Antibody mimetic drug conjugate manufactured by high-yield Escherichia coli expression and non-covalent binding system.

Antibody-drug conjugates (ADCs) are a major therapeutic tool for the treatment of advanced cancer. Malignant cells in advanced cancer often display multiple genetic mutations and become resistant to monotherapy. Therefore, a therapeutic regimen that simultaneously targets multiple molecules with multiple payloads is desirable. However, the development of ADCs is hampered by issues in biopharmaceutical manufacturing and the complexity of the conjugation process of low-molecular-weight payloads to biologicals. Here, we report antibody mimetic-drug conjugates (AMDCs) developed by exploiting the non-covalent binding property of payloads based on high-affinity binding of mutated streptavidin and modified iminobiotin. Miniprotein antibodies were fused to a low immunogenic streptavidin variant, which was then expressed in Escherichia coli inclusion bodies, solubilized, and refolded into functional tetramers. The AMDC developed against human epidermal growth factor receptor 2 (HER2) effectively killed cultured cancer cells using bis-iminobiotin conjugated to photo-activating silicon phthalocyanine. The HER2-targeting AMDC was also effective in vivo against a mouse KPL-4 xenograft model. This AMDC platform provides rapid, stable, and high-yield therapeutics against multiple targets.

A strategy for iron oxide nanoparticles to adhere to the neuronal membrane in the substantia nigra of mice.

Effective attachment of magnetic nanoparticles to neuronal membranes has far-reaching significance in activating ion channels and treating neurodegenerative diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) synthesized by the polyol pyrolysis method have the advantages of rich surface functional groups, excellent magnetic properties, controllable particle size and water dispersibility. We propose that perfusion of biotin into the targeted brain area should be initially performed because it tends to be adsorbed by cell membranes, followed by injection of streptavidin (SA)-modified SPIONs into the same area of the brain. By means of the strong binding force between SA and biotin, the SPIONs may subsequently adhere to the cell surfaces in the brain area. In this work, fluorescein isothiocyanate-streptavidin (FITC-SA) was modified on the surface of polyethylene imine (PEI)-SPIONs by the EDC-NHS method and stereotaxically injected into the biotin-supplemented substantia nigra of mice. The combination of fluorescence detection with transmission electron microscopy (TEM) confirmed that FITC-SA/PEI-SPIONs adhered to neuronal membranes in the substantia nigra of mice 24 h after injection. The results show that our strategy can promote the attachment of SPIONs to neuronal membranes.

Mimicking the human environment in mice reveals that inhibiting biotin biosynthesis is effective against antibiotic-resistant pathogens.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets1. In Mycobacteria tuberculosis and Francisella tularensis, biotin biosynthesis is a key fitness determinant during infection2-5, making it a high-priority target. However, biotin biosynthesis has been overlooked for priority pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. This can be attributed to the lack of attenuation observed for biotin biosynthesis genes during transposon mutagenesis studies in mouse infection models6-9. Previous studies did not consider the 40-fold higher concentration of biotin in mouse plasma compared to human plasma. Here, we leveraged the unique affinity of streptavidin to develop a mouse infection model with human levels of biotin. Our model suggests that biotin biosynthesis is essential during infection with A. baumannii, K. pneumoniae and P. aeruginosa. Encouragingly, we establish the capacity of our model to uncover in vivo activity for the biotin biosynthesis inhibitor MAC13772. Our model addresses the disconnect in biotin levels between humans and mice, and explains the failure of potent biotin biosynthesis inhibitors in standard mouse infection models.

Interference of High Dose Biotin Supplementation with Thyroid Parameters in Immunoassays Utilizing the Interaction between Streptavidin and Biotin: a Case Report and Review of Current Literature.

BACKGROUND: Automated immunoassays utilizing the interaction between streptavidin and biotin are widely used. Nonetheless, biotin remains an often overlooked confounder. METHODS: We report the case of a 54-year-old female patient with progressive multiple sclerosis and Hashimoto's thyroiditis who presented herself for a follow-up. Measurements on Roche's cobas® 8000 modular analyzer series suggested severe hyperthyroidism. Initially, no relevant confounders could be identified. RESULTS: All requested thyroid parameters were measured with alternative methods, yielding plausible results. CONCLUSIONS: Biotin is a significant confounder in many immunoassays. Alternative measurement methods or methods of biotin neutralization need to be implemented for certain situations.

A facile approach to functionalizing cell membrane-coated nanoparticles with neurotoxin-derived peptide for brain-targeted drug delivery.

The blood brain barrier separates the circulating blood from the extracellular fluid in the central nervous system and thus presents an essential obstacle to brain transport of therapeutics. Herein, we report on an effective brain-targeted drug delivery system that combines a robust red blood cell membrane-coated nanoparticle (RBCNP) with a unique neurotoxin-derived targeting moiety. The RBCNPs retain the complex biological functions of natural cell membranes while exhibiting physicochemical properties that are suitable for effective drug delivery. CDX peptide is derived from candoxin and shows high binding affinity with nicotinic acetylcholine receptors (nAChRs) expressed on the surface of brain endothelial cells. Through a facile yet robust approach, we successfully incorporate DCDX peptides onto the surface of RBCNPs without compromising the peptide's brain targeting ability. The resulting DCDX-RBCNPs show promising brain targeting efficiency both in vitro and in vivo. Using a glioma mouse model, we demonstrate that doxorubicin-loaded DCDX-RBCNPs have superior therapeutic efficacy and markedly reduced toxicity as compared to the nontargeted drug formulations. While RBCNPs are used as a model system to evaluate the surface modification approach, the reported method can be readily generalized to various types of cell membrane-derived nanocarriers for broad medical applications.

PBCA-based polymeric microbubbles for molecular imaging and drug delivery.

Microbubbles (MB) are routinely used as contrast agents for ultrasound (US) imaging. We describe different types of targeted and drug-loaded poly(n-butyl cyanoacrylate) (PBCA) MB, and demonstrate their suitability for multiple biomedical applications, including molecular US imaging and US-mediated drug delivery. Molecular imaging of angiogenic tumor blood vessels and inflamed atherosclerotic endothelium is performed by modifying the surface of PBCA MB with peptides and antibodies recognizing E-selectin and VCAM-1. Stable and inertial cavitation of PBCA MB enables sonoporation and permeabilization of blood vessels in tumors and in the brain, which can be employed for direct and indirect drug delivery. Direct drug delivery is based on US-induced release of (model) drug molecules from the MB shell. Indirect drug delivery refers to US- and MB-mediated enhancement of extravasation and penetration of co-administered drugs and drug delivery systems. These findings are in line with recently reported pioneering proof-of-principle studies showing the usefulness of (phospholipid) MB for molecular US imaging and sonoporation-enhanced drug delivery in patients. They aim to exemplify the potential and the broad applicability of combining MB with US to improve disease diagnosis and therapy.

Annexin V imaging detects diabetes-accelerated apoptosis and monitors the efficacy of benfotiamine treatment in ischemic limbs of mice.

The role of apoptosis imaging for monitoring treatment response in ischemic limbs has not been properly explored. In this study, we investigated the ability of annexin V (AnxV) imaging to assess the efficacy of antiapoptotic treatment in ischemic limbs of diabetic mice. Normal C57BL/6 mice and streptozotocin-induced diabetic mice were subject to hindlimb ischemia. AnxV-conjugated fluorescent streptavidin probes were intravenously injected, and optical imaging was performed. Tissue apoptosis was quantified by histochemistry and Western blotting. The AnxV probes showed specific targeting to apoptotic cells on confocal microscopy and flow cytometry. Intravenous AnxV probes displayed substantially greater accumulation in ischemic limbs of diabetic mice. Benfotiamine (BFT) treatment of diabetic mice led to better perfusion recovery on laser Doppler imaging and reduced AnxV binding on optical imaging. TUNEL staining and cleaved caspase-3 Western blots confirmed accelerated apoptosis by diabetes and its suppression by BFT treatment. Furthermore, AnxV-SAv-PEcy5.5 uptake in the ischemic limbs closely correlated to cleaved caspase-3 expression. Thus, AnxV imaging may be useful for monitoring the efficacy of therapeutic agents designed to suppress ischemia-induced apoptosis.

Streptavidin: a novel immunostimulant for the selection and delivery of autologous and syngeneic tumor vaccines.

Induction of antitumor immunity using autologous tumor proteins is an attractive approach to cancer therapy. However, better methods and stimulants to present these autologous proteins back to the immune system are needed. Here, we identify streptavidin as a novel carrier protein and stimulant, and test the efficacy of both syngeneic (rat) and autologous vaccines (dogs) using streptavidin in combination with reduced soluble tumor proteins. Initial syngeneic vaccine studies in the 9L rat glioma model were used to optimize vaccine dose and selectivity. Cytokine and blood analysis was used to monitor the response. Rats receiving two vaccinations of syngeneic tumor vaccine demonstrated a statistically significant (P < 0.05) survival advantage compared with controls (adjuvant only). Notably, vaccination also led to remission rates of between 30% and 60% in the aggressive 9L glioma model. Antibodies to streptavidin were detected in the serum of vaccinated rats; however, antibody levels did not correlate with the response. The cytokine TNF-α was upregulated in vaccine-treated rats, whereas ICAM1 was downregulated. After engraftment, vaccinated rats maintained CD4(+), CD8(+) T cells, and total lymphocyte levels closer to normal baseline than those in the controls. Twenty-five dogs treated with autologous vaccine preparations using streptavidin as a stimulant showed no adverse reactions, irrespective of additional chemotherapy and other medications. In this study, we developed a novel method for producing syngeneic and autologous vaccines using streptavidin selectivity and immunogenicity. These vaccines show efficacy in the 9L glioma rat model. Safety was also demonstrated in canine patients presenting with cancer treated with autologous vaccine.

Rapid and serum-insensitive endocytotic delivery of proteins using biotinylated polymers attached via multivalent hydrophobic anchors.

We have designed biotinylated polymers as synthetic receptors that have multiple alkyl groups for endocytotic delivery of target proteins. The polymers were stably attached to a cell surface via multivalent anchoring. The presented biotin was bound to streptavidin (SA) on the cell surface, and, via an endocytotic pathway, the cell rapidly internalized the biotinylated polymer/SA complex. The cell's uptake of the complex was not inhibited by the presence of 10% fetal bovine serum, and its efficacy for the uptake of SA was the highest when compared with commercial reagents and single-anchored-type synthetic receptors. The synthetic receptor-mediated endocytosis can be used generally for other kind of protein by using SA as an adaptor molecule between a target protein and the cell-surface presented biotin.

Killed Bacillus subtilis spores expressing streptavidin: a novel carrier of drugs to target cancer cells.

Carriers of drugs in cancer therapy are required to reduce side-effects of the drugs to normal cells. Here we constructed killed recombinant Bacillus subtilis spores (SA1) that expressed streptavidin as a chimeric fusion to the spore coat protein CotB and used the spores as bioparticle carrier. When bound with biotinylated cetuximab these spores could specifically target to the epidermal growth factor receptor on HT 29 colon cancer cells, thereby delivered paclitaxel to the cells with 4-fold higher efficiency, as indicated by fluorescent intensity of paclitaxel Oregon Green 488 bound to HT29 cells. Based on real-time monitoring of cell index, the IC50 of growth of HT29 cells by paclitaxel-SA1-cetuximab was estimated to be 2.9 nM approximately 5-fold lower than water-soluble paclitaxel (14.5 nM). Instability of DNA content was observed when cells were treated with 16 nM paclitaxel-SA1-cetuximab, resulting in a 2-fold enhancement in polyploidy cells. Thus, by targeting the release of paclitaxel to HT29 cells, spore-associated cetuximab augmented the inhibitory effect of paclitaxel on cell division and proliferation. The SA1 could be used as a "universal" drug carrier to target specific biomarkers on cancer cells by conjugating with suitable biotinylated antibodies.

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.

Her2/neu small interfering RNA delivered in culture by a streptavidin nanoparticle.

A three-component nanoparticle consisting of biotinylated Trastuzumab antiHer2 antibody, tat transferring peptide and radiolabeled antisense oligomer, linked together through streptavidin, have shown promise in the delivery to Her2+ tumor in mice following intravenous administration and with evidence of radiotherapeutic efficacy. These results have encouraged us to consider the nanoparticle as a delivery vehicle for RNA interference therapy in which the radiolabeled antisense oligomer is replaced with an unlabeled siRNA duplex. The siRNA stability within the nanoparticle was first confirmed by incubation with RNase A. The interferon responses, that indicate off-target cytotoxicity, were evaluated by quantitative real-time RT-PCR in BT-474 (Her2+) human breast cancer cells by measuring the mRNA expression of 2', 5'-oligoadenylate synthetase (OAS1) and Stat-1, two key interferon-responsive genes. Thereafter the cytotoxicity induced by the siRNA nanoparticle was evaluated by a clonogenic survival assay in BT-474 cells while the Her2 expression of these target cells was evaluated for evidence of specific gene silencing. The siRNA within the three-component anti- Her2/neu siRNA nanoparticle was largely protected from RNase-dependent degradation and did not activate an interferon response. The nanoparticle effectively and significantly inhibited colony formation of the target cells and silenced the Her2 gene expression at 5 nM compared with the identical nanoparticle with a scrambled siRNA. Our delivery nanoparticle, with tumor targeting provided by the antibody and its accumulation without entrapment, possibly due to the transfecting peptide, delivered an siRNA duplex to the proper subcellular localization for specific and effective gene silencing in culture by what appears to be an siRNA mechanism.

Comparing the intracellular fate of components within a noncovalent streptavidin nanoparticle with covalent conjugation.

INTRODUCTION: Auger radiotherapy requires adequate tumor delivery and high nuclear accumulation and retention. We hypothesize that the noncovalent nature of a streptavidin/biotin three-component nanoparticle possessing these qualities may be required for dissociation of the radiolabeled oligomer and its accumulation into the cell nucleus. METHODS: As a test of our hypothesis, the intracellular fate of an antisense oligomer when incubated as the nanoparticle and when incubated while covalently conjugated to the antibody was compared. The three-component noncovalent nanoparticle consisted of streptavidin linking three biotinylated components: a Cy3-labeled anti-RIα antisense phosphorodiamidate morpholino (MORF) oligomer, a tat transfecting peptide and the anti-Her2 herceptin antibody. The covalent constructs included an anti-RIα antisense DNA conjugated to a radiolabeled herceptin and a fluorescent DNA conjugated to native herceptin. Fluorescence microscopy in SK-BR-3 (Her2+) cells was used to evaluate the fate of the fluorescent Cy5.5-DNA and Cy3-MORF, while the subcellular accumulation of the (111)In-labeled herceptin and herceptin-DNA in both SK-BR-3 and MDA-MB-231 (Her2) cells was determined by isolating and counting the nuclear fractions. RESULTS: Previously, we demonstrated that when incubated as the three-component nanoparticle consisting of herceptin and streptavidin and (99m)Tc-labeled antisense MORF, only the MORF accumulated in the nucleus of Her2+ cells. In this investigation, clear evidence was observed of nuclear accumulation of the antisense oligomer within the noncovalent nanoparticle as before, but when incubated as the covalent construct, by both fluorescence microscopy and nuclear counting, no evidence of nuclear accumulation was observed. CONCLUSION: The weaker noncovalent biotin-streptavidin bond may be essential for adequate delivery of the radiolabeled antisense oligomer to the nucleus of tumor cells.

Modulation of the humoral immune response by targeting CD40 and FcγRII/III; delivery of soluble but not particulate antigen to CD40 enhances antibody responses with a Th1 bias.

Targeted delivery of antigen improves immunogenicity and can obviate the use of adjuvants. In addition to molecular targeting based on affinity interactions, particle-based antigen targeting to myeloid cells is also an efficient means to enhance immune responses. We compared the efficiency of targeting a model antigen, streptavidin, to CD40 and low affinity Fc gamma receptors II and III, either in a soluble or in a particulate form. Single chain fragments targeting these receptors were used to generate soluble tetramers with streptavidin or to decorate streptavidin coated nanobeads, and mice were immunized with the different formulations. Whereas particulate presentation of streptavidin enhanced total IgG1 and IgG2a levels, overall antigen specific antibody production increased in the case of targeted soluble antigen only, as assessed by reverse protein arrays and ELISPOT. In particular, soluble CD40 targeted antigen induced the strongest IgG2a responses, suggesting a Th1 bias compared to FcgammaRII/III targeting. Combined targeting to these receptors did not further increase immunogenicity. Thus, in our model, affinity targeting of soluble antigen to CD40 proved to be superior to particle-mediated delivery both in terms of antibody quantity and quality.

Controlled release of substances bound to fibrin-anchors or of DNA.

Fibrin sealants have been proposed as depot matrices for substances due to their biocompatibility, advantageous biological properties, and widespread use in wound healing. This study showed possibilities for a continuous and controlled release of pharmaceutically active substances out of a fibrin matrix. Substances of interest were linked to naturally occuring fibrin-anchors, (i) thrombin, (ii) fibronectin, and (iii) DNA. Fibronectin and thrombin bind fibrin by a specific binding moiety and DNA by charge. Fibrin clots were prepared from Tisseel Fibrin Sealant (Baxter AG, Vienna) by mixing 100 mg/ml fibrinogen, the substance of interest, and 4 U/ml of thrombin. Chemical crosslinking of proteins was performed with EDC using standard reaction conditions. Modification of proteins with biotin and PPACK was performed with N-hydroxysuccinimid activated compounds. With fibrin-anchors pharmaceutically active substances, i.e., tumor necrosis factor (TNF), albumin, and plasmid-DNA, were continously released over 10 days. In conclusion, the naturally occuring proteins fibronectin and thrombin with a fibrin binding moiety or DNA can be used as fibrin-anchors.

Auger radiation-induced, antisense-mediated cytotoxicity of tumor cells using a 3-component streptavidin-delivery nanoparticle with 111In.

UNLABELLED: When antisense oligomers are intracellular, they migrate to and are retained in the nucleus of tumor cells and therefore may be used to carry Auger electron-emitting radionuclides such as (111)In for effective tumor radiotherapy. METHODS: Our nanoparticle consists of streptavidin that links 3 biotinylated components: the antiHer2 antibody trastuzumab (to improve pharmacokinetics), the tat peptide (to improve cell membrane transport), and the (111)In-labeled antiRIalpha messenger RNA antisense morpholino (MORF) oligomer. RESULTS: As evidence of unimpaired function, tumor cell and nuclear accumulations were orders of magnitude higher after incubation with (99m)Tc-MORF/tat/trastuzumab than after incubation with free (99m)Tc-MORF and significantly higher with the antisense than with the sense MORF. In mice, tumor and normal-tissue accumulations of the (99m)Tc-MORF/tat/trastuzumab nanoparticle were comparable to those of free (99m)Tc-trastuzumab, confirming the improved pharmacokinetics due to the trastuzumab component. Although kidneys, liver, and other normal tissues also accumulated the nanoparticle, immunohistochemical evaluation of tissue sections in mice receiving the Cy3-MORF/tat/trastuzumab nanoparticle showed evidence of nuclear accumulation only in tumor tissue. In a dose escalation study, as measured by the surviving fraction, the nanoparticle significantly increased the kill of SK-BR-3 breast cancer Her2+/RIalpha+ cells, compared with all controls. CONCLUSION: Significant radiation-induced antisense-mediated cytotoxicity of tumor cells in vitro was achieved using an Auger electron-emitting antisense MORF oligomer administered as a member of a 3-component streptavidin-delivery nanoparticle.

Carbohydrate dependent targeting of cancer cells by bleomycin-microbubble conjugates.

Biotinylated bleomycin A(5) was attached to streptavidin-derivatized microbubbles, and a solution containing the conjugate was passed over a monolayer of cultured MCF-7 cells. The bleomycin-derivatized microbubbles adhered to the MCF-7 cells, and the association could be monitored by the use of a microscope. Three other cancer cell lines gave similar results. The bleomycin-microbubble conjugate did not bind to a normal breast cell line (MCF-10A) or to the matched noncancer cell lines corresponding to the other cancer cell lines targeted by bleomycin. No binding to any tested cell line was observed when the microbubbles lacked conjugated bleomycin A(5) or when the microbubble contained a bleomycin A(5) analogue lacking the carbohydrate moiety.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules.

BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.

Targeted magnetic resonance imaging with intraperitoneal and intravenous streptavidin (SA)-DTPA-Gd: a comparative study in tumor-bearing nude mice.

OBJECTIVE: To investigate the effect of streptavidin (SA)-DTPA-Gd after intraperitoneal and intravenous administration for tumor enhancement in targeted magnetic resonance imaging (MRI). METHODS: Biotinylated monoclonal antibody CL3 (600 microg) was intravenously injected into 12 BALB/c nude mice with subcutaneous inoculation of LoVo cells, followed by administration of 80 microg avidin as the chaser 24 h later and then SA-DTPA-Gd was injected intravenously or intraperitoneally after another 30 min. MRI was performed before and 20, 60 min and 3, 6, 9, 12 h after the injection of the contrast agents, and the MR signal intensity of the tumor and liver was determined. RESULT: The maximum enhancement ratio of the tumor was 70.2% in the intravenous injection group and 46.4% in the intraperitoneal group, showing significant difference between them. The maximal enhancement rate of the liver was 23.7% in the intraperitoneal group and 20.4% in the intravenous group, showing no significant difference. CONCLUSION: MR targeted imaging with biotinylated monoclonal antibody CL3 and SA-DTPA-Gd has specific enhancement effect. Higher blood level of SA-DTPA-Gd in the intravenous group facilitates the tumor enhancement in MRI in subcutaneous tumor model.