IL-15 enhanced antibody-dependent cellular cytotoxicity mediated by NK cells and macrophages.

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.

Tumor-Shed Antigen Affects Antibody Tumor Targeting: Comparison of Two 89Zr-Labeled Antibodies Directed against Shed or Nonshed Antigens.

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.

Pharmacokinetics and microbiodistribution of 64Cu-labeled collagen-binding peptides in chronic myocardial infarction.

OBJECTIVES: The aim of the study is to evaluate the pharmacokinetics and microbiodistribution of Cu-labeled collagen-binding peptides. METHODS: 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. RESULT: 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. CONCLUSION: 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 (t1/2<2 min) of the Cu-peptides from collagen.

64Cu-DOTA as a surrogate positron analog of Gd-DOTA for cardiac fibrosis detection with PET: pharmacokinetic study in a rat model of chronic MI.

OBJECTIVES: 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. METHODS: 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. RESULTS: (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. CONCLUSION: (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.

Tumor and organ uptake of (64)Cu-labeled MORAb-009 (amatuximab), an anti-mesothelin antibody, by PET imaging and biodistribution studies.

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)CuCl2 in 0.25 M 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, 80-300 mm(3)) one day after iv injection of (64)Cu-NOTA-amatuximab (10 µCi) containing a total amatuximab dose of 2, 30, or 60 µg. The BD and PET imaging were also investigated 3, 24 and 48 h after injecting a total dose of 30 µg (10 µCi for BD), and 2 or 60 µg (300 µCi 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 60 µg doses were greater than those from 2 µg 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 60 µg amatuximab, whereas the injection of 2 µg 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 60 µg) 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.

(99m)Tc-labeled therapeutic inhaled amikacin loaded liposomes.

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.

Augmented IL-15Rα expression by CD40 activation is critical in synergistic CD8 T cell-mediated antitumor activity of anti-CD40 antibody with IL-15 in TRAMP-C2 tumors in mice.

IL-15 has potential as an immunotherapeutic agent for cancer treatment because it is a critical factor for the proliferation and activation of NK and CD8(+) T cells. However, monotherapy of patients with malignancy with IL-15 that has been initiated may not be optimal, because of the limited expression of the private receptor, IL-15Rα. We demonstrated greater CD8 T cell-mediated therapeutic efficacy using a combination regimen of murine IL-15 administered with an agonistic anti-CD40 Ab (FGK4.5) that led to increased IL-15Rα expression on dendritic cells (DCs), as well as other cell types, in a syngeneic established TRAMP-C2 tumor model. Seventy to one hundred percent of TRAMP-C2 tumor-bearing wild-type C57BL/6 mice in the combination group manifested sustained remissions, whereas only 0-30% in the anti-CD40-alone group and none in the murine IL-15-alone group became tumor free (p < 0.001). However, the combination regimen showed less efficacy in TRAMP-C2 tumor-bearing IL-15Rα(-/-) mice than in wild-type mice. The combination regimen significantly increased the numbers of TRAMP-C2 tumor-specific SPAS-1/SNC9-H(8) tetramer(+)CD8(+) T cells, which were associated with the protection from tumor development on rechallenge with TRAMP-C2 tumor cells. Using an in vitro cytolytic assay that involved NK cells primed by wild-type or IL-15Rα(-/-) bone marrow-derived DCs, we demonstrated that the expression of IL-15Rα by DCs appeared to be required for optimal IL-15-induced NK priming and killing. These findings support the view that anti-CD40-mediated augmented IL-15Rα expression was critical in IL-15-associated sustained remissions observed in TRAMP-C2 tumor-bearing mice receiving combination therapy.

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.

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.

Preclinical evaluation of an anti-CD25 monoclonal antibody, 7G7/B6, armed with the beta-emitter, yttrium-90, as a radioimmunotherapeutic agent for treating lymphoma.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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 (p < 0.001). 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. CONCLUSIONS: Our findings suggested that (90)Y-7G7/B6 is a potentially useful radioimmunotherapeutic agent for the treatment of patients with CD25-expressing lymphomas.

Interleukin-15 combined with an anti-CD40 antibody provides enhanced therapeutic efficacy for murine models of colon cancer.

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-alpha 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.

Effective therapy of murine models of human leukemia and lymphoma with radiolabeled anti-CD30 antibody, HeFi-1.

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 anti-CD25 monoclonal antibody 7G7/B6, armed with the alpha-emitter 211At, provides effective radioimmunotherapy for a murine model of leukemia.

Radioimmunotherapy of cancer with radiolabeled antibodies has shown promise. alpha-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 alpha (IL-2R alpha)] monoclonal antibody, 7G7/B6, armed with (211)At 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 (211)At-7G7/B6 from the circulation was virtually identical to (125)I-7G7/B6. The biodistributions of (211)At-7G7/B6 and (125)I-7G7/B6 were also similar with the exception of a higher stomach uptake of radioactivity with (211)At-7G7/B6. Therapy using 15 microCi of (211)At-7G7/B6 prolonged survival of the karpas299 leukemia-bearing mice significantly when compared with untreated mice and mice treated with (211)At-11F11, a radiolabeled nonspecific control antibody (P < 0.01). All of the mice in the control and (211)At-11F11 groups died by day 46 whereas >70% of the mice in the (211)At-7G7/B6 group still survived at that time. In summary, (211)At-7G7/B6 could serve as an effective therapeutic agent for patients with CD25-expressing leukemias.

Effective therapy for a murine model of human anaplastic large-cell lymphoma with the anti-CD30 monoclonal antibody, HeFi-1, does not require activating Fc receptors.

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.

Pretargeted alpha emitting radioimmunotherapy using (213)Bi 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-biotin.

PURPOSE: The use of an alpha emitter for radioimmunotherapy has potential advantages compared with beta emitters. When administered systemically optimal targeting of intact antibodies requires >24 h, therefore limiting the use of short-lived alpha 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 alpha emitter, (213)Bi-labeled biotin. EXPERIMENTAL DESIGN: Biotinidase-resistant 7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin was radiolabeled with (205,206)Bi or (213)Bi. Treatment of tumor-bearing mice began by administration of B3-SA (400 micro g) 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: (213)Bi-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.

Effect of albumin fusion on the biodistribution of interleukin-2.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Effect of molecular size of pegylated peptide on the pharmacokinetics and tumor targeting in lymphoma-bearing mice.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: As molecular size increased, blood and body clearances decreased (P < 0.001). 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 (P < 0.01). CONCLUSIONS: 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.

Synthesis of dendrimer-based biotin radiopharmaceuticals to enhance whole-body clearance.

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 (p<0.0001). Lysine co-injection further lowered renal uptake.

Radioimmunotherapy of A431 xenografted mice with pretargeted B3 antibody-streptavidin and (90)Y-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-biotin.

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 had <2.6% 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 to >163 days in the 37 MBq (90)Y group (P < 0.005). In a second therapy group, 7 of 10 mice receiving 37 MBq of (90)Y and B3-SA were cured, and remained healthy for >180 days after therapy, compared with control groups, with

Pretargeting radioimmunotherapy of a murine model of adult T-cell leukemia with the alpha-emitting radionuclide, bismuth 213.

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 ((213)Bi)-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 microg) was administered intravenously (i.v.) to bind to the interleukin 2 receptor alpha (IL-2R alpha; CD25)-expressing tumor cells. After 24 hours, 100 microg of a synthetic clearing agent was administered i.v. to remove unbound circulating HAT-SA conjugate from the circulation. Four hours later, (213)Bi-DOTA-biotin was administered i.v. for therapy. Tumor growth was significantly inhibited in 3 trials by using 250 microCi (9.25 MBq) of (213)Bi-DOTA-biotin with a pretargeting technique as monitored by serum levels of soluble IL-2R alpha and/or human beta-2-microglobulin (P <.05, t test) and by survival of tumor-bearing mice in the treatment groups (P <.02, log rank test) as compared with the control groups. No prolongation of survival was observed with a nonspecific antibody-SA conjugate or in the absence of the radionuclide. Additionally, no prolongation of survival resulted from administration of (213)Bi directly linked to intact HAT. Furthermore, there was no prolongation of survival when the beta-emitting radionuclide yttrium 90 instead of the alpha-emitting radionuclide (213)Bi was used. The pretargeting approach with (213)Bi inhibited tumor growth more effectively than did immunotherapy with unmodified HAT. The best results were obtained with combination therapy that involved (213)Bi-DOTA-biotin with a pretargeting technique supplemented by 4 weekly doses of HAT. The findings of this study support the use of this combination approach in a clinical trial in patients with IL-2R alpha-expressing leukemias.