Micropeptide MIAC inhibits the tumor progression by interacting with AQP2 and inhibiting EREG/EGFR signaling in renal cell carcinoma.

BACKGROUND: Although, micropeptides encoded by non-coding RNA have been shown to have an important role in a variety of tumors processes, there have been no reports on micropeptide in renal cell carcinoma (RCC). Based on the micropeptide MIAC (micropeptide inhibiting actin cytoskeleton) discovered and named in the previous work, this study screened its tumor spectrum, and explored its mechanism of action and potential diagnosis and treatment value in the occurrence and development of renal carcinoma. METHODS: The clinical significance of MIAC in RCC was explored by bioinformatics analysis through high-throughput RNA-seq data from 530 patients with kidney renal clear cell carcinoma (KIRC) in the TCGA database, and the detection of clinical samples of 70 cases of kidney cancer. In vitro and in vivo experiments to determine the role of MIAC in renal carcinoma cell growth and metastasis; High-throughput transcriptomics, western blotting, immunoprecipitation, molecular docking, affinity experiments, and Streptavidin pulldown experiments identify MIAC direct binding protein and key regulatory pathways. RESULTS: The analysis of 600 renal carcinoma samples from different sources revealed that the expression level of MIAC is significantly decreased, and corelated with the prognosis and clinical stage of tumors in patients with renal carcinoma. Overexpression of MIAC in renal carcinoma cells can significantly inhibit the proliferation and migration ability, promote apoptosis of renal carcinoma cells, and affect the distribution of cells at various stages. After knocking down MIAC, the trend is reversed. In vivo experiments have found that MIAC overexpression inhibit the growth and metastasis of RCC, while the synthetized MIAC peptides can significantly inhibit the occurrence and development of RCC in vitro and in vivo. Further mechanistic studies have demonstrated that MIAC directly bind to AQP2 protein, inhibit EREG/EGFR expression and activate downstream pathways PI3K/AKT and MAPK to achieve anti-tumor effects. CONCLUSIONS: This study revealed for the first time the tumor suppressor potential of the lncRNA-encoded micropeptide MIAC in RCC, which inhibits the activation of the EREG/EGFR signaling pathway by direct binding to AQP2 protein, thereby inhibiting renal carcinoma progression and metastasis. This result emphasizes that the micropeptide MIAC can provide a new strategy for the diagnosis and treatment of RCC.

Biotinylated Streptavidin Surface Coating Improves the Efficacy of a PLGA Microparticle-Based Cancer Vaccine.

Triple-negative breast cancer (TNBC) is an immune-enriched subset of breast cancer that has recently demonstrated clinical responsiveness to combinatorial immunotherapy. However, the lack of targeted interventions against hormone receptors or HER2 continues to limit treatment options for these patients. To begin expanding available interventions for patients with metastatic TNBC, we previously reported a therapeutic vaccine regimen that significantly reduced spontaneous lung metastases in a preclinical TNBC model. This heterologous vaccine approach "primed" mice with tumor lysate antigens encapsulated within poly(lactic-co-glycolic) acid microparticles (PLGA MPs), and then "boosted" mice with tumor lysates plus adjuvant. The use of the PLGA MP prime as monotherapy demonstrated no efficacy, suggesting that improving this component of our therapy would achieve greater vaccine efficacy. Here, we functionally improved the PLGA MP prime by coating microparticles with biotinylated streptavidin-conjugated using 1-ethyl-3-(3-dimethylaminoproplyl) carbodiimide/N-hydroxysuccinimide (EDC/Sulfo-NHS) linkers. This modification enhanced the immunostimulatory potential of our PLGA MPs, as evidenced by increased phagocytosis, maturation, and stimulatory ligand expression by antigen-presenting cells (APCs). Therapeutic prime/boost vaccination of TNBC-bearing mice with surfaced-coated PLGA MPs significantly reduced spontaneous lung metastases by an average of 56% relative to mice primed with unmodified PLGA MPs, and a significant 88% average reduction in spontaneous lung metastases relative to untreated control mice. These findings illustrate that relatively common biotin-streptavidin conjugation formulations can positively affect microparticle-based vaccine immunogenicity resulting in enhanced therapeutic efficacy against established preclinical mammary tumors.

Development of an ELISA-LC-MS hybrid assay for quantification of biotherapeutics.

BACKGROUND: Magnetic bead immunocapture-LC-MS has been widely used for bioanalysis of biotherapeutic proteins. However, magnetic beads are difficult to be fully automated and more costly than ELISA plates. AIM: Develop an ELISA-LC-MS hybrid assay as an alternate platform. RESULTS: Among seven ELISA plates tested, Pierce streptavidin plates, which did not require time-consuming capture antibody precoating steps, provided the best sensitivity and assay dynamic range (5-2500 ng/ml or 10-5000 ng/ml), similar to magnetic bead immunocapture-LC-MS assay and better than an ELISA (50-500 ng/ml). The entire procedures could be fully automated using a liquid handling system. CONCLUSION: This study demonstrates that ELISA-LC-MS hybrid approach using streptavidin plates represents a promising platform for bioanalysis of biotherapeutics.

Comparative Analysis of Bispecific Antibody and Streptavidin-Targeted Radioimmunotherapy for B-cell Cancers.

Streptavidin (SA)-biotin pretargeted radioimmunotherapy (PRIT) that targets CD20 in non-Hodgkin lymphoma (NHL) exhibits remarkable efficacy in model systems, but SA immunogenicity and interference by endogenous biotin may complicate clinical translation of this approach. In this study, we engineered a bispecific fusion protein (FP) that evades the limitations imposed by this system. Briefly, one arm of the FP was an anti-human CD20 antibody (2H7), with the other arm of the FP an anti-chelated radiometal trap for a radiolabeled ligand (yttrium[Y]-DOTA) captured by a very high-affinity anti-Y-DOTA scFv antibody (C825). Head-to-head biodistribution experiments comparing SA-biotin and bispecific FP (2H7-Fc-C825) PRIT in murine subjects bearing human lymphoma xenografts demonstrated nearly identical tumor targeting by each modality at 24 hours. However, residual radioactivity in the blood and normal organs was consistently higher following administration of 1F5-SA compared with 2H7-Fc-C825. Consequently, tumor-to-normal tissue ratios of distribution were superior for 2H7-Fc-C825 (P < 0.0001). Therapy studies in subjects bearing either Ramos or Granta subcutaneous lymphomas demonstrated that 2H7-Fc-C825 PRIT is highly effective and significantly less myelosuppressive than 1F5-SA (P < 0.0001). All animals receiving optimal doses of 2H7-Fc-C825 followed by 90Y-DOTA were cured by 150 days, whereas the growth of tumors in control animals progressed rapidly with complete morbidity by 25 days. In addition to demonstrating reduced risk of immunogenicity and an absence of endogenous biotin interference, our findings offer a preclinical proof of concept for the preferred use of bispecific PRIT in future clinical trials, due to a slightly superior biodistribution profile, less myelosuppression, and superior efficacy. Cancer Res; 76(22); 6669-79. ©2016 AACR.

Osseoconductivity of a Specific Streptavidin-Biotin-Fibronectin Surface Coating of Biotinylated Titanium Implants - A Rabbit Animal Study.

BACKGROUND: Biofunctionalized implant surfaces may accelerate bony integration and increase long-term stability. PURPOSE: The aim of the study was to evaluate the osseous reaction toward biomimetic titanium implants surfaces coated with quasicovalent immobilized fibronectin in an in vivo animal model. MATERIALS AND METHODS: A total of 84 implants (uncoated [control 1, n = 36], streptavidin-biotin coated [test 1, n = 24], streptavidin-biotin-fibronectin coated [test 2, n = 24]) were inserted 1 mm supracortically in the proximal tibia of 12 rabbits. The samples were examined after 3 and 6 weeks. Total bone-implant contact (tBIC; %), bone-implant contact in the cortical (cBIC; %) and in the spongious bone (sBIC; %) as well as the percentage of linear bone fill (PLF; %) were evaluated. RESULTS: After 3 weeks, streptavidin-biotin-fibronectin implants had a significant higher sBIC (p = .043) and PLF (p = .007) compared with the uncoated samples. After 6 weeks, this difference was significant for tBIC (p = .016) and cBIC (p < .001). Additionally, uncoated screws showed a significant higher sBIC when compared with the fibronectin coating (p < .001). Streptavidin-biotin-coated implants showed less bone growth at both time points of all examined parameters when compared with their counterparts (all p < .001). CONCLUSIONS: Quasicovalent immobilization of biotinylated fibronectin with the streptavidin-biotin-fibronectin system on smooth surface titanium shows a beneficial faster osseous healing in vivo. Besides, an antifouling effect of the streptavidin-biotin coating was proven.

Targeted cell uptake of a noninternalizing antibody through conjugation to iron oxide nanoparticles in primary central nervous system lymphoma.

BACKGROUND: At present there is no standard of care for patients with primary central nervous system lymphoma (PCNSL) because of the difficulty in delivering therapeutically effective doses of drugs to the intracellular site of the target PCNSL. Here we report the use of an iron oxide nanoparticle to promote the internalization of a PCNSL targeting antibody by target cells. METHODS: Iron oxide nanoparticles coated with a copolymer of chitosan-grafted polyethylene glycol (NPs) were conjugated with an anti-CD20 single-chain variable fragment-streptavidin fusion protein (FP), and optically activated with Oregon Green 488. The ability of NP-FP to target PCNSL cells was assessed using flow cytometry and the ferrozine assay. Cell internalization of NP-FP was examined by confocal fluorescence microscopy. RESULTS: The antibody-conjugated NPs had a near-neutral zeta potential and remained stable in biological media for more than 1 week, which may minimizes nonspecific cell uptake. The diameter of the NPs was about 70 nm, which is in an optimal range for maximizing cell uptake. The selective binding of these NPs was demonstrated with binding to PCNSL cells 3- to 4-fold higher than binding to control cells. Z-stack imaging by confocal microscopy revealed the NPs were internalized by PCNSL cells. CONCLUSIONS: The high-degree specific binding and cell uptake of NP-FP in PCNSL suggests this NP formulation can be further developed to improve therapy of PCNSL.

[Intravesical anchoring of streptavidin-tagged interleukin-4 fusion protein for immunotherapy of mouse superficial bladder cancer].

OBJECTIVE: To evaluate the antitumor efficacy of streptavidin-tagged interleukin-4 (IL-4-SA) bifunctional fusion protein in the immunotherapy of mouse model of superficial bladder cancer. METHODS: IL-4-SA fusion protein was prepared and its biological activity was determined. One day after MB49 cell implantation, 100 µl of 1 mg/ml NHS-PEO4-biotin was instilled into the bladder for 30 minutes, followed by intravesical instillation of 100 µl PBS, GFP-SA+IL-4 or IL-4-SA and incubation for 1 hour. The bladder irrigation was performed twice a week for three weeks. The CTL cytotoxicity and profile of CD8(+) tumor-infiltrating lymphocytes were analyzed. RESULTS: The IL-4-SA fusion protein was durably anchored to the biotinylated mucosal surface of bladder wall for up to 5 days.On day 80 after the implantation of MB49 cells, all of PBS-treated mice died, and 8 out of 10 mice in the GFP-SA-treated group died from tumor burden.In contrast, 5 out of 10 mice in the IL-4-SA-treated group were tumor-free. The MB49 tumor-specific cytotoxicity from mice in the IL-4-SA group was (11.3 ± 1.2)%, (22.7 ± 1.5)% and (31.0 ± 3.0)% at the effector to target ratios of 1:1, 25:1 and 50:1, respectively. But the corresponding cytotoxicity was (4.3 ± 0.6)%, (9.0 ± 1.0)% and (14.3 ± 1.5)% in the GFP-SA+IL-4 group, and (3.3 ± 0.6)%, (7.3 ± 0.6)%, (12.7 ± 2.1)% in the PBS group. The tumor-specific cytotoxicity in the SA-CD40L group was significantly higher than that in the control groups (P < 0.05). The infiltrating CD8(+) T cells in tumors in the IL-4-SA-treated group were increased compare with those in other groups. CONCLUSION: Intravesical anchoring of IL-4-SA elicites strong and long-lasting immunoprotection against superficial bladder cancer, and the novel immunotherapy may be an attractive therapeutic alternative in future.

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.

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro.

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.

[Immobilization of streptavidin-tagged bioactive hTNF-alpha on biotinylated mucosal surface of the bladder wall for treatment of superficial bladder cancer in mice].

OBJECTIVE: To investigate a novel immunotherapy through immobilization of streptavidin-tagged hTNF-alpha on the biotinylated mucosal surface of the bladder wall for bladder cancer treatment in mice. METHODS: A total of 120 female C57BL/6j mice were randomized into 5 equal groups, namely blank control, PBS, soluble hTNF-alpha, SA-GFP, and SA-hTNF-alpha treatment groups. Twenty-four hours after establishment of a mouse model of orthotopic superficial bladder cancer, SA-hTNF-alpha fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall, which was repeated every 4 days for a total of 6 sessions. Immunohistochemistry was performed to detect the retention time of SA-hTNF-alpha fusion protein in the biotinylated mouse bladder mucosa and the distribution of CD4(+) and CD8(+) lymphocytes in the mucosa and tumor tissues, with the tumor growth and mouse survival also observed. The cytotoxiciy of the tumor-specific lymphocytes was evaluated. The mice responding well to the treatment were re-challenged by MB49 and monitored for survival. RESULTS: SA-hTNF-alpha could be efficiently and stably immobilized on the bladder mucosal surface for as long as 7 days. On day 60 after MB49 implantation, 18 out of 22 SA- hTNF-alpha-treated mice survived, with 9 appearing tumor-free, but all the mice in PBS control group died. Five out of 9 tumor-free mice in SA-hTNF-alpha group showed resistance to a re-challenge with intravesical MB49. The numbers of CD4(+) and CD8(+) lymphocytes were significantly greater in SA-hTNF-alpha group than in the other groups (P<0.05). The cytotoxicity of the tumor-specific lymphocytes was significantly stronger in SA-hTNF-alpha group than in the other groups (P<0.05). CONCLUSION: SA-hTNF-alpha immobilized on the biotinylated mucosal surface of the bladder wall can significantly inhibit the tumor growth and promote the survival of the mice bearing orthotopic superficial bladder cancer.

A novel immunotherapy for superficial bladder cancer by the immobilization of streptavidin-tagged bioactive IL-2 on the biotinylated mucosal surface of the bladder wall.

BACKGROUND AND OBJECTIVE: Intravesical administration of Bacillus Calmette-Guerin (BCG) after transurethral resection is by far the most effective local therapy for superficial bladder cancer, the fifth most common cancer in the world. However, approximately one-third of patients fail to respond and most patients eventually relapse. In addition, there are pronounced side effects of BCG therapy, such as BCG sepsis and a high frequency of BCG-induced cystitis. This study established a novel immunotherapy through immobilization of streptavidin-tagged human IL-2 (SA-hIL-2) on the biotinylated mucosal surface of bladder wall. METHODS: A mouse orthotopic model of MB49 bladder cancer was established by perfusing MB49 cells into mouse bladders. The SA-hIL-2 fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall. Treatment began on day 1 after MB49 implantation, once every 3 days for 6 times. Immunohistochemical assay was performed to assess the persistence of SA-hIL-2 immobilized on the biotinylated mucosal surface of the bladder wall. The mice were monitored for tumor growth and survival. On day 60 after MB49 implantation, the SA-hIL-2-cured mice, which were found to have no hematuria or palpable tumors, were challenged with wild-type MB49 cells implanted into the pretreated bladder and monitored for survival. RESULTS: SA-hIL-2 could be immobilized efficiently and durably on the bladder mucosal surface as long as 7 days. On day 60 after MB49 implantation, 9 out of 20 SA-hIL-2-treated mice survived, but all mice in PBS control group died. More importantly, 5 out of 9 tumor-free mice in the SA-hIL-2 group were protected against a second intravesical wild-type MB49 tumor challenge. CONCLUSIONS: SA-hIL-2 fusion protein could significantly inhibit tumor growth and extend the survival time in the orthotopic model of MB49 bladder cancer.

In vivo delivery of antisense MORF oligomer by MORF/carrier streptavidin nanoparticles.

Tumor targeting by oligomers is largely limited by the pharmacokinetics and cell-membrane transport obstacles. In this article, we describe the use of a delivery nanoparticle, in which streptavidin served as a convenient bridge between a biotinylated oligomer and a biotinylated cell-membrane-penetrating peptide, to improve the delivery of an antisense phosphorodiamidate morpholino (MORF) oligomer in vivo. A biotinylated (99m)Tc-radiolabeled MORF oligomer with a base sequence antisense to the RIalpha mRNA and its sense control were incorporated separately into nanoparticles, along with biotinylated tat or polyarginine carrier. The streptavidin nanoparticles were administrated intravenously to both normal and nude mice bearing SUM149 breast tumor xenografts. The biodistributions showed much higher normal tissue levels for the radiolabeled MORFs, independent of antisense or sense or tat or polyarginine, when administered as the nanoparticles, compared to naked. A statistically significant higher accumulation of both antisense nanoparticles, compared to the respective sense control nanoparticles, was observed, along with much higher tumor accumulations, compared to historical naked controls. This study has provided evidence that the in vivo function of an antisense oligomer within the streptavidin nanoparticle is not impeded, and, as such, the MORF/streptavidin/carrier nanoparticles may be suitable for in vivo tumor delivery of antisense MORF and other oligomers.

Pretargeted radioimmunotherapy.

This brief review covers the concept of pretargeted radioimmunotherapy and summarize the results obtained in preclinical animal models and initial phase I clinical trials. Reagents studied have been a bifunctional antibody prepared by crosslinking Fab' fragments from two antibodies with different specificity, one binding the target antigen expressed on tumors and the other binding a radiolabeled peptide. The alternative system is a conjugate of streptavidin linked to the pretargeting agent and radiolabeled biotin. After reaching optimal tumor targeting of the pretargeting agent, a synthetic mono-biotin poly N-acetyl-galactosamine compound was used to clear unbound targeting agent from the circulation before the injection of radiolabeled biotin. Promising therapeutic responses were obtained in various tumor xenograft models in athymic nude mice. A phase I study of an anti-CD20/streptavidin pretargeting agent and 15 mCi/m(2)(90)Y-biotin produced objective responses with minimal toxicity among lymphoma patients, with an average tumor-to-whole-body radiation dose ratio of 49. Pretargeting radioimmunotherapy approaches have shown higher tumor-to-whole-body ratios than that usually obtained with one-step radioimmunotherapy.

Novel preparation and characterization of a trastuzumab-streptavidin conjugate for pre-targeted radionuclide therapy.

INTRODUCTION: This study describes a novel and convenient route for the preparation of a trastuzumab-streptavidin conjugate such as might be used in a pre-targeting system and its in-vitro and in-vivo evaluation. METHODS: Trastuzumab was irradiated with UV light in the presence of stannous ions to reduce a number of the disulfide bridges to free thiol groups. A range of irradiation times were studied in order to quantify the number of thiols produced and to optimize the reduction process. The conjugate was then prepared by reaction with succinimidyl 4-(N-maleimidomethy cyclohexane)-1-carboxylate (SMCC)-linked streptavidin. RESULTS: Initial conjugation reactions in phosphate buffer were inefficient, producing low conjugate yields, but conjugation reactions in triethanolamine-based buffer showed greatly increased conjugation yields. A high purity product (approximately 100%) was obtained following purification by gel-filtration HPLC as determined by subsequent size exclusion HPLC analysis. The conjugate was shown to possess an essentially identical immunoreactivity to that of the native, unconjugated antibody and an unaltered biotin binding stoichiometry. Shedding and internalization by Her-2-expressing cells were low and the uptake in vivo by Her-2-expressing xenografts in nude mice was similar to that of labelled antibody. CONCLUSION: Our results demonstrate a new, simple and effective method for the successful synthesis of antibody-streptavidin conjugates which could also be applied to many other heterodimeric protein conjugation reactions.

Pretargeted radioimmunotherapy with a single-chain antibody/streptavidin construct and radiolabeled DOTA-biotin: strategies for reduction of the renal dose.

UNLABELLED: Multistep immune targeting holds great promise for radioimmunodiagnosis and therapy of cancer. Pretargeting of the tetrameric single-chain, variable-fragment streptavidin construct of the tetrameric monoclonal antibody CC49 with subsequent administration of radiolabeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-biotin has yielded promising results in TAG-72-expressing tumor xenograft models. A potential limitation of this approach, however, has been high and prolonged renal uptake of radioactivity. The objective of the current study, therefore, was to evaluate the reduction of kidney uptake of radiolabeled DOTA-biotin achieved by each of 4 different methods. METHODS: A human pancreatic adenocarcinoma xenograft model (HPAC) in nude mice was used. The animals were intravenously injected with the antibody-streptavidin construct and a synthetic clearing agent (biotinylated N-acetyl-galactosamine) 24 and 4 h, respectively, before the administration of (67)Ga-DOTA-biotin. For reduction of the renal uptake, different groups of mice were treated with streptavidin saturated with biotin, with several administrations of lysine or colchicine or with a succinylated antibody-streptavidin construct (resulting in a decreased electrical charge). All animals were sacrificed 24 h after injection of the (67)Ga-DOTA-biotin for biodistribution and quantitative autoradiography (QAR) studies and selected animals underwent microSPECT/microCT studies. RESULTS: There was marked targeting of the radiolabeled DOTA-biotin to tumor in all groups except in negative-control animals. Only succinylation of the scFv-CC49-streptavidin fusion protein significantly reduced ( approximately 30%) kidney uptake without affecting tumor activity. QAR corroborated these results and demonstrated that radiolabeled DOTA-biotin localized selectively in the renal cortex. Among the other experimental groups, there was no change in kidney uptake of the radiolabeled biotin. CONCLUSION: In contrast to directly labeled antibodies and antibody fragments, administration of the negatively charged amino acid lysine was largely ineffective in pretargeting strategies with a single-chain-immuno-streptavidin fusion protein. Succinylation of the scFv-CC49-streptavidin construct, on the other hand, reduces kidney uptake of subsequently administered radiolabeled biotin, presumably by inhibiting reuptake of the fusion protein in the proximal renal tubules, and, therefore, could significantly reduce renal doses and improve therapeutic indices associated with multistep immune targeting approaches to radioimmunotherapy.

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.

Effect of streptavidin on cardiac allograft prolongation is due to host T-Cell suppression.

AIM: The aim of this study was to evaluate the effectiveness of streptavidin immunomodulation in the high-responder WF-to-Lewis combination. METHODS/RESULTS: We examined the effects of streptavidin on the proliferative response of T cells in coculture studies. Two to 200 microg/mL streptavidin significantly (P < .001) suppressed the proliferation of Lewis T cells to WF by 76%-83% compared with untreated responders. Next, we studied the survival of WF cardiac allografts in Lewis recipients pretreated with streptavidin. A 5-day course of peritransplantation recipient treatment with streptavidin doses of 8, 12, 20, 40, and 60 mg/kg combined with single dose of 0.5 mL antilymphocyte serum (ALS) significantly (P < .001) prolonged cardiac allograft survival from MST of 7 +/- 0.5 and 8 +/- 0.5 days in naive and ALS-treated controls to 15 +/- 1, 20 +/- 3, 16 +/- 3, 17 +/- 3, and 23 +/- 2 days, respectively. In contrast, posttransplantation administration of 80 mg/kg streptavidin resulted in animal death, suggesting toxicity of this dose. Additionally, 10 mg/kg or 20 mg/kg streptavidin administration for 10 consecutive days resulted in significant graft prolongation (MST of 18 +/- 1 and 21 +/- 1 days, respectively; P < .001). CONCLUSION: Although peritransplantation streptavidin treatment is effective in prolonging rat cardiac allografts in the high-responder WF-to-Lewis combination, it does not induce permanent graft survival as observed in the low-responder combination of Lewis-to-ACI. Our finding of in vitro immunomodulatory effect of streptavidin on T-cell proliferation suggests that its in vivo effect is partly due to prevention of T-cell activation following antigen exposure.

Recent advances in pretargeted radioimmunotherapy.

Pretargeted delivery of radionuclides is based upon bispecific immunoconjugates that bind a target tumor antigen and a small molecule carrying the active payload. This strategy is supposed to combine the advantage of antibodies to track tumor cells in vivo and of small radiolabeled molecules that clear rapidly from normal organs and minimize toxicity. Many pretargeting approaches have been proposed, but only those using the biotin/avidin recognition system and those using bispecific anti-tumor x anti-hapten antibodies have been tested in the clinic for both immunoscintigraphy and radioimmunotherapy. Their respective advantages and drawbacks, as well as hurdles in the way of an effective therapy against solid tumors, are discussed. In the light of the encouraging results obtained so far in the clinic, pretargeting remains a most promising challenge for chemistry and biotechnology.

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.