David A. Scheinberg

A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia

Long-term survival still eludes most patients with leukemia and non-Hodgkin’s lymphoma. No approved therapies target the hallmark of the B cell, its mIgM, also known as the B-cell receptor (BCR). Aptamers are small oligonucleotides that can specifically bind to a wide range of target molecules and offer some advantages over antibodies as therapeutic agents. Here, we report the rational engineering of aptamer TD05 into multimeric forms reactive with the BCR that may be useful in biomedical applications. Systematic truncation of TD05 coupled with modification with locked nucleic acids (LNA) increased conformational stability and nuclease resistance. Trimeric and tetrameric versions with optimized polyethyleneglycol (PEG) linker lengths exhibited high avidity at physiological temperatures both in vitro and in vivo. Competition and protease studies showed that the multimeric, optimized aptamer bound to membrane-associated human mIgM, but not with soluble IgM in plasma, allowing the possibility of targeting leukemias and lymphomas in vivo. The B-cell specificity of the multivalent aptamer was confirmed on lymphoma cell lines and fresh clinical leukemia samples. The chemically engineered aptamers, with significantly improved kinetic and biochemical features, unique specificity and desirable pharmacological properties, may be useful in biomedical applications.

Structure and activity of human mitochondrial peptide deformylase, a novel cancer target.

Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 A), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 A) identified the substrate-binding site. A defined S1 pocket, but no S2 or S3 substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2 and S3 binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2and P3 positions of a formylated peptide substrate to turnover.

A TCR-mimic antibody to WT1 bypasses tyrosine kinase inhibitor resistance in human BCR-ABL+ leukemias

Acute and chronic leukemias, including CD34(+) CML cells, demonstrate increased expression of the Wilms tumor gene 1 product (WT1), making WT1 an attractive therapeutic target. However, WT1 is a currently undruggable, intracellular protein. ESKM is a human IgG1 T-cell receptor mimic monoclonal antibody directed to a 9-amino acid sequence of WT1 in the context of cell surface HLA-A*02. ESKM was therapeutically effective, alone and in combination with tyrosine kinase inhibitors (TKIs), against Philadelphia chromosome-positive acute leukemia in murine models, including a leukemia with the most common, pan-TKI, gatekeeper resistance mutation, T315I. ESKM was superior to the first-generation TKI, imatinib. Combination therapy with ESKM and TKIs was superior to either drug alone, capable of curing mice. ESKM showed no toxicity to human HLA-A*02:01(+) stem cells under the conditions of this murine model. These features of ESKM make it a promising nontoxic therapeutic agent for sensitive and resistant Ph(+) leukemias.

Current applications of monoclonal antibodies for the therapy of hematopoietic cancers.

Recent trials of monoclonal antibodies in patients with leukemias or lymphomas have demonstrated the remarkable potency of these agents to kill tumor cells specifically and safely. New molecular biological and radiochemical techniques are also allowing rapid inroads into the remaining obstacles to this mode of therapy.

Therapeutic antibodies to intracellular targets in cancer therapy

Therapeutic monoclonal antibodies (mAbs) are a proven therapeutic platform, but they cannot readily cross the cell membranes to bind intracellular antigens, while some of the most important disease-associated proteins are intracellular, protected from direct mAb attack. However, the cellular processes of necrosis and major histocompatibility complex (MHC) class I antigen presentation expose epitopes from intracellular proteins to the extracellular environment or cell surface. Antibodies that exploit these processes can therefore specifically target diseased cells based on their intracellular protein content. These strategies expose important new targets for mAb therapy and expand the potential for effective therapies.

A Self-Assembling Short Oligonucleotide Duplex Suitable for Pretargeting

Monoclonal antibodies (mAbs) have naturally evolved as suitable, high affinity and specificity targeting molecules. However, the large size of full-length mAbs yields poor pharmacokinetic properties. A solution to this issue is the use of a multistep administration approach, in which the slower clearing mAb is administered first and allowed to reach the target site selectively, followed by administration of a rapidly clearing small molecule carrier of the cytotoxic or imaging ligand, which bears a cognate receptor for the mAb. Here, we introduce a novel pretargetable RNA based system comprised of locked nucleic acids (LNA) and 2O-Methyloligoribonucleotides (2OMe-RNA). The duplex shows fast hybridization, high melting temperatures, excellent affinity, and high nuclease stability in plasma. Using a prototype model system with rituximab conjugated to 2OMe-RNA (oligo), we demonstrate that LNA-based complementary strand (c-oligo) effectively hybridizes with rituximab-oligo, which is slowly circulating in vivo, despite the high clearance rates of c-oligo.

Immune reconstitution and remission in CML

In a comprehensive and provocative report in this issue of Blood, with findings that were not necessarily expected, Hughes and colleagues report on the immune reconstitution that occurs as a consequence of the deepening remission achieved by patients with chronic myelogenous leukemia (CML) on tyrosine kinase inhibitors (TKIs). A significant fraction of patients with CML treated with BCR-ABL TKI achieve long-term remissions and maintain this state even after stopping the TKI. Why is this? Although the TKI itself may be contributing to this immune restoration, it is not entirely responsible as best effects are observed in those patients no longer on these drugs.1