In Soo Shin

Multimodal Nanoprobes for Radionuclide and Five-Color Near-Infrared Optical Lymphatic Imaging

Current contrast agents generally have one function and can only be imaged in monochrome; therefore, the majority of imaging methods can only impart uniparametric information. A single nanoparticle has the potential to be loaded with multiple payloads. Such multimodality probes have the ability to be imaged by more than one imaging technique, which could compensate for the weakness or even combine the advantages of each individual modality. Furthermore, optical imaging using different optical probes enables us to achieve multicolor in vivo imaging, wherein multiple parameters can be read from a single image. To allow differentiation of multiple optical signals in vivo, each probe should have a close but different near-infrared emission. To this end, we synthesized nanoprobes with multimodal and multicolor potential, which employed a polyamidoamine dendrimer platform linked to both radionuclides and optical probes, permitting dual-modality scintigraphic and five-color near-infrared optical lymphatic imaging using a multiple-excitation spectrally resolved fluorescence imaging technique.

In vivo Diagnosis of Epidermal Growth Factor Receptor Expression using Molecular Imaging with a Cocktail of Optically Labeled Monoclonal Antibodies

Purpose: Epidermal growth factor receptors (EGFR) play an important role in tumorigenesis and, therefore, have become targets for new molecular therapies. Here, we use a “cocktail” of optically labeled monoclonal antibodies directed against EGFR-1 (HER1) and EGFR-2 (HER2) to distinguish tumors by their cell surface expression profiles. Experimental Design:In vivo imaging experiments were done in tumor-bearing mice following s.c. injection of A431 (overexpressing HER1), NIH3T3/HER2+ (overexpressing HER2), and Balb3T3/DsRed (non-expression control) cell lines. After tumor establishment, a cocktail of optically labeled antibodies: Cy5.5-labeled cetuximab (anti-HER1) and Cy7-labeled trastuzumab (anti-HER2) was i.v. injected. In vivo and ex vivo fluorescence imaging was done. For comparison with radionuclide imaging, experiments were undertaken using 111Indium-labeled antibodies. Additionally, a “blinded” diagnostic study was done for mice bearing one tumor type. Results:In vivo spectral fluorescent molecular imaging of 14 mice with three tumor types clearly differentiated tumors using the cocktail of optically labeled antibodies both in vivo and ex vivo. Twenty-four hours after injection, A431 and NIH3T3/HER2+ tumors were detected distinctly by their peak on Cy5.5 and Cy7 spectral images, respectively; radionuclide imaging was unable to clearly distinguish tumors at this time point. In blinded single tumor experiments, investigators were able to correctly diagnose a total of 40 tumors. Conclusion: An in vivo imaging technique using an antibody cocktail simultaneously differentiated two tumors expressing distinct EGFRs and enabled an accurate characterization of each subtype.

Noninvasive in vivo imaging of CD4 cells in simian-human immunodeficiency virus (SHIV)–infected nonhuman primates

Since the earliest days of the HIV epidemic, the number of CD4(+) T cells per unit volume of blood has been recognized as a major prognostic factor for the development of AIDS in persons with HIV infection. It has also been generally accepted that approximately 2% of total body lymphocytes circulate in the blood. In the present study, we have used a nondepleting humanized anti-CD4 monoclonal antibody labeled with the gamma emitter indium-111 to visualize the CD4(+) T-cell pool in vivo in nonhuman primates with simian HIV infection. A strong correlation was noted between radiotracer uptake in spleen, tonsil, axillary lymph nodes, and peripheral blood CD4 T-cell counts (rho = 0.75, 0.93, and 0.85, respectively, P < .005). The relationship between radiotracer retention in lymphoid tissues and CD4(+) T-cell counts in the circulation was governed by an exponential law. These data provide an estimate for the total number of lymphocytes in the body as being between 1.9 and 2.9 x 10(12) and suggest that the partition between peripheral blood and lymphoid tissue is between 0.3% and 0.5%.

Pulsed high intensity focused ultrasound increases penetration and therapeutic efficacy of monoclonal antibodies in murine xenograft tumors

The success of radioimmunotherapy for solid tumors remains elusive due to poor biodistribution and insufficient tumor accumulation, in part, due to the unique tumor microenvironment resulting in heterogeneous tumor antibody distribution. Pulsed high intensity focused ultrasound (pulsed-HIFU) has previously been shown to increase the accumulation of (111)In labeled B3 antibody (recognizes Lewis(y) antigen). The objective of this study was to investigate the tumor penetration and therapeutic efficacy of pulsed-HIFU exposures combined with (90)Y labeled B3 mAb in an A431 solid tumor model. The ability of pulsed-HIFU (1 M Hz, spatial averaged temporal peak intensity=2685 W cm(-2); pulse repetition frequency=1 Hz; duty cycle=5%) to improve the tumor penetration and therapeutic efficacy of (90)Y labeled B3 mAb ((90)Y-B3) was evaluated in Le(y)-positive A431 tumors. Antibody penetration from the tumor surface and blood vessel surface was evaluated with fluorescently labeled B3, epi-fluorescent microscopy, and custom image analysis. Tumor size was monitored to determine treatment efficacy, indicated by survival, following various treatments with pulsed-HIFU and/or (90)Y-B3. The pulsed-HIFU exposures did not affect the vascular parameters including microvascular density, vascular size, and vascular architecture; although 1.6-fold more antibody was delivered to the solid tumors when combined with pulsed-HIFU. The distribution and penetration of the antibodies were significantly improved (p-value<0.05) when combined with pulsed-HIFU, only in the tumor periphery. Pretreatment with pulsed-HIFU significantly improved (p-value<0.05) survival over control treatments.

Effect of chelator conjugation level and injection dose on tumor and organ uptake of 111In-labeled MORAb-009, an anti-mesothelin antibody.

INTRODUCTION Radiolabeling of a monoclonal antibody (mAb) with a metallic radionuclide requires the conjugation of a bifunctional chelator to the mAb. The conjugation, however, can alter the physical and immunological properties of the mAb, consequently affecting its tumor-targeting pharmacokinetics. In this study, we investigated the effect of the amount of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriamine-pentaacetic acid (CHX-A″) conjugated to MORAb-009, a mAb directed against mesothelin, and the effect of MORAb dose on the biodistribution of (111)In-labeled MORAb-009. METHODS We used nude mice bearing the A431/K5 tumor as a mesothelin-positive tumor model and the A431 tumor as a mesothelin-negative control. To find the optimal level of CHX-A″ conjugation, CHX-A″-MORAb-009 conjugates with 2.4, 3.5 and 5.5 CHX-A″ molecules were investigated. To investigate the effect of injected MORAb-009 dose on neutralizing the shed mesothelin in the circulation, biodistribution studies were performed after the intravenous co-injection of (111)In-labeled MORAb-009 (2.4 CHX-A″/MORAb-009) with three different doses: 0.2, 2 and 30 μg of MORAb-009. RESULTS The tumor uptake in A431/K5 tumor was four times higher than that in A431 tumor, indicating that the tumor uptake in A431/K5 was mesothelin mediated. The conjugate with 5.5 CHX-A″ showed a lower isoelectric point (pI) and lower immunoreactivity (IR) than the 2.4 CHX-A″ conjugate. These differences were reflected in the biodistribution of the (111)In label. The (111)In-labeled MORAb-009 conjugated with 2.4 CHX-A″ produced higher tumor uptake and lower liver and spleen uptakes than the 5.5 CHX-A″ conjugate. The biodistribution studies also revealed that the tumor uptake was significantly affected by the injected MORAb-009 dose and tumor size. The 30-μg dose produced higher tumor uptake than the 0.2- and 2-μg doses, whereas the 30-μg dose produced lower liver and spleen uptakes than the 0.2-μg dose. CONCLUSION This study demonstrates that the number of chelate conjugation and the injected dose are two important parameters to achieve high tumor and low non-target organ uptake of (111)In-labeled MORAb-009. This study also suggests that the injected dose of mAb could be individualized based on the tumor size or the blood level of shed antigen in a patient to achieve the ideal tumor-to-organ radioactivity ratios.

Combined-modality radioimmunotherapy: synergistic effect of paclitaxel and additive effect of bevacizumab

INTRODUCTION This study was undertaken to investigate the effect of paclitaxel and bevacizumab on the therapeutic efficacy of (90)Y-labeled B3 monoclonal antibody, directed against Le(y) antigen, for the treatment of Le(y)-positive A431 tumors implanted subcutaneously in the right hind flank of nude mice. METHODS When the tumor size reached ~200 mm(3), the mice received a single dose of intravenous (iv) (90)Y-labeled B3 (60 μCi/150 μg or 100 μCi/150 μg B3), intraperitoneal paclitaxel (40 mg/kg) or iv bevacizumab (5 mg/kg) for monotherapy. To investigate the effect of combined therapies on survival, the mice were treated with two or three agents in the following combinations: (90)Y-B3 on day 0 and paclitaxel on day 1; bevacizumab on -1 day and (90)Y-B3 on day 0; bevacizumab on -1 day and paclitaxel on day 1; bevacizumab, (90)Y-B3 and paclitaxel each at 1-day intervals. The mice with no treatment were used as a control. The tumor volume at 1000 mm(3) was used as a surrogate end point of survival. RESULTS Compared to control animals, paclitaxel delayed tumor growth with a significantly longer median survival time (P<.001), whereas bevacizumab alone showed a less pronounced effect on a median survival time (P=.18). (90)Y-B3 increased the median survival time in a dose-dependent manner (P<.05). The combined therapy of bevacizumab with paclitaxel produced a trend toward an increase of the median survival time compared to paclitaxel alone (P=.06), whereas bevacizumab combined with (90)Y-B3 showed a statistically insignificant increase in the median survival time compared to (90)Y-B3 alone (P=.25). The tumor sizes of all animals in these groups reached the surrogate end point of survival by day 35. In contrast, the combined therapy involving (90)Y-B3 with paclitaxel showed a striking synergistic effect in shrinking tumors and prolonging the survival time (P<.001); on day 120, three of nine mice (33%) and six of six mice (100%) were alive without tumor when treated with 60 μCi (90)Y-B3 and 100 μCi (90)Y-B3, respectively. The addition of bevacizumab treatment 1 day before the combined therapy of 60 μCi (90)Y-B3 with paclitaxel did not produce a statistically significant increase in survival when compared to the (90)Y-B3 with paclitaxel (P>.10). Fluorescence microscopy analysis indicated that paclitaxel increased, whereas bevacizumab decreased, the accumulation and penetration of Alexa Fluor 647-B3 into tumor microenvironment compared to the control (P<.05). CONCLUSION Our findings on the paclitaxel effect support a hypothesis that the increased tumor accumulation and penetration of (90)Y-B3 as well as the high radiosensitization of tumor cells by paclitaxel may be the major factors responsible for the synergistic effect of the combined therapy involving (90)Y-B3 with paclitaxel.

99mTc-labeling of Peptidomimetic Antagonist to Selectively Target αvβ3 Receptor-Positive Tumor: Comparison of PDA and EDDA as co-Ligands

Objectives: The aim of this research was to synthesize radiolabeled peptidomimetic integrin αvβ3 antagonist with 99mTc for rapid targeting of integrin αvβ3 receptors in tumor to produce a high tumor to background ratio. Methods: The amino terminus of 4-[2-(3,4,5,6-tetra-hydropyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N-(3-amino-- neopenta-1-carbamyl)]-aminoethylsulfonyl- amino-β-alanine hydrochloride (IAC) was conjugated with N-hydroxysuccinimide ester of HYNIC and labeled with 99mTc using tricine with either 1,5-pyridinedicarboxylic acid (PDA) or ethylenediamine-N,N-diacetic acid (EDDA) as the co-ligand. The products, 99mTc EDDA2/HYNIC-IAC (P1) and 99mTc PDA (tricin)/HYNIC-IAC (P2) were subjected to in vitro serum stability, receptor-binding, biodistribution and imaging studies. Results: P1 and P2 were synthesized with an overall yield of > 80%. P1 was slightly more stable than P2 when incubated in serum at 37 °C for 18 hrs (84 vs 77% intact). The In vitro receptor-binding of P1 was higher than that of P2 (78.02 ± 13.48 vs 51.05 ± 14.05%) when incubated with αvβ3 at a molar excess (0.8 μM). This receptor binding was completely blocked by a molar excess of an unlabeled peptidomimetic antagonist. Their differences shown in serum stability and the receptor-binding appeared to be related to their biological behaviors in tumor uptake and retention; the 1 h tumor uptakes of P1 and P2 were 3.17±0.52 and 2.13±0.17 % ID/g, respectively. P1 was retained in the tumor longer than P2. P1 was excreted primarily through the renal system whereas P2 complex was excreted equally via both renal and hepatobiliary systems. Thus, P1 was retained in the whole-body with 27.25 ± 3.67% ID at 4 h whereas 54.04 ± 3.57% ID of P2 remained in the whole-body at 4 h. This higher whole-body retention of P2 appeared to be resulted from a higher amount of radioactivity retained in liver and intestine. These findings were supported by imaging studies showing higher tumor-to-abdominal contrast for P1 than for P2 at 3 h postinjection. Conclusions: P1 showed good tumor targeting properties with a rapid tumor uptake, prolonged tumor retention and fast whole-body clearance kinetics. These findings warrant further investigation of the HYNIC method of 99mTc labeling of other peptidomimetic antagonists using EDDA as a coligand.

Biodistribution of 99m Tc Labeled Integrin Antagonist

The selective targeting of an integrin αvβ3 receptor using radioligands may enable the assessment of angiogenesis and integrin αvβ3 receptor status in tumors. The aim of this research was to label a peptidomimetic integrin αvβ3 antagonist (PIA) with 99mTc(CO)3 and to test its receptor targeting properties in nude mice bearing receptor-positive tumors. PIA was reacted with tris-succinimidyl aminotriacetate (TSAT) (20 mM) as a PIA per TSAT. The product, PIA-aminodiacetic acid (ADA), was radiolabeled with [99mTc(CO)3(H2O)3]+1, and purified sequentially on a Sep-Pak C-18 cartridge followed by a Sep-Pak QMA anion exchange cartridge. Using gradient C-18 reverse-phase HPLC, the radiochemical purity of 99mTc(CO)3-ADA-PIA (retention time, 10.5 min) was confirmed to be > 95%. Biodistribution analysis was performed in nude mice (n = 5 per time point) bearing receptor-positive M21 human melanoma xenografts. The mice were administered 99mTc(CO)3-ADA-PIA intravenously. The animals were euthanized at 0.33, 1, and 2 hr after injection for the biodistribution study. A separate group of mice were also co-injected with 200 μg of PIA and euthanized at 1 hr to quantify tumor uptake. 99mTc(CO)3-ADA-PIA was stable in phosphate buffer for 21 hr, but at 3 and 6 hr, 7.9 and 11.5% of the radioactivity was lost as histidine, respectively. In tumor bearing mice, 99mTc(CO)3-ADA-PIA accumulated rapidly in a receptor-positive tumor with a peak uptake at 20 min, and rapid clearance from blood occurring primarily through the hepatobiliary system. At 20 min, the tumor-toblood ratio was 1.8. At 1 hr, the tumor uptake was 0.47% injected dose (ID)/g, but decreased to 0.12% ID/g when co-injected with an excess amount of PIA, indicating that accumulation was receptor mediated. These results demonstrate successful 99mTc labeling of a peptidomimetic integrin antagonist that accumulated in a tumor via receptor-specific binding. However, tumor uptake was very low because of low blood concentrations that likely resulted from rapid uptake of the agent into the hepatobiliary system. This study suggests that for 99mTc(CO)3-ADA-PIA to be useful as a tumor detection agent, it will be necessary to improve receptor binding affinity and increase the hydrophilicity of the product to minimize rapid hepatobiliary uptake.