RESUMEN
Radioimmunotherapy (RIT) with α-emitting radionuclides is an attractive approach for the treatment of minimal residual disease because the short path lengths and high energies of α-particles produce optimal cytotoxicity at small target sites while minimizing damage to surrounding normal tissues. Pretargeted RIT (PRIT) using antibody-streptavidin (Ab-SA) constructs and radiolabeled biotin allows rapid, specific localization of radioactivity at tumor sites, making it an optimal method to target α-emitters with short half-lives, such as bismuth-213 (²¹³Bi). Athymic mice bearing Ramos lymphoma xenografts received anti-CD20 1F5(scFv)(4)SA fusion protein (FP), followed by a dendrimeric clearing agent and [²¹³Bi]DOTA-biotin. After 90 minutes, tumor uptake for 1F5(scFv)4SA was 16.5% ± 7.0% injected dose per gram compared with 2.3% ± .9% injected dose per gram for the control FP. Mice treated with anti-CD20 PRIT and 600 µ Ci [²¹³Bi]DOTA-biotin exhibited marked tumor growth delays compared with controls (mean tumor volume .01 ± .02 vs. 203.38 ± 83.03 mm³ after 19 days, respectively). The median survival for the 1F5(scFv)4SA group was 90 days compared with 23 days for the control FP (P < .0001). Treatment was well tolerated, with no treatment-related mortalities. This study demonstrates the favorable biodistribution profile and excellent therapeutic efficacy attainable with ²¹³Bi-labeled anti-CD20 PRIT.
Asunto(s)
Antígenos CD20/metabolismo , Bismuto/uso terapéutico , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/radioterapia , Neoplasia Residual/tratamiento farmacológico , Radioinmunoterapia/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Antineoplásicos/inmunología , Biotina/efectos adversos , Biotina/análogos & derivados , Biotina/farmacocinética , Biotina/farmacología , Biotina/uso terapéutico , Bismuto/efectos adversos , Bismuto/farmacocinética , Bismuto/farmacología , Recuento de Células Sanguíneas , Línea Celular Tumoral , Humanos , Región Variable de Inmunoglobulina/inmunología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/fisiopatología , Pruebas de Función Hepática , Linfoma no Hodgkin/patología , Linfoma no Hodgkin/fisiopatología , Ratones , Neoplasia Residual/inmunología , Compuestos Organometálicos/efectos adversos , Compuestos Organometálicos/farmacocinética , Compuestos Organometálicos/farmacología , Compuestos Organometálicos/uso terapéutico , Radiometría , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Supervivencia , Distribución Tisular/efectos de los fármacosRESUMEN
The efficacy of radioimmunotherapy (RIT) for patients with relapsed non-Hodgkin lymphoma (NHL) is limited by nonspecific delivery of radiation to normal tissues due to the long circulating half-life of radiolabeled anti-CD20 antibodies (Abs). Pretargeted RIT using a covalent conjugate of the 1F5 anti-CD20 Ab with streptavidin (SA) has been shown to augment the efficacy of RIT and decrease toxicity compared with a directly labeled 1F5 Ab. We have engineered a tetravalent singlechain 1F5 (scFv)4SA fusion protein and compared it to the 1F5-SA conjugate. Athymic mice bearing Ramos lymphoma xenografts received either the conjugate or fusion protein, followed 20 hours later by a biotin-N-acetyl-galactosamine clearing agent, followed 4 hours later by 111In-DOTA-biotin. After 24 hours, 11.4% +/- 2.1% of the injected dose of radionuclide was present per gram of tumor (% ID/g) using 1F5 (scFv)4SA compared with 10.8% +/- 2.5% ID/g with 1F5 Ab-SA. Superior tumor-to-normal organ ratios of radioactivity were consistently seen using the fusion protein compared with the chemical conjugate (eg, tumor-to-blood ratio > 65:1 after 48 hours with the fusion protein, but < 7:1 with the conjugate). More than 90% of lymphomabearing mice could be cured with minimal toxicity using either reagent followed by 1200 muCi (44.4 MBq) 90Y-DOTA-biotin.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD20/inmunología , Antineoplásicos/uso terapéutico , Linfoma de Células B/radioterapia , Radioinmunoterapia , Proteínas Recombinantes de Fusión/uso terapéutico , Estreptavidina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
One pretargeting approach to cancer radioimmunotherapy utilizes an antibody-streptavidin conjugate that is first localized to the tumor. A "clearing agent" is then administered to remove the excess bioconjugate from blood, followed by injection of the radiolabeled biotin therapeutic. In this study, the role of streptavidin-biotin affinity in this pretargeting system was investigated for the first time in vivo, with a reduced affinity, site-directed streptavidin mutant and with radiolabeled bis-biotin reagents. The S45A streptavidin mutant (SA-S45A), which displays a faster off-rate for biotin, was utilized with a bivalent biotin carrier that retains high avidity for the streptavidin mutant. Mice were fed either a normal or biotin-deficient diet, yielding serum endogenous biotin concentrations of 31 nM and 5 nM, respectively. Lymphoma-bearing nude mice pretargeted with 1F5 Antibody-SA-Wild Type (WT) bioconjugates produced (125)I-bis-biotin tumor concentrations of 2.2%ID/g and 7.0%ID/g in mice fed normal diets vs biotin-deficient diets. (125)I-bis-biotin tumor concentrations of mice pretargeted with 1F5-SA-S45A were 12%ID/g and 10%ID/g for mice fed normal and biotin-deficient diets, respectively. However, poor clearance of the 1F5-SA-S45A with the biotinylated clearing agent led to high normal organ concentrations of (125)I-bis-biotin. A galactosylated human serum albumin (HSA) modified with bis-biotin was then tested, and normal organ (125)I-bis-biotin concentrations were significantly reduced. Tumor-to-organ ratios achieved for 1F5-SA-S45A with the HSA-bis-biotin clearing agent in mice with high serum biotin were similar to those achieved with 1F5-SA-WT in mice with low serum biotin. These results demonstrate that exchange of bound endogenous biotin with lower affinity streptavidin mutants is possible, and that corresponding use of bis-biotin carriers can nearly eliminate the differences in therapeutic radioactivity at the tumor site in animals on normal vs biotin-deficient diets. The results also interestingly demonstrate, however, that improved clearance agents capable of removing the lower affinity streptavidin-antibody conjugate are needed to achieve comparable specificity in tumor to blood or normal organ ratios.
Asunto(s)
Biotina/farmacología , Linfoma/tratamiento farmacológico , Radioinmunoterapia , Estreptavidina/farmacología , Animales , Sitios de Unión , Biotina/análogos & derivados , Biotina/uso terapéutico , Humanos , Linfoma/metabolismo , Ratones , Ratones Desnudos , Mutagénesis Sitio-Dirigida , Neoplasias Experimentales/tratamiento farmacológico , Especificidad de Órganos , Estreptavidina/uso terapéutico , Fracciones SubcelularesRESUMEN
UNLABELLED: Pretargeted radioimmunotherapy (RIT) using streptavidin (sAv)-conjugated antibodies before radiolabeled-biotin is a promising approach to improve absorbed dose ratios and achieve high durable remission rates with diminished systemic toxicity. This study compared the immunoscintigraphy, toxicity, and therapeutic efficacy of pretargeted RIT with conventional RIT using an anti-CD20 antibody. METHODS: Athymic mice bearing Ramos human Burkitt's lymphoma xenografts were injected intraperitoneally with a 1F5-sAv conjugate followed 24 h later by a galactosylated, biotinylated clearing agent (CA) and, finally, 3 h later by (111)In- or (90)Y-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin. The comparison groups consisted of mice injected with conventional, directly labeled (111)In- or (90)Y-1F5. RESULTS: Rapid tumor uptake of radioactivity within 2 h was observed with the pretargeting approach, resulting in high-contrast tumor images at 24 h with minimal blood-pool radioactivity. Although conventional radiolabeled antibodies produced clear tumor images at 24 h, a large amount of radioactivity was present in the blood pool. The tumor-to-blood ratio was 3.5:1 with pretargeting compared with 0.4:1 with conventional (111)In-1F5. Pretargeted RIT with 29.6 MBq (800 micro Ci) (90)Y-DOTA-biotin cured 100% of mice with tolerable toxicity, whereas conventional RIT with (90)Y-1F5 at a dose of 14.8 MBq (400 micro Ci) produced no cures, induced profound pancytopenia, and was lethal to all mice. CONCLUSION: These results suggest that anti-CD20 pretargeted RIT may be superior to conventional radiolabeled antibodies in terms of radioimmunoscintigraphy, toxicity, and therapeutic efficacy for treatment of B-cell lymphomas.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD20/inmunología , Inmunoconjugados/uso terapéutico , Linfoma de Células B/radioterapia , Radioinmunoterapia , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/toxicidad , Antígenos CD20/análisis , Avidina , Femenino , Humanos , Inmunoconjugados/toxicidad , Linfoma de Células B/diagnóstico por imagen , Linfoma de Células B/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Cintigrafía , Estreptavidina , Distribución Tisular , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.