Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)

<p>Abstract</p> <p>Purpose</p> <p>In a departure from conventional strategies to improve treatment outcome for myeloid malignancies, we report the isolation of leukemia-specific peptides using a phage display library screened with freshly obtained human myeloid leukemia cells.</p> <p>Results</p> <p>A phage display library was screened by 5 rounds of biopanning with freshly isolated human AML cells. Individual colonies were randomly picked and after purification, biologic activity (growth and differentiation) on fresh AML cells was profiled. Ten peptides were synthesized for further biological studies. Multiple peptides were found to selectively bind to acute myeloid leukemia (AML) cells. The peptides bound to leukemia cells, were internalized and could induce proliferation and/or differentiation in the target patient cells. Two of the peptides, HP-A2 and HP-G7, appeared to have a novel mechanism of inducing differentiation since they did not cause G1 arrest in cycling cells even as the expression of the differentiation marker CD11b increased.</p> <p>Conclusion</p> <p>Peptide induced differentiation of leukemia cells offers a novel treatment strategy for myeloid malignancies, whereas their ability to induce proliferation could be harnessed to make cells more sensitive to chemotherapy. Conceptually, these leukemia specific peptides can also be used to refine diagnosis, document minimal residual disease, and selectively deliver toxins to malignant cells.</p

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme β-glucuronidase. The sequences encoding C28 and human enzyme β-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGκ signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-β-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme β-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug

Secreted and tumour targeted human carboxylesterase for activation of irinotecan

Irinotecan (CPT-11) is an anticancer agent for the treatment of colon cancer. CPT-11 can be considered as a prodrug, since it needs to be activated into the toxic drug SN-38 by the enzyme carboxylesterase. An approach to achieve tumour specific activation of CPT-11 is to transduce the cDNA encoding carboxylesterase into tumour cells. A secreted form of carboxylesterase may diffuse through a tumour mass and may activate CPT-11 extracellularly. This could enhance the antitumour efficacy by exerting a bystander effect on untransduced cells. In addition a secreted tumour-targeted form of carboxylesterase should prevent leakage of the enzyme from the site of the tumour into the circulation. We have constructed a secreted form of human liver carboxylesterase-2 by deletion of the cellular retention signal and by cloning the cDNA downstream of an Ig kappa leader sequence. The protein was secreted by transfected cells and showed both enzyme activity and efficient CPT-11 activation. To obtain a secreted, tumour-targeted form of carboxylesterase-2 the cDNA encoding the human scFv antibody C28 directed against the epithelial cell adhesion molecule EpCAM, was inserted between the leader sequence and carboxylesterase-2. This fusion protein showed CPT-11 activation and specific binding to EpCAM expressing cells. Importantly, in combination with CPT-11 both recombinant carboxylesterase proteins exerted strong antiproliferative effects on human colon cancer cells. They are, therefore, promising new tools for gene directed enzyme prodrug therapy approaches for the treatment of colon carcinoma with CPT-11

Liver cell proliferation after partial hepatectomy in rats with liver metastases

OBJECTIVE: To validate proliferating cell nuclear antigen (PCNA) expression and flow cytometry as proliferation markers in regenerating rat liver containing metastases. STUDY DESIGN: Rats containing colorectal liver metastases were killed at various days after 70% partial hepatectomy or a sham operation. [H-3]thymidine and 5-bromo-2 'deoxyuridine (BrdU) incorporation, PCNA expression and flow cytometry were used to evaluate liver cell proliferation. RESULTS: The assessment of proliferating liver cells by PCNA expression and BrdU incorporation was more reliable than autoradiography. PCNA expression correlated well with BrdU incorporation (r=.68, P=.003) and autoradiography (r=.57, P=.02) in regenerating liver. BrdU incorporation and PCNA expression were higher in hepatectomized rats as compared to sham-operation rats at days 1-4 after hepatectomy. Flow cytometry of propidium-stained nuclei from livers of hepatectomized rats showed a higher proportion of S-phase nuclei as compared to S-phase nuclei in control rats. The correlation coefficients of the number of S-phrase nuclei, BrdU-positive nuclei and PCNA-positive nuclei were .39 (P CONCLUSION: Flow cytometry and PCNA expression are simple and reliable methods of studying proliferation in metastases containing rat liver after partial hepatectomy

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in: in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen