Search

Although metastatic rhabdomyosarcoma (RMS) is chemotherapy and radiotherapy responsive, few patientsare cured. 8H9, a murine IgG1 monoclonal antibody, recognizes a unique cell surface tumor antigenbroadly distributed on neuroectodermal, epithelial, and mesenchymal tumors, including RMS. We nowreport on the in vitro characterization of radiolabeled 8H9 and its in vivo immunotargeting potential inmice with subcutaneous human RMS. Saturation-binding studies carried out to determine 125I-8H9 affinityto the RMS cell line HTB82 demonstrated that 125I-8H9 had a Kd of 10.3nM with an estimated 115,000binding sites on every HTB82 cell. 125I-8H9 was retained on the cell surface without significant internalization.Biodistribution of 125I-8H9 was studied in athymic mice bearing HTB82 xenografts. Followingintravenous injection of 4.44MBq of 125I-8H9, selective tumor uptake was evident 4 to 172 hours afterinjection. Average tumor uptake was 7.0 ± 1.8, 11.5 ± 3.9, 15.1 ± 3.7, and 5.4 ± 1.2% injected doseper gram at 4, 24, 48, and 172 hours, respectively. Mean tumor: tissue ratios were maximal at 172 hours(for lung, 4, kidney, 6, liver, 7, spleen, 11, femur, 14, muscle, 18, and brain, 48). Established RMSxenografts treated with a single injection of 18.5 MBq 131I-8H9 were significantly suppressed comparedto controls. Radiolabeled 8H9 effectively targeted RMS xenografts and may have a potential clinical rolein radioimmunotherapy. [ABSTRACT FROM AUTHOR]

Chimeric immune receptors (CIR) transduced into lymphocytes link target recognition by single chain antibody Fv (scFv) to activation through CD28/TCRζ signaling. As surrogate antigens, anti-idiotypic antibodies may facilitate gene-transduction and clonal expansion of human lymphocytes for in vivo tumor therapy. The murine monoclonal antibody (MAb) 8H9 reacts with a novel antigen widely expressed on solid tumors. A CIR consisting of human CD8-leader sequence, 8H9-scFv, CD28 (transmembrane and cytoplasmic domains), and TCR-ζ chain was constructed, ligated into the pMSCVneo vector, and used to transfect the packaging line GP + envAM12 bearing an amphotropic envelope. Rat anti-idiotypic MAb 2E9 (IgG2a) was used to clone retroviral producer line as well as to expand gene-modified primary human lymphocytes. Sequential enrichments using either affinity chromatography or cell sorting using anti-idiotypic MAb 2E9 significantly improved the percentage of producer clones positive for surface 8H9-scFv and the efficiency of their supernatant in transducing the indicator cell line K562. By 3 weeks of in vitro culture, >95% of transduced primary human lymphocytes were CIR-positive. Upon periodic stimulation with 2E9, these lymphocytes underwent >106-fold expansion by 6 months in culture. They mediated antigen-specific non-MHC restricted cytokine release and tumor cytotoxicity, and inhibited human xenograft engraftment in SCID mice. Anti-idiotypic antibody may provide a useful tool for optimizing gene transduction of CIR fusion constructs into primary human lymphocytes and their continual expansion in vitro. [ABSTRACT FROM AUTHOR]

B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation, and epitope mapping of 8H9 based on structure determination, modeling, and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5-Å resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (scFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescence-activated cell sorting. Six mutations (three in the complementarity-determining region and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nM KD) compared with parental hu8H9 scFv (144 nM KD). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to subnanomolar KD) had 2-9-fold enhancements in affinity compared with their parental forms, potent in vitro antibody-dependent cell-mediated cytotoxicity (0.1-0.3 µg/ml EC50), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the molecular epitope of 8H9 was determined to be dependent on the FG loop of B7-H3, a region critical to its function in immunologic blockade and unique among anti-B7-H3 antibodies published to date. [ABSTRACT FROM AUTHOR]

Cellular heterogeneity is a hallmark of human neuroblastoma tumors and cell lines. Within a single neuroblastoma are cells from distinct neural crest lineages whose relative abundance is significant for prognosis. We postulate that a self-renewing multipotent tumor stem cell, which gives rise to diverse cell lineages, is the malignant progenitor of this cancer. To test this hypothesis, we have established 22 cloned, phenotypically homogeneous populations of the three major cell types from 17 neuroblastoma cell lines. In vitro, malignant neuroblastoma stem cells, termed I-type (intermediate type), have distinct morphologic, biochemical, differentiative, and tumorigenic properties. I-type cells express features of both neuroblastic (N) cells (scant cytoplasm, neuritic processes, neurofilaments, pseudoganglia, and granin and neurotransmitter enzyme expression) and substrate-adherent (S) cells (extensive cytoplasm and vimentin and CD44 expression). Moreover, they show bidirectional differentiation to either N or S cells when induced by specific agents. I-type cells are significantly more malignant than N- or S-type cells, with four- to five-fold greater plating efficiencies in soft agar and six-fold higher tumorigenicity in athymic mice. Differences in malignant potential are unrelated to N-myc amplification/ overexpression or the ability to digest and migrate through the extracellular matrix. Immunocytochemical analyses of a small series of tumors reveal that frequency of cells coexpressing N and S cell markers correlates with poor prognosis. Thus, I-type stem cells may be instrumental in the genesis and growth of tumors in the patient. Their unique biology deserves attention and further investigation. [ABSTRACT FROM AUTHOR]