MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

Abstract

BACKGROUND: Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). METHODS: We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. RESULTS: Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). CONCLUSIONS: Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.

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Citation

Published Version (Please cite this version)

10.1186/1471-2407-10-468

Publication Info

Kuan, Chien-Tsun, Kenji Wakiya, James E Herndon, Eric S Lipp, Charles N Pegram, Gregory J Riggins, Ahmed Rasheed, Scott E Szafranski, et al. (2010). MRP3: a molecular target for human glioblastoma multiforme immunotherapy. BMC Cancer, 10. p. 468. 10.1186/1471-2407-10-468 Retrieved from https://hdl.handle.net/10161/4357.

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Scholars@Duke

Chien-Tsun Kuan

Adjunct Associate Professor in the Department of Pathology

Research Interests:
Conventional therapy for malignant brain tumors is ineffective. Targeted therapy using tumor-specific antibodies (MAb) alone or MAbs armed with radionuclides or toxins is a promising alternative approach for increasing therapeutic efficacy and decreasing toxicity to normal tissue. The major factors that influence antibody-targeted therapy for cancer treatment, including glioma therapy, are specificity, affinity, tumor penetration, toxicity and immunogenicity. The effective use of radioimmunotherapy (RAIT) for the treatment of solid malignancies has been limited by inadequate tumor penetration and non-targeted myelotoxicity resulting from the presence of radioimmunoconjugates in circulation. We believe that these limitations to direct RAIT can be overcome by using smaller engineered antibody-based molecules as vehicles and by selecting therapeutic radioisotopes with physical properties that complement the pharmacokinetics and pharmacodynamics of the antibody.

Our research is focused upon exploiting engineered antibody fragments to treat brain tumors by targeting to glioma-associated, oncofetal epitopes such as tenascin, glioma variant epidermal growth factor variant III (EGFRvIII), medulloblastoma-associated developmental markers, as well as the newly identified glioma-associated antigens, GPNMB and MRP3, by serial analysis of gene expression (SAGE). Projects performed in the current years have: 1) produced and evaluated the monovalent single-chain Fv (scFv) against EGRvIII in athymic mice bearing human glioma xenografts; 2) begun the development of divalent form of scFv, including diabody and minibody, to increase the efficacy of therapeutic agents in vivo; 3) generated CH2 domain-deleted Ch81C6 vs tenascin and evaluated the pharmacokinetics in mice and canines; 4) begun an extensive analysis of GPNMB and MRP3 protein expression correlated with measurement of RNA transcript levels and degree of DNA amplification.

Unarmed antibody can be effective against both subcutaneous and intracranial tumor models. The unarmed antibody approach with Mab Y10 vs EGFRvIII is very similar to the successful use of HerceptinTM. The mechanism is most likely both a direct antiproliferative effect with the induction of apoptosis and an indirect effect through the mobilization of antibody-mediated immune effector functions, such as complement and antibody-dependent cell-mediated cytotoxicity (ADCC). We also have begun to construct human/mouse chimeric Y10 to reduce immunogenicity of the Mab reagent and possibly enhance ADCC.

Our objectives for the coming years are to continue the optimization of engineered-antibody systems for in vivo application, namely; a) development of human/mouse chimeric anti-EGFRvIII murine Y10 with the same affinity and specificity but reduced immunogenicity and enhanced ADCC for in vivo application; b) to generate a totally human scFv specific to EGFRvIII but with anti-proliferative activity via screening from human phage libraries; c) generation of monomeric and dimeric anti-GPNMB/MRP3 scFvs and construction of immunoconjugate toxins or radiolabeled to determine the efficacy of therapeutic reagents in athymic rodent in athymic rodent in vivo models of intracranial glioma.

Herndon

James Emmett Herndon

Professor of Biostatistics & Bioinformatics

Current research interests have application to the design and analysis of cancer clinical trials. Specifically, interests include the use of time-dependent covariables within survival models, the design of phase II cancer clinical trials which minimize some of the logistical problems associated with their conduct, and the analysis of longitudinal studies with informative censoring (in particular, quality of life studies of patients with advanced cancer).

B. K. Ahmed Rasheed

Assistant Professor in Pathology

Our lab is interested in identifying the specific genetic alterations associated with the genesis and progression of glial malignancies. Studies from our and other laboratories have shown that in adult glioblastomas, approximately 80% of the cases show loss of alleles on chromosome 10, and to a lesser extent on 9p, 17p, 19q and 22q. Amplification of epidermal growth factor receptor gene is detected in about a third of glioblastomas. The high incidence of loss of chromosome 10 alleles suggests the presence of a tumor suppressor gene on this chromosome important in glial tumorigenesis. In an attempt to identify the putative tumor suppressor gene on chromosome 10, we have carried out a detailed deletion mapping of a series of gliomas using RFLP and microsatellite markers. The allelic loss pattern is indicative of a smallest common deletion region on chromosome 10, located between loci D10S587 and D10S216. We are isolating transcribed sequences from this region of chromosome 10 and screening them for presence of somatic mutations in the brain tumors.


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