Imaging doxorubicin and polymer-drug conjugates of doxorubicin-induced cardiotoxicity with bispecific anti-myosin-anti-DTPA antibody and Tc-99m-labeled polymers.

BACKGROUND: Radiolabeled anti-myosin imaging is well-established for imaging doxorubicin-induced cardiotoxicity. However, to enable imaging of drug-induced cardiotoxicity in small experimental animals, pretargeting with bispecific anti-myosin-anti-DTPA-Fab-Fab' and targeting with high-specific radioactivity Tc-99m-DTPA-succinylated-polylysine (DSPL) was developed. METHODS: Mice were injected biweekly with 10 mg/kg Dox or its equivalent as D-Dox-PGA. Tc-99m-DSPL myocardial activity after pretargeting with bsAb-Fab-Fab' was determined after gamma imaging performed at day 7 for Dox-treated mice and day 39 for all others. RESULTS: Mice treated with 10 mg/kg Dox lost 10% total body weight in 1 week and 20% after a second dose. Pretargeted mice treated with 30 mg/kg cumulative D-Dox-PGA dose showed no loss of body weight for the duration of the study. Cardiotoxicity was confirmed by gamma imaging and scintillation counting (1.9 ± 0.25 [mean% ID/g ± SD]) after 1 dose of Dox. Mice injected with 3 × 10 mg/kg Dox equivalent as D-Dox-PGA (0.4 ± 0.04, P < .01) and untreated 2 control groups (0.20 ± 0.05 and 0.19 ± 0.04, P < .01) showed significantly lower myocardial anti-myosin radioactivity relative to the 10 mg/kg Dox group. CONCLUSION: Pretargeting with bsAb-Fab-Fab' and targeting with Tc-99m labeled high-specific activity polymers enabled early visualization of doxorubicin induce cardiotoxicity in mice. Tolerated dose of D-Dox-PGA was greater than to 30 mg/kg Dox-equivalent dose with minimal cardiotoxicity.

Bispecific antibody complex pre-targeting and targeted delivery of polymer drug conjugates for imaging and therapy in dual human mammary cancer xenografts: targeted polymer drug conjugates for cancer diagnosis and therapy.

INTRODUCTION: Doxorubicin, a frontline chemotherapeutic agent, limited by its cardiotoxicity and other tissue toxicities, was conjugated to N-terminal DTPA-modified polyglutamic acid (D-Dox-PGA) to produce polymer pro-drug conjugates. D-Dox-PGA or Tc-99 m labeled DTPA-succinyl-polylysine polymers (DSPL) were targeted to HER2-positive human mammary carcinoma (BT-474) in a double xenografted SCID mouse model also hosting HER2-negative human mammary carcinoma (BT-20). METHODS: After pretargeting with bispecific anti-HER2-affibody-anti-DTPA-Fab complexes (BAAC), anti-DTPA-Fab or only phosphate buffered saline, D-Dox-PGA or Tc-99 m DSPL were administered. Positive therapeutic control mice were injected with Dox alone at maximum tolerated dose (MTD). RESULTS: Only BT-474 lesions were visualized by gamma imaging with Tc-99 m-DSPL; BT-20 lesions were not. Therapeutic efficacy was equivalent in mice pretargeted with BAAC/targeted with D-Dox-PGA to mice treated only with doxorubicin. There was no total body weight (TBW) loss at three times the doxorubicin equivalent MTD with D-Dox-PGA, whereas mice treated with doxorubicin lost 10% of TBW at 2 weeks and 16% after the second MTD injection leading to death of all mice. CONCLUSIONS: Our cancer imaging and pretargeted therapeutic approaches are highly target specific, delivering very high specific activity reagents that may result in the development of a novel theranostic application. HER/2 neu specific affibody-anti-DTPA-Fab bispecific antibody pretargeting of HER2 positive human mammary xenografts enabled exquisite targeting of polymers loaded with radioisotopes for molecular imaging and doxorubicin for effective therapy without the associating non-tumor normal tissue toxicities.

In vitro demonstration of enhanced prostate cancer toxicity: pretargeting with Bombesin bispecific complexes and targeting with polymer-drug-conjugates.

BACKGROUND: Bombesin has been used to target Bombesin receptor, a growth receptor, which is over-expressed in many cancers, including prostate cancer. Polymer-anti-neoplastic-drug-conjugates (PDC) were also developed to reduce non-specific toxicity and increase tumor toxicity utilizing the enhanced permeability and retention effect, benefitting treatment of large tumors with well-established vasculature. PURPOSE: If PDCs were delivered by targeted delivery to cancer cells, tumor toxicity would be enhanced and non-specific toxicity decreased. METHODS: Cardiocyte toxicity was assessed in H9c2 cardiocytes with doxorubicin (Dox) or N-terminal DTPA-modified-Doxorubicin-loaded-polyglutamic acid polymers (D-Dox-PGA). Therapeutic efficacy of targeted D-Dox-PGA after pretargeting with Bombesin-conjugated anti-DTPA-antibody Bispecific Complexes (Bom-BiSpCx) was compared to that of Dox in PC3 cells. Bom-BiSpCx was generated by thioether bond between Bombesin to Anti-DTPA antibody. RESULTS: D-Dox-PGA was demonstrated to have less cardiocyte toxicity (IC50 = 20 µg/ml) than free Dox (1.55 µg/ml, p < 0.001). However, after pre-targeting of human prostate cancer PC3 cells with Bom-BiSpCx and targeting with D-Dox-PGA, IC50 (13.2 µg/ml) was about two times less than that of Dox (28.5 µg/ml, p < 0.0001). DISCUSSION: Targeted delivery of PDCs having lower cardiocyte toxicity enabled higher efficiency cancer cell therapy. CONCLUSION: This study may allow development of very efficient targeted prostate cancer pro-drug therapy.

Imaging small human prostate cancer xenografts after pretargeting with bispecific bombesin-antibody complexes and targeting with high specific radioactivity labeled polymer-drug conjugates.

PURPOSE: Pretargeting with bispecific monoclonal antibodies (bsMAb) for tumor imaging was developed to enhance target to background activity ratios. Visualization of tumors was achieved by the delivery of mono- and divalent radiolabeled haptens. To improve the ability to image tumors with bsMAb, we have combined the pretargeting approach with targeting of high specific activity radiotracer labeled negatively charged polymers. The tumor antigen-specific antibody was replaced with bombesin (Bom), a ligand that binds specifically to the growth receptors that are overexpressed by many tumors including prostate cancer. Bomanti- diethylenetriaminepentaacetic acid (DTPA) bispecific antibody complexes were used to demonstrate pretargeting and imaging of very small human prostate cancer xenografts targeted with high specific activity ¹¹¹In- or 99mTc-labeled negatively charged polymers. METHODS: Bispecific antibody complexes consisting of intact anti-DTPA antibody or Fab' linked to Bom via thioether bonds (Bom-bsCx or Bom-bsFCx, respectively) were used to pretarget PC-3 human prostate cancer xenografts in SCID mice. Negative control mice were pretargeted with Bom or anti-DTPA Ab. 111In-Labeled DTPA-succinyl polylysine (DSPL) was injected intravenously at 24 h (7.03 ± 1.74 or 6.88 ± 1.89 MBq ¹¹¹In-DSPL) after Bom-bsCx or 50 ± 5.34 MBq of 99mTc-DSPL after Bom-bsFCx pretargeting, respectively. Planar or single photon emission computed tomography (SPECT)/CT gamma images were obtained for up to 3 h and only planar images at 24 h. After imaging, all mice were killed and biodistribution of 111In or 99mTc activities were determined by scintillation counting. RESULTS: Both planar and SPECT/CT imaging enabled detection of PC-3 prostate cancer lesions less than 1-2 mm in diameter in 1-3 h post 111In-DSPL injection. No lesions were visualized in Bom or anti-DTPA Ab pretargeted controls. 111In-DSPL activity in Bom-bsCx pretargeted tumors (1.21 ± 0.36 %ID/g) was 5.4 times that in tumors pretargeted with Bom or anti-DTPA alone (0.22 ± 0.08, p = 0.001). PC-3 xenografts pretargeted with Bom-bsFCx and targeted with 99mTc-DSPL were visualizable by 1-3 h. Exquisite tumor uptake at 24 h (6.54 ± 1.58 %ID/g) was about 15 times greater than that of Bom pretargeted controls (0.44 ± 0.17, p = 0.002). CONCLUSION: Pretargeting prostate cancer with Bom-bsCx or Bom-bsFCx enabled fast delivery of high specific radioactivity ¹¹¹In- or 99mTc-labeled polymer-drug conjugates resulting in visualization of lesions smaller than 1- 2 mm in diameter within 3 h.

Bispecific antibody complex pre-targeted delivery of polymer-drug conjugates for cancer therapy.

The pretargeting approach using bispecific affibody-antibody complex (BAAC) and targeting of chemotherapeutic drug loaded polymers have been used in breast cancer cell cultures to demonstrate targeted chemotherapy and reduce toxicity to non-pretargeted cancer and cardiac cells. HER2/neu-positive BT-474 and -negative BT-20 human mammary carcinoma cell lines were pretargeted with BAAC and targeted with multi-doxorubicin (Dox) loaded polyglutamic acid (PGA) site specifically modified with diethylene triamine pentaacetic acid (DTPA) (D-Dox-PGA) at the N-terminal of PGA. Toxicity to embryonic cardiocytes and human mammary carcinoma cells were assessed. BAAC was prepared by covalent conjugation of anti-HER2/neu affibody and anti-DTPA Fab via the thioether linkage. N-terminal DTPA modified polyglutamic acid was conjugated with doxorubicin via the amide bonds. Reduction in cardiotoxicity and IC50 of D-Dox-PGA and free Dox were determined in embryonic cardiocyte H9C2 cultures. Enhanced targeted tumor toxicity was demonstrated in BT-474 human mammary carcinoma cell line pretargeting with BAAC followed by targeting with D-Dox-PGA and compared to D-Dox-PGA alone with no pretargeting or free Dox. No enhanced targeted tumor toxicity was observed in HER2/neu negative BT-20 cells. IC50 of D-Dox-PGA and free Dox on embryonic cardiocytes was 15.75 and 1.20 µg/ml, respectively. When BT-474 and BT-20 cells were pretargeted with BAAC followed by targeting with D-Dox-PGA, higher tumor cell-killing was seen only in BT-474 cells. Pretargeting with BAAC resulted in greater tumor cell death in BT-474 human breast cancer cells due to specific targeted delivery of D-Dox-PGA than cancer cells treated with D-Dox-PGA without pretargeting or treatment with free doxorubicin. In vitro targeted delivery of polymer drug conjugate resulted in highly specific, targeted HER2/neu positive BT-474 cancer cell death. Such a pretargeting and targeting approach using prodrug polymers may allow development of very efficient, lower non-target toxicity, and image-guided targeted therapy since these polymers can also be labeled with radioisotopes.

Pretargeted gamma imaging of murine metastatic melanoma lung lesions with bispecific antibody and radiolabeled polymer drug conjugates.

INTRODUCTION: Bispecific monoclonal antibodies (bsMAbs) have been developed as a pretargeting tool to reduce background activity, thereby increasing target to background (T : B) ratios. To enhance visualization of small lesions in vivo, we have used the pretargeting approach of bsMAb and negatively charged polymers radiolabeled with high-specific radioactivity. METHODS: Imaging of metastatic melanoma lesions localized in lung tissues pretargeted with bsMAb and targeted with high-specific radioactivity polymers was carried out. The bsMAb was prepared by covalent conjugation of an antinucleosomal antibody (2C5) recognizing a nucleosomal pan cancer antigen and an anti-diethylene triaminepentaacetic acid antibody (6C31H3) by means of thioether linkage. BsMAb was injected intravenously 10 days after the initiation of the induction of murine melanoma metastasized to the lungs. The next day, 37 MBq 99mTc-diethylene triaminepentaacetic acid-succinylated polylysine were injected intravenously and in-vivo imaging was carried out after the injection. In-vivo and ex-vivo target (T) to background (B) activity ratios were assessed by computer planimetry and biodistribution studies. RESULTS: Lesions were visualized unequivocally in 3 h by gamma scintigraphy. Ex-vivo gamma-scintillation counting corrected for the lesion mass showed that the mean lesion activity was 24.85 ± 13.53 percent injected dose per gram when pretargeted with bsMAb, whereas it was 0.977 ± 0.465 percent injected dose per gram (P<0.01) in the control group injected only with radioactive polymers also corrected similarly. CONCLUSION: The use of bsMAb complexes and 99mTc-diethylene triaminepentaacetic acid-succinylated polylysine enabled early in-vivo visualization of small metastatic melanoma lesions in the lungs.

Cytoskeletal-antigen specific immunoliposomes: preservation of myocardial viability.

Pathological conditions such as hypoxia and inflammation can lead to the development of cell membrane-lesions. The presence of these membrane-lesions leads to egress of intracellular macromolecules as well as exposure of intracellular microenvironment to the extracellular milieu resulting in necrotic cell death. An intracellular structure that becomes exposed to the extracellular environment is myosin, a cytoskeletal antigen. We had hypothesized that cell viability can be preserved in nascent necrotic cells if the cell membrane lesions were sealed and the injurious conditions removed. Cell membrane lesion sealing and preservation of cell viability were achieved by the application of Cytoskeletal-antigen Specific ImmunoLiposomes (CSIL) as molecular "Band-Aid" that initially plugs the holes with subsequent sealing of the lesions. Anti-myosin antibody was chosen as the cytoskeleton-antigen specific antibody to develop CSILs, because antimyosin antibody is highly specific for targeting myosin exposed through myocardial cell membrane lesions in various cardiomyopathies. Liposomes are biocompatible lipid bilayer vesicles that have been used in many biological applications for several decades. This chapter will be limited to the description of CSIL therapy to ex vivo studies in adult mammalian hearts. Due to page limitations, cell culture, gene delivery and in vivo studies will not be included. Therapeutic efficacy of CSIL in preservation of myocardial viability as well as function (by left ventricular developed pressure measurements) as assessed in globally ischemic Langendorff instrumented hearts is both dose and time dependent. This approach of cell membrane lesion repair and sealing may have broader applications in other cell systems.

External magnet improves antitumor effect of vinblastine and the suppression of metastasis.

The use of magnetic drug targeting (MDT) to selectively deliver chemotherapeutic drugs to tumor cells is a widely investigated approach; however, the notion of targeting tumor endothelial cells by this method is a fairly new concept. Positively-charged (cationic) liposomes have an extraordinarily high affinity for tumor vessels, but heterogeneous targeting is frequently observed. In order to improve on the overall efficiency of targeting tumor vessels, we investigated the use of an externally applied magnetic field together with magnetic cationic liposomes (MCLs) for cancer treatment. We examined the antitumor effect of the chemotherapeutic agent vinblastine loaded in MCLs, using a murine model of melanoma. Two hours following i.v. administration of MCLs, we observed significant tumor vascular uptake with use of an external magnet (15.9 +/- 6.3%) compared to no magnet (5 +/- 1.3%). The administration of vinblastine-loaded MCLs with the magnet produced a significant antitumor effect, reducing the presence of tumor nodules in preferential sites of metastasis compared to untreated and free drug control groups. CD31 immunostaining revealed a decrease in the general length of tumor blood vessels, altered vascular morphology and interruptions in the tumor vascular lining for the vinblastine-loaded MCL groups. Drug-loaded MCLs with magnetic fields may represent a promising combination approach for cancer treatment.