Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

Summary The object of our current investigations is to explore the potential of antibodies for localisation and treatment of disseminated disease, using as a model rat monoclonal antibodies (mAbs) raised against syngeneic tumourspecific antigens. As part of this study, antibodies of differing isotypes with specificity for either HSN or MC24 sarcoma were labelled with¹²⁵iodine and injected intravenously into normal rats or those bearing paired tumours in contralateral flanks. The blood clearance rates of the radiolabelled antibodies were found to be influenced by immunoglobulin subclass (IgG2b > IgG2a > IgG1) and to be increased non-specifically by the presence of growing tumours. The tumour and normal tissue distributions of the antibodies tested were also found to vary according to their apparent degree of interaction with host Fc-receptor-bearing cells, to the extent that tumour specificity in vitro was not necessarily reflected in selectivity of localisation in vivo. Three IgG2b monoclonal antibodies showed preferential uptake in the spleens of syngeneic rats and non-specific accumulation in tumours. This effect was not observed with antibodies of IgG2a or IgG1 subclass, and was abolished by the use of IgG2b F(ab)₂ preparations. In spite of the use of immunoglobulin fragments, varying the assay time and testing tumours of different sizes, specific tumour localisation was low with all seven monoclonal antibodies tested. The maximum uptake achieved was less than 1% of the injected dose of antibody per gram of tumour. Much higher levels of antibody localisation have been reported for human tumour xenografts growing in nude mice, but these are rarely achieved in other systems. We propose that the use of autologous monoclonal antibodies recognising tumour-associated antigens of relatively low epitope density in syngeneic hosts provides a valid alternative model in which to investigate the factors limiting more effective, specific immunolocalisation of malignant disease.     

ADVENTURES IN TARGETING

ABSTRACT

An overview of our experiences in the field of immunoliposomal anticancer drugs is provided with respect to choice of ligand, and choice of model system, in order to provide some guidance as to the rational use of this new technology. Liposomes targeted by either peptide or monoclonal antibodies showed significantly higher binding to their respective target cells in vitro compared to non-targeted liposomes in all model systems examined. This higher binding led to higher cytotoxicities relative to non-targeted liposomes. For the immunoliposomes to deliver their entrapped drug to target cell in vivo, long circulations half-lives are required. We have evaluated the pharmacokinetics of liposomes prepared by several different coupling techniques, and have found significant differences in the clearance of these immunoliposomes from the circulation. Immunoliposomes prepared with whole anti-CD19 IgG coupled by the Mal-PEG-DSPE method demonstrated a short plasma half-life, which may reflect the random orientation of the MAb on the liposome surface. Coupling methods that mask or eliminate the Fc region result in immunoliposomes that have clearance rates more similar to untargeted liposomes. Insertion of peptides or antibodies into pre-formed liposomes through incubation with ligand-coupled PEG micelles resulted in immunoliposomes, termed post-insertion liposomes, that demonstrated comparable in vitro binding, pharmacokinetic profiles and in vivo therapeutic efficacy to liposomes made by conventional coupling methods. The therapeutic efficacy of liposomes, prepared by various coupling methods and targeted by different ligands, was compared in several different animal models of either haematological malignancies, pseudometastatic disease or solid tumours. In our hands, successful in vivo targeting has been obtained when the target is either small or readily accessible from the vasculature, where the liposomes have longer circulating half-lives and/or where a ligand against an internalizing epitope has been chosen. These results should aid in the rational design of applications for immunoliposomal drugs in the future.     

Adventures in targeting

An overview of our experiences in the field of immunoliposomal anticancer drugs is provided with respect to choice of ligand, and choice of model system, in order to provide some guidance as to the rational use of this new technology. Liposomes targeted by either peptide or monoclonal antibodies showed significantly higher binding to their respective target cells in vitro compared to non-targeted liposomes in all model systems examined. This higher binding led to higher cytotoxicities relative to non-targeted liposomes. For the immunoliposomes to deliver their entrapped drug to target cell in vivo, long circulations half-lives are required. We have evaluated the pharmacokinetics of liposomes prepared by several different coupling techniques, and have found significant differences in the clearance of these immunoliposomes from the circulation. Immunoliposomes prepared with whole anti-CD19 IgG coupled by the Mal-PEG-DSPE method demonstrated a short plasma half-life, which may reflect the random orientation of the MAb on the liposome surface. Coupling methods that mask or eliminate the Fc region result in immunoliposomes that have clearance rates more similar to untargeted liposomes. Insertion of peptides or antibodies into pre-formed liposomes through incubation with ligand-coupled PEG micelles resulted in immunoliposomes, termed post-insertion liposomes, that demonstrated comparable in vitro binding, pharmacokinetic profiles and in vivo therapeutic efficacy to liposomes made by conventional coupling methods. The therapeutic efficacy of liposomes, prepared by various coupling methods and targeted by different ligands, was compared in several different animal models of either haematological malignancies, pseudometastatic disease or solid tumours. In our hands, successful in vivo targeting has been obtained when the target is either small or readily accessible from the vasculature, where the liposomes have longer circulating half-lives and/or where a ligand against an internalizing epitope has been chosen. These results should aid in the rational design of applications for immunoliposomal drugs in the future.     

Stimulation of intestinal Na+/d-glucose cotransport by monoclonal antibodies

Summary The small intestinal brush border membrane is endowed with a number of transport systems. Monoclonal antibodies were produced against integral membrane proteins and tested for their ability to bind to such membranes. For this purpose papain-digested, deoxycholate-extracted BBMVs from rabbit small intestine were used to immunize mice. Of the 765 hybridoma supernatants tested, 119 gave a significantly higher extent of binding to the crude antigen preparation as compared with the background. The monoclonal antibodies were also tested for their ability to influence the sodium-dependent uptake of solutes into intact BBMVs. Two monoclonal antibodies clearly showed stimulation of secondary actived-glucose transport, whereas sodium-dependent uptake ofl-alanine andl-proline was not affected. Hydrophobically labeled, i.e. intrinsic, membrane proteins of 175, 78 and 65 kilodaltons could be immunoprecipitated by both monoclonal antibodies, the 78 kDa band corresponding in all likelihood to the Na⁺/glucose cotransporter.     

Interactions of immunoliposomes with target cells

We have covalently attached a monoclonal antibody (11-4.1) against the murine major histocompatibility antigen, H-2Kk, on the surface of liposomes. The interaction of these antibody-coated liposomes (immunoliposomes) with target cells, RDM-4 lymphoma (H-2Kk), was investigated. About 90% of the immunoliposomes taken up by target cells at 4 degrees C could be removed by a mild protease treatment of the cells, whereas only 30% of the uptake at 37 degrees C was labile to the same treatment. Furthermore, the uptake of immunoliposomes at 37 degrees C was inhibitable by cytochalasin B or by a combination of 2-deoxyglucose and NaN3. These results suggest that immunoliposome binding to the target cell surface is the primary uptake event at 4 degrees C and that the surface-bound liposomes are rapidly internalized by the cells at 37 degrees C, probably via an endocytic pathway. Studies with fluorescence microscopy of target cells treated with immunoliposomes containing carboxyfluorescein also supported this conclusion. If endocytosis is the mechanism by which immunoliposomes gain entry into target cells, the efficacy of a cytotoxic drug encapsulated would depend on the resistance of the drug to lysosomal inactivation and its ability to escape from the lysosomal system. Consistent with this notion, we observed that methotrexate encapsulated in liposomes bearing 11-4.1 antibody specifically inhibited deoxy[6-3H]uridine incorporation into DNA in target RDM-4 cells but not in P3-X63-Ag8 myeloma cells (H-2Kd) at the same doses. The observed cytotoxic effect of encapsulated methotrexate could be reversed by the treatment of cells with a lysosomotropic amine, chloroquine, which has been shown to increase the intralysosomal pH of mammalian cells. On the other hand, cytosine-beta-D-arabinofuranoside encapsulated in immunoliposomes showed no target-specific killing, probably because the drug is readily inactivated in the lysosomal system. These results are discussed in terms of the drug carrier potential of immunoliposomes.     

Sterically stabilized liposomes bearing anti-HLA-DR antibodies for targeting the primary cellular reservoirs of HIV-1

The ability of liposomes bearing anti-HLA-DR Fab' fragments at the end termini of polyethyleneglycol chains (sterically stabilized immunoliposomes) to target HLA-DR expressing cells and increase the accumulation of liposomes into lymphoid organs has been evaluated and compared to that of conventional liposomes, sterically stabilized liposomes and conventional immunoliposomes after a single subcutaneous injection to mice. The accumulation of sterically stabilized liposomes in lymph nodes was higher than that of conventional liposomes. Sterically stabilized immunoliposomes accumulated much better than conventional immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system. Fluorescence microscopy studies showed that sterically stabilized liposomes are mainly localized in macrophage-rich areas such as the subcapsular region of lymph nodes and in the red pulp and marginal zone of the spleen. In contrast, sterically stabilized immunoliposomes mostly accumulated in the cortex in which follicles are located and in the white pulp of the spleen. As the human HLA-DR determinant of the major histocompatibility complex class II is expressed on activated CD4+ T lymphocytes and antigen presenting cells such as monocyte/macrophages and dendritic cells, known as the cellular reservoirs of HIV-1, liposomes bearing anti-HLA-DR antibodies constitute an attractive approach to concentrate drugs in HIV-1 reservoirs and improve their therapeutic effect.     

Comparison of the biodistribution of 75Se- and 131I-labelled monoclonal antibodies in nude mice

A monoclonal antibody, C-215, against colon cancer, was internally labelled with [⁷⁵Se]methionine. The biodistribution was studied in tumour-bearing nude mice and compared with the biodistribution of [¹³¹I]C-215. The tissue uptake was divided into three parts: antibody bound to the antigen, antibody in the extracellular space and uptake of the released radionuclide. [⁷⁵Se]C-215 showed a greater amount of antigen-bound antibody in the tumour, but also a greater unspecific uptake both in tumour and normal tissue.     

Simple preparation and characterization of cationic liposomes associated with a monoclonal antibody against glioma-associated antigen (immunoliposomes)

Abstract

In this study we prepared and characterized monoclonal antibody associated cationic liposomes (immunoliposomes) to be used as a vehicle for human gene therapy of malignant glioma. This association method is especially amenable to mass production. The immunoliposomes consist of N-(a-trimethylammonio-acetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroyl phosphatidylcholine (DLPC), and dioleoyl phosphatidyl- ethanolamine (DOPE) in a molar ratio of 1:2:2 as TMAG:DLPC:DOPE. Their preparation required only the addition of a solution containing plasmid DNA and a monoclonal antibody against glioma-associated antigen to a lipid film of the above three lipids. The association of antibody on the surface of immunoliposomes was confirmed by an immunochemical procedure. Liposome-mediated LacZ gene transfection of human glioma cells resulted in p-galactosidase activity about 2- to 3-fold higher when immunoliposomes were used as compared to control liposomes that were not associated to antibody. Also, the production of human (3-interferon (HuIFN-P) into the medium was 2- to 7-fold higher when HuIFN-P gene was transfected. Based on the present results, the immunoliposomes associating a monoclonal antibody against glioma-associated antigen may become effective carriers for gene transfer to human glioma cells.     

UPTAKE AND INTRACELLULAR PROCESSING OF PEG-LIPOSOMES AND PEG-IMMUNOLIPOSOMES BY KUPFFER CELLS IN VITRO*

Specific targeting of drugs to for instance tumors or sites of inflammation may be achieved by means of immunoliposomes carrying site-specific antibodies on their surface. The presence of these antibodies may adversely affect the circulation kinetics of such liposomes as a result of interactions with cells of the mononuclear phagocyte system (MPS), mainly represented by macrophages in liver and spleen. The additional insertion of poly(ethylene glycol) chains on the surface of the immunoliposomes may, however, attenuate this effect.

We investigated the influence of surface-coupled rat or rabbit antibodies and of PEG on the uptake of liposomes by rat Kupffer cells in culture with ³H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular processing of the liposomes by the Kupffer cells, based on a double-label assay using the ³H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-¹⁴C-oleate as relative measure of degradation.

Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3–4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes.

In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface.

These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity.     

Changing tumour antigen expression in metastatic hepatocellular carcinoma cells of the guinea pig

Hepatocellular carcinoma cells (Line-10), obtained from ascitic fluid after diethylnitrosamine treatment of Sewall Wright strain-2 guinea pigs, produce solid (primary) tumours, lymph-node and lung metastases and malignant ascites when reinjected into animals of the same strain. Monoclonal antibodies were raised against the tumour cells by immunizing BALB/c mice with viable ascitic hepatocellular Line-10 tumour cells. Three hybridomas producing anti-Line-10 monoclonal antibodies were selected for further studies (10TL1, 10TL40 and 10TL43) and compared with monoclonal antibodies against intermediate filament keratins. The anti-Line-10 monoclonal antibodies did not cross react with Line-1 hepatocellular carcinoma cells, nor with normal guinea pig hepatocytes. When ascitic Line-10 cells form high papillary projections on the peritoneal surface, they significantly reduced their antigen expression of 10TL40 and of 10TL43 defined antigens, while the expression of 10TL1 defined antigens remained unaltered. Invading Line-10 cells in the deep submesothelial stromal tissue, however, lost reactivity with MoAb 10TL43 but not with the MoAb's 10TL40 and 10TL1. The antigens on lung- and lymphnode metastases remained largely unaffected. The reactivity with MoAb's 10TL40 and with 10TL43 was also lost upon prolonged culturing of Line-10 cells. The reactivity of Line-10 and Line-1 cells with all monoclonal antibodies against keratin filaments remained unaltered. Line-1 cells could be distinguished from Line-10 cells by the absence of any reactivity with the MoAb's 10TL1, -40, -43, but also by the fact that 100% of the Line-1 cells were positive with antibodies against keratins 5/8, 18, and 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

In the preceding paper it was suggested that the tumour localisation of 125I-labelled syngeneic rat monoclonal antibodies (mAbs) may be limited in immunocompetent hosts by the presence of competing endogenous serum antibodies. In syngeneic congenitally athymic (nu/nu) and cyclosporin-A-treated rats (both of which fail to mount immune responses to tumour antigens) increased uptake of mAbs in tumour tissue was obtained compared with that in immunocompetent animals. However, in the case of IgG2b and IgG1 mAbs, this appeared to be due primarily to enhanced "non-specific" localisation mediated by Fc binding, since it was abolished by the use of F(ab')2 fragments with two out of three mAbs tested. Normal tissue distribution was also influenced by host immune status: in nu/nu rats the uptake of IgG2b mAbs in the spleen was up to fivefold higher than that previously found in normal animals and the levels in liver were also increased. This effect was not seen in cyclosporin-A-treated hosts, suggesting that the reticuloendothelial system of congenitally athymic animals contains cells with enhanced IgG2b-FcR activity. This hypothesis was strengthened by the observation that splenic uptake was reduced by either the use of F(ab')2 fragments, or prior "blockade" of Fc receptors by "cold competition" with excess unlabelled IgG2b mAbs. This blockade could not be effected by mAbs of any other isotype or by IgG2b F(ab')2 fragments. The former manoeuvre resulted in higher tumour specificity ratios but usually at the expense of reduced levels of tumour associated radiolabelled mAb. The latter was found to increase "absolute" tumour localisation by up to 35%. In an attempt to characterise further and compare the Fc receptor activity of intratumour and intrasplenic host cells. The distribution of IgG2b mAbs was assayed in 3-week, 8-week and 12-week-old rats. We were able operationally to distinguish the activity of these two categories of cells, suggesting that they represent either different lineages or differentially activated subpopulations: the splenic IgG2b binding was fully expressed in weanling nu/nu rats whereas the FcR activity of cells infiltrating MC24 sarcoma was limited in 3-week-old compared with 8-12-week-old hosts. A further difference was apparent in the subclass "preference" of FcR binding: in immunodeprived rats both IgG1 and IgG2b mAbs were able to bind to tumour-infiltrating host cells, but uptake of IgG1 mAbs in the spleen was always low and not reduced further by the use of F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effective suppression of tumour cells by oligoclonal HER2-targeted delivery of liposomal doxorubicin

Synergistic effect of combined antibodies targeting distinct epitopes of a particular tumour antigen has encouraged some clinical trial studies and is now considered as an effective platform for cancer therapy. Providing several advantages over conventional antibodies, variable domain of heavy chain of heavy chain antibodies (V_HH) is now major tools in diagnostic and therapeutic applications. Active targeting of liposomal drugs is a promising strategy, resulting in enhanced binding and improved cytotoxicity of tumour cells. In the present study, we produced four anti-HER2 recombinant VHHs and purified them via native and refolding method. ELISA and flow cytometry analysis confirmed almost identical function of VHHs in refolded and native states. Using a mixture of four purified VHHs, PEGylated liposomal doxorubicin was targeted against HER2-overexpressing cells. The drug release was analyzed at pH 7.4, 6.4 and 5.5 and dynamic light-scattering detector and TEM micrograph was applied to characterize the produced nanoparticles. The binding efficiency of these nanoparticles to BT474 and SKBR3 as HER2-positive and MCF10A as HER2-negative cell line was examined by flow cytometry. Our results indicated effective encapsulation of about 94% of the total drug in immunoliposomes. Flow cytometry results verified receptor-specific binding of targeted liposomes to SKBR3 and BT474 cell lines and more efficient binding was observed for liposomes conjugated with oligoclonal VHHs mixture compared with monoclonal VHH-targeted liposomes. Oligoclonal nanoparticles also showed more cytotoxicity compared with non-targeted liposomes against HER2-positive tumour cells. Oligoclonal targeting of liposomes was represented as a promising strategy for the treatment of HER2-overexpressing breast cancers.     

Targeting of drug loaded immunoliposomes to herpes simplex virus infected corneal cells: an effective means of inhibiting virus replication in vitro

The goal of our studies was to develop liposomes containing antiviral drugs and targeted with antiviral antibody (immunoliposomes) that would be effective at inhibiting replication of herpes simplex virus (HSV) in vitro. To achieve this, a monoclonal antibody to glycoprotein D of HSV was derivatized with palmitic acid and was incorporated into the lamellae of dehydration-rehydration vesicles. The gD containing immunoliposomes were shown to bind specifically to HSV-infected rabbit corneal cells in vitro, whereas control immunoliposomes prepared with a monoclonal antibody of the same class as the anti-gD failed to preferentially bind to virus-infected cells. The gD immunoliposome binding was inhibitable by pretreatment with rabbit anti-HSV serum but not by aggregated normal serum. Thus liposome binding was judged to represent an antigen-antibody reaction not binding to Fc receptors expressed by cells infected with HSV. Immunoliposomes loaded with iododeoxyuridine (IUDR) leaked drug rapidly at 37 degrees C, whereas acyclovir (ACV)-loaded liposomes still contained 48% of drug after 24 hr at 37 degrees C. The ACV-liposomes retained 44% of drug after 14 days at 4 degrees C. The ability of immunoliposomes to inhibit virus replication was compared with that of untargeted and empty liposomes by means of virus yield assays in vitro, Immunoliposomes loaded with either IUDR or ACV inhibited virus replication, although ACV-containing immunoliposomes were the most efficacious. The implications of our in vitro results for the development of immunoliposomes suitable for the treatment of ocular herpes infection are briefly discussed.     

Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells

Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18⁺/PSA^– HT29 colon carcinoma cells. CK18⁺ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hypertrophy. In conclusion, the approach presented appears to be a reliable method to phenotype individual prostatic carcinoma cells.     

Development and effects of immunoliposomes carrying an antisense oligonucleotide against DHFR RNA and directed toward human breast cancer cells overexpressing HER2

The development and the effect of immunoliposomes directed against human breast cancer cells overexpressing p185/HER2 are described. These immunoliposomes carry an antisense oligonucleotide directed toward the translational start site of dihydrofalate reductase (DHFR) RNA, which causes high cytotoxicity. To prepare the immunoliposomes, we followed two methodologies based on the high affinity between streptavidin and biotin and the use of biotinylated antibodies. In the first approach, the streptavidin molecule is covalently attached to the phospholipid DOPE, which is mixed with the cationic liposome DOTAP complexed with the antisense oligonucleotide. The second approach, which is much easier to perform, involves the binding of streptavidin to antibody and oligonucleotide, both biotinylated, and the latter complexed with DOTAP. The formation of the intermediary complexes of this immunoliposome was studied sequentially by gel electrophoresis. The uptake of the oligonucleotide carried by the immunoliposome was monitored by flow cytometry and confocal microscopy. As a model, we used SKBR3 cells that overexpress p185. The full immunoliposomes were more toxic than the antisense oligonucleotide in the absence of the antibody, thus increasing the sensitivity of the treatment.     

Targeted delivery of insulin-modified immunoliposomes in vivo

The purpose of this study was to investigate the effect of liposomes conjugated with insulin to the surface on circulation time, biodistribution, and antitumor activity after intravenous injection in tumor-bearing mice. Immunoliposomes were constructed with insulin, which was covalently linked to liposomes containing anticancer drugs. In order to investigate the targeting performance of insulin-modified immunoliposomes (SILs) in vivo, plasma pharmacokinetics, biodistribution, and antitumor activity were tested. In comparison with nontargeted liposomes (SLs), SILs were cleared faster from circulation as a result of greater liver and tumor uptake. In addition, SILs retarded the growth of the tumor effectively, compared with the ZTO injection or SL. This is the first time for selective in vivo targeting of tumor vessels using insulin-modified immunoliposomes. SILs are candidate drug-delivery systems for therapeutic anticancer approaches.     

Criteria for selecting monoclonal antibodies with respect to accumulation in melanoma tissue

Summary Immunohistology provides a necessary but insufficient criterion for selecting monoclonal antibodies (MAbs) capable of tumour targeting in vivo. Additional selection procedures have been evaluated using a panel of anti-melanoma MAbs, including immunoreactivity of (labelled) MAbs, antibody affinity, kinetics of binding and release, apparent antigen density and accumulation in nude mouse transplants. According to these criteria, MAbs M.2.7.6 and M.2.9.4 showed the most favourable properties, i.e. high immunoreactivity and pronounced internalization into melanoma cells. With MAbs M.2.10.15 and KG 6–56, moderate immunoreactivity and a binding pattern characterized by temperature dependence in the absence of internalization was observed. According to the paired label assay, all four MAbs showed specific accumulation into solid melanoma tissue. However, application in the patient still requires evaluation of the side effects of antigen cross-expression on normal human tissues.     

Immunospecific targeting of immunoliposomes, F(ab')2 and IgG to red blood cells in vivo

In this report a model to study the fate of target cells in the blood circulation after injection of appropriate immunoliposomes is discussed. The effect of intravenous administration of antimouse RBC immunoliposomes, F(ab')2 or IgG on the fate of intravenously injected 51Cr-labelled mouse RBC (Cr-mRBC) in the mouse and, particularly, in the rat was studied. The immunoliposome was of the Fab'-MPBPE-REV type (Fab'-fragments covalently linked to reverse phase evaporation vesicles by maleimido-4-(p-phenylbutyrate)phosphatidylethanolamine). In the rat model a high blood level (80%) of the injected dose of target cells, Cr-mRBC, was maintained for several hours. The elimination by Fab'-liposomes, F(ab')2 or IgG of Cr-mRBC, and subsequent uptake into liver and spleen was dose dependent. Administration of Fab'-liposomes or F(ab')2 resulted in a preferential uptake into the spleen (above a certain dose also, but much lower, uptake into the liver was observed), while after IgG administration 51Cr-label was mainly recovered in the liver. At equal protein doses (+/- 130 micrograms) Fab'-liposomes induced a faster elimination of the Cr-mRBC and a higher uptake into the spleen than F(ab')2. The potential advantage of the use of drug-loaded immunoliposomes to eliminate target cells from the blood stream and to induce a certain pharmacological effect in the target cells, in comparison with the free antibody administration of F(ab')2 or IgG is discussed.     

Tumor-directed targeting of liposomes

Tumor-specific targeting is a critical goal in the research area of liposomal drug delivery. Identification of the specific interactions between ligands and target tumor cells is a principle prerequisite in achieving this goal. Generally, tumor cells aberrantly express tumor-associated antigens that can be utilized as appropriate target molecules. Monoclonal antibodies against tumor-associated antigens have been successfully adopted for targeting to various types of cancer cells. The incorporation of humanized monoclonal antibodies or single chain human antibodies, instead of rodent antibodies into immunoliposomes has resulted in better clinical applicability. Tumor-specific ligands other than monoclonal antibodies have also been investigated as in vivo tumor-directing molecules. However, the number of pre-clinical studies of anticancer treatments using tumor-specific liposomal drugs reporting successful targeting and enhanced therapeutic efficacy has been limited. Further refinement of tumor-specific interactions and liposomal formulations will be necessary for the application of the tumor-specific liposomal drug strategy for anticancer chemotherapy or gene therapy.     

Three-dimensional morphological analysis of antigen-antibody reaction in hepatic sinusoids preserved in hypothermic UW solution.

ICAM-1 antigen-antibody reaction was visualized by three-dimensional immunoscanning electron microscopy of hepatic sinusoids in rat liver treated with hypothermic University of Wisconsin (UW) organ preservation solution. The results were compared with similar antigen-antibody reactions carried out with immunoliposomes injected in vivo. Morphologically, the hepatic sinusoids were preserved well during the hypothermic procedure. Endothelial cells had a large number of fenestrations, which partly aggregated and formed sieve plates. ICAM-1 expression was induced by injection of LPS and detected by monoclonal antibody in the UW solution followed by gold-labeled secondary antibody. ICAM-1 was restricted mostly to the unique areas of sieve plates with immature, small fenestrations. A similar distribution of ICAM-1 was present when detected by in vivo injection of immunoliposomes containing the monoclonal ICAM-1 antibody. The results showed that antigen-antibody reactions can take place in livers preserved in hypothermic UW solution. Further, the reaction is similar to that which could occur in vivo during transplantation. This suggests that it may be possible to block potentially harmful antigen-antibody reactions by addition of appropriate antibodies to hypothermic UW solution prior to transplantation.