Toshiaki Tagawa

Immunoliposomes bearing polyethyleneglycol-coupled Fab' fragment show prolonged circulation time and high extravasation into targeted solid tumors in vivo.

We have developed a new type of long‐circulating immunoliposome (Fab′–PEG immunoliposomes) which is efficiently extravasated into the targeted solid tumor in vivo. Small unilamellar liposomes (100–130 nm in diameter) were prepared from distearoylphosphatidylcholine (DSPC), cholesterol (CHOL) and a dipalmitoylphosphatidylethanolamine derivative of PEG with a terminal maleimidyl group (DPPE‐PEG‐Mal), and conjugated Fab′ fragment of antibody. Inclusion of DPPE‐PEG‐Mal and linkage of the Fab′ fragment instead of intact antibody to PEG terminals allowed the liposomes to evade RES uptake and remain in the circulation for a long time, resulting in enhanced accumulation of the liposomes in the solid tumor. Because of the ability of such Fab′–PEG immunoliposomes to target solid tumors, they appear highly attractive as carriers of not only chemotherapeutic agents, but also of macromolecular drugs.

Targeting efficiency of PEG-immunoliposome-conjugated antibodies at PEG terminals

Abstract We have developed a new type of PEG-immunoliposome carrying monoclonal antibodies or their fragments (F(ab′)2, Fab′) at the distal ends of the PEG chains (Type C). Distearoylphosphatidylethanolamine derivatives of PEG with car☐yl group (DSPE-PEG-COOH) or dipalmitoyl phosphatidylethanolamine derivatives of PEG with maleimidyl group (DPPE-PEG-Mal) at the PEG terminal were newly synthesized. Small unilamellar liposomes (90–130 nm in diameter) were prepared from distearoyl phosphatidylcholine and cholesterol (2:1, m/m) containing 6 mol% of DSPE-PEG-COOH or DPPE-PEG-Mal. To target to the vascular endothelial lung surface as a model accessible site, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to PEG-liposome (34A-Type C). The degree of lung binding of 34A-Type C in BALB/c mice was significantly higher than that of the 34A-Type A which is an ordinary type immunoliposome (without PEG derivatives). To target to the solid tumor tissue as a model of the less accessible site, 21B2 antibody which is anti-human CEA and its Fab′ fragment were used. The targeting ability of Fab′-Type C was examined by using CEA-positive human gastric cancer strain MKN-45 cells inoculated into BALB/c nu/nu mice. Fab′-Type C showed the low RES uptake and the long circulation time, and resulted in enhanced accumulation of the liposomes in the solid tumor. The small Fab′-Type C could predominantly pass through the leaky tumor endothelium by passive convective transport.

Antitumor effect of MCC-465, pegylated liposomal doxorubicin tagged with newly developed monoclonal antibody GAH, in colorectal cancer xenografts.

MCC‐465 is an immunoliposome‐encapsulated doxorubicin. The liposome is tagged with polyethylene glycol and the F(ab)2 of a monoclonal antibody named GAH, a human antibody obtained by the hybridoma technique. The epitope recognized by GAH is not well characterized, but human gastric, colorectal, and mammary cancer cells were GAH‐positive, while the normal counterparts were GAH‐negative. Pegylated liposome doxorubicin (PLD) and MCC‐465 did not show significant antitumor activity against GAH‐negative Caco‐2 xenografts. On the other hand, MCC‐465 exhibited significantly superior antitumor effects against GAH‐positive WiDr‐Tc and SW837 xenografts, compared with PLD. Immunohis‐tochemistry with GAH revealed that 94% (100 of 106) of surgical specimens of colorectal cancer were GAH‐positive. These results warrant a phase I clinical trial of MCC‐465 for patients with metastatic colorectal cancer.

Establishment and Evaluation of Cancer-Specific Human Monoclonal Antibody GAH for Targeting Chemotherapy Using Immunoliposomes

To establish human monoclonal antibodies suitable for targeting chemotherapy, we prepared a panel of human-mouse hybridomas, using mouse myelomas and lymphocytes of regional lymph nodes excised from cancer patients, and selected antibodies on the basis of their specificity of binding to the surface of viable cancer cells derived from fresh cancer tissues. A selected antibody, named GAH, was found to react with viable cancer cells from 21/22 stomach and 13/20 colon cancer tissues. As for further analysis, complementary DNAs encoding GAH were cloned and recombinant GAH (rGAH) was obtained from established CHO cells transfected with GAH expression vectors. rGAH selectively stained cancer cells in human tissue sections from 13/14 stomach, 4/11 colon, 5/11 mammary, and 0/7 lung cancers, while no positive staining was observed in those of non-tumor and various normal specimens. Notably, using confocal fluorescence microscopy, rGAH was not only bound to the surface of cancer cells, but was also internalized by the cells. The potential of rGAH for intracellular drug delivery was subsequently evaluated using rGAH-conjugated, doxorubicin (DXR)-encapsulated immunoliposomes. The immunoliposomes were also internalized into the cancer cells and finally DXR was delivered to the cell nucleus. Furthermore, the immunoliposomes could inhibit the growth of DXR-insensitive stomach cancer cells (B37) in an in vivo model. These results suggest that a GAH-utilized liposome-targeting technique will provide a potent and useful cancer chemotherapy with broad applications for cancer patients.

Improvement of pharmacokinetics and antitumor activity against human hepatoma cell line by using adriamycin‐entrapped stealth liposomes

Preferential accumulation in the reticuloendothelial system is one of the major obstacles to the use of liposomes as a drug carrier for targeting therapy. To reduce their uptake, ganglioside GM1 was introduced into the components of conventional liposomes that had been used in our targeting experiments. Two types of such liposomes were prepared. Tissue distribution studies on Adriamycin entrapped in both types of liposomes clearly indicated that the uptake of Adriamycin by liver and spleen decreased to the level comparable to that of free Adriamycin administration. By contrast, the level of Adriamycin in the serum remains high, and some increase was observed in the accumulation to the tumor. Furthermore, Adriamycin in these liposomes, which were conjugated with anti‐α‐fetoprotein (AFP) antibody, inhibited the growth of AFP‐positive human hepatoma Li‐7 more efficiently than free Adriamycin or Adriamycin in antibody‐conjugated conventional liposomes.