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The aim of the study is to evaluate the pharmacokinetics and microbiodistribution of Cu-labeled collagen-binding peptides.The affinity constant (KD), association (ka), and dissociation rate constant (kd) for the peptide collagelin or its analog (named CRPA) binding to collagen were measured by biolayer interferometric analysis. Rats (n=4-5) with myocardial infarction or normal were injected intravenously with the Cu-labeled peptides or Cu-DOTA as a control. Dynamic PET imaging was performed for 60 min at 7-8 weeks after infarct. Fluorine-18 fluorodeoxyglucose PET imaging was performed to identify the viable myocardium. To validate the PET images, slices of heart samples from the base to the apex were analyzed using autoradiography and histology.The peptides bound to collagen with a KD of ∼0.9 µmol/l. The Cu-peptides and Cu-DOTA accumulated in the infarct area (confirmed by autoradiography and histology images) within 1 min of injection and were excreted rapidly through the renal system. The blood clearance curves were biphasic with elimination half-lives of 21.9±2.4, 26.2±4.6, and 21.2±2.1 min for Cu-CRPA, Cu-collagelin, and the control Cu-DOTA, respectively. The clearance half-lives from the focal fibrotic tissue (24.1±1.5, 25.6±8.0, and 21.4±1.3 min, respectively) and remote myocardium (20.8±0.7, 21.0±5.5, and 19.1±2.4 min, respectively) were not significantly different. The uptake ratios of infarct-to-remote myocardium (1.93±0.18, 2.15±0.38, and 1.88±0.08, respectively) for Cu-CRPA, Cu-collagelin, and Cu-DOTA remained stable for the time period between 10 and 60 min.The distribution of the Cu-collagelin probes corresponds to the heterogeneous distribution of expanded extracellular space in the setting of myocardial infarction. The overall washout rate from the fibrous tissue was determined by the slow washout rate (t1/2≥20 min) of the peptides from the extracellular space to the vasculature, not by the dissociation rate (t1/22 min) of the Cu-peptides from collagen.

Objectives To investigate the effect of the injection dose of MORAb-009 (amatuximab, an anti-mesothelin monoclonal antibody), the tumor size and the level of shed mesothelin on the uptake of the antibody in mesothelin-positive tumor and organs by biodistribution (BD) and positron emission tomography (PET) imaging studies. Methods 2- S -(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid ( p -SCN-Bn-NOTA) was conjugated to amatuximab and labeled with 64 CuCl 2 in 0.25M acetate buffer, pH4.2. The resulting 64 Cu-NOTA-amatuximab was purified with a PD 10 column. To investigate the dose effect or the effect of tumor size, the BD was performed in groups of nude mice (n=5) with mesothelin-expressing A431/H9 tumors (range, 80–300mm 3 ) one day after iv injection of 64 Cu-NOTA-amatuximab (10μCi) containing a total amatuximab dose of 2, 30, or 60μg. The BD and PET imaging were also investigated 3, 24 and 48h after injecting a total dose of 30μg (10μCi for BD), and 2 or 60μg (300μCi for PET), respectively. Results Comparing the results of the BDs from three different injection doses, the major difference was shown in the uptake (%ID/g) of the radiolabel in tumor, liver and blood. The tumor uptake and blood retention from 30 and 60μg doses were greater than those from 2μg dose, whereas the liver uptake was smaller. The BD studies also demonstrated a positive correlation between tumor size (or the level of shed mesothelin in blood) and liver uptake. However, there was a negative correlation between tumor size (or the shed mesothelin level) and tumor uptake and between tumor size and blood retention. These findings were confirmed by the PET imaging study, which clearly visualized the tumor uptake with the radiolabel concentrated in the tumor core and produced a tumor to liver ratio of 1.2 at 24h post-injection with 60μg amatuximab, whereas the injection of 2μg amatuximab produced a tumor to liver ratio of 0.4 at 24h post-injection. Conclusion Our studies using a nude mouse model of A431/H9 tumor demonstrated that the injection of a high amatuximab dose (30 to 60μg) could provide a beneficial effect in maximizing tumor uptake while maintaining minimum liver and spleen uptakes of the radiolabel, and in facilitating its penetration into the tumor core.

The goal of cancer immunotherapy is to stimulate the host immune system to attack malignant cells. Antibody-dependent cellular cytotoxicity (ADCC) is a pivotal mechanism of antitumor action of clinically employed antitumor antibodies. IL-15 administered to patients with metastatic malignancy by continuous i.v. infusion at 2 μg/kg/d for 10 days was associated with a 38-fold increase in the number and activation status of circulating natural killer (NK) cells and activation of macrophages which together are ADCC effectors. We investigated combination therapy of IL-15 with rituximab in a syngeneic mouse model of lymphoma transfected with human CD20 and with alemtuzumab (Campath-1H) in a xenograft model of human adult T cell leukemia (ATL). IL-15 greatly enhanced the therapeutic efficacy of both rituximab and alemtuzumab in tumor models. The additivity/synergy was shown to be associated with augmented ADCC. Both NK cells and macrophages were critical elements in the chain of interacting effectors involved in optimal therapeutic responses mediated by rituximab with IL-15. We provide evidence supporting the hypothesis that NK cells interact with macrophages to augment the NK-cell activation and expression of FcγRIV and the capacity of these cells to become effectors of ADCC. The present study supports clinical trials of IL-15 combined with tumor-directed monoclonal antibodies.

We investigated the effect of shed antigen mesothelin on the tumor uptake of amatuximab, a therapeutic anti-mesothelin mAb clinically tested in mesothelioma patients. The B3 mAb targeting a nonshed antigen was also analyzed for comparison. The mouse model implanted with A431/H9 tumor, which expresses both shed mesothelin and nonshed Lewis-Y antigen, provided an ideal system to compare the biodistribution and PET imaging profiles of the two mAbs. Our study demonstrated that the tumor and organ uptakes of 89Zr-B3 were dose-independent when 3 doses, 2, 15, and 60 μg B3, were compared at 24 h after injection. In contrast, tumor and organ uptakes of 89Zr-amatuximab were dose-dependent, whereby a high dose (60 μg) was needed to achieve tumor targeting comparable to the low dose (2 μg) of 89Zr-B3, suggesting that shed mesothelin may affect amatuximab tumor targeting as well as serum half-life. The autoradiography analysis showed that the distribution of 89Zr-B3 was nonuniform with the radioactivity primarily localized at the tumor periphery independent of the B3 dose. However, the autoradiography analysis for 89Zr-amatuximab showed dose-dependent distribution profiles of the radiolabel; at 10 μg dose, the radiolabel penetrated toward the tumor core with its activity comparable to that at the tumor periphery, whereas at 60 μg dose, the distribution profile became similar to those of 89Zr-B3. These results suggest that shed antigen in blood may act as a decoy requiring higher doses of mAb to improve serum half-life as well as tumor targeting. Systemic mAb concentration should be at a severalfold molar excess to the shed Ag in blood to overcome the hepatic processing of mAb-Ag complexes. On the other hand, mAb concentration should remain lower than the shed Ag concentration in the tumor ECS to maximize tumor penetration by passing binding site barriers.

We investigated the effect of shed antigen mesothelin on the tumor uptake of amatuximab, a therapeutic anti-mesothelin mAb clinically tested in mesothelioma patients. The B3 mAb targeting a nonshed antigen was also analyzed for comparison. The mouse model implanted with A431/H9 tumor, which expresses both shed mesothelin and nonshed Lewis-Y antigen, provided an ideal system to compare the biodistribution and PET imaging profiles of the two mAbs. Our study demonstrated that the tumor and organ uptakes of 89Zr-B3 were dose-independent when 3 doses, 2, 15, and 60 μg B3, were compared at 24 h after injection. In contrast, tumor and organ uptakes of 89Zr-amatuximab were dose-dependent, whereby a high dose (60 μg) was needed to achieve tumor targeting comparable to the low dose (2 μg) of 89Zr-B3, suggesting that shed mesothelin may affect amatuximab tumor targeting as well as serum half-life. The autoradiography analysis showed that the distribution of 89Zr-B3 was nonuniform with the radioactivity primarily localized at the tumor periphery independent of the B3 dose. However, the autoradiography analysis for 89Zr-amatuximab showed dose-dependent distribution profiles of the radiolabel; at 10 μg dose, the radiolabel penetrated toward the tumor core with its activity comparable to that at the tumor periphery, whereas at 60 μg dose, the distribution profile became similar to those of 89Zr-B3. These results suggest that shed antigen in blood may act as a decoy requiring higher doses of mAb to improve serum half-life as well as tumor targeting. Systemic mAb concentration should be at a severalfold molar excess to the shed Ag in blood to overcome the hepatic processing of mAb-Ag complexes. On the other hand, mAb concentration should remain lower than the shed Ag concentration in the tumor ECS to maximize tumor penetration by passing binding site barriers.

The radiolabeling of the liposome surface can be a useful tool for in vivo tracking of therapeutic drug loaded liposomes. We investigated radiolabeling therapeutic drug (i.e. an antibiotic, amikacin) loaded liposomes with (99m)Tc, nebulization properties of (99m)Tc-labeled liposomal amikacin for inhalation ((99m)Tc-LAI), and its stability by size exclusion low-pressure liquid chromatography (LPLC). LAI was reacted with (99m)Tc using SnCl2 dissolved in ascorbic acid as a reducing agent for 10 min at room temperature. The labeled products were then purified by anion exchange resin. The purified (99m)Tc-LAI in 1.5% NaCl solution was incubated at 4 °C to assess its stability by LPLC. The purified (99m)Tc-LAI was subjected to studies with a clinically used nebulizer (PARI eFlow®) and the Anderson Cascade Impactor (ACI). The use of ascorbic acid at 0.91 mM resulted in a quantitative labeling efficiency. The LPLC profile showed that the liposomal peak of LAI detected by a UV monitor at both 200 nm and 254 nm overlapped with the radioactivity peak of (99m)Tc-LAI, indicating that (99m)Tc-LAI is suitable for tracing LAI. The ACI study demonstrated that the aerosol droplet size distribution determined gravimetrically was similar to that determined by radioactivity. The liposome surface labeling method using SnCl₂ in 0.91 mM ascorbic acid produced (99m)Tc-LAI with a high labeling efficiency and stability that are adequate to evaluate the deposition and clearance of inhaled LAI in the lung by gamma scintigraphy.

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.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.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).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.

Radioimmunotherapy of cancer with radiolabeled antibodies has shown promise. We evaluated an anti-CD25 monoclonal Antibody, 7G7/B6, armed with (90)Y as a potential radioimmunotherapeutic agent for CD25-expressing lymphomas.The lymphoma model was established by subcutaneous injection of 1 x 10(7) SUDHL-1 cells into nude mice. The biodistribution of (111)In-7G7/B6 and therapeutic studies with (90)Y-7G7/B6 were performed in the tumor-bearing mice.Therapy using (90)Y-7G7/B6 prolonged survival of the SUDHL-1 lymphoma-bearing mice significantly, as compared with either untreated mice or the mice treated with (90)Y-11F11, a radiolabeled isotype-matched control antibody (p0.001). All of the mice in the control and the (90)Y-11F11 treatment groups died by days 18 and 24, respectively. In contrast, 30% of the mice in the low-dose group (75 microCi of (90)Y-7G7/B6/mouse) and 75% in the high-dose group (150 microCi of (90)Y-7G7/B6/mouse) became tumor free and remained healthy for greater than 6 months.Our findings suggested that (90)Y-7G7/B6 is a potentially useful radioimmunotherapeutic agent for the treatment of patients with CD25-expressing lymphomas.

IL-15 has potential as an immunotherapeutic agent for cancer treatment because it is a critical factor for the proliferation and activation of natural killer (NK) and CD8 + T cells. Administration of anti-CD40 antibodies has shown anti-tumor effects in vivo through a variety of mechanisms. Furthermore, activation of CD40 led to increased expression of IL-15 receptor-α by dendritic cells, an action that is critical for trans-presentation of IL-15 to NK and CD8 + T cells. In this study, we investigated the therapeutic efficacy of the combination regimen of murine IL-15 (mIL-15) with an agonistic anti-CD40 antibody (FGK4.5) in murine lung metastasis models involving CT26 and MC38, which are murine colon cancer cell lines syngeneic to BALB/c and C57BL/6 mice, respectively. Treatment with mIL-15 or the anti-CD40 antibody alone significantly prolonged survival of both CT26 and MC38 tumor-bearing mice compared with the mice in the PBS solution control group ( P < 0.01). Furthermore, combination therapy with both mIL-15 and the anti-CD40 antibody provided greater therapeutic efficacy as demonstrated by prolonged survival of the mice compared with either mIL-15 or the anti-CD40 antibody-alone groups ( P < 0.001). We found that NK cells isolated from the mice that received the combination regimen expressed increased levels of intracellular granzyme B and showed stronger cytotoxic activity on the target cells. The findings from this study provide the scientific basis for clinical trials using the combination regimen of IL-15 with an anti-CD40 antibody for the treatment of patients with cancer.

CD30 is a member of the TNF receptor superfamily. Overexpression of CD30 on some neoplasms versus limited expression on normal tissues makes this receptor a promising target for antibody-based therapy. Radioimmunotherapy of cancer with radiolabeled antibodies has shown promise. In this study, we evaluated the therapeutic efficacy of an anti-CD30 antibody, HeFi-1, armed with 211 At in a leukemia (karpas299) model and with 90 Y in a lymphoma (SUDHL-1) model. Furthermore, we investigated the combination therapy of 211 At-HeFi-1 with unmodified HeFi-1 in the leukemia model. Treatment with unmodified HeFi-1 significantly prolonged the survival of the karpas299-bearing mice compared with the controls ( P < 0.001). Treatment with 211 At-HeFi-1 showed greater therapeutic efficacy than that with unmodified HeFi-1 as shown by survival of the mice ( P < 0.001). Combining these two agents further improved the survival of the mice compared with the groups treated with either 211 At-HeFi-1 ( P < 0.05) or unmodified HeFi-1 ( P < 0.001) alone. In the lymphoma model, the survival of the SUDHL-1-bearing mice was significantly prolonged by the treatment with 90 Y-HeFi-1 compared with the controls ( P < 0.001). In summary, radiolabeled HeFi-1 is very promising for the treatment of CD30-expressing leukemias and lymphomas, and the combination regimen of 211 At-HeFi-1 with unmodified HeFi-1 enhanced the therapeutic efficacy.

The aim of this study was to investigate the pharmacokinetics of (64)Cu-DOTA (1,4,7,10-azacyclododecane-N,N',N'',N'''-tetraacetic acid), a positron surrogate analog of the late gadolinium (Gd)-enhancement cardiac magnetic resonance agent, Gd-DOTA, in a rat model of chronic myocardial infarction (MI) and its microdistribution in the cardiac fibrosis by autoradiography.DOTA was labeled with (64)Cu-acetate. CD rats (n=5) with MI by left anterior descending coronary artery ligation and normal rats (n=6) were injected intravenously with (64)Cu-DOTA (18.5 MBq, 0.02 mmol DOTA/kg). Dynamic PET imaging was performed for 60 min after injection. (18)F-Fluorodeoxyglucose ([(18)F]-FDG) PET imaging was performed to identify the viable myocardium. For the region of interest analysis, the (64)Cu-DOTA PET image was coregistered to the [(18)F]-FDG PET image. To validate the PET images, slices of heart samples from the base to the apex were analyzed using autoradiography and by histological staining with Masson's trichrome.(64)Cu-DOTA was rapidly taken up in the infarct area. The time-activity curves demonstrated that (64)Cu-DOTA concentrations in the blood, fibrotic tissue, and perfusion-rich organs peaked within a minute post injection; thereafter, it was rapidly washed out in parallel with blood clearance and excreted through the renal system. The blood clearance curve was biphasic, with a distribution half-life of less than 3 min and an elimination half-life of ∼21.8 min. The elimination half-life of (64)Cu-DOTA from the focal fibrotic tissue (∼22.4 min) and the remote myocardium (∼20.1 min) was similar to the blood elimination half-life. Consequently, the uptake ratios of focal fibrosis-to-blood and remote myocardium-to-blood remained stable for the time period between 10 and 60 min. The corresponding ratios obtained from images acquired from 30 to 60 min were 1.09 and 0.59, respectively, indicating that the concentration of (64)Cu-DOTA in the focal fibrosis was 1.85 (1.09/0.59) times greater than that in the remote myocardium. Thus, this finding indicates that the extracellular volume fraction was 1.85 times greater in the focal fibrosis than in the remote myocardium. The accumulation of (64)Cu-DOTA in fibrotic tissue was further supported by autoradiography and histology images. The autoradiography images of (64)Cu-DOTA in the fibrotic tissues were qualitatively superimposed over the histology images of the fibrotic tissues. The histology images of the infarct areas were characterized by a heterogeneous distribution of thin bands of fibrotic collagen, myocytes, and expanded extracellular space.(64)Cu-DOTA is a useful surrogate positron analog of Gd-DOTA, enabling quantitative measurement of the uptake values in fibrotic tissues by dynamic PET imaging and calculation of the extracellular volume fractions of the fibrotic tissues. At a microscopic level, the distribution of (64)Cu-DOTA is nonuniform, corresponding to the heterogeneous distribution of expanded extracellular space in the setting of MI.

Radioimmunotherapy of cancer with radiolabeled antibodies has shown promise. α-Particles are very attractive for cancer therapy, especially for isolated malignant cells, as is observed in leukemia, because of their high linear energy transfer and short effective path length. We evaluated an anti-CD25 [interleukin-2 receptor α (IL-2Rα)] monoclonal antibody, 7G7/B6, armed with 211At as a potential radioimmunotherapeutic agent for CD25-expressing leukemias and lymphomas. Therapeutic studies were done in severe combined immunodeficient/nonobese diabetic mice bearing the karpas299 leukemia and in nude mice bearing the SUDHL-1 lymphoma. The results from a pharmacokinetic study showed that the clearance of 211At-7G7/B6 from the circulation was virtually identical to 125I-7G7/B6. The biodistributions of 211At-7G7/B6 and 125I-7G7/B6 were also similar with the exception of a higher stomach uptake of radioactivity with 211At-7G7/B6. Therapy using 15 μCi of 211At-7G7/B6 prolonged survival of the karpas299 leukemia–bearing mice significantly when compared with untreated mice and mice treated with 211At-11F11, a radiolabeled nonspecific control antibody (P < 0.01). All of the mice in the control and 211At-11F11 groups died by day 46 whereas >70% of the mice in the 211At-7G7/B6 group still survived at that time. In summary, 211At-7G7/B6 could serve as an effective therapeutic agent for patients with CD25-expressing leukemias. (Cancer Res 2006; 66(16): 8227-32)

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

Adhesion molecules play a major role in the processes of invasion and metastasis of malignant tumors. Their expression within tumors has been reported to be quantitatively and qualitatively altered according to the invasiveness and metastatic potential of the tumor. The present study tested whether the intratumoral expression of integrin alpha 3 can be detected by a radiolabeled monoclonal antibody. The in vitro binding study with four different human cancer cells showed that radioiodinated GA17 antibody recognizing integrin alpha 3 bound specifically to these cells to varying degrees, according to the antigen density on each cell. The biodistribution study with 125I- and 111In-labeled antibodies showed specific localization of radiolabeled GA17 to the xenografts. However, the in vivo tumor localization was not proportional to the antigen density calculated in vitro, and antibody metabolism varied among the tumors, as was also confirmed by in vitro radionuclide retention assay. The intratumoral distribution of radioactivities varied reflecting the antigen expression within the tumor. These results indicate that 1) integrin alpha 3 was expressed in various kinds of tumors and could be localized by the radiolabeled antibody, and 2) the expression of integrin alpha 3 and the metabolism of the radiolabeled antibody after binding to the antigen within the tumor were variable among the tumors, which affected the radionuclide distribution characteristics. The expression of adhesion molecules within these tumors was noninvasively detected by a radiolabeled antibody. It may be possible to use integrin alpha 3, when it is overexpressed, as a target of therapy with antibodies radiolabeled with alpha or beta emitters.

Although development of human anti-murine immunoglobulin antibody (HAMA) is often seen in patients receiving murine antibodies, the variety of methods used for detecting HAMA makes it difficult to compare directly the HAMA responses measured by different assays. In the present study, several parameters of the HAMA response to two murine monoclonal antibodies were evaluated. The anti-sialosyl Tn antibody MLS102 and anti-CA125 antibody 145-9, which were labeled with 111In, were injected intravenously into 17 colorectal cancer patients and 11 ovarian cancer patients for immunoscintigraphy, respectively. HAMA was measured by enzyme-linked immunosorbent assay. There was no difference in baseline HAMA levels before antibody injection between the two groups. HAMA developed more frequently in ovarian cancer patients receiving the 145-9 antibody than in colorectal cancer patients receiving the MLS102 antibody (9/11 vs. 6/17, P < 0.05). No significant difference was observed in maximal HAMA levels between the two groups of patients. However, time to reach the maximal levels was delayed and the duration of the response seemed longer in ovarian cancer patients. Among 11 patients receiving the 145-9 antibody three patients became positive for HAMA more than 2 months after antibody injection and the other two had HAMA activity in their sera for more than 17 months. HAMA response was different between the two antibodies, and late onset or long duration of HAMA response against the 145-9 antibody suggests the importance of HAMA measurement in patients who receive a second injection of murine antibodies even after a long interval.

To clarify the mechanism of in vivo proliferation of adult T cell leukemia (ATL) cells, we examined the organ distribution of ATL-43T cell line cells derived from original leukemic cells in severe combined immunodeficiency (SCID) mice using radiometric techniques. First, we injected 111In-oxine-labeled ATL-43T cells into SCID and CB17 mice. On day 6, significant accumulation of radioactivity was found in the spleen and thymus of SCID mice (33.3 +/- 9.4 and 10.0 +/- 3.6% injected dose/g of tissue [%ID/g], respectively) in comparison with that in CB17 mice (19.1 +/- 2.5 and 3.7 +/- 0.9%ID/g, respectively). Next, we injected radiolabeled anti-Tac monoclonal antibody (MoAb) recognizing human interleukin-2 receptor (IL-2R) alpha chain or isotype-matched control MoAb RPC5 in SCID mice bearing ATL-43T cells 4 weeks after cell inoculation. The amounts of radioactivity found in the spleen and thymus of SCID mice injected with 125I-labeled anti-Tac MoAb (22.5 +/- 6.9 and 22.8 +/- 9.6 %ID/g, respectively) were significantly higher than those in the corresponding organs of SCID mice injected with 125I-labeled RPC5 MoAb (12.0 +/- 5.1 and 7.5 +/- 4.6 %ID/g, respectively). Similar results were obtained with 111In-labeled anti-Tac MoAb. These results were consistent with the histological findings of SCID mice bearing ATL-43T cells, indicating that ATL-43T cells infiltrated preferentially into the lymphoid organs, such as the spleen and thymus, and proliferated there. Thus, the radiometric techniques employed in this study were very useful to evaluate the proliferation sites of ATL-43T cells in SCID mice. Furthermore, this murine model could give us an opportunity to test the feasibility of therapeutic application of radiolabeled anti-Tac MoAb.

Summary In this paper, we present an algorithm for seismic source wavelet estimation that is based on seismic time frequency spectral decomposition with matching pursuit technique. The main assumption of this algorithm is that the source wavelet is stationary for single wavelet estimation in a selected time window and that the source wavelet has normalized energy to avoid scale ambiguity between reflectivity and source wavelet amplitude.

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.

Seismic trace interpolation can be formulated as an underdetermined least squares inverse problem. In order to force interpolation to follow the directions of seismic events, the weighting function is designed to build the interpolated energy along the desired direction. Usually, such a weighting function is built in the Fourier domain. However, when Fourier spectra are aliased, especially in the case of up-sampling seismic traces (creating more output than input), the design of the weighting function faces problems. In this paper, we discuss these problems and a proposed solution using the f-p domain.

CD30 is a member of the tumor necrosis factor receptor family. Overexpression of CD30 on some neoplasms versus its limited expression on normal tissues makes this receptor a promising target for antibody-based therapy. Anaplastic large-cell lymphoma (ALCL) represents a heterogeneous group of aggressive non-Hodgkin lymphomas characterized by the strong expression of CD30. We investigated the therapeutic efficacy of HeFi-1, a mouse IgG1 monoclonal antibody, which recognizes the ligand-binding site on CD30, and humanized anti-Tac antibody (daclizumab), which recognizes CD25, in a murine model of human ALCL. The ALCL model was established by intravenous injection of karpas299 cells into nonobese diabetic/severe combined immuno-deficient (SCID/NOD) wild-type or SCID/NOD Fc receptor common gamma chain-deficient (FcRgamma(-/-)) mice. HeFi-1, given at a dose of 100 microg weekly for 4 weeks, significantly prolonged survival of the ALCL-bearing SCID/NOD wild-type and SCID/NOD FcRgamma(-/-) mice (P.01) as compared with the control groups. In vitro studies showed that HeFi-1 inhibited the proliferation of karpas299 cells, whereas daclizumab did not inhibit cell proliferation. We demonstrated that the expression of FcRgamma on polymorphonuclear leukocytes and monocytes was not required for HeFi-1-mediated tumor growth inhibition in vivo, although it was required for daclizumab.

The phase migration provides an interpretation method for wide-angle data which is an alternative to more traditional methods such as modelling or inversion of arrival time information picked e.g. by visual inspection, or classical migration. The phase migration extracts detailed kinematic information from even one wide-angle shot gather by estimation of phase coherency along travel-time curves corresponding to a grid of medium points. Our results indicate that the new method can produce results in some ways superior to those from the other methods, and it is simple and not particularly computationally demanding.

Rapid blood and body clearances have hampered effective tumor targeting by small molecules. We used branched poly(ethylene glycol) (pegylated) polymers (M(r) 40,000, M(r) 70,000, M(r) 100,000, and M(r) 150,000) conjugated to tumor-specific and control peptides to assess the effect of both molecular weight and tumor specificity on pharmacokinetics and biodistribution.Pegylated specific lymphoma-binding peptide and control peptide (containing stereoisomers of proline and aspartate) were synthesized, radiolabeled with (111)In, fractionated by size, and injected into Raji lymphoma-bearing athymic mice (4-6 mice/group). Pharmacokinetics were followed for 2 days to evaluate effects of specificity and molecular size on blood clearance, body clearance, and biodistribution.As molecular size increased, blood and body clearances decreased (P0.001). The effect of molecular size on blood clearance was not altered by ligand binding specificity (P = 0.21), with t(1/2) ranging from 5.4 h (M(r) 40,000) to 17.7 h (M(r) 150,000). However, ligand specificity did alter body clearance, with pegylated control peptides clearing the body more slowly than pegylated specific peptides [P = 0.03; range, 19.1-91.3 h (specific peptides) versus 23.6-115.7 h (control peptides)]. At 24 h, there was more uptake of specific versus control pegylated peptides in tumor, liver, and marrow, but there was less uptake in kidneys, with a more pronounced difference for the higher molecular weight peptides (P0.01).These results demonstrate that the pharmacokinetics and biodistribution of peptides and resultant uptake in tumor and normal tissues can be altered by both molecular size and ligand specificity, with molecular size affecting pharmacokinetics and organ uptake in a predictable manner.

To synthesize a biotin radiopharmaceutical that clears rapidly, dendrimer was used as a carrier and conjugated with succinimidyl 3-[(125)I]iodobenzoate and tetrafluorophenyl norbiotinamidosuccinate. Then, succinic anhydride was used to reduce its pI. In mice, the non-succinylated product showed high liver (67% ID/g) and kidney (44% ID/g) uptakes and whole-body retention (94% ID) at 20 min that persisted for 12 hr. The corresponding organ uptakes (22% and 11% ID/g) and the whole-body retention (47% ID) were drastically reduced by succinylation (p0.0001). Lysine co-injection further lowered renal uptake.

We investigated and compared the biodistribution of Albuleukin, a human serum albumin (HSA)-interleukin-2 (IL-2) fusion protein, with those of IL-2 and HSA. The objective was to determine whether Albuleukin distributes differently to normal organs and lymphoid tissues than IL-2 by virtue of its genetic fusion with HSA.The chelating agent 2-( p-isothiocyanato-benzyl)-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-A(II) was selected for radiolabeling with (111)In, and conjugation with CHX-A(II) did not alter bioactivities of IL-2 and Albuleukin on proliferation of CTLL-2 cells. The radiolabeled proteins were injected intravenously into mice, uptake in organs was measured, and whole-body autoradiography was performed.Striking differences in the biodistribution of IL-2 and Albuleukin were noted. (111)In-IL-2 cleared from blood rapidly, with less than 1% ID/g (percentage of injected dose per gram of tissue) at 20 min after injection. At this time, the kidneys showed more than 120% ID/g uptake, and these high levels persisted through 6 h. (111)In-Albuleukin, by contrast, showed significantly longer circulation (14% ID/g at 6 h), lower kidney uptake (6% ID/g), and higher localization in liver, spleen, and lymph nodes (maximal uptake approximately 22% ID/g for all three organs). Uptake in liver, spleen, and lymph nodes appears to be mediated in part by the IL-2 component of Albuleukin because (111)In-HSA showed significantly lower accumulation in those tissues despite more prolonged circulation in blood.These data support the hypothesis that Albuleukin targets tissues where lymphocytes reside to a much greater extent than does IL-2, and suggest that Albuleukin may exhibit improved efficacy and reduced toxicity in the treatment of solid tumors. Clinical trials underway will determine whether the improved targeting in the mice translates into a better therapeutic index in humans.

We investigated the biodistribution of (88)Y/(111)In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin and therapy with (90)Y-labeled DOTA-biotin in tumor-bearing mice after B3-streptavidin antibody conjugate (B3-SA) pretargeting. B3 antibody, recognizing Lewis(y) antigen, was conjugated to streptavidin (B3-SA). For pretargeting, 400 micro g of the B3-SA was injected i.v. into mice bearing A431 tumor xenografts. After tumor localization of B3-SA, 100 micro g of synthetic clearing agent was injected i.v. to clear the unbound B3-SA from the circulation. Four h later, 1 micro g of radiolabeled DOTA-biotin was injected i.v. Radioimmunotherapy was performed with doses of 9.25 to 37 MBq of (90)Y-labeled DOTA-biotin. As a result, radiolabeled DOTA-biotin cleared rapidly. All of the normal tissues had2.6% of the injected dose per gram, whereas tumor uptake reached approximately 15% ID/g. The total tumor uptake of radioactivity remained similar for 96 h or longer. In the first study, the median survival of the control group was 8 days, whereas it increased to163 days in the 37 MBq (90)Y group (P0.005). In a second therapy group, 7 of 10 mice receiving 37 MBq of (90)Y and B3-SA were cured, and remained healthy for180 days after therapy, compared with control groups, with/=29.2 days mean survival time (P0.001). Tumor pretargeting with B3-SA and radiolabeled DOTA-biotin has shown favorable, specific, and fast targeting that has resulted in good tumor responses and, thus, serves as a rationale for human studies with the B3-SA pretargeting approach.

We used a pretargeting technique to treat a nonobese diabetic/severe combined immunodeficient murine model of human adult T-cell leukemia with an anti-Tac antibody-streptavidin (HAT-SA) conjugate, which recognizes CD25, followed by bismuth 213 (213Bi)-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)- biotin. In the 3-step pretargeting radioimmunotherapy protocol, HAT-SA (140 or 400 μg) was administered intravenously (i.v.) to bind to the interleukin 2 receptor α (IL-2Rα; CD25)–expressing tumor cells. After 24 hours, 100 μg of a synthetic clearing agent was administered i.v. to remove unbound circulating HAT-SA conjugate from the circulation. Four hours later,213Bi–DOTA-biotin was administered i.v. for therapy. Tumor growth was significantly inhibited in 3 trials by using 250 μCi (9.25 MBq) of 213Bi–DOTA-biotin with a pretargeting technique as monitored by serum levels of soluble IL-2Rα and/or human β-2-microglobulin (P

This study was undertaken to improve the renal clearance and tumor targeting properties of 99mTc-labeled humanized anti-Tac (HuTac) monoclonal antibody Fab fragments using two chemical approaches: 1) labeling with a renal secretion agent 99mTc-mercaptoacetyltriglycine (MAG3) and 2) lowering its isoelectric point (pI) by acylation. HuTac Fab (3.3 mg/mL) was reacted with a trifluorophenyl ester (TFP) of 99mTc-MAG3 alone or was additionally reacted with TFP-glycolate to reduce the pI. In Balb/c mice, 99mTc-MAG3-Fab (pI9.3) rapidly accumulated in the kidneys (177% injected dose [ID]/g at 15 min) and then gradually cleared out of the kidneys. In contrast, the glycolation (pI 4.6 approximately 6.6) drastically reduced the renal uptake (31% ID/g) and also the whole-body retention (82% ID vs 101% for the nonglycolated) at 15 min, indicating that the glycolated 99mTc-MAG3-Fab (pI 4.6 approximately 6.6) was rapidly excreted. The glycolated remained in the blood longer than the nonglycolated (1.2% vs 0.3% ID/g at 360 min), but this effect was less drastic than the effect shown on the renal uptake. In nude mice bearing receptor-positive (ATAC4) tumors, the glycolated 99mTc-MAG3-Fab increased the peak tumor uptake to 14.8% ID/g from 8.3% ID/g for 99mTc-MAG3-Fab, whereas the glycolation resulted in a drastic reduction of the renal uptake at 15 min. We demonstrated that the renal clearance and the tumor targeting of Fab could be optimized by chemical modifications.

The detailed synthesis of the bifunctional chelating agent 2-(p-isothiocyanatobenzyl)-1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (BF_PEPA) is reported. This ligand was conjugated to monoclonal antibody B3 and the resultant immunoconjugate radiolabeled with (205,206)Bi. The in vivo stability of the radiolabeled immunoconjugate, and targeting characteristics were determined by biodistribution studies in A431 xenograft tumor-bearing mice sacrificed at 0.5, 1, 2, 4, and 24 hr. Results indicate that BF_PEPA appears to not be a suitable bifunctional chelating agent for sequestering isotopes of Bi(III) for radioimmunotherapy applications.

Using separation of variables, the wave equation in laterally inhomogeneous media can be spatially separated into two eigenequations corresponding to the direction of extrapolation and the transverse direction. The two equations are connected via eigenvalues, which leads to an eigenvalue problem represented by a second order differential equation. This equation can be solved either approximately or exactly in the Fourier domain. The solutions obtained with different approximations can lead to different methods in seismic exploration seismology.

Radioiodinated biocytin derivatives are potentially useful for multi-step tumour targeting using the avidin-biotin system. We synthesized a radioiodinated biocytin derivative and evaluated its properties in vivo. We labelled biocytin with 125 I by coupling biocytin to radioiodinated N-succinimidyl 3-(tri-n-butyl-stannyl) benzoate, and assessed its binding to avidin and its biodistribution in normal and tumour-bearing mice. When the synthesized biocytin was incubated with immobilized avidin, more than 94% of the radioactivity was bound. However, after 2 h incubation in serum, only 40% of the radioactivity was bound to the avidin. The iodinated biocytin derivative was characteristically taken up by the liver and the kidneys when injected intravenously into mice. In mice bearing an intraperitoneal tumour xenograft, 125 I-biocytin and 111 In-biotin were co-injected intraperitoneally 4 h after the intraperitoneal administration of avidin, which accumulated in the intraperitoneal tumours. At 2 and 24 h, the tumour uptake of 125 I-biocytin was 8.2 and 3.8% ID/g respectively, whereas that of 111 In-biotin was 20.0 and 18.7% ID/g respectively. When radioiodinated, biocytin retains its binding capacity to avidin, and it localizes well with high tumour-to-normal tissue ratios early post-injection using the two-step method, but compared to 111 In-biotin it is unstable. We conclude that the stability of the product in serum needs to be improved prior to in-vivo applications.

To develop chelating molecules that provide 99mTc-labeled polypeptides of high in vivo stability and high specific activities under mild reaction conditions, an asymmetrical bis(benzohydroxamamide) compound with an amine group, 4'-aminomethyl-N,N'-trimethylenedibenzohydroxamamide [NH2-C3(BHam)2], was designed and synthesized. The amine residue of NH2-C3(BHam)2 was converted to a maleimide group by reaction with N-succinimidyl-6-maleimidohexanoate, and the conjugation product was coupled to thiol groups of a monoclonal antibody against osteogenic sarcoma (OST7, IgG1) pretreated with 2-iminothiolane to prepare C3(BHam)2-OST7. 99mTc radiolabeling of C3(BHam)2-OST7 was performed by the exchange reaction with [99mTc]glucoheptonate. [99mTc]C3(BHam)2-OST7 was further characterized using directly radioiodinated OST7 ([125I]OST7) and [111In]labeled OST7 with 1-[4-[(5-maleimidopentyl)amidobenzyl]ethylenediamine-N,N, N'N'-tetraacetic acid (EMCS-Bz-EDTA) as references. [99mTc]C3(BHam)2-OST7 was obtained with radiochemical yields of over 94% at protein concentrations as low as 0.2 mg/mL at room temperature for 1 h. [99mTc]C3(BHam)2-OST7 remained stable after incubation in freshly prepared murine plasma and in the presence of cysteine. Similar binding affinities to tumor cells were observed between [99mTc]C3(BHam)2-OST7 and [125I]OST7. When injected into normal mice, [99mTc]C3(BHam)2-OST7 exhibited radioactivity levels in the blood similar to [111In]-EMCS-Bz-EDTA-OST7 up to 24 h postinjection with significantly faster elimination rate of the radioactivity from the liver. In nude mice bearing osteogenic sarcoma, no significant differences were observed in the radioactivity levels in the blood and the tumor between [99mTc]C3(BHam)2-OST7 and [125I]OST7 at 24 h postinjection. These findings indicated that C3(BHam)2 provided 99mTc chelate of high stability at low concentrations even when conjugated to an intact antibody. Such characteristics render bis(hydroxamamide) compounds useful as chelating molecules for preparation of 99mTc-labeled polypeptides.

Radiolabeled streptavidin accumulated in tumors pretargeted with biotinylated antibody. However, the absolute delivery of radioactivity was limited. To increase the tumor uptake of radioactivity further, we conjugated streptavidin with a mouse monoclonal antibody (MAb) fragment, OST6Fab, which recognizes antigen on human osteosarcoma. Another mouse MAb, OST7, which also reacts with the same tumor but recognizes an epitope different from the OST6 epitope, was biotinylated. The radioiodinated streptavidin-OST6Fab conjugate was administered to tumor-bearing mice after the biotinylated OST7 pretargeting. The uptake of the conjugate in tumors pretargeted with the biotinylated antibody was significantly higher than that of streptavidin and that of the conjugate of streptavidin and irrelevant Fab fragment. Renal uptake of radioactivity was decreased markedly, and the blood clearance was retarded by the conjugation with Fab fragment. In conclusion, the conjugate of streptavidin with specific Fab fragment increased the accumulation of radioactivity in tumors pretargeted with biotinylated antibody.

99mTc labeled galactosyl serum albumin (GSA) has been used clinically as a receptor-binding agent for the assessment of liver function. The aim of this study was to investigate the usefulness of 99mTc-GSA in intraperitoneal (i.p.) tumor imaging. A tumor model was established by i.p. inoculating nude mice with human ovarian cancer cell SHIN-3, or colon cancer cell LS 180. Radiolabels were i.p. injected into the tumor-bearing mice and the biodistribution of radioactivity was examined. After administration, 99mTc-GSA rapidly accumulated in the tumor. The tumor uptake was 5.82-8.46 %ID/g from 30 min to 6 h after the injection. Radioactivity in the blood was very low, less than 0.3 %ID/g, resulting in high tumor-to-blood ratio. Tumors could be clearly seen by scintigraphic imaging. Accumulation of i.p.-injected 99mTc labeled human serum albumin (HSA) in i.p. tumors was similar to that of 99mTc-GSA, but radioactivity of 99mTc-HSA in the circulation was high, resulting in a significantly lower tumor-to-blood ratio. In conclusion, 99mTc-GSA, when i.p. injected, accumulated in i.p. tumors and cleared from circulation rapidly, which would make it useful for the imaging of i.p. tumors.

Background: Lectins (proteins that bind specific sugar molecules on glycoproteins and glycolipids) are expressed at various levels on the surface of tumor cells. Conjugation of cytotoxic agents to glycoproteins recognized by lectins could be useful in the treatment of tumors. Avidin (a highly glycosylated, positively charged protein found in egg white) contains terminal N-acetylglucosamine and mannose residues that bind to some lectins. In this study, we tested the ability of avidin, labeled through conjugation to radioactive biotin (a B vitamin), to target intraperitoneal tumors. Methods : Biotin was radioactively labeled with 111 In. Four tumor models (one ovarian, one lung, and two colon) were established in nude mice by intraperitoneal injection of cultured cancer cells. The following two approaches were used in the intraperitoneal administration of avidin: 1) radioactive biotin-avidin conjugates were injected and 2) avidin was injected 1-24 hours before the injection of radioactive biotin (avidin pretargeting; avidin-biotin conjugates formed in vivo). The distribution of injected radioactivity in the tissues of treated animals was assessed. Results: Radiolabeled avidin localized highly and rapidly in the tumors. More than 50% of the administered dose of avidin-biotin conjugate accumulated per gram of tumor tissue 2 hours after injection; high tumor uptake of radioactivity was observed up to 24 hours after conjugate injection. In contrast, accumulation of radioactivity in normal tissues was low, yielding high tumor to nontumor ratios. With avidin pretargeting, accumulation of radioactivity in the liver, kidney, and spleen was reduced to a greater extent than that in the tumor, and tumor to nontumor ratios were increased. Conclusions: Avidin may be a promising vehicle for the delivery of radioisotopes, drugs, toxins, or therapeutic genes to intraperitoneal tumors.

In the present study, we examined the effect of avidin administered intravenously (i.v.) on the biodistribution of radiolabeled streptavidin in mice bearing intraperitoneal (IP) xenografts pretargeted with biotinylated antibody. Tumors were established in nude mice by IP inoculation of LS180 human colon cancer cells. Monoclonal antibody MLS128, which recognizes Tn antigen on mucin, was biotinylated and injected IP into the IP tumor-bearing mice. Radioiodinated streptavidin was administered IP or i.v. 48 h after pretargeting of biotinylated antibody. Avidin was administered i.v. 30 min prior to streptavidin injection. The localization of radioiodinated streptavidin in the tumor pretargeted with biotinylated antibody was significantly higher than that without pretargeting and that of radioiodinated MLS128 by the one-step method. Avidin administration significantly accelerated the clearance of radioiodinated streptavidin in blood and other normal tissues and increased the tumor-to-blood radioactivity ratio regardless of administration route of streptavidin. The i.v. avidin chase improved tumor localization of radiolabeled streptavidin in the IP xenografts pretargeted with biotinylated antibody.

The murine monoclonal antibody (mAb) 145-9 recognizes an epitope present on CA125 but different from the epitope defined by the mAb OC125. To evaluate the clinical usefulness of the 145-9 antibody, immunoscintigraphy was performed in ovarian cancer patients and the effect of circulating CA125 on tumor imaging was investigated. Two milligrams (74 MBq) of 111In-labeled 145-9 was injected intravenously into 11 patients with ovarian cancer. Pre-injection serum CA125 concentrations were between 166 U/ml and 7414 U/ml. Tumors were visualized in 10 of 11 patients. In two patients, lymph nodes that were not detected by other imaging modalities but were clinically suspected as metastases were visualized. There was no correlation between serum CA125 level and antibody uptake in the tumors. Immune complexes between the antibody and circulating antigen were observed in sera of all the patients, but the fraction of radioactivity in complex form did not correlate well with serum CA125 levels. The immune complexes survived in the circulation and the circulating radiolabel, including immune complexes, was still bound to solid-phase CA125. The plasma clearance rate and hepatic uptake of the antibody were not significantly affected by circulating CA125. In conclusion, the antibody 145-9 formed complexes with CA125 in vivo but this did not Compromise the outcome of antibody imaging. The antibody 145-9 can be used in immunoscintigraphy of ovarian cancer irrespective of serum CA125 level.

We tried to apply the avidin chasing system to the inflammation-seeking scintigraphy using radiolabeled nonspecific polyclonal IgG. We studied the pharmacokinetics of technetium-99m and iodine-125-labeled biotinylated murine polyclonal IgG followed by an avidin chase injection in model mice with inflammatory foci. Avidin chase decreased the circulating radioactivity of 99mTc and 125I, which was a major problem for inflammation-seeking scintigraphy using radiolabeled nonspecific polyclonal IgG, to 9.3% and 19.3% of that without an avidin chase injection, respectively. Inflammation-seeking scintigraphy with the aforementioned method would be better than that with conventional method.

Anti-Tac monoclonal antibody recognizes human interleukin-2 receptor, which is overexpressed in leukemic cells of most adult T-cell leukemia (ATL) patients. To examine the potency of anti-Tac for targeting of ATL, biodistributions of intravenously administered 125I- and 111In-labeled anti-Tac were examined in severe combined immunodeficiency (SCID) mice inoculated with ATL cells. Significant amounts of radiolabeled anti-Tac were found in the spleen and thymus. The trafficking of ATL cells in SCID mice was detected using 111In-oxine-labeled ATL cells. These results were coincident with the histologically confirmed infiltration of ATL cells. The radiolabeled anti-Tac seemed potent for targeting of ATL.

Immunoscintigraphy using radiolabelled biotinylated monoclonal antibodies followed by infusion of avidin as a “chase” has been recently reported to improve the biodistribution for both immunoscintigraphy and radioimmunotherapy. In this study the circulating protein-bound and avidin-binding fractions of radiolabelled biotinylated antibodies were determined serially after injection of an avidin “chase”, and the effect of repeating the avidin chase was also studied. Nude mice bearing KT005 human osteogenic sarcoma were injected with radiolabelled biotinylated antitumour monoclonal antibody (OST7). After injection of an avidin chase, the protein-bound and avidin-binding fractions in plasma were determined serially using the trichloroacetate method and avidin-Sepharose gel. The biodistribution of radiolabelled biotinylated OST7 was compared after single and double avidin chases with no chase. At 6 h after the first avidin chase in mice injected with radioiodinated and technetiumlabelled biotinylated OST7, 67.7% and 67.8%, respectively, of the plasma radioactivity was available for binding to avidin and was cleared from the circulation. Reinjection of avidin decreased the plasma radioactivity and improved the biodistribution of the radiolabelled biotinylated antibodies. Repeating the avidin chase markedly improved the biodistribution of the radioiodine-labelled biotinylated antibody when compared with the use of a single avidin chase. This new method for radioimmunotherapy is sure to protect the critical organs from radiation injury without decreasing the therapeutic effect.

A murine monoclonal antibody MLS102 recognizes sialosyl-Tn antigen in mucin and immunohistochemically reacts with more than 80% of colorectal cancer tissues. The purpose of this study was to assess the usefulness of this monoclonal antibody for the immunoscintigraphy of colorectal cancer. Planar and SPECT images were obtained on day 2 or day 3 after injection of 2 mg and 74 MBq 111In-labeled MLS102 antibody into 17 patients with colorectal cancer. Nine of 11 primary tumors and 4 of 6 locally recurrent tumors were detected. Positive images were obtained in all tumors larger than 4.5 x 2.7 cm. Three tumors of less than 2.5 cm and 1 recurrent tumor, which was missed by other imaging modalities, were negative. There were no adverse reactions. Human anti-(mouse Ig) antibody developed in 4 patients. Although improvement of detectability for smaller tumors needs to be pursued, the antibody MLS102 is potentially promising for use in immunoscintigraphy of colorectal cancer.

It has previously been reported that almost all of the trivalent technetium-99m dimercaptosuccinic acid (99mTc (III) DMSA) present in the labelling product of pentavalent technetium-99m DMSA (99mTc (V) DMSA) can be changed into 99mTc (V) DMSA by bubbling with pure oxygen. We therefore performed studies in animals (mice) and humans to investigate the effect of such oxygen bubbling on the labelling efficiency of and on the renal uptake of 99mTc. The method of labelling of 99mTc (V) DMSA was that of Hirano. It was found that oxygen bubbling oxidized the contaminated 99mTc (III) DMSA into 99mTc (V) DMSA in vitro and decreased the uptake of radioactivity in the kidney in both animals and humans.

Injection of avidin can decrease the background radioactivity due to a radiolabeled biotinylated monoclonal antibody. We compared the chase effects of avidin, streptavidin, neutravidin, and avidin-conjugated ferritin on a radiolabeled antitumor monoclonal antibody in tumor-bearing nude mice. A radioiodine-labeled biotinylated monoclonal antibody (OST7) was administered to athymic mice bearing osteogenic sarcomas. After 24 h, an avidin, streptavidin, neutravidin or avidin-conjugated ferritin chaser was intravenously injected into the mice. At 2 h after the chase, the biodistribution of the radiolabeled monoclonal antibody was determined. Clearance from the blood was dose-dependently accelerated by avidin and its effect was 10-fold stronger than that of neutravidin or avidin-ferritin. Streptavidin did not promote clearance of the biotinylated antibody. Avidin was the most effective chasing agent for improving the biodistribution of the radiolabeled biotinylated monoclonal antibody among the four avidin derivatives tested.