ALBERT

Description: Abstract 2711 Poster Board II-687 Non-Hodgkins lymphomas affect 450,000 patients in the United States and even with recent advances in antibody-based therapies; more than 20,000 people will die of their disease annually. The goal of this work was to develop a high affinity, stable aptamer selective for B-cell leukemias and lymphomas. Aptamers are small DNA molecules that have the ability to bind to proteins with high affinity and specificity. They are also ideal candidates as therapeutic carriers. Aptamer binding is based on the ability of small oligonucleotide polymers (typically 20–80mers) to fold into unique three-dimensional structures that can interact with a specific target. Based on nature of this interaction, aptamers could be considered to be antibody analogs. Compared to antibodies however, one of the inherent properties of aptamers is that their small size (typically 10–20,000 daltons for aptamers vs 150,000 daltons for antibodies,) might address some difficult pharmacologic issues of antibodies, which penetrate slowly into tumors and clear the blood slowly. Recently, the TDO5 aptamer was identified and it was found to bind to membrane bound human IgM, a component of the BCR complex in B-Cell neoplasms. In contrast to currently available monoclonal antibodies, TD05 binds to membrane bound human IgM only and not with soluble IgM, eliminating the possibility of competitive inhibition by soluble IgM in the serum. The specificity of the aptamer was confirmed by screening with 24 cell lines and fresh clinical leukemia samples. Out of 24 cell lines, the IgM-negative cells, including T-cell leukemias and solid tumor lines such as breast, kidney, and colon and ovarian, showed no binding with TD05 indicating there is no non-specific adhesion with cell lines. One of the challenges of using the current form of TD05 as a drug carrier is that it is not yet suitable for use in vivo because of low avidity (〉10uM) and stability (t ½= 1min) at physiological conditions. In order to increase the affinity of this aptamer, a new truncated multivalent and nuclease stable aptamer was designed. First, truncation of the original version of TD05 was considered because reduced size may lead to more efficient chemical syntheses and better pharmacologic properties. The resulting TD05.1 has a 5-fold increased affinity compared to original version of TD05. Second, bivalent (BV) aptamers utilizing TD05.1 with various polyethylene glycol (PEG) linker lengths were designed. Linker length is critical in designing multivalent aptamers to avoid loss of binding due to steric hindrance and to optimize the binding geometry, both of which would affect binding affinity. An optimal linker length of ∼16nm was chosen after empiric binding studies. Nuclease stability was also addressed by the introduction of chemical functionalities into TD05.1. The introduction of non-natural bases such as LNA bases (Locked Nucleic Acids), have been demonstrated to be effective in this regard. The stability of LNA stems from the bicyclic furanose unit locked in a sugar conformation. In order to retain the specific recognition and 3-dimensional nature of the aptamer's folding, LNA analogues were only introduced at the regions that are not involved in binding. Additionally, to further improve nuclease resistance, increase circulation time in vivo and to prevent non-specific adhesion to serum proteins and cells, the LNA modified BV aptamer TD05.1 was modified with polyethylene glycol at the 3' and 5' ends. The introduction of LNA and polyethylene glycol further stabilized the secondary structure, increasing the affinity and nuclease resistance in serum from one minute to seven hours(t ½〉 420 min). We then constructed a trivalent analog of the aptamer with multiple functionalities, including chelators and fluorophores, which showed ∼25-fold higher affinity compared to monomeric aptamer at physiological conditions. In conclusion, this study demonstrates the development of a trivalent, high affinity aptamer selective for the membrane bound human IgM found in B-cell leukemias and lymphoma. Studies to assess the biological activity and the use of this construct as a drug carrier to treat B-NHL and B-CLL are in progress. Disclosures: No relevant conflicts of interest to declare.