A Therapeutic Antibody for Cancer, Derived from Single Human B Cells.

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

Development and Characterization of Monovalent and Bivalent RNA Aptamers Targeting the Common Pathway of Coagulation

Anticoagulant agents are commonly used drugs to reduce blood coagulation in acute and chronic clinical settings. Many of these drugs target the common pathway of coagulation because it is critical for thrombin generation and disruption of this portion of the pathway has profound effects on the hemostatic process. Currently available drugs for these indications struggle with balancing desired activity with immunogenicity and poor reversibility or irreversibility in the event of hemorrhage. While improvements are being made with the current drugs, new drugs with better therapeutic indices are needed for surgical intervention and chronic indications to prevent thrombosis from occurring.

A class of therapeutics known as aptamers may be able to meet the need for safer anticoagulant agents. Aptamer are short single-stranded RNA oligonucleotides that adopt specific secondary and tertiary structures based upon their sequence. They can be generated to both enzymes and cofactors because they derive their inhibitory activity by blocking protein-protein interactions, rather than active site inhibition. They inhibit their target proteins with a high level of specificity and bind with high affinity to their target. Additionally, they can be reversed using two different antidote approaches, specific oligonucleotide antidotes, or with cationic, “universal” antidotes. The reversal of their activity is both rapid and durable.

The ability of aptamers to be generated to cofactors has been conclusively proven by generating an aptamer targeting the common pathway coagulation cofactor, Factor V (FV). We developed two aptamers with anticoagulant ability that bind to both FV and FVa, the active cofactor. Both aptamers were truncated to smaller functional sizes and had specific point mutant aptamers developed for use as controls. The anticoagulant activity of both aptamer-mutant pairs was characterized using plasma-based clotting assays and whole blood assays. The mechanism of action resulting in anticoagulant activity was assessed for one aptamer. The aptamer was found to block FVa docking to membrane surfaces, a mechanism not previously observed in any of our other anticoagulant aptamers.

To explore development of aptamers as anticoagulant agents targeting the common pathway for surgical interventions, we fused two anticoagulant aptamers targeting Factor X and prothrombin into a single molecule. The bivalent aptamer was truncated to a minimal size while maintaining robust anticoagulant activity. Characterization of the bivalent aptamer in plasma-based clotting assays indicated we had generated a very robust anticoagulant therapeutic. Furthermore, we were able to simultaneously reverse the activity of both aptamers with a single oligonucleotide antidote. This rapid and complete reversal of anticoagulant activity is not available in the antithrombotic agents currently used in surgery.

Characterization of a Full-Length TTP Family Member Association with RNA Sequence Elements

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.