Radioimmunotherapy with alpha-particle emitting 213Bi-C-functionalized trans-cyclohexyl-diethylenetriaminepentaacetic acid-humanized 3S193 is enhanced by combination with paclitaxel chemotherapy.

PURPOSE: Previous experience in solid tumor radioimmunotherapy studies has indicated that greatest therapeutic efficacy is achieved in the treatment of small-volume disease. alpha-Particle-emitting radioisotopes possess several physical characteristics ideally suited to the treatment of minimal residual disease. Therefore, we have investigated the efficacy of the alpha-particle-emitting bismuth-213 (213Bi) radioimmunotherapy using the humanized anti-Lewis Y (Ley) monoclonal antibody humanized 3S193 (hu3S193). EXPERIMENTAL DESIGN: The intracellular localization of hu3S193 in Ley-positive MCF-7 breast carcinoma cells was assessed by confocal microscopy. Cytotoxicity of 213Bi-hu3S193 and apoptosis was assessed using [3H]thymidine incorporation assay and ELISA, respectively. Immunoblotting for gamma-H2AX assessed DNA strand breaks. In vivo efficacy of 213Bi-hu3S193 was assessed using a minimal residual disease model in BALB/c nude mice, with radioconjugate [15, 30, and 60 microCi (9.2 microg)] injected 2 days after s.c. implantation of MCF-7 cells. Radioimmunotherapy was also combined with a single injection of 300 microg paclitaxel to explore improved efficacy. Further, mice with established tumors received 30, 60, or 120 microCi (14.5 microg) of 213Bi-hu3S193 to assess the effect of tumor volume on treatment efficacy. RESULTS: hu3S193 is internalized via an endosomal and lysosomal trafficking pathway. Treatment with 213Bi-hu3S193 results in >90% cytotoxicity in vitro and induces apoptosis and increased gamma-H2AX expression. 213Bi-hu3S193 causes specific and significant retardation of tumor growth even in established tumors, and efficacy was enhanced by paclitaxel to produce defined complete responses. CONCLUSIONS: These studies show the potency of alpha-particle radioimmunotherapy and warrant its further exploration in the treatment of micrometastatic disease in Ley-positive malignancies.

Soluble Robo4 receptor inhibits in vivo angiogenesis and endothelial cell migration.

Roundabout receptors are molecular guidance molecules that function by interaction with Slit proteins to regulate axon guidance, neuronal migration, and leukocyte chemotaxis. We recently isolated a novel roundabout gene, called Robo4, which is restricted in expression to the endothelium, notably in areas of angiogenesis. The aim of this study was to use the soluble extracellular domain of Robo4 as a probe of function in angiogenesis and endothelial biology. Thus, the soluble extracellular domain of the receptor (Robo4Fc) showed diverse in vivo and in vitro activities including 1) inhibition of angiogenesis in vivo in the rodent subcutaneous sponge model, 2) inhibition of tube formation in the rat aortic ring assay, 3) inhibition of VEGF- and bFGF-stimulated endothelial cell migration, and 4) inhibition of endothelial proliferation. To assess whether Robo4Fc was inhibiting Slit-mediated effects, we determined whether Robo4 and Slit interact. Recombinant Slits-1, -2, and -3 were shown by immunoprecipitation and BiaCore analysis to bind to Robo1 but not Robo4. Further study of the role of Robo4 in angiogenesis appears justified.

Expression and targeting of human fibroblast activation protein in a human skin/severe combined immunodeficient mouse breast cancer xenograft model.

Antigens and receptors that are highly expressed on tumor stromal cells, such as fibroblast activation protein (FAP), are attractive targets for antibody-based therapies because the supporting stroma and vessel network is essential for a solid neoplasm to grow beyond a size of 1-2 mm. The in vivo characterization of antibodies targeting human stromal or vessel antigens is hindered by the lack of an appropriate mouse model system because xenografts in standard mouse models express stromal and vessels elements of murine origin. This limitation may be overcome by the development of a human skin/mouse chimeric model, which is established by transplanting human foreskin on to the lateral flank of severe combined immunodeficient mice. The subsequent inoculation of breast carcinoma MCF-7 cells within the dermis of the transplanted human skin resulted in the production of xenografts expressing stromal and vessel elements of human origin. Widespread expression of human FAP-positive reactive stromal fibroblasts within xenografts was seen up to 2 months posttransplantation and postinjection of cells. Human blood vessel antigen expression also persisted at 2 months posttransplantation and postinjection of cells with murine vessels coexisting with the human vascular supply. The model was subsequently used to evaluate the biodistribution properties of an iodine-131-labeled humanized anti-FAP monoclonal antibody (BIBH-7). The results showed high specific targeting of the stromal compartment of the xenograft, indicating that the model provides a useful and novel approach for the in vivo assessment of the immunotherapeutic potential of molecules targeting human stroma and angiogenic systems.

Tumor targeting by a multivalent single-chain Fv (scFv) anti-Lewis Y antibody construct.

The use of single-chain variable fragment (scFv) constructs has been investigated in cancer radioimmunotherapy (RIT) and radioimmunodetection, as these molecules permit rapid tumor penetration and clearance from the serum relative to whole IgG. Multimerization of scFv constructs has demonstrated improvements in functional affinity (i.e., avidity) and maximal tumor uptake. In this paper, we report the first biodistribution and pharmacokinetics studies of a noncovalent, direct-linked scFv (V(L)-0-V(H)) trimeric/tetrameric "multimer" of the anti-Lewis Y monoclonal antibody, hu3S193. The in vitro binding and in vivo biodistribution of the hu3S193 multimer was characterized alongside the hu3S193 F(ab')(2) following radiolabeling with the Indium-111 ((111)In) radioisotope. Immunoreactivities of the radiolabeled multimer and F(ab')(2) were 73% and 53.2%, and binding affinities (K(a)) were 1.58 x 10(7) M(1) and 4.31 x 10(6) M (1) for the multimer and F(ab')(2), respectively. Maximal tumor uptake in Le(y)-positive MCF-7 breast cancer xenografted BALB/c nude mice was 12.6 +/- 2.5 percent injected dose/per gram (%ID/g) at 6 hours postinjection for the multimer and 15.7 +/- 2.1 %ID/g at 24 hours postinjection for the F(ab')(2). However, limited in vitro stability and high renal localization of radiolabeled constructs were observed, which, despite the observed tumor targeting of the hu3S193 multimer, most likely preclude its use in RIT and imaging modalities.