Herceptin-directed nanoparticles activated by an alternating magnetic field selectively kill HER-2 positive human breast cells in vitro via hyperthermia.

PURPOSE: HER-2 is in the EGF tyrosine kinase receptor family, overexpressed by many human cancers and minimally expressed by normal adult tissues. HER-2 expression in human cancers is correlated with reduced survival, increased metastasis, reduced apoptosis and increased proliferation. Herceptin is a humanised mouse antibody that targets and inactivates HER-2. In the present study, Herceptin was used to deliver ferric oxide-enriched nanoparticles to HER-2(+) cancer cells. If exposed to alternating magnetic field (AMF), the nanoparticles heat. We tested the ability of AMF-activated Herceptin-directed nanoparticles to selectively kill HER-2(+) human cancer cells. METHODS: Herceptin-conjugated nanoparticles were incubated with normal human mammary epithelial cells (HMEC)(HER-2(-)) or malignant human mammary epithelial cells (SK-BR-3)(HER-2(+)). Cells were stained to detect Herceptin or iron and the kinetics of binding quantified. Once conditions were optimised for binding, cells were exposed to either antibody-directed or non-antibody-conjugated nanoparticles, washed and sham-treated or exposed to AMF and cell death quantified. RESULTS: SK-BR-3 cells bound Herceptin-directed nanoparticles in increasing amounts over 3 h but did not retain non-antibody conjugated nanoparticles. HMECs did not retain either nanoparticles. SK-BR-3 cells with bound Herceptin-directed-nanoparticles, exposed to AMF, died by apoptosis, quantifiable by Live/Dead and nuclear morphology assays and released LDH. Sham-treated SK-BR-3 cells with Herceptin-directed nanoparticles, HMECs with either nanoparticles, with or without AMF treatment, exhibited no increase in toxicity above baseline cell death using these three assays. CONCLUSIONS: These studies demonstrate Herceptin-directed nanoparticles can selectively kill HER-2(+) cancer cells via hyperthermia after AMF activation.

Rituximab-mediated cell signaling and chemo/immuno-sensitization of drug-resistant B-NHL is independent of its Fc functions.

PURPOSE: Rituximab [chimeric anti-CD20 monoclonal antibody], alone or combined with chemotherapy, is used in the treatment of non-Hodgkin's lymphoma (NHL). Rituximab binds to CD20 and inhibits intracellular survival/growth pathways leading to chemo/immunosensitization of tumor cells in vitro. The contribution of rituximab Fc-FcR interaction in signaling is not known. This study examined the role of Fc-FcR interactions in rituximab-induced signaling using rituximab (Fab')(2) fragments as well as rituximab devoid of the CH2 Fc-binding domain (CH2(-)). EXPERIMENTAL DESIGN: Rituximab (CH2(-)) and rituximab (Fab')(2) were tested for their activity on B-NHL cell lines. Cell signaling and sensitization to chemotherapy and immunotherapy were examined. The in vitro studies were validated in mice bearing tumor xenografts. RESULTS: Although the modified antibodies were defective in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity functions, they retained all other biological activities such as inhibition of cell proliferation, induction of cell aggregation, and apoptosis induction. In addition, similar to rituximab, the modified antibodies inhibited the activity of cell survival/growth pathways and their associated transcription factors (e.g., NF-kappaB, YY1, SP-1), and signal transducers and activators of transcription 3 (STAT-3), and downregulated the expression of antiapoptotic gene products, such as Bcl-2/Bcl(xl), which regulate drug resistance. The modified antibodies, similar to rituximab, sensitized resistant B-NHL cells to both CDDP and Fas ligand-induced apoptosis. Furthermore, treatment of nude mice bearing Raji tumor cell xenografts with the combination of rituximab (Fab')(2) or rituximab and CDDP resulted in similar and significant inhibition of tumor growth. CONCLUSION: These findings reveal that rituximab-mediated inhibition of intracellular signaling pathways and leading to chemo/immuno-sensitization of resistant B-NHL is Fc independent.

Pharmacokinetics and tumor localization of (111)in-labeled HuCC49DeltaC(H)2 in BALB/c mice and athymic murine colon carcinoma xenograft.

The primary limitation of IgG antibodies for radioimmunotherapy of solid tumors is their prolonged serum half-life, leading to dose-limiting bone marrow toxicity at doses providing inadequate radiation to the tumor. A humanized C(H)2 domain-deleted variant of the anti-TAG-72 antibody CC49 (HuCC49DeltaC(H)2) has faster blood clearance, compared to the IgG, while retaining tumor targeting. We compared the pharmacokinetics and tumor uptake of (111)In-HuCC49DeltaC(H)2 in BALB/c mice and a colon carcinoma (LS-174T) mouse xenograft with that of (111)In-labeled chimeric CC49 (cCC49), an antibody with pharmacokinetics similar to the humanized CC49 parent. Immuno-conjugates of HuCC49DeltaC(H)2 and cCC49 prepared with the (111)In chelator Mx-DTPA (1-isothiocyantobenzyl-3-methyldiethylenetriaminepentaacetic acid) retained low nM affinity and radiolabeling protocols provided greater than 95% radio-incorporation with (111)In while retaining greater than 80% immunoreactivity. Blood clearance of (111)In-HuCC49DeltaC(H)2 in BALB/c mice was monoexponential (t(1/2) 5.4 hours) and faster than (111)In-cCC49 (biexponential clearance; t1/2Delta 1.5 hours; t1/2beta 162 hours). The (111)In-HuCC49DeltaC(H)2 also cleared more rapidly from the blood in the murine xenograft. At 1 hour postinjection, blood concentrations for (111)In-HuCC49DeltaC(H)2 and (111)In-cCC49 were comparable (25.5 injected dose per g [%ID/g] and 21.3 %ID/g, respectively); tumor uptake for (111)In- HuCC49DeltaC(H)2 was 7.9 %ID/g, compared to 7.5 %ID/g for (111)In-cCC49. However, at 24 hours, blood concentration for (111)In-HuCC49DeltaC(H)2 was less than (111)In-cCC49 (0.9 %ID/g versus 5.2 %ID/g, respectively) with comparable tumor retention (14.4 %ID/g versus 19.0 %ID/g, respectively). Faster blood clearance of (111)In-HuCC49DeltaC(H)2 and tumor localization comparable to that of (111)In-cCC49 provided a fourfold improved tumor-to-blood ratio for (111)In-HuCC49DeltaC(H)2 at 24 hours postinjection.

Antibody therapy of non-Hodgkin's B-cell lymphoma.

Engineering antibodies with reduced immunogenicity and enhanced effector functions, and selecting antigen targets with the appropriate specificity, density, and/or functionality, have contributed to the recent clinical successes in using unconjugated "naked" antibody therapies of B-cell lymphoma (rituximab) and breast carcinoma (Herceptin). The non-overlapping toxicities of naked antibodies and chemotherapy, together with their potential synergy, which is based on unique and complementary mechanisms of action, have contributed to the creation of new standards of care in cancer therapy and management. Clinical trial results supporting these concepts are presented. Furthermore, the exquisite specificity of antibodies renders them ideal vehicles for selective delivery of toxic payloads such as drugs or radionuclides. Although successful in therapy of hematological cancers (Zevalin, Mylotarg), the broader application of these technologies to carcinomas still remains to be proven in clinical testing. Engineering of antibody constructs with optimal blood clearance and tumor-targeting kinetics, and selecting the radionuclide that may deliver sufficient radiation energy to kill the more radio-resistant carcinomas, are discussed. With the advent of genomics and proteomics, new membrane-associated tumor antigens are being discovered and will provide novel targets for future antibody therapy of cancer.