Comparative immunohistochemistry of L19 and F16 in non-small cell lung cancer and mesothelioma: two human antibodies investigated in clinical trials in patients with cancer

The antibody-mediated targeted delivery of therapeutics to tumor sites is an attractive avenue for combating cancer while sparing normal tissues. Indeed, five derivatives of the human monoclonal antibodies L19 and F16, specific to splice isoforms of fibronectin and tenascin-C, are currently being investigated in clinical trials in patients with malignancies. Until now, a comparative immunohistochemical analysis of these antibodies, which recognize components of the modified extracellular matrix, was missing. Here, we report that the majority of NSCLC and mesothelioma specimens are stained with both antibodies in the stroma, while non-tumoral lung and mesothelium samples rarely exhibit reactivity with either L19 or F16. In our analysis, the anti-tenascin F16 antibody was found to generally exhibit a stronger staining of desmoplastic stroma surrounding tumor. This superior performance was found to be particularly striking in the case of low-grade non-small cell lung cancer.     

Non‐internalizing antibody–drug conjugates display potent anti‐cancer activity upon proteolytic release of monomethyl auristatin E in the subendothelial extracellular matrix

Antibody–drug conjugates (ADCs) represent a promising class of biopharmaceuticals with the potential to localize at the tumor site and improve the therapeutic index of cytotoxic drugs. While it is generally believed that ADCs need to be internalized into tumor cells in order to display optimal therapeutic activity, it has recently been shown that non‐internalizing antibodies can efficiently liberate disulfide‐linked drugs at the extracellular tumor site, leading to potent anti‐cancer activity in preclinical animal models. Here, we show that engineered variants of the F16 antibody, specific to a splice isoform of tenascin‐C, selectively localize to the subendothelial tumor extracellular matrix in three mouse models of human cancer (U87, A431, MDA‐MB‐231). A site‐specific coupling of F16 in IgG format with a monomethyl auristatin E (MMAE) derivative, featuring a valine‐citrulline dipeptide linker equipped with a self‐immolative spacer, yielded an ADC product, which cured tumor‐bearing mice at a dose of 7 mg/Kg. The observation of an efficient extracellular proteolytic cleavage of the valine‐citrulline linker was surprising, as it has generally been assumed that this peptidic structure would be selectively cleaved by cathepsin B in intracellular compartments. The products described in this article may be useful for the treatment of human malignancies, as their cognate antigen is strongly expressed in the majority of human solid tumors, lymphomas and aggressive leukemias, while being virtually undetectable in most normal adult tissues.     

Clinical impact and functional aspects of tenascin-C expression during glioma progression

The extracellular matrix protein tenascin-C is expressed in processes like embryogenesis and wound healing and in neoplasia. Tenascin-C expression in gliomas has been described previously; however, the relation to clinical data remains inconsistent. Generally, analysis of tenascin-C function is difficult due to different alternatively spliced isoforms. Our studies focus on changes in tenascin-C expression in human gliomas, correlating these changes with tumor progression and elucidating the functional role of the glioma cell-specific tenascin-C isoform pool. Eighty-six glioma tissues of different World Health Organization (WHO) grades were analyzed immunohistochemically for tenascin-C expression. The influence of the specific tenascin-C isoforms produced by glioblastoma cells on proliferation and migration was examined in vitro using blocking antibodies recognizing all isoforms. In general, tenascin-C expression increased with tumor malignancy. Perivascular staining of tenascin-C around tumor-supplying blood vessels was observed in all glioblastoma tissues, whereas in WHO II and III gliomas, perivascular tenascin-C staining appeared less frequently. The appearance of perivascular tenascin-C correlated significantly with a shorter disease-free time. Analysis of proliferation and migration in the presence of blocking antibodies revealed an inhibition of proliferation by around 30% in all 3 glioblastoma cell cultures, as well as a decrease in migration of 30.6-46.7%. Thus we conclude that the endogenous pool of tenascin-C isoforms in gliomas supports both tumor cell proliferation and tumor cell migration. In addition, our data on the perivascular staining of tenascin-C in WHO II and III gliomas and its correlation with a shorter disease-free time suggest that tenascin-C may be a new and potent prognostic marker for an earlier tumor recurrence.     

Expression and degeneration of tenascin-C in human lung cancers.

Tenascin-C is an extracellular matrix glycoprotein produced in response to epithelial-mesenchymal interactions during organogenesis and tissue remodelling. It has therefore been proposed as a stromal marker for epithelial malignancy. To test this hypothesis, 30 human lung cancers, presenting a variety of clinicopathological features, and six specimens of normal tissue were examined by Western and Northern blotting of tenascin-C protein and mRNA. The results obtained were: (1) elevated tenascin-C expression was detected in all 30 cases by Western blotting, with mRNA increase in 22 of them; (2) mRNA for a large isoform of tenascin-C, including an alternatively spliced sequence, was expressed in lung cancer tissues but not in normal lungs; and (3) metastasis to lymph nodes was frequently found in cases whose tenascin-C was degraded into small fragments. These results suggest that tenascin-C degradation can be used as a marker for metastatic potential of a tumour.     

Preparation of monoclonal antibody to P53 and its clinical application

Objective:The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initial y in clinical immunoassay. Methods:Monoclonal antibody wa...     

NHS76/PEP2, a fully human vasopermeability-enhancing agent to increase the uptake and efficacy of cancer chemotherapy.

PURPOSE: Previously, we have shown that the attachment of interleukin 2 (IL-2) to a tumor-targeting antibody can produce a 4-fold enhancement in the uptake of antibodies and drugs in tumors. More recently, we discovered that a 37-amino-acid linear sequence of IL-2 designated vasopermeability-enhancing peptide (PEP), contained the vasopermeability activity of IL-2, and could be used after linkage to tumor-targeting antibodies to produce the same enhancement of drugs and antibodies in tumors. We now describe the generation of a fully human antibody fusion protein, designated NHS76/PEP(2), which can be used in patients to enhance the therapeutic potential of chemotherapy. METHODS: NHS76/PEP(2) was expressed in NS0 cells using the glutamine synthetase gene amplification system. To show its clinical potential as a pretreatment to chemotherapy, NHS76/PEP(2) was given i.v. 2 hours before the injection of suboptimal doses of etoposide, doxorubicin, Taxol, Taxotere, 5-fluorouracil, or vinblastine in mice bearing established solid tumors. Results were recorded by measuring tumor volumes thrice per week. RESULTS: Compared with drug treatment alone, NHS76/PEP(2) pretreatment substantially improved the effectiveness of chemotherapeutic agents in solid tumor models. Tumor suppression was most pronounced in those groups of mice bearing tumors known to be sensitive to the specific drug under study. However, in certain instances, tumors previously known to be resistant to specific single chemotherapeutic agents were shown to respond by the addition of NHS76/PEP(2) pretreatment. CONCLUSIONS: NHS76/PEP(2) seems an excellent candidate to improve the value of standard chemotherapy drug treatment by virtue of its ability to increase the uptake of drugs in solid tumors selectively.     

The clinicopathological and prognostic impact of 14-3-3 protein isoforms expression in human cholangiocarcinoma by immunohistochemistry

The 14-3-3 proteins are highly conserved, ubiquitous molecules involved in a variety of biologic phenomena, such as cell cycle control, and apoptosis. However, their expression in cholangiocarcinoma has not been previously characterized. In this paper, immunohistochemistry using specific anti-14-3-3 monoclonal antibodies was performed on formalin-fixed;, paraffin embedded archival tissue from 86 patients of cholangiocarcinoma. We also examined the correlation between expression and survival rate and clinicopathologic factors such as tumor location, tumor size, pathologic differentiation, lymphatic permeation, lymph node metastasis, and tumor stage. Positive 14-3-3 proteins expression was observed for 6 isoforms (β, σ, γ, θ, β, η) of these proteins in 86 patients of cholangiocarcinoma. β and σ isoform immunoreactivity was correlated with lymph node metastasis, tumor stage and patients' survival rate. In addition, δ isoform immunoreactivity showed trends with tumor location, tumor size, pathologic differentiation and tumor stage, while the θ isoform was correlated with pathologic differentiation. These results indicated that upregulated expression of some isoforms of 14-3-3 may be a common mechanism for evading apoptosis in cholangiocarcinoma, so that targeting 14-3-3 may be a novel promising strategy for the treatment of this tumor.     

Preclinical studies on the pharmacokinetic properties of human monoclonal antibodies to colorectal cancer and their use for detection of tumors

We studied the pharmacokinetic properties of two human monoclonal antibodies to colon carcinoma cells and their ability to detect tumors in nude mice bearing primary human colon carcinoma xenografts. The 16-88 and 28A32 monoclonal antibodies are immunoglobulin M class human antibodies produced by cell lines derived from peripheral blood lymphocytes from patients with colon carcinoma. The patients received an autologous tumor cell vaccine as part of an active specific immunotherapy protocol. The 125I-labeled antibodies were cleared from the circulation of non-tumor-bearing and tumor-bearing nude mice with a 6-8-h half-life. The half-life of the antibodies in tumor tissue was 48 to 72 h compared to 8 to 12 h for normal tissues. Tumor:normal tissue ratios were highest 4 to 7 days postinjection with tumor:blood ratios of 12:1 for 16-88 and 10:1 for 28A32 antibody. Experiments with a control human immunoglobulin M myeloma protein confirmed the specificity of the human monoclonal antibodies. Radioimmunoscintigraphic studies using nude mice bearing contralateral antibody-reactive and nonreactive colon tumor xenografts further confirmed that the antibodies specifically localized in tumor tissues. The antibody-reactive tumors were clearly visible by radioimmunoscintigraphy within 4 days of injection. These experiments, undertaken as a preliminary step to clinical trials, demonstrated for the first time that i.v. administered human immunoglobulin M monoclonal antibodies could be taken up by human colon tumor tissue and retained to a sufficient extent to easily permit tumor detection by external radioimmunoscintigraphy. These studies also demonstrated that the nude mouse human colon tumor xenograft model is a useful in vivo system for comparison studies of human monoclonal antibodies as part of a selection process for clinical trials and for evaluating immunoconjugates containing these antibodies for relative pharmacokinetic properties and potential diagnostic or therapeutic efficacy.     

Localization of GD2-specific monoclonal antibody 3F8 in human osteosarcoma

3F8 is a murine IgG3 monoclonal antibody specific for the antigen disialoganglioside GD2. Immunofluorescence staining showed strong binding of 3F8 to 15 of 17 human osteosarcomas, including primary and metastatic tumors. The targeting potentials of the native monoclonal antibody (3F8) and the F(ab')2 fragment (p-3F8) were tested in BALB/c athymic nude mice xenografted with human osteosarcomas. After radiolabeling with iodine using the chloramine T method, both 3F8 and p-3F8 retained immunoreactivities. The irrelevant IgG3 antibody TIB114 and its F(ab')2 fragment were used as negative controls. A Ewing's sarcoma xenograft, which was low in GD2 antigen, was also studied for comparison. Mice were sacrificed 1 day and 4 days after i.v. antibody injection. 3F8 and p-3F8 showed preferential accumulation in osteosarcoma over normal tissues with tumor:nontumor ratios of 2.7-58:1 and 1.4-82:1, respectively, on Day 1. These ratios improved to 10-163:1 and 6.0-75:1 on Day 4. The intact antibody 3F8 showed selective tumor uptake with a much higher percentage of injected dose per g than the fragment p-3F8 and exhibited a longer tissue half-life than p-3F8. These data indicate that anti-GD2 monoclonal antibodies may be useful for imaging and targeted therapy of human osteogenic sarcoma. The F(ab')2 fragment has the advantage of achieving favorable tumor:nontumor ratios sooner after antibody injection while the intact antibody shows better retention by tumor tissues.     

Immunohistochemical localization of prostate carcinoma-associated antigens

The immunoperoxidase technique was used to study the localization and distribution of antigens reactive with two monoclonal antibodies, D83.21 and P6.2, produced against cultured prostate tumor cells, in formalin-fixed, paraffin-embedded histological sections of human tissues. Monoclonal D83.21 reacted with 11 of 19 (58%) primary prostate carcinomas and 1 of 6 (17%) metastatic tumors, whereas monoclonal P6.2 reacted with 14 of 19 (68%) primary and 4 of 6 (67%) metastatic prostate tumors. Neither antibody reacted with five primary prostate tumors and one metastatic prostate tumor. In some tumor cells, the antigens recognized by these monoclonals were localized in either the cytoplasm or cell membrane, while in other tumor cells, both diffuse cytoplasmic and membrane or focal staining patterns were observed. In addition to the variable staining patterns, antigenic heterogeneity was also noted within most prostate tumors examined. Two types of staining variability were observed: (a) tumor cells in one area of the tissue section stained positive, but in another area they did not react with the antibody; and (b) both stained and unstained tumor cells were adjacent to each other. These results would suggest that a panel of monoclonals will be required to detect the different subpopulations of prostate tumor cells. Neither antibody reacted with 6 normal or 12 benign prostate tissues, nor any of a variety of other normal human tissues except for staining of the proximal tubules of normal kidneys. The antigen detected by P6.2 demonstrated a wider tissue distribution being found on bladder, breast, lung, and pancreatic tumors, whereas the antigen recognized by D83.21 was restricted to prostate and bladder carcinomas. These antibodies may have clinical applicability for the identification of prostate tumor cells in biopsy specimens and for immunohistopathological classification of prostate carcinomas.     

Detection of tenascin-C isoforms in colorectal mucosa, ulcerative colitis, carcinomas and liver metastases.

The glycoprotein tenascin-C is up-regulated in inflammatory and neoplastic diseases. Most available data on tissue tenascin-C content do not distinguish its various isoforms. We have quantified tissue tenascin-C signals in colorectal mucosa, ulcerative colitis, colorectal carcinomas and liver metastases using 5 monoclonal antibodies (MAbs) with different binding sites. Tenascin-C of tissue extracts was analyzed by a standardized Western blot technique and densitometry. As a reference MAb, K8 displayed tenascin-C tissue concentrations of 4.1 +/- 2.3 microgram/mg total protein in normal mucosa, 13.8 +/- 4.7 microgram/mg in ulcerative colitis, 28.8 +/- 14.5 microgram/mg in colorectal carcinomas and 25.6 +/- 8.9 microgram/mg in liver metastases. The optical density values per microgram protein tissue extract of the 5 MAbs reflect the levels of the corresponding tenascin-C epitopes. Various signal intensities indicate a distinct diagnostic usefulness of the MAbs in detecting colorectal carcinomas. The binding characteristics of MAb J1/tn2 point to an under-representation of the TNfnD domain in metastasizing colorectal carcinomas, while MAb 19H12 showed an increased binding rate on the TNfnA1,2,4 region. Our comparative study of tenascin-C in inflammatory and neoplastic diseases of the colon mucosa substantiates the occurrence of large differences in the diagnostic value of tenascin-C MAbs. The detected alterations of tenascin-C in metastasizing colorectal carcinomas might indicate a prognostic value of specific tenascin-C isoforms.     

Prognostic significance of tenascin-C expression in clear cell renal cell carcinoma

Tenascin-C is an extracellular matrix protein that plays an important role in cell proliferation, migration and tumor invasion in various types of cancer. However, few reports exist on tenascin-C expression in renal cell carcinoma (RCC). This study aimed to assess the prognostic significance of tenascin-C in clear cell RCC. Using immunohistochemistry, 137 formalin-fixed, paraffin-embedded tissue sections obtained from patients with clear cell RCC were examined for tenascin-C expression. Tenascin-C expression was observed in 55 (40.1%) of the 137 clear cell RCC sections. Tumor cells displayed membranous and/or cytoplasmic staining for tenascin-C. Tenascin-C expression was more prominent near the pseudocapsule of the tumor and around the tumor vessels. Tenascin-C expression was significantly associated with a higher stage (P=0.0065) and higher nuclear grade (P=0.0001). However, there was no correlation between the tenascin-C expression and venous involvement. The cancer-specific survival rate in patients with a tenascin-C-positive primary tumor was significantly lower than that in those with a tenascin-C-negative primary tumor in univariate analysis (P=0.0017). However, tenascin-C expression did not exhibit a significant value for cancer-related death in the Cox regression analysis. In patients with stage 1-3 disease, the 5-year metastasis-free rate in patients with the tenascin-C-positive primary tumor was significantly lower than that in those with the tenascin-C-negative primary tumor (67.8 vs. 88.5%, respectively; P=0.0038). The Cox regression analysis showed that tenascin-C expression is a significant predictor of metastasis (P=0.0345). The tenascin-C expression was strongly related to the stage, nuclear grade and 5-year metastasis-free rate. Therefore, tenascin-C expression may be a possible marker for the metastatic potential of clear cell RCC.     

Limitations of radiolabeled monoclonal antibodies for localization of human neoplasms

Tumor-associated monoclonal antibodies were radiolabeled with 125I and 131I and given i.v. in pairs to 19 patients 1-26 days prior to surgical excision of primary and metastatic breast, ovarian, and gastrointestinal tumors. For individual patients each monoclonal antibody was designated as specific or nonspecific according to prior immunoperoxidase staining results on the appropriate target neoplastic tissues. Quantitation of antibody uptake was performed on resected normal and neoplastic tissues. Although good tumor:non-tumor ratios were obtained with the specific antibodies (maximal tumor:blood ratio, 35.8:1 at 12 days postadministration), the absolute amount of radiolabel detected in tumors was small (mean value of 0.015% of total injected amount per g of tumor occurring 1 day postadministration). Furthermore, both specific and nonspecific antibodies accumulated in normal lymph nodes to a significant extent (mean value of 0.0026% of total injected amount per g of tissue occurring 1 day postadministration). Knowledge of such data is essential prior to considering therapeutic uses of radiolabeled monoclonal antibodies.     

Chemical engineering of the monoclonal antibody A7 by polyethylene glycol for targeting cancer chemotherapy

The murine monoclonal antibody A7 (Mab A7) against human colon cancer was chemically modified with methoxypolyethylene glycol (PEG) (Mr 5000). A high substitution of PEG molecules on Mab A7 produced a progressive reduction in antibody-binding activity. The pharmacokinetic and immunological properties of PEG-modified monoclonal antibody A7 (Mab A7) and the PEG-modified F(ab')2 fragment, which retained their antibody-binding activity, were assessed and compared with the parent Mab A7 and the parent F(ab')2 fragment. Blood clearance of PEG-modified antibodies appeared to be diminished by PEG modification and was fitted by a two-compartment model. Low PEG-substituted Mab A7 showed less organ uptake in the liver and spleen and similar uptake in the lung and kidney, compared with the parent Mab A7. PEG-F(ab')2 showed less uptake in the liver and kidney. Both preparations exhibited less tissue:blood ratios in all resected organs as compared with parent antibodies. Tumor localization was enhanced by PEG modification for the F(ab')2 fragment, but not by PEG modification for the whole Mab A7. Multiple i.v. administration of PEG-modified antibody to rabbit did not appear to elicit a measurable immune response to the antibody portion of the conjugate. In conclusion, PEG-modified antibodies are promising reagents as drug carriers to the target tumor.     

Detection of membrane-bound alpha-fetoprotein in human hepatoma cell lines by monoclonal antibody 19F12

Monoclonal antibodies against human alpha-fetoprotein (AFP) were obtained by the hybridoma technique and studied with regard to their reactivities with the human hepatoma cell lines PLC/PRF/5 and KN, and a spontaneously immortalized cell line derived from fetal liver, NuE, all of which synthesize AFP. One of the monoclonal antibodies, 19F12 (IgG2b) became bound to free AFP which was used as the immunogen with an affinity constant of 3.4 X 10(8) M-1. This value was not much higher than those of two other antibodies, 19B1 (IgG1) and 9D12 (IgG2b). However, only antibody 19F12 showed definite reactivity with AFP-producing cells in analysis using flow cytometry. Immunofluorescence microscopy showed that antibody 19F12 detected AFP over the surface of NuE and PLC/PRF/5 cells with a uniform distribution, whereas definite reactivities of antibodies 19B1 and 9D12 to these cells were not detected. These antibodies did not show the specific binding to a non-AFP-producing human lung cancer cell line, PC-9, or to human peripheral blood lymphocytes. The binding ability of 19F12 to hepatoma cells was shown in both viable and fixed cells. Addition of free AFP inhibited the binding of antibody 19F12 to PLC/PRF/5 cells in a concentration-dependent manner. The specific reactivity of 19F12 to human AFP was also confirmed by immunostaining of a tissue section of human cancer proved to be AFP positive with AFP-specific antisera. In two-dimensional polyacrylamide gel electrophoresis of the antigen (from membrane fraction of PLC/PRF/5 cells)-antibody (19F12) complex, spots derived from the antibody and a spot (pI 4.7, Mr 65,000) corresponding in pI and molecular weight to AFP were detected. Western blot analysis showed that material in the membrane fraction of PLC/PRF/5 cells recognized by antibody 19F12 has the same molecular weight as human AFP derived from placenta. In a study of reactivities to PLC/PRF/5 cells treated with various enzymes, the reactivity of this antibody decreased when cells were treated with protease and trypsin and increased when lipase was used. The binding of 19F12 to AFP was not inhibited by concanavalin A. The antibody 19F12 appeared to recognize an epitope that is considered to be part of the peptide area of AFP. These results indicate that the reactivity, the amount of bound antibodies, and the distribution of monoclonal antibodies on antigen-producing cells vary, respectively, even though these antibodies were produced using the same antigen as an immunogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoclonal antibodies to cell surface antigens shared by chemically induced mouse bladder carcinomas

Rats were immunized with cultured cells from chemically induced transitional cell carcinomas of the mouse urinary bladder, and their spleen cells were hybridized with NS-1 mouse myeloma cells. Following initial screening of antibodies made by hybridoma clones, the tissue distribution of antigens defined by the antibodies was established by using a peroxidase-antiperoxidase technique with frozen sections of a variety of mouse tumors, as well as normal adult and embryonic tissues. Two antibodies were identified which detected antigens with bladder carcinoma specificity. One antibody (3B12) reacted weakly with epithelial cells from several sources, including normal bladder, while the second antibody (6.10), which bound strongly to bladder carcinoma cells, was negative on bladder epithelium and bound (weakly) to only a small fraction of all epithelial cells tested except for epidermal cells and periosteum from embryos. Both antibodies should be useful to assess the immunotherapeutic and immunoprophylactic effects of monoclonal antibodies to tumor-type specific oncofetal antigens.     

Three clinical-stage tumor targeting antibodies reveal differential expression of oncofetal fibronectin and tenascin-C isoforms in human lymphoma

The antibody-based targeted delivery of bioactive molecules to tumor vasculature is an attractive avenue to concentrate therapeutic agents at cancer sites, while sparing normal organs. L19, F8 and F16 are clinical-stage human monoclonal antibodies, which selectively recognize splice isoforms of fibronectin and tenascin-C in the modified extracellular matrix of neoplastic lesions. Here, we report the first comparative immunohistochemical analysis of L19, F8 and F16 in human Hodgkin and non-Hodgkin lymphomas. F16 was found to strongly stain the majority of lymphomas but also specimens of nonspecific lymphadenitis. By contrast, L19 exhibited a better discrimination between tumoral and inflammatory processes, yet at the expense of a weaker staining of the majority of lymphoma specimens investigated. The staining patterns observed for F8 were intermediate between the ones observed for L19 and F16. This study provides a rationale basis for the clinical investigation of therapeutic derivatives of the three antibodies in lymphoma patients.     

Pharmacokinetics and microdistribution of polyethylene glycol-modified humanized A33 antibody targeting colon cancer xenografts

Therapeutic proteins have been conjugated with polyethylene glycol (PEGylation) to reduce immunogenicity and enhance circulating dose. Here we have investigated the effect of PEGylation on immunogenicity, pharmacokinetics, and histologic microdistribution of tumor-targeting antibodies with humanized A33 antibody (huA33) as a model system. Conjugation of huA33 with methoxy-PEG of M(r) 5,000 (32%-34% of primary amines modified) or M(r) 20,000 (16%-18% modification) preserved >50% of native huA33 binding to SW1222 colon cancer cells. In mice, both PEGylated forms cleared from serum moderately slower than native huA33. After repeated immunization with PEG-huA33, antiantibody titers in immunocompetent mice were <5% of those in huA33-treated controls. Both PEG-huA33 forms reached approx. 75% of the maximum tumor dose of huA33 in SW1222-xenografted mice, but their tumor:blood ratios were considerably reduced. To demonstrate immunologic specificity of PEG-huA33 targeting in SW1222 tumor-bearing mice, antigenic sites were presaturated by injecting excess native huA33. This reduced subsequent uptake of PEG-huA33 by up to 80%, whereas presaturation with hu3S193 control antibody had no significant effect. To assess the microdistribution of antibody uptake in the same xenograft model, tumor tissue resected at different time points after antibody administration was examined for human IgG by immunohistochemistry. Both PEG preparations achieved the same peak staining intensity and homogeneity as native huA33 with a delay of several hours. Given the measured reduction in immunoreactivity in vitro, these results demonstrate that the tumor targeting potential of huA33 in vivo is preserved at PEGylation levels sufficient to suppress immunogenicity.     

Selective targeting of liver cancer with the endothelial marker CD146

Hepatocellular carcinomas are well-vascularized tumors; the endothelial cells in these tumors have a specific phenotype. Our aim was to develop a new approach for tumor-specific drug delivery with monoclonal antibody targeting of endothelial ligands. CD146, a molecule expressed on the endothelial surface of hepatocellular carcinoma, was identified as a promising candidate for targeting. In the present study, endothelial cells immediately captured circulating anti-CD146 (ME-9F1) antibody, while antibody binding in tumors was significantly higher than in hepatic endothelium. Macroscopically, after intravenous injection, there were no differences in the mean accumulation of anti-CD146 antibody in tumor compared to liver tissue, due to a compensating higher blood vessel density in the liver tissue. Additional blockade of nontumoral epitopes and intra-arterial administration, improved selective antibody capture in the tumor microvasculature and largely prevented antibody distribution in the lung and liver. The potential practical use of this approach was demonstrated by imaging of radionuclide-labeled ME-9F1 antibody, which showed excellent tumor-selective uptake. Our results provide a promising principle for the use of endothelial markers for intratumoral drug delivery. Tumor endothelium–based access might offer new opportunities for the imaging and therapy of hepatocellular carcinoma and other liver malignancies.     

Enhanced delivery of a monoclonal antibody F(ab')2 fragment to subcutaneous human glioma xenografts using local hyperthermia

The purpose of this study was to investigate the effects of tumor-localized hyperthermia at 42 degrees C on the tissue distribution of radioiodinated monoclonal antibody F(ab')2 fragments. Paired-label biodistribution measurements were performed in athymic mice bearing D-54 MG human glioma xenografts on one leg. Mice received both the 131I-labeled F(ab')2 fragment of Mel-14, reactive with human gliomas and melanomas, and nonspecific 125I-labeled RPC 5 F(ab')2. Tumor-bearing legs were placed in a 42 degrees C water bath or a 37 degrees C water bath (control) for 2 or 4 h. In mice sacrificed immediately after 2 h of heating, no hyperthermia-induced differences in the distribution of either fragment were observed. In the 4-h groups, tumor uptake of Mel-14 F(ab')2 increased from 7.04 +/- 1.59% injected dose (ID)/g at 37 degrees C to 20.65 +/- 4.53% ID/g at 42 degrees C (P less than 0.0001), and tumor localization of the control fragment rose from 5.23 +/- 1.35% ID/g to 14.51 +/- 1.37% ID/g (P less than 0.0001). In another experiment, F(ab')2 fragments were injected, tumors were heated for 4 h, and groups were sacrificed at 4, 8, and 16 h after injection. Statistically significant 2- to 3-fold higher uptake of both fragments in tumor were observed at all time points. Hyperthermic conditions also resulted in higher tumor:tissue ratios for both fragments. These results suggest that it may be possible to use tumor-localized hyperthermia to increase the therapeutic utility of radiolabeled monoclonal antibodies, particularly when labeled with short lived nuclides such as the 7.2-h alpha-emitter 211At.