An N-Acetylgalactosamino Dendron-Clearing Agent for High-Therapeutic-Index DOTA-Hapten Pretargeted Radioimmunotherapy.

Clearing agents (CAs) can rapidly remove nonlocalized targeting biomolecules from circulation for hepatic catabolism, thereby enhancing the therapeutic index (TI), especially for blood (marrow), of the subsequently administered radioisotope in any multistep pretargeting strategy. Herein we describe the synthesis and in vivo evaluation of a fully synthetic glycodendrimer-based CA for DOTA-based pretargeted radioimmunotherapy (DOTA-PRIT). The novel dendron-CA consists of a nonradioactive yttrium-DOTA-Bn molecule attached via a linker to a glycodendron displaying 16 terminal α-thio-N-acetylgalactosamine (α-SGalNAc) units (CCA α-16-DOTA-Y3+; molecular weight: 9059 Da). Pretargeting [177Lu]LuDOTA-Bn with CCA α-16-DOTA-Y3+ to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [177Lu]LuDOTA-Bn for blood, tumor, liver, spleen, and kidneys of 11.7, 468, 9.97, 5.49, and 13.3 cGy/MBq, respectively. Tumor-to-normal tissues absorbed-dose ratios (i.e., TIs) ranged from 40 (e.g., for blood and kidney) to about 550 for stomach.

Intraperitoneal pretarget radioimmunotherapy with CC49 fusion protein.

PURPOSE: This study examined a pretarget radioimmunotherapy strategy for treatment of an i.p. tumor model (LS174T). EXPERIMENTAL DESIGN: The strategy used regional administration (i.p.) of a novel targeting molecule composed of four CC49 anti-tumor-associated glycoprotein 72 (TAG-72) single-chain antibodies linked to streptavidin as a fusion protein (CC49 fusion protein); 24 hours later, a synthetic clearing agent was administered i.v. to produce hepatic clearance of unbound CC49 fusion protein/synthetic clearing agent complexes. Four hours later, a low molecular weight radiolabeled reagent composed of biotin conjugated to the chelating agent 7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) complexed with (111)In-, (90)Y-, or (177)Lu-DOTA-biotin was injected. RESULTS: Radiolocalization to tumor sites was superior with i.p. administration of radiolabeled DOTA-biotin as compared with i.v. administration. Imaging and biodistribution studies showed excellent tumor localization of radioactivity with (111)In- or (177)Lu-DOTA-biotin. Tumor localization of (111)In-DOTA-biotin was 43% ID/g and 44% ID/g at 4 and 24 hours with the highest normal tissue localization in the kidney with 6% ID/g at 48 and 72 hours. Therapy studies with (90)Y-DOTA-biotin at doses of 400 to 600 microCi or (177)Lu-DOTA-biotin at doses of 600 to 800 microCi produced significant prolongation of survival compared with controls (P = 0.03 and P < 0.01). CONCLUSIONS: Pretarget radioimmunotherapy using regional administration of CC49 fusion protein and i.p. (90)Y- or (177)Lu-DOTA-biotin represents a successful therapeutic strategy in the LS174T i.p. tumor model and this strategy may be applicable to human trials in patients with i.p. ovarian cancer.

Pretargeted radioimmunotherapy of mesothelin-expressing cancer using a tetravalent single-chain Fv-streptavidin fusion protein.

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.

Single-chain Fv-streptavidin substantially improved therapeutic index in multistep targeting directed at disialoganglioside GD2.

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.

Pretargeted alpha emitting radioimmunotherapy using (213)Bi 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-biotin.

PURPOSE: The use of an alpha emitter for radioimmunotherapy has potential advantages compared with beta emitters. When administered systemically optimal targeting of intact antibodies requires >24 h, therefore limiting the use of short-lived alpha emitters. This study investigated the biodistribution of bismuth-labeled biotin in A431 tumor-bearing mice pretargeted with antibody B3-streptavidin (B3-SA) and examined the therapeutic efficacy of the alpha emitter, (213)Bi-labeled biotin. EXPERIMENTAL DESIGN: Biotinidase-resistant 7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin was radiolabeled with (205,206)Bi or (213)Bi. Treatment of tumor-bearing mice began by administration of B3-SA (400 micro g) to target the tumor sites for 24 h. Then, an agent containing biotin and galactose groups was used to clear the conjugate from the circulation. Four h later, bismuth-radiolabeled DOTA-biotin was given, and biodistribution or therapy was evaluated. Dose escalation treatment from 3.7-74 MBq was performed, and the effects on tumors of different sizes were investigated. Tumor growth, complete blood cell counts, toxicity, and survival were monitored. RESULTS: Radiolabeled biotin cleared rapidly. Rapid tumor uptake resulted in much higher tumor:nontumor targeting ratios than achieved with the directly labeled monoclonal antibody. Dose escalation revealed that 74 MBq caused acute death of mice, whereas 0.37-37 MBq doses inhibited tumor growth and prolonged survival significantly. Evidence of mild hematological toxicity was noted. At therapeutically effective doses renal toxicity was observed. CONCLUSIONS: (213)Bi-DOTA-biotin, directed by the Pretarget method to tumor-targeted B3-SA, showed a therapeutic effect, although the therapeutic index was low. The source of the toxicity was most likely related to the renal toxicity.

Biological comparison of 149Pm-, 166Ho-, and 177Lu-DOTA-biotin pretargeted by CC49 scFv-streptavidin fusion protein in xenograft-bearing nude mice.

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.

In vivo evaluation of pretargeted 64Cu for tumor imaging and therapy.

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.

Pretarget radiotherapy with an anti-CD25 antibody-streptavidin fusion protein was effective in therapy of leukemia/lymphoma xenografts.

Although radioimmunotherapy with radiolabeled intact monoclonal antibodies has demonstrated efficacy in the treatment of lymphoma, it provides low tumor-to-normal-tissue radionuclide target ratios and unwanted prolonged radiation exposure to the bone marrow. To overcome these obstacles, the administration of the radionuclide was separated from that of the antibody by using an anti-IL-2 receptor alpha antibody single chain Fv-streptavidin fusion protein, followed by radiolabeled biotin to treat lymphoma or leukemia xenografted mice. This Pretarget approach provided extremely rapid and effective tumor targeting, permitting the use of short-lived alpha-emitting radionuclides. With the beta-emitter (90)Y, all of the 10 lymphoma-xenografted mice were cured. With the alpha-emitter (213)Bi, significant efficacy was obtained in treating leukemic mice, and, furthermore, when combined with immunotherapy, 7 of 10 leukemic mice were cured. Thus, Pretarget radioimmunotherapy is very promising and could represent the next generation in the treatment of lymphoma and leukemia.

Radioimmunotherapy of A431 xenografted mice with pretargeted B3 antibody-streptavidin and (90)Y-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin.

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

Pretargeting radioimmunotherapy of a murine model of adult T-cell leukemia with the alpha-emitting radionuclide, bismuth 213.

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.