RESUMEN
The therapeutic efficacies of radiolabeled biotin, pretargeted by monoclonal antibody (mAb)-streptavidin fusion protein CC49 scFvSA, were compared to those of radiolabeled mAb CC49, using the three radiolanthanides in an animal model of human colon cancer. The purpose of the present study was to compare antibody pretargeting to conventional radioimmunotherapy using (149)Pm, (166)Ho, or (177)Lu. Nude mice bearing LS174T colon tumors were injected sequentially with CC49 scFvSA, the blood clearing agent biotin-GalNAc(16), and (149)Pm-, (166)Ho-, or (177)Lu-DOTA-biotin. Tumor-bearing mice were alternatively administered (149)Pm-, (166)Ho-, or (177)Lu-MeO-DOTA-CC49. Therapy with pretargeted (149)Pm-,(166)Ho-, and (177)Lu-DOTA-biotin increased the median time of progression to a 1 g tumor to 50, 41, and 50 days post-treatment, respectively. Therapy with (149)Pm-,(166)Ho-, and (177)Lu-MeO-DOTA-CC49 increased the median time to progression to 53, 24, and 67 days post-treatment, respectively. In contrast, saline controls showed a median time to progression of 13 days postinjection. Treatment with pretargeted (149)Pm-, (166)Ho-, and (177)Lu-biotin or (149)Pm-, (166)Ho-, and (177)Lu-CC49 increased tumor doubling time to 18-36 days, compared to 3 days for saline controls. Among treated mice, 23% survived >84 days post-therapy, and 11% survived 6 months, but controls survived <29 days. Long-term survivors showed tumor growth inhibition or partial regression, extensive necrosis in residual masses, and no evidence of nontarget tissue toxicity at necropsy. Both pretargeted and conventional RIT demonstrated considerable efficacy in an extremely aggressive animal model of cancer. Our results identified (177)Lu as an optimal radiolanthanide for future evaluation of these agents in toxicity and multiple-dose therapy studies.
Asunto(s)
Anticuerpos Antineoplásicos/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias del Colon/radioterapia , Sistemas de Liberación de Medicamentos , Elementos de la Serie de los Lantanoides/uso terapéutico , Radioinmunoterapia/métodos , Radiofármacos/uso terapéutico , Estreptavidina/uso terapéutico , Animales , Biotina/análogos & derivados , Biotina/química , Línea Celular Tumoral , Neoplasias del Colon/patología , Femenino , Humanos , Ratones , Ratones Desnudos , Compuestos Organometálicos/química , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
UNLABELLED: Mesothelin is a glycoprotein that is overexpressed in several human tumors, including mesotheliomas and ovarian cancers, and has been identified as a potential target for therapy. We evaluated the biodistribution and tumor-targeting ability of an antimesothelin tetravalent single-chain Fv-streptavidin fusion protein (SS1scFvSA) in mice. METHODS: SS1scFvSA was labeled with 125I or 111In for evaluation of internalization in vitro and for optimization of its biodistribution. The A431-K5 mesothelin transfected cell line was used as the target. We used a 3-step pretargeting approach consisting of injections of (i) SS1scFvSA, followed 20 h later by (ii) a synthetic clearing agent, and (iii) 4 h later, radiolabeled (111In, 88Y/90Y, or 177Lu) 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-biotin. To optimize the tumor uptake, the effect of the specific activity of 111In-DOTA-biotin was evaluated. RESULTS: Approximately 60% of SS1sc FvSA internalized within 6 h. The optimal dose of SS1scFvSA for pretargeting was 600 microg. Decreasing the specific activity of DOTA-biotin by administering 0.1-5 microg of DOTA-biotin resulted in tumor uptake decreasing from 31.8 to 5.5 %ID/g (percentage injected dose per gram) at 2 h. Pretargeted therapy of A431-K5 tumor with 90Y doses of 11.1-32.4 MBq resulted in a dose-dependent tumor response. With 32.4 MBq, 86% of mice survived tumor free for 110 d. All nontreated mice died, with a median survival of 16 d. CONCLUSION: SS1scFvSA localized in the mesothelin-expressing tumor, resulting in a high accumulation of radiolabeled DOTA-biotin. The specific activity of DOTA-biotin had a significant effect on its tumor uptake. Therapeutic tumor doses were obtained without dose-limiting toxicity.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Radioisótopos de Yodo/uso terapéutico , Glicoproteínas de Membrana/metabolismo , Radioinmunoterapia/métodos , Radiofármacos/uso terapéutico , Estreptavidina/uso terapéutico , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Femenino , Proteínas Ligadas a GPI , Humanos , Fragmentos de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas/uso terapéutico , Radioisótopos de Yodo/farmacocinética , Mesotelina , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Especificidad de Órganos , Radiofármacos/farmacocinética , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/uso terapéutico , Estreptavidina/farmacocinética , Análisis de Supervivencia , Distribución Tisular , Resultado del TratamientoRESUMEN
UNLABELLED: Multistep targeting can improve the therapeutic index of antibody-based targeting, particularly relevant to pediatric tumors where acute toxicity and late effects of treatment are major concerns. Neuroblastoma is uniquely suited for such investigations because of its abundance of surface ganglioside GD2. METHODS: 5F11scFv (scFv = single-chain variable fragment) was constructed from the variable regions of the heavy (V(H)) and kappa-light (V(L)) chain complementary DNA (cDNA) of anti-GD2 IgM hybridoma 5F11 and ligated to full-length streptavidin cDNA for expression in Escherichia coli. Purified 5F11-scFv-streptavidin (5F11-scFv-SA) was a homotetramer and showed comparable avidity to 5F11 IgM and a 30-fold improvement over monomeric scFv. Biodistribution of 5F11-scFv-SA was studied in nude mice xenografted with neuroblastoma LAN-1. Twenty-four hours after intravenous injection of 300-900 microg 5F11-scFv-SA, 150-450 microg of a thiogalactoside-containing clearing agent, (Gal-NAc)(16)-alpha-S-C(5)H(10)-NH-LC-N-Me-biotin (molecular weight, 8652), were administered intravenously, followed by approximately 2.5 microg (1.85-3.7 MBq) (111)In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin ((111)In-DOTA-biotin) intravenously 4 h later and clocked as time 0. RESULTS: Tumor uptake (percentage of injected dose per gram [%ID/g]) at 2 h was 7 %ID/g and decayed with a half-life of 72 h, whereas blood %ID/g rapidly decreased to <1/500 of that of tumor after the first 24 h. The tumor-to-nontumor (T/NT) ratio at 72 h was high (median, 106; range, 3.4 [kidney] to 1660 [blood]). When the area under the radioactivity curve was computed, the T/NT organ ratio was favorable (4.8 for kidney and 162 for blood). When human and murine tumors were surveyed, the T/NT ratio of (111)In-DOTA-biotin uptake correlated with their levels of GD2 expression as assayed by flow cytometry. Biotinylated polypeptides (bovine serum albumin and vasointestinal peptides) achieved selective tumor targeting when the multistep strategy was applied. CONCLUSION: Improvement in the T/NT ratio using pretargeting strategy may increase the efficacy and safety of scFv-based approaches in cancer therapy. Additionally, since biotinylated polypeptides can be rendered tumor selective, a large repertoire of agents can potentially be explored.
Asunto(s)
Gangliósidos/metabolismo , Neoplasias Experimentales/terapia , Estreptavidina/farmacología , Animales , Humanos , Radioisótopos de Indio , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones SCID , Células Tumorales CultivadasRESUMEN
The radiolanthanides (149)Pm, (166)Ho, and (177)Lu possess a range of half-lives and alpha(-) beta(-) energies for targeted radiotherapy of cancer. (149)Pm-, (166)Ho-, and (177)Lu-DOTA-biotin were pretargeted to LS174T colorectal tumors in nude mice with CC49 scFvSA antibody-streptavidin fusion protein. Tumor uptakes of (149)Pm (22.9% ID/g), (166)Ho (30.2% ID/g), and (177)Lu (35.4% ID/g) peaked at 1-4 h. Rapid blood disappearance was accompanied by urinary excretion of 59-66% ID within 1 h. Biodistributions of these agents show promise for pretargeted radioimmunotherapy of cancer.
Asunto(s)
Anticuerpos Antineoplásicos/metabolismo , Biotina/análogos & derivados , Biotina/farmacocinética , Neoplasias del Colon/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Compuestos Organometálicos/farmacocinética , Radioinmunoterapia/métodos , Radioisótopos/farmacocinética , Estreptavidina/farmacocinética , Animales , Anticuerpos Antineoplásicos/administración & dosificación , Biotina/administración & dosificación , Línea Celular Tumoral , Neoplasias del Colon/radioterapia , Holmio/administración & dosificación , Holmio/farmacocinética , Humanos , Fragmentos de Inmunoglobulinas/administración & dosificación , Fragmentos de Inmunoglobulinas/metabolismo , Inyecciones Intravenosas , Lutecio/administración & dosificación , Lutecio/farmacocinética , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Especificidad de Órganos , Compuestos Organometálicos/administración & dosificación , Prometio/administración & dosificación , Prometio/farmacocinética , Radioisótopos/administración & dosificación , Radiofármacos/administración & dosificación , Radiofármacos/farmacocinética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacocinética , Estreptavidina/administración & dosificación , Distribución TisularRESUMEN
UNLABELLED: Pretargeting involves administration of a tumor-targeting monoclonal antibody (mAb) covalently linked to a molecule having a high-affinity binding site for a rapidly distributed radiolabeled effector molecule. The aim of this study was to compare pretargeting to a conventionally labeled antibody for tumor targeting of the intermediate-lived radionuclide (64)Cu, which has shown promise for PET imaging and radioimmunotherapy of cancer. METHODS: DOTA-biotin (where DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid) and the intact immunoconjugate DOTA-NR-LU-10 were labeled to high specific activities with (64)Cu, and the serum stabilities and target binding capabilities of each agent were assayed in vitro. Nude mice bearing SW1222 human colorectal carcinoma xenografts were administered (64)Cu-DOTA-biotin, with and without pretreatment with the mAb-streptavidin conjugate NR-LU-10/SA and the synthetic clearing agent Biotin-GalNAc(16), or injected with (64)Cu-DOTA-NR-LU-10. Biodistributions of both agents were obtained from 5 min to 48 h after injection. RESULTS: Both (64)Cu-DOTA-biotin and (64)Cu-DOTA-NR-LU-10 were 100% stable in serum in vitro. (64)Cu-DOTA-biotin exhibited >98% specific binding to immobilized streptavidin, whereas the immunoreactivity of (64)Cu-DOTA-NR-LU-10 averaged nearly 80%. Biodistributions in SW1222-bearing mice showed that NR-LU-10/SA-pretargeted (64)Cu-DOTA-biotin attained a peak tumor uptake of 18.9 percentage injected dose per gram (%ID/g) at 1 h, with concomitant rapid disappearance from blood and renal excretion. In the absence of pretargeting, (64)Cu-DOTA-biotin had very similar biodistribution and clearance properties, except with extremely low nonspecific tumor uptake. In contrast, (64)Cu-DOTA-NR-LU-10 reached 80.3 %ID/g in tumor tissue, after 48 h, whereas blood clearance was considerably slower than pretargeted (64)Cu-DOTA-biotin. Comparison of the time-activity curves for tumor uptake and blood clearance of pretargeted (64)Cu and the (64)Cu-labeled antibody revealed that the maximum tumor accumulations of radioactivity were similar for each agent, 17.9 percentage injected activity per gram (%IA/g) and 20.7 %IA/g, respectively. However, the tumor-to-blood ratio of areas under the curves was 14 times higher for pretargeted (64)Cu-DOTA-biotin because of the substantial increase in blood clearance of the small effector molecule. CONCLUSION: The extremely rapid tumor uptake and blood clearance of pretargeted (64)Cu-DOTA-biotin should afford markedly superior PET imaging contrast and therapeutic efficacy, compared with conventionally labeled (64)Cu-DOTA-NR-LU-10. Further comparison of the therapeutic efficacy, toxicity, and dosimetry of these 2 agents is warranted.
Asunto(s)
Biotina/farmacocinética , Neoplasias Colorrectales/metabolismo , Radioisótopos de Cobre/farmacocinética , Inmunoconjugados/farmacocinética , Compuestos Organometálicos/farmacocinética , Animales , Anticuerpos Monoclonales , Biomarcadores de Tumor/metabolismo , Biotina/análogos & derivados , Biotina/sangre , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/diagnóstico por imagen , Radioisótopos de Cobre/sangre , Sistemas de Liberación de Medicamentos/métodos , Humanos , Inmunoconjugados/sangre , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Especificidad de Órganos , Compuestos Organometálicos/sangre , Radioinmunodetección/métodos , Radioinmunoterapia/métodos , Radiofármacos/sangre , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución TisularRESUMEN
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
Radiolabeled anti-CD20 antibodies produce responses in 60% to 95% of patients with relapsed non-Hodgkin lymphoma (NHL); however, absorbed radiation ratios between tumors and normal organs are relatively low, and many patients have relapses. In this study we compared the abilities of anti-CD45 (BC8) and anti-CD20 (1F5) antibodies to target human Ramos lymphoma xenografts in athymic mice. When direct radioiodination was performed with conventional methods, BC8 delivered 2- to 4-fold more radioiodine to tumors than 1F5, with tumor-to-normal organ ratios as high as 20:1 using radiolabeled BC8 compared with a maximal ratio of 9.8:1 using radioiodinated 1F5. To optimize the biodistribution of radioactivity, we performed studies following a pretargeting method using streptavidin (SA)-conjugated BC8 and 1F5. Injection of a synthetic clearing agent decreased the circulating level of conjugates by 80% to 90% within 1 hour. Pretargeting with BC8-SA resulted in a 2- to 4-fold greater tumor uptake of radiolabeled biotin than with 1F5-SA, with maximal tumor-to-normal organ ratios of more than 80:1 and approximately 16:1, respectively. Therapy experiments demonstrated that 400 microCi (14.8 MBq) or more of yttrium-90-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin cured 100% of mice treated with BC8-SA and more than 90% of mice pretargeted with 1F5-SA, with complete remission occurring 8 to 10 days sooner in mice receiving BC8-SA. After treatment with 200 microCi (7.4 MBq) (90)Y-DOTA-biotin, 70% of the mice treated with BC8-SA were cured, but no mice were cured using 1F5-SA. Doses up to 800 microCi (29.6 MBq) (90)Y-DOTA-biotin were delivered with minor toxicity using either antibody conjugate. These lymphoma xenograft data suggest that pretargeted radioimmunotherapy using either anti-CD20 or anti-CD45 conjugates is highly effective and minimally toxic.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD20/inmunología , Biotina/análogos & derivados , Antígenos Comunes de Leucocito/inmunología , Linfoma de Células B/radioterapia , Radioinmunoterapia/métodos , Animales , Biotina/farmacocinética , Femenino , Humanos , Radioisótopos de Indio/farmacocinética , Radioisótopos de Yodo/análisis , Radioisótopos de Yodo/farmacocinética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Compuestos Organometálicos/farmacocinética , Radioinmunoterapia/efectos adversos , Inducción de Remisión , Estreptavidina , Células Tumorales Cultivadas , Radioisótopos de Itrio/farmacocinéticaRESUMEN
We investigated the biodistribution of (88)Y/(111)In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin and therapy with (90)Y-labeled DOTA-biotin in tumor-bearing mice after B3-streptavidin antibody conjugate (B3-SA) pretargeting. B3 antibody, recognizing Lewis(y) antigen, was conjugated to streptavidin (B3-SA). For pretargeting, 400 micro g of the B3-SA was injected i.v. into mice bearing A431 tumor xenografts. After tumor localization of B3-SA, 100 micro g of synthetic clearing agent was injected i.v. to clear the unbound B3-SA from the circulation. Four h later, 1 micro g of radiolabeled DOTA-biotin was injected i.v. Radioimmunotherapy was performed with doses of 9.25 to 37 MBq of (90)Y-labeled DOTA-biotin. As a result, radiolabeled DOTA-biotin cleared rapidly. All of the normal tissues had <2.6% of the injected dose per gram, whereas tumor uptake reached approximately 15% ID/g. The total tumor uptake of radioactivity remained similar for 96 h or longer. In the first study, the median survival of the control group was 8 days, whereas it increased to >163 days in the 37 MBq (90)Y group (P < 0.005). In a second therapy group, 7 of 10 mice receiving 37 MBq of (90)Y and B3-SA were cured, and remained healthy for >180 days after therapy, compared with control groups, with
Asunto(s)
Biotina/análogos & derivados , Biotina/farmacología , Biotina/farmacocinética , Inmunoconjugados/farmacología , Compuestos Organometálicos/farmacología , Compuestos Organometálicos/farmacocinética , Radioinmunoterapia/métodos , Radiofármacos/farmacología , Radiofármacos/farmacocinética , Estreptavidina/farmacología , Iterbio/uso terapéutico , Animales , Anticuerpos Monoclonales/farmacología , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Sinergismo Farmacológico , Femenino , Humanos , Radioisótopos de Indio/uso terapéutico , Ratones , Ratones Desnudos , Cintigrafía , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We used a pretargeting technique to treat a nonobese diabetic/severe combined immunodeficient murine model of human adult T-cell leukemia with an anti-Tac antibody-streptavidin (HAT-SA) conjugate, which recognizes CD25, followed by bismuth 213 ((213)Bi)-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)- biotin. In the 3-step pretargeting radioimmunotherapy protocol, HAT-SA (140 or 400 microg) was administered intravenously (i.v.) to bind to the interleukin 2 receptor alpha (IL-2R alpha; CD25)-expressing tumor cells. After 24 hours, 100 microg of a synthetic clearing agent was administered i.v. to remove unbound circulating HAT-SA conjugate from the circulation. Four hours later, (213)Bi-DOTA-biotin was administered i.v. for therapy. Tumor growth was significantly inhibited in 3 trials by using 250 microCi (9.25 MBq) of (213)Bi-DOTA-biotin with a pretargeting technique as monitored by serum levels of soluble IL-2R alpha and/or human beta-2-microglobulin (P <.05, t test) and by survival of tumor-bearing mice in the treatment groups (P <.02, log rank test) as compared with the control groups. No prolongation of survival was observed with a nonspecific antibody-SA conjugate or in the absence of the radionuclide. Additionally, no prolongation of survival resulted from administration of (213)Bi directly linked to intact HAT. Furthermore, there was no prolongation of survival when the beta-emitting radionuclide yttrium 90 instead of the alpha-emitting radionuclide (213)Bi was used. The pretargeting approach with (213)Bi inhibited tumor growth more effectively than did immunotherapy with unmodified HAT. The best results were obtained with combination therapy that involved (213)Bi-DOTA-biotin with a pretargeting technique supplemented by 4 weekly doses of HAT. The findings of this study support the use of this combination approach in a clinical trial in patients with IL-2R alpha-expressing leukemias.