferative disorders that over-express AID. Some key questions involving AID enzyme and tumoral B-cells development are raised: How AID enzyme is regulated during development of B lymphocyte? How AID contributes to lymphomagenesis? Which are the partners of AID involved in the deamination process? How transcribed non Ig-genes escape to deamination?
  • Effects of constant regions in the antigen binding site and their applications in the recombinant antibodies used in cancer immunotherapy.

Functional role of the mutagenic B-cell specific enzyme, Activation-Induced Cytidine Deaminase (AID) in the tumoral process

At this moment we need to learn more about how, Ig gene diversification occurs, and how it is regulated in order to prevent the mutagenic action of AID enzyme. The different haematopoietic B-cell malignancies are excellent models to dissect and understand the different pathways involved in the deamination event and in the tumoral origins. In this context our laboratory has established a successful interaction with the Hematological Service of Maciel Hospital and put forward two main diagnostic methods used as predictors in the CLL progression. This collaboration is coordinated with the national bank of cancer in Uruguay allowing the establishment of a CLL cellular bank and the continuation of different scientific projects on this leukemia.

We have made progress most notably in the functional characterization (Oppezzo, Vuillier et al. 2003) and differential expression of AID enzyme in CLL (Oppezzo, Dumas et al. 2004). Despite of Somatic Hypermutation (SHM) and Class Switch Recombination (CSR) are physiologically triggered in B-lymphocyte by AID, undesirable actions such as lymphomagenesis has been associated to its over-expression (Oppezzo and Dighiero 2005) (Perez-Duran, de Yebenes et al. 2007). More recently an interesting role for AID in the epigenetic reprogramming including demethylation of DNA was demonstrated (Popp, Dean et al. 2010) (Tran, Nakata et al. 2010). Thus, in addition to AID over-expression and lymphomas development, this works highlighted the putative importance of this enzyme in the tumoral origins. In this context we have started a collaborative research with Dr. J Di Noia (Institut de Recherches Cliniques de Montréal), concerning the characterization of CLL B-cells and another lymphomas over-expressing AID. In this line recombinant AID protein has being produced in order to characterize putative cofactors of this enzyme that could be implicated in the specificity of deamination process.

Different solubilization methods have been optimized in order to obtain functional AID enzyme in collaboration with Dr. Vincentelli in the laboratory of Cloning, Expression & Protein purification from CNRS, Universités Aix-Marseille. Despite of known problems to obtain soluble AID, the results from this high-throughput analysis in this collaboration have been very successful. At the present two constructions were selected from 250 conditions test and preliminary functional Biacore analysis are being carried out with AID enzyme and different single and double strand DNA molecules.

Genomic and proteomic characterization of a proliferating CLL B-cells subset over-expressing AID enzyme with a typically profile to an aggressive tumoral disease

To understand how AID over-expression could be responsible in the tumoral development of CLL B-cell, we isolated a particular proliferative subset of CLL B-cells with active CSR described in (Oppezzo, Dumas et al. 2004). Since AID expression results from interaction with activated tissue microenvironment, we speculated that the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in peripheral blood (PB) of 50 CLL patients and we characterized the expression of different molecules related to microenvironment interaction. Our results show that among unmutated (UM) patients: 1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; 2) this small subset expresses high levels of proliferation, anti-apoptotic and progression CLL markers (Ki-67, c-myc, Bcl-2, CD49d and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

This research line has resulted in the first high impact article made in our laboratory, including most of the work performed at the IP Montevideo (Palacios, Moreno et al. 2010). At present, new collaborations are being developed in this field with the laboratory of Dr. Giordano at the Academy of Medicine in Bs As (Argentina).

Effects of constant regions in the antigen binding site and their applications in the recombinant antibodies used in cancer immunotherapy

Although the CSR process is frequently associated with affinity maturation, one of the immunological dogmas is that the constant region does not assume any role in the binding with the antigen. However in the last decade several works suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form. (Pritsch, Hudry-Clergeon et al. 1996; Dam, Torres et al. 2008). Although these data support clear evidence for this hypothesis, no structural proofs are available at this time. In the context of structural platform of IP Montevideo (Protein Crystallographic Unit, Biophysics Protein Unit and Recombinant Protein Unit), our group decided to provide crystallographic and biophysical data in order to confirm whether the constant regions of the Ig play a role in the recognition of the antigen.

The three-dimensional structure of Igs has provided essential information about antigen recognition. Despite the fact that there are over 300 crystal structures of human IgG Fabs currently available, there are no crystal structures of a human IgA1 fab fragment. The major difficulty in obtaining crystal structures from human IgA1 is related to the presence of several heterogeneous O-linked glycosylation sites in the hinge region of IgA1. To solve this we produced Fab fragments (FabA) by cleavage with a recombinant protease from Clostridium ramosum which specifically cuts the IgA1 molecule at the very end of the hinge region, releasing a Fab fragment without any glycan residues. With this FabA, we were able to solve two different crystal structures at high resolution (1.5 and 2.3 Å), resulting in the first crystal structure of a human IgA1 Fab fragment. (Correa, Trajtenberg F et al. 2009) (Manuscript in preparation).

Additionally to this we have also solved the Fab from a monoclonal IgG1 with identical variable region of the IgA described above. Comparison and crystallographic studies from these two structures with identical variable regions but with different CH1 domain might finally support clear evidence on the relevance of CH1 fragment in the antigenic binding site. At present, we are carrying out BiaCore analysis in order to characterize the kinetic constants for both Igs and a second manuscript is envisaged this year.

Part of this work corresponds to the degree thesis of Correa Agustin (MSc student – staff Research Assistant) which will be defended next April.

Research lines perspectives

Tasks for the next future involves the consolidation of the research lines “1” and “2.” mentioned above. We planned to carry out two doctoral theses on these subjects. We have recently applied for funding (Lady TATA Foundation, England 2010 – under revision) with the project entitled “Genomic characterization of a proliferating B-cell subset in CLL: Could AID expression be implicated in the development and progression of this disease?”.

Additionally, in collaboration with Dr. Giordano from Immunologic Department in the Academy of Medicine of Bs As, Argentina we applied for funding (PICT 2010, Argentine – under revision) with the project entitled “Origins and clinical evolution in CLL: Role of auto antigen stimulation”.

Finally, a project in the CLL field developing a recombinant antibody in order to generate a new prognostic marker entitled “Lipoprotein Lipase expression in Chronic Lymphocytic Leukemia (CLL): Towards the development of a new prognostic marker” has been presented to the “Fondo Maria Viñas 2010 and Comision de Lucha contra el Cancer” Uruguay - under revision).

Regarding point “3.” of the research lines, it will probably become a more general project including other IP Montevideo laboratories. In this line we expect the publication of the two manuscripts above mentioned and we will subsequently focus on the antibodies engineered design for the therapeutic tumoral area. We believe that we have the knowledge and the necessary equipment for the development of this area in order to provide these tools to the academic area. (see (PASCALE;, SEBASTIAN; et al.)). In this context a collaborative project with Dr. Osinaga and Dr. Pritsch laboratories entitled “Producción por ingeniería genética de diabodies e inmunotoxinas anti-antígeno tumoral Tn. Aplicación en imagenología molecular y tratamiento del cáncer” has been presented in the “Fondo Sectorial de Salud” Uruguay - under revision)

Bibliography

  • Correa, A., Trajtenberg F, et al. (2009). First crystallographic structure of a human IgA1 Fab fragment. Immunochile, Viñas del Mar-Chile, Sociedad Latinoamericana de Inmunología.
  • Dam, T. K., M. Torres, et al. (2008). "Isothermal titration calorimetry reveals differential binding thermodynamics of variable region-identical antibodies differing in constant region for a univalent ligand." J Biol Chem.
  • Oppezzo, P. and G. Dighiero (2005). "What do somatic hypermutation and class switch recombination teach us about chronic lymphocytic leukaemia pathogenesis?" Curr Top Microbiol Immunol 294: 71-89.
  • Oppezzo, P., G. Dumas, et al. (2004). "Different isoforms of BSAP regulate expression of AID in normal and chronic lymphocytic leukemia B-cells." Blood.
  • Oppezzo, P., F. Vuillier, et al. (2003). "Chronic lymphocytic leukemia B cells expressing AID display a dissociation between class switch recombination and somatic hypermutation." Blood 9: 9.
  • Palacios, F., P. Moreno, et al. (2010). "High expression of AID and active class switch recombination might accounts for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease." Blood.
  • PASCALE;, H.-H., A. SEBASTIAN;, et al. (2009). AN ANTIBODY SPECIFIC FOR THE TN ANTIGEN FOR THE TREATMENT OF CANCER I. M. C. (France), I. N. d. S. e. R. M. (France) and F. M. o. M. (Uruguay). France and Uruguay. C07K16/28Z; C07K16/30.
  • Perez-Duran, P., V. G. de Yebenes, et al. (2007). "Oncogenic events triggered by AID, the adverse effect of antibody diversification." Carcinogenesis 28(12): 2427-33.
  • Popp, C., W. Dean, et al. (2010). "Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency." Nature.
  • Pritsch, O., G. Hudry-Clergeon, et al. (1996). "Can immunoglobulin C(H)1 constant region domain modulate antigen binding affinity of antibodies?" J Clin Invest 98(10): 2235-43.
  • Tran, T. H., M. Nakata, et al. (2010). "B cell-specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers." Nat Immunol 11(2): 148-54.