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Biomedical subjects

Martin Trepel

Publications and source records attributed to Martin Trepel.

11 recordsLinked to original sources
PubMed2006

The epitope recognized by rituximab.

Rituximab is a monoclonal antibody widely used in the treatment of malignant lymphoma and autoimmunity. Its epitope within the B-cell antigen CD20 is largely unknown. We used phage display libraries to select peptides binding to rituximab. Enriched peptides showed 2 sequence patterns: one motif (CALMIANSC) is related to (170)ANPS(173) within CD20, while another motif (WEWTI) may mimic the CD20 segment (182)YCYSI(185). Phages displaying either motif specifically bound rituximab. Binding to rituximab by the CD20 peptides ANPS and YCYSI was weak when used separately and enhanced when both peptides were linked. Recombinant CD20 extracellular loop proteins blocked binding of the selected CWWEWTIGC phage to rituximab, suggesting that CWWEWTIGC mimics the epitope. Blocking capacity was strongly reduced upon mutation of the CD20 strings ANPS or YCYSI. We conclude that rituximab binds a discontinuous epitope in CD20, comprised of (170)ANPS(173) and (182)YCYSI(185), with both strings brought in steric proximity by a disulfide bridge between C(167) and C(183).

Amino Acid Sequence↗
PubMed2006

A hybrid vector for ligand-directed tumor targeting and molecular imaging.

Merging tumor targeting and molecular-genetic imaging into an integrated platform is limited by lack of strategies to enable systemic yet ligand-directed delivery and imaging of specific transgenes. Many eukaryotic viruses serve for transgene delivery but require elimination of native tropism for mammalian cells; in contrast, prokaryotic viruses can be adapted to bind to mammalian receptors but are otherwise poor vehicles. Here we introduce a system containing cis-elements from adeno-associated virus (AAV) and single-stranded bacteriophage. Our AAV/phage (AAVP) prototype targets an integrin. We show that AAVP provides superior tumor transduction over phage and that incorporation of inverted terminal repeats is associated with improved fate of the delivered transgene. Moreover, we show that the temporal dynamics and spatial heterogeneity of gene expression mediated by targeted AAVP can be monitored by positron emission tomography. This new class of targeted hybrid viral particles will enable a wide range of applications in biology and medicine.

Animals↗
PubMed2006

Isolation of targeted AAV2 vectors from novel virus display libraries.

Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.

Animals↗
PubMed2005

Targeting pancreatic islets with phage display assisted by laser pressure catapult microdissection.

Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.

Amino Acid Sequence↗
PubMed2004

Aminopeptidase A is a functional target in angiogenic blood vessels.

We show that a membrane-associated protease, aminopeptidase A (APA), is upregulated and enzymatically active in blood vessels of human tumors. To gain mechanistic insight, we evaluated angiogenesis in APA null mice. We found that, although these mice develop normally, they fail to mount the expected angiogenic response to hypoxia or growth factors. We then isolated peptide inhibitors of APA from a peptide library and show that they specifically bind to and inhibit APA, suppress migration and proliferation of endothelial cells, inhibit angiogenesis, and home to tumor blood vessels. Finally, we successfully treated tumor-bearing mice with APA binding peptides or anti-APA blocking monoclonal antibodies. These data show that APA is a regulator of blood vessel formation, and can serve as a functional vascular target.

Amino Acid Motifs↗
PubMed2004

Hematologic and molecular spontaneous remission following sepsis in acute monoblastic leukemia with translocation (9;11): a case report and review of the literature.

Spontaneous remission in patients with acute myeloid leukemia (AML) is a rarely reported phenomenon of usually short duration. The etiology remains unclear, but an association with preceding blood transfusions or bacterial infections has been reported. Triggered immune responses are suggested to play a potential role in the development of spontaneous remission. Acute monocytic leukemia was diagnosed in a 61-yr-old male patient. Cytogenetic analysis revealed a sole translocation (9;11) (q22;q23) and RT-PCR the MLL/AF9 fusion gene. As a result of the patient's reduced performance status and septic condition, cytostatic therapy was withheld. No microorganisms could be detected. Hematologic and molecular remission occurred after initiating antibiotic therapy without any cytostatic treatment; 29 months after the initial diagnosis, he is in complete remission, and excellent physical condition. Our report includes a review of the literature since 1985, reporting cases of patients with AML and spontaneous remission together with informative cytogenetics. Balanced translocations such as in core binding factor (CBF) leukemias appear somewhat overrepresented. We speculate that AML-specific T cells might be relevant for induction of spontaneous remission and need to be further investigated.

Anti-Bacterial Agents↗
PubMed2003

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.

Amino Acid Sequence↗
PubMed2003

Peptides targeting caspase inhibitors.

Here we report on the identification of peptides targeting the X-inhibitor of apoptosis protein (XIAP). XIAP functions as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. IAPs are often overexpressed in cancers and leukemias and are associated with an unfavorable clinical prognosis. We have selected peptides from a phage library by using recombinant full-length human XIAP or a fragment containing only the baculovirus IAP repeat 2 (BIR2) domain. A consensus motif, C(D/E/P)(W/F/Y)-acid/basic-XC, was recovered from two independent screenings by using different libraries. Phage-displaying variations of the consensus sequence bound specifically to the BIR2 domain of XIAP but not to other IAPs. The interaction was specific as it could be blocked by the cognate synthetic peptides in a dose-dependent manner. Phage displaying the XIAP-binding motif CEFESC bound to the BIR2 domain of XIAP with an estimated dissociation constant of 1.8 nm as determined by surface plasmon resonance. Protein-protein interaction assays revealed that caspase-3 and caspase-7 (but not caspase-8) blocked the binding of the CEFESC phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. In fact, the sequence EFES is homologous to a loop unique to the executioner caspase-3 and caspase-7 that are targeted by XIAP. Finally, we demonstrated that an internalizing version of the XIAP-binding peptide identified in our screenings (PFKQ) can induce programmed cell death in leukemia cells. Peptides interacting with XIAP could serve as prototypes for the design of low molecular weight modulators of apoptosis.

Amino Acid Motifs↗
PubMed2002

In vivo phage display and vascular heterogeneity: implications for targeted medicine.

The vascular endothelium expresses differential receptors depending on the functional state and tissue localization of its cells. A method to characterize this receptor heterogeneity with phage display random peptide libraries has been developed. Using this technology, several peptide ligands have been isolated that home to tissue-specific endothelial cell receptors following intravenous administration. Such peptide ligands, or antibodies directed against specific vascular receptors, can be used to target therapeutic compounds or imaging agents to endothelial cells in vitro and in vivo. Recent advances in the field include identification of novel endothelial receptors expressed differentially in normal and pathological conditions and the isolation of peptides or antibody ligands to such receptors in in vitro assays, in animal models and in a human patient. These milestones, which extend the 'functional map' of the vasculature, should lead to clinical applications in diseases such as cancer and other conditions that exhibit distinct vascular characteristics.

Endothelium, Vascular↗
PubMed2002

Steps toward mapping the human vasculature by phage display.

The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.

Blood Vessels↗