Evaluation of glycodendron and synthetically modified dextran clearing agents for multistep targeting of radioisotopes for molecular imaging and radioimmunotherapy.

A series of N-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific antitumor antibody streptavidin fusion protein (scFv4-SA) in vivo, to improve tumor-to-normal tissue concentration ratios for multistep targeted (MST) radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with an average molecular weight of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogues (e.g., CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. In vivo studies included dynamic positron-emission tomography (PET) imaging of (124)I-labeled-scFv4-SA clearance and dual-label biodistribution studies following MST directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a scFv4-SA specific for an antitumor-associated glycoprotein (TAG-72); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of the (124)I-labeled-scFv4-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e., biotin linked with an extended spacer, referred to herein as long-chain (LC)) can impact the binding to the antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor-to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv4-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin.

Comparison of pretargeted and conventional CC49 radioimmunotherapy using 149Pm, 166Ho, and 177Lu.

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.

Comparative biodistributions of pretargeted radioimmunoconjugates targeting CD20, CD22, and DR molecules on human B-cell lymphomas.

Pretargeted radioimmunotherapy (PRIT) using streptavidin (SA)-conjugated antibodies (Abs), followed by clearing agent and radiolabeled biotin is a promising method that can increase the effectiveness of RIT, while decreasing the toxicities associated with directly labeled Abs. Although CD20 has been the traditional target antigen for RIT of non-Hodgkin lymphoma (NHL), studies targeting HLA DR and CD22 have yielded promising results. Targeting all 3 antigens at once may further augment the effect of PRIT. This study compares the targeting of Ramos, Raji, and FL-18 lymphoma xenografts with either anti-CD20 Ab/SA (1F5/SA), anti-HLA DR Ab/SA (Lym-1/SA), anti-CD22 Ab/SA (HD39/SA), or all 3 conjugates in combination, followed 24 hours later by a biotin-N-acetyl-galactosamine clearing agent, and 3 hours after that by (111)In-DOTA-biotin. The Ab/SA conjugate yielding the best tumor uptake and tumor-to-normal organ ratios of radioactivity varied depending on the target antigen expression on the cell line used, with 1F5/SA and Lym-1/SA yielding the most promising results overall. Also, the best tumor-to-normal organ ratios of absorbed radioactivity were obtained using single conjugates optimized for target tumor antigen expression rather than the combination therapy. This study highlights the importance of screening the antigenic expression on lymphomas to select the optimal reagent for PRIT.

Pretargeted radioimmunotherapy in tumored mice using an in vivo 212Pb/212Bi generator.

OBJECTIVE: Pretargeting is the concept that combines optimal delivery of the antibody and rapid capture and elimination of the radioactivity. In this study, we evaluated the potential of antibody pretargeting to enable the tumor-targeting (212)Pb for in vivo generation of (212)Bi for alpha particle radiotherapy. METHODS: The (212)Pb/(212)Bi chelate of DOTA-biotin, as well as their gamma-emitting analogues, (203)Pb and (205)Bi, was prepared and characterized. The radiolabeled compounds were injected in animals for evaluation of tumor targeting and normal tissue uptake and retention. In the pretargeting protocol, injection of 400 microg of NR-LU-10 antibody-streptavidin conjugate was given at t = 0 h, then 100 microg of N-acetyl-galatosamine-biotin clearing agent was injected at t = 20-24 h; finally, 1 microg of (212)Pb/(212)Bi-DOTA-biotin was injected 6 h later. RESULTS: Both (203)Pb and (205)Bi-DOTA-biotin were stable for at least 4 days in the different challenging solutions including PBS, 10 mM DTPA and serum. Contrary to its gamma-emitting analogues, radiolabeled (212)Pb-DOTA-biotin was not stable. There was greater than 30% of free (212)Bi released 4 h after (212)Pb-labeled DOTA-biotin. The results of pretargeting protocol of (203)Pb and (205)Bi-DOTA-biotin showed that the tumor target reached 20% injected dose (ID)/g at 4 h postinjection and remained high for 5 days. The %ID/g in the whole blood and other nontarget organs was low after administration of labeled (203)Pb and (205)Bi-DOTA-biotin similar to the biodistribution of labeled DOTA-biotin alone. In the animals administered (212)Pb-DOTA-biotin, radioactivity in nontarget organs was low except the kidneys. The %ID/g in the kidney for (212)Bi was 14.5 at 2 h, higher than (212)Pb, but dropped to about 6% ID/g by 4 h. However, tumor uptake for (212)Pb and (212)Bi was >25% ID/g at 1 h postinjection and remained so through 24 h. CONCLUSIONS: Antibody pretargeting system with Mab-streptavidin, clearing agent and DOTA-biotin provides the potential of (212)Bi for solid tumor radiotherapy despite the release of (212)Bi after (212)Pb decay. Dosimetry calculations resulted in tumor dose at 93 rad/muCi and ratios of tumor to marrow and kidney at 386:1 and 12:1, respectively.

Pretargeted radioimmunotherapy (RIT) with a novel anti-TAG-72 fusion protein.

Pretargeted radioimmunotherapy (RIT) increases the dose of radionuclide delivered to tumor sites while limiting radiation to normal tissues. The three components in Pretarget include a streptavidin-containing targeting molecule, a synthetic clearing agent (sCA), and (90)Y and/or (111)In-DOTA-biotin. This trial determined the feasibility and safety of using a genetically engineered fusion protein directed to TAG-72 as the targeting agent. Nine (9) patients with metastatic colorectal cancer (TAG-72+) received 160 mg/m(2) of CC49Fusion protein intravenously (i.v.), followed by the sCA, 45 mg/m(2) i.v. Twenty-four (24) hours later, patients received radiolabeled DOTA-biotin (either 0.65 or 1.3 mg/m(2)). All patients received 5 mCi of (111)In-DOTA-biotin for imaging and dosimetry purposes and patients 4-9 received 10 mCi/m2 of (90)Y-DOTA-biotin as well. The mean plasma T1/2 of CC49Fusion protein was 23 +/- 6 hours. Greater than 95% of the circulating CC49Fusion protein was eliminated from the circulation within 6 hours of sCA administration. The radiolabeled DOTA-biotin rapidly localized to tumor sites while the unbound fraction was rapidly excreted. The mean tumor-to-marrow radiation dose ratio was 139:1 and mean tumor: whole body was 56:1. No infusion-related, renal, hepatic, or hematologic toxicities were noted. CC49Fusion protein performs well in a pretargeted RIT schema, and further study with escalating doses of (90)Y should be pursued. This strategy has the potential to deliver effective radiation tumor doses to TAG- 72+ tumors.

Role of biotin-binding affinity in streptavidin-based pretargeted radioimmunotherapy of lymphoma.

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.

Phase 1 trial of a novel anti-CD20 fusion protein in pretargeted radioimmunotherapy for B-cell non-Hodgkin lymphoma.

Pretargeted radioimmunotherapy (PRIT) has the potential to increase the dose of radionuclide delivered to tumors while limiting radiation to normal tissues. The purpose of this phase 1 trial is to assess safety of this multistep approach using a novel tetrameric single-chain anti-CD20-streptavidin fusion protein (B9E9FP) as the targeting moiety in patients with B-cell non-Hodgkin lymphoma (NHL), and to characterize its pharmacokinetics and immunogenicity. All patients received B9E9FP (160 mg/m(2) or 320 mg/m(2)); either 48 or 72 hours later, a synthetic clearing agent (sCA) was administered (45 mg/m(2)) to remove circulating unbound B9E9FP. (90)Yttrium ((90)Y; 15 mCi/m(2))/(111)In (5 mCi)-DOTA-biotin was injected 24 hours later. There were 15 patients enrolled in the study. B9E9FP had a mean plasma half-life (T(1/2)) of 25 +/- 6 hours with a reduction in plasma level of more than 95% within 6 hours of sCA administration. (90)Y/(111)In-DOTA-biotin infusion resulted in rapid tumor localization and urinary excretion. The ratio of average tumor to whole-body radiation dose was 49:1. No significant hematologic toxicities were noted in 12 patients. There were 2 patients who had hematologic toxicity related to progressive disease. There were 2 complete remissions (90 and 325 days) and one partial response (297 days). B9E9FP performs well as the targeting component of PRIT with encouraging dosimetry, safety, and efficacy. A dose escalation trial of (90)Y-DOTA-biotin in this format is warranted.

Combination therapy with Pretarget CC49 radioimmunotherapy and gemcitabine prolongs tumor doubling time in a murine xenograft model of colon cancer more effectively than either monotherapy.

Pretarget radioimmunotherapy (RIT) is a multistep strategy for cancer therapy designed to reduce nontarget organ exposure by uncoupling the tumor targeting moiety from the radioactive ligand. Using this approach, we and others have demonstrated objective responses to therapy among patients with non-Hodgkin's lymphoma, with less hematological toxicity than is typically seen at equivalent doses of conventional RIT in the same patient population. In the present study, we show that combination therapy with gemcitabine (200 mg/kg on days -1 and +1) and Pretarget RIT (400 micro Ci (90)Y-labeled DOTA-biotin on day +1) is superior to Pretarget monotherapy (400 or 800 micro Ci (90)Y) as well as to gemcitabine monotherapy in nude mice bearing established human LS174T colon cancer xenografts. For the targeting moiety, we used a murine anti-TAG-72 (CC49) single-chain Fv-streptavidin (scFvSA) fusion protein that has been shown to be safe and well-tolerated in humans. The median number of days to tumor volume doubling in the gemcitabine-only studies (200 mg/kg) was 10.4 +/- 5.5 days; in the Pretarget 400 micro Ci dose-only studies, tumor doubling time was 6.7 +/- 4.9 days; and in combination therapy studies, it was 23.9 +/- 7.2 days (P

Comparison of immunoscintigraphy, efficacy, and toxicity of conventional and pretargeted radioimmunotherapy in CD20-expressing human lymphoma xenografts.

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.