Genetically designing a more potent antipancreatic cancer agent by simultaneously co-targeting human IL13 and EGF receptors in a mouse xenograft model.

OBJECTIVE: Investigators are currently interested in the epidermal growth factor receptor (EGFR) and interleukin 13 receptor (IL13R) as potential targets in the development of new biologicals for pancreatic cancer. Attempts to develop successful agents have met with difficulty. The novel approach used here was to target these receptors simultaneously with EGF and IL13 cloned on the same bispecific single-chain molecule with truncated diphtheria toxin (DT(390)) to determine if co-targeting with DTEGF13 had any advantages. DESIGN: Proliferation experiments were performed to measure the potency and selectivity of bispecific DTEGF13 and its monospecific counterparts against pancreatic cancer cell lines PANC-1 and MiaPaCa-2 in vitro. DTEGF13 was then administered intratumourally to nude mice with MiaPaCa-2 flank tumours to measure efficacy and toxicity (weight loss). RESULTS: In vitro, bispecific DTEGF13 was 2800-fold more toxic than monospecific DTEGF or DTIL13 against PANC-1. A similar enhancement was observed in vitro when MiaPaCa-2 pancreatic cancer cells or H2981-T3 lung adenocarcinoma cells were studied. DTEGF13 activity was blockable with recombinant EGF13. DTEGF13 was potent (IC(50) = 0.00017 nM) against MiaPaCa-2, receptor specific and significantly inhibited MiaPaCa-2 tumours in nude mice (p<0.008). CONCLUSIONS: In vitro studies show that the presence of both ligands on the same bispecific molecule is responsible for the superior activity of DTEGF13. Intratumoural administration showed that DTEGF13 was highly effective in checking aggressive tumour progression in mice. Lack of weight loss in these mice indicated that the drug was tolerated and a therapeutic index exists in an "on target" model in which DTEGF13 is cross-reactive with native mouse receptors.

Combination of cytosine deaminase suicide gene expression with DR5 antibody treatment increases cancer cell cytotoxicity.

Combined treatment using adenoviral-directed enzyme/prodrug therapy and immunotherapy has the potential to become a powerful alternative method of cancer therapy. We have developed adenoviral vectors encoding the cytosine deaminase gene (Ad-CD) and cytosine deaminase:uracil phosphoribosyltransferase fusion gene (Ad-CD:UPRT). A monoclonal antibody, TRA-8, specifically binds to death receptor 5, one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of Ad-CD:UPRT and TRA-8 against human pancreatic cancer and glioma cell lines. The present study demonstrates that Ad-CD:UPRT infection resulted in increased 5-FC-mediated cell killing, compared with Ad-CD. Furthermore, a significant increase of cytotoxicity following Ad-CD:UPRT/5-FC and TRA-8 treatment of cancer cells in vitro was demonstrated. Animal studies showed significant inhibition of tumor growth of MIA PaCa-2 pancreatic and D54MG glioma xenografts by the combination of Ad-CD:UPRT/5-FC plus TRA-8 as compared with either agent alone or no treatment. The results suggest that the combination of Ad-CD:UPRT/5-FC with TRA-8 produces an additive cytotoxic effect in cancer cells in vitro and in vivo. These data indicate that combined treatment with enzyme/prodrug therapy and TRAIL immunotherapy provides a promising approach for cancer therapy.

Experimental approaches to increase radiolabeled antibody localization in tumors.

Approaches have been developed to improve the localization of radiolabeled monoclonal antibodies (MAbs) in experimental tumors, to reduce their uptake in normal tissues, and, thus, to improve the time-dependent tumor: normal tissue (T:NT) ratios so that higher and more frequent doses of radionuclide could be used for radioimmunotherapy. These approaches involve three general strategies: (a) modifying antibodies or radiolabeling techniques; (b) increasing the clearance of radiolabeled MAbs; and (c) modifying tumor delivery, tumor antigen expression, or increasing tumor vascular permeability or blood flow. The use of animal models permits the assessment of a wide range of MAbs, radiolabeling conditions, and the efficacy of administration methods before their initial use in clinical trials. MAbs with specificity for binding to tumor-associated antigens or growth factor receptors expressed on tumor cells have been utilized in experimental studies of radiolabeled antibody targeting. Tumor-associated targets present on endothelial cells should be highly accessible to systemically administered radiolabeled MAbs. The use of indirect radio-iodination techniques and labile linker-chelates may provide an improvement in tumor retention and T:NT ratios. The addition or deletion of glycosylation to MAbs by alteration of recombinant immunoglobulin genes or by biochemical modification can alter the pharmacokinetics of blood and whole body clearance of radiolabeled MAbs. Genetically engineered chimeric or humanized MAbs have shown equivalent or greater tumor localization compared to murine MAbs. By using MAbs with greater affinity and avidity, an increase in the uptake and retention of radiolabeled MAbs in tumors and an increase in their therapeutic efficacy may be achieved. Several approaches in the administration methods of MAbs have been developed in an attempt to improve tumor localization and therapeutic results and to reduce toxicity. These approaches include: (a) predosing with unlabeled antibody before administering a radiolabeled MAb; (b) using a mixture or "cocktail" of MAbs rather than a single radiolabeled antibody; and (c) administering multiple doses of radiolabeled MAbs. Various approaches have been tested for increasing the blood clearance of radiolabeled MAbs and, thus, for increasing the T:NT ratio. It has been found that compared to intact antibody, the smaller antibody fragments (F(ab')2, Fab, or single-chain Fv) can bind to tumor cells with a more homogeneous distribution. The antibody fragments and domain deletions often have a more rapid catabolism in blood, in tumors, and in normal tissues than an intact antibody does. In general, the use of antibody fragments leads to higher T:NT ratios but a lower percentage of injected dose delivered to the tumor.

Reconciliation of tumor dose response to external beam radiotherapy versus radioimmunotherapy with 131iodine-labeled antibody for a colon cancer model.

Reported doses of external beam radiotherapy and radioimmunotherapy (RIT) to produce equivalent therapeutic effects are inconsistent, with many proposed causes. Calculations of effective dose were performed for the case of LS174T human colon cancer xenografts, where a 60Co single fraction exposure (6 Gy) was matched with 131I-labeled 17-1A monoclonal antibody therapy (300 microCi injection, 19 +/- 2 Gy using the Medical Internal Radiation Dose uniform isotropic model). Measured three-dimensional dose-rate distributions were used to form a time-dependent description of the dose-rate nonuniformity. Included in the calculation of RIT effective dose was energy loss, dose nonuniformity, dose-rate dependence, hypoxic fraction, and cell proliferation. The calculations assumed the linear quadratic model for cell survival with alpha = 0.3 Gy-1, alpha/beta = 15 to 25 Gy, and mu = 0.46 h-1. The biologically effective dose for the single fraction 60Co exposure was 7.4 to 8.4 Gy. Estimates of dose efficiency factors consecutively applied to the RIT dose estimate were: (a) energy loss external to the tumor (x0.85); (b) effect of dose nonuniformity on cell survival (x0.65); and (c) effect of correlation of dose nonuniformity with cell proliferation rate (x1.08). The resulting effective dose for RIT was 11.4 Gy for tumor regrowth. This analysis substantially reconciles external beam radiotherapy/RIT dose-response results for this tumor model to within experimental uncertainties.

Distribution of radiolabeled alloantibodies in mice bearing 3-methylcholanthrene-induced sarcoma.

The distribution of purified 125I-labeled alloantibodies, prepared from the serum of DBA/2J mice obtained after immunization with C3H/HeJ spleen cells, was studied in immunosuppressed DBA/2J mice bearing either allogeneic C3H/HeJ 3-methylcholanthrene sarcomas growing s.c. or syngeneic SaD2 3-methylcholanthrene sarcomas. Once purified radiolabeled antibody was isolated from 125I-labeled immune gamma-globulin by a single adsorption onto C3H/HeJ RBC and elution from stroma prepared from these cells, by using 0.1 M glycine buffer (pH 3.0). Twice-purified alloantibody was then produced by Bio-Gel P-200 or Sephadex G-200 gel filtration chromatography or DEAE A-50 ion-exchange chromatography. In vitro, such purified antibodies bound specifically to C3H/HeJ RBC. In vivo, they localized to a significant extent following i.p. injection, preferentially in C3H/HeJ 3-methylcholanthrene sarcomas (4.4 to 8.9% of the injected dose per g of tumor equal to 1% of mouse weight), with mean tumor/blood ratios of 4.0 to 7.8, at 24 or 48 hr after injection. The percentage of injected dose localized in tumor and the tumor/blood ratio did not show significant differences with respect to time or method of antibody purification. Normal tissue/blood ratios in C3H/HeJ or SaD2 sarcoma-bearing mice were less than 0.9. The tumor/blood ratios in SaD2 sarcomas were approximately 0.6. Injection of 131I-labeled normal DBA/2J gamma-globulin resulted in normal tissue/blood and tumor/blood ratios of less than 0.9 in C3H/HeJ tumor-bearing mice.

Therapy with unlabeled and 131I-labeled pan-B-cell monoclonal antibodies in nude mice bearing Raji Burkitt's lymphoma xenografts.

Clinical trials of radioimmunotherapy (RIT) of lymphoma have produced frequent tumor regressions and remissions, but it has been difficult to determine to what extent these tumor responses have been due to antibody-specific targeted radiation, nontargeted radiation, and/or cytotoxicity mediated by the carrier monoclonal antibody (MoAb). In this report, RIT was studied in athymic nude mice bearing s.c. Raji human Burkitt's lymphoma xenografts using two different pan-B-cell MoAbs, MB-1 (anti-CD37) and anti-B1 (anti-CD20), which differ in isotype (and thus the potential for interaction with host effector mechanisms) and isotype-matched control antibodies either in the unlabeled state or labeled with 131I. When a single i.p. injection of 300 microCi 131I-labeled MB-1 (IgG1) was compared to treatment with unlabeled MB-1 or 300 microCi 131I-labeled MYS control IgG1 MoAb, an antibody-specific targeted radiation effect of RIT was seen. 131I-labeled MB-1 produced a 44 +/- 19% (SEM) reduction in tumor size at 3 weeks posttreatment, while unlabeled MB-1 or 300 microCi 131I-labeled MYS control IgG1 antibody treatment resulted in continued tumor growth over this period of time. In vitro studies demonstrated that MB-1 was incapable of mediating antibody-dependent cellular cytotoxicity using Raji tumor cell targets and human peripheral blood mononuclear cells. Similar to the MB-1 studies, treatment with 300 microCi 131I-labeled anti-B1 produced a 64% reduction in mean tumor size, while 300 microCi of control antibody resulted in a 58% increase in tumor size over the same 3-week period. In contrast to MB-1, however, unlabeled anti-B1 (an IgG2a MoAb which in vitro studies showed to be capable of antibody-dependent cellular cytotoxicity) also had a substantial antitumor effect. Indeed, 300 microCi 131I-labeled anti-B1 and unlabeled anti-B1 treatment (using an equivalent amount of total protein in the treatment dose) produced a similar specific reduction in tumor size. Increasing the radionuclide dose of anti-B1 to 450 microCi in another experiment did not produce a significant difference in tumor regression compared to a 300-microCi dose. These results suggest that the antitumor effects of 131I-labeled anti-B1 treatment were dominated by antibody-mediated cytotoxicity mechanisms, such that an antibody-specific targeted radiation effect could not be distinguished. In contrast, antibody-specific targeting of radiation was the dominant mechanism of tumor killing with 131I-labeled MB-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytotoxic effects of anti-CD5 radioimmunotoxins on human tumors in vitro and in a nude mouse model.

An immunoconjugate, consisting of both toxin and radionuclide on the same antibody molecule, was synthesized by cross-linking the phytotoxin ricin to the T101 monoclonal antibody recognizing the CD5 cluster expressed on normal and malignant T-cells. The hybrid molecule was then labeled with iodine-125 by an iodine monochloride procedure. This radioimmunotoxin (RIT), which selectively bound to the CD5-positive CEM human leukemia cell line, was selectively inhibitory to antigen-positive cells in protein synthesis inhibition assays. RIT was only 3.0-7.8-fold less toxic and was 1.1-1.6-fold slower than unlabeled immunotoxin in inhibiting protein synthesis. Because of the radionuclide moiety, the RIT also provided information related to biodistribution and pharmacokinetics. Four days following intratumoral injection, more than 125-fold greater activity was found in CEM tumors implanted in nude mice as compared to normal tissues. The mean blood half-life for RIT was 25.7 h and for radiolabeled antibody, 91.3 h. Intratumoral injections of RIT selectively induced regression of established CEM tumors. To our knowledge, these studies are the first to demonstrate that a single immunoconjugate can combine the advantages of both a catalytic toxin and radionuclide for cancer therapy.

Radiotherapy in mice with yttrium-90-labeled anti-Ly1 monoclonal antibody: therapy of the T cell lymphoma EL4.

Yttrium-90 is a potent beta-emitting radionuclide with potential for therapy of lymphoma. A monoclonal antibody against Ly1, the murine homologue of human CD5, was labeled with 90Y and found to selectively bind to Ly1-positive, radiation-sensitive, EL4 mouse lymphoma cells. When tested in this aggressive model of T cell lymphoma, in vivo studies in C57BL/6 mice showed that a single 140-microCi i.p. dose of 90Y-anti-Ly1, given 1 day after i.v. injection of a lethal dose of EL4 cells, resulted in significant but transient improvement in survival. Protection was selective, since a 90Y-labeled irrelevant control antibody did not prolong survival. Biodistribution studies showed that protection was likely limited by inadequate localization of labeled antibody to tumor. Importantly, labeled anti-Ly1 specifically localized in the immunological tissue (spleen and thymus) and lowered the WBC count, perhaps limiting the tolerated dose. Myelosuppression, which is considered one of the major side effects associated with 90Y usage, was not a lethal complication, since WBC counts recovered in mice given a 140-microCi dose of 90Y-anti-Ly1 without EL4 cells and 100% of these animals survived. The maximum tolerated dose was less than 200 microCi. Despite the high localization of 90Y-anti-Ly1 in spleen, splenectomies of tumor-injected mice did not improve the antitumor efficacy of radiolabeled antibody. Further evidence for inadequate delivery of radionuclide to tumor was shown when external total-body irradiation was given to mice given injections of a lethal dose of EL4 tumor cells. Comparison of internal and external irradiation studies indicated that the partially protective effect of 140 microCi 90Y-Ly1 was equivalent to external radiation of only 100-200 cGy. Because this model reflects the current clinical limitations of radiolabeled antibodies for therapy, including partial antitumor efficacy, delivery of labeled anti-T cell antibodies to the immune system, and low maximum tolerated dose, the model may be useful for examining strategies which could increase the tolerated dose and therapeutic efficacy.

Radiotherapy in mice with yttrium-90-labeled anti-Ly1 monoclonal antibody: therapy of established graft-versus-host disease induced across the major histocompatibility barrier.

A monoclonal antibody recognizing Ly1, the murine homologue of CD5, was labeled with 90Y. In vivo biodistribution studies showed that 90Y-anti-Ly1 selectively localized in lymphoid tissue. Groups of B10,BR mice (H-2k) were lethally irradiated and given major histocompatibility complex-disparate C57BL/6 (H-2b) bone marrow and spleen cells to induce graft-versus-host disease (GVHD). Eight days later, mice with active GVHD were administered a single i.p. injection of 50 microCi90Y-anti-Ly1. Fifty % of these mice were alive 2 months after treatment. Long term (greater than 4-month) survival was significantly higher than in phosphate-buffered saline-treated mice. Survival was slightly improved in groups of mice receiving control irrelevant antibody labeled with 90Y or mice receiving free 90Y. However, survival in these groups was not significantly different from the phosphate-buffered saline-treated control group. The improved survival was supported by data showing improved mean animal weight. An anti-GVHD effect was confirmed by histopathological analysis. Unlabeled anti-Ly1 monoclonal antibody at comparable doses to 90Y-anti-Ly1 was not effective. Animals that died following 50-microCi treatment did not die of radiation toxicity, since all mice receiving 50 microCi 90Y-anti-Ly1 plus syngeneic bone marrow survived. The window of therapy was narrow in our studies, since 100 microCi 90Y-anti-Ly1 did not confer any survival advantage. Animals that did survive long term were studied for evidence of alloengraftment and found to have high levels of circulating donor mononuclear cells. 90Y-Anti-Ly1 localized in the spleen, thymus, liver, kidney and bone marrow but not in the bowel, lung, muscle, or skin. Animals given similar doses of free 90Y, 90Y-anti-Ly1, or labeled irrelevant antibody eliminated free 90Y fastest, followed by 90Y-anti-Ly1 and then labeled irrelevant antibody. Hematological analysis of peripheral blood from 90Y-anti-Ly1-treated mice showed reduction in total WBC counts, absolute lymphocyte numbers, and absolute neutrophil numbers on day 24 after treatment. Myelosuppression recovered by day 38. These findings indicate that Ly1-positive cells are involved in the effector phase of GVHD and that radiolabeled antibodies may be useful as cell-specific probes for studying the GVHD network. 90Y-Anti-Ly1 protected recipients long term from lethal GVHD, and the fact that it had a rather remarkable inhibitory and selective effect on the lymphoid system of mice suggests that these agents may have broader application in the field of transplantation.

Improved delivery of radiolabeled anti-B1 monoclonal antibody to Raji lymphoma xenografts by predosing with unlabeled anti-B1 monoclonal antibody.

A human B-cell lymphoma xenograft model was used to test whether the administration of unlabeled MoAb prior to injection of radiolabeled monoclonal antibody (MoAb) improves delivery of the radiolabeled MoAb to tumor prior to testing in clinical radioimmunotherapy trials. The anti-B1/CD20 pan-B-cell MoAb reactive with human B-cell lymphomas and leukemias but not reactive with mouse B-cells was used in this study. Athymic nude mice bearing human Raji Burkitt lymphoma xenografts were given injections of 2.5 muCi (0.3 microgram) 131I-labeled anti-B1 with or without a 2-h prior single injection of 100 micrograms of unlabeled anti-B1 antibody. Four days later the animals given injections of 131I-labeled anti-B1 and the unlabeled anti-B1 predose had a tumor uptake of 12.72 +/- 1.17% (SEM) of injected dose/g which was 44% greater than the animals receiving the 131I-labeled anti-B1 alone (P = 0.014). The uptake in most normal tissues was unchanged, although the blood level of 131I-labeled anti-B1 appeared to be greater following unlabeled anti-B1 predosing (P = 0.067). Predosing with isotype matched irrelevant MoAb did not result in a greater tumor uptake or blood concentration of 131I-labeled anti-B1 compared to the administration of 131I-labeled anti-B1 alone. In studies using 111In-labeled anti-B1, the effect of unlabeled antibody predosing was more pronounced. For animals given injections of 4.5 muCi (0.4 microgram) 111In-labeled anti-B1 and the unlabeled anti-B1 predose, the uptake in tumor was 12.37 +/- 2.07% of injected dose/g which was 162% greater than the animals receiving the 111In-labeled anti-B1 alone (P = 0.009). Predosing decreased 111In-labeled anti-B1 uptake in spleen, while the blood level was significantly greater. Predosing was more effective than simultaneous injection in improving tumor delivery. When tumor-bearing mice were either simultaneously given injections of 36 micrograms of unlabeled anti-B1 and 4 micrograms 111In-labeled anti-B1 or were given preinjections of 36 micrograms unlabeled anti-B1 3 h prior to injection of 4 micrograms 111In-labeled anti-B1, tumor uptake 3 days later was 1.3-fold higher in the animals which received the preinjection of unlabeled antibody (P = 0.011). As the quantity of unlabeled anti-B1 was increased (36, 96, 996 micrograms) in the predose, significantly greater uptake in tumor was observed, although this uptake appeared to plateau at the highest predoses.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo.

Cholangiocarcinoma is a virtually incurable tumor, resistant to current surgical, chemotherapy, and radiotherapy interventions. We applied the gene therapy strategy of toxin gene conversion of nontoxic prodrug to chemotherapeutic drug in combination with radiation therapy to the treatment of cholangiocarcinoma. In this regard, 5-fluorouracil (5-FU) is an accepted radiosensitizing and chemotherapeutic agent presently used in cancer therapy. The Escherichia coli enzyme cytosine deaminase (CD) converts the prodrug 5-fluorocytosine (5-FC) to 5-FU. Therefore, our goal was to express the CD gene in the human cholangiocarcinoma cell line, SK-ChA-1, assess the cytotoxicity of intracellular production of 5-FU, and determine any enhanced cell killing by the addition of external beam radiation. The susceptibility of SK-ChA-1 cells to recombinant adenoviral infection was determined by fluorescence-activated cell sorting analysis. We used the recombinant adenoviral vector AdCMVLacZ, encoding the E. coli beta-galactosidase reporter gene under control of the human cytomegalovirus (CMV) promoter, to infect SK-ChA-1 and HeLa cells at 10 and 100 plaque forming units (pfu)/cell, followed by FACS analysis. To evaluate CD-mediated conversion of 5-FC to 5-FU and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes CD. Cells were then plated in 96-well microtiter plates and exposed to varying concentrations of 5-FC. Cell proliferation assays (tetrazolium salt conversion to formazan colorimetric assay) were performed beginning 2-8 days after plating. We evaluated the effects of external beam radiation using a single 8 Gy 60Co dose to AdCMVCD infected cells, with prior exposure to 5-FC for 2-3 days. MTS assays were performed following radiation treatment. Radiation dose-response analysis, via clonogenic assay, was used as a more sensitive assay to confirm the interaction of the treatment conditions. s.c. SK-ChA-1 tumors in athymic nude mice were established, which then received three intratumoral injections of 1 x 10(9) pfu AdCMVCD. Mice received i.p. injections of 400 mg/kg of 5-FC twice daily for 7 days beginning the day of initial AdCMVCD injection (day -2). The radiation treatment group received 10 Gy of 60Co exposure to their tumor on day 0. SK-ChA-1 cells were efficiently transduced (48.7 and 99.2%) by 10 and 100 pfu/cell of AdCMVLacZ, respectively. From 37.9 to 84.4% of SK-ChA-1 cells were killed following infection with 10 pfu/cell AdCMVCD and 8 days of exposure to various concentrations of 5-FC (5, 10, 30, 50, and 100 microg/ml). Higher 5-FC concentrations and longer duration of exposure resulted in greater cell killing. Radiation treatment (8 Gy) enhanced cell killing by greater than 70% when combined with 10 or 20 microg/ml of 5-FC. Radiation dose-response analysis with clonogenic assay confirmed enhanced SK-ChA-1 cell cytotoxicity as a result of radiation treatment following AdCMVCD infection and 5-FC exposure, with radiobiological parameters alpha = 0.44 and D0 = 0.96. Combined treatment of SK-ChA-1 tumors with AdCMVCD, 5-FC, and radiation in animals resulted in significantly greater survival, time to tumor regrowth, and doubling time compared to the nonradiation treatment group (P = 0.03, 0.015, and 0.002, respectively). Significantly greater change in tumor size, smaller ratio of final tumor size to original tumor size, and smaller final tumor size were observed in the radiation treatment group compared to the no radiation treatment group (P = 0.02, 0.03, and 0.03, respectively). Human cholangiocarcinoma cells were transduced with a recombinant adenovirus in vitro at high efficiency and were susceptible to CD-mediated intracellular 5-FU production. Radiobiological survival curve parameters confirmed an interactive cytotoxic effect when viral infection and prodrug therapy were combined with external beam radiation exposure. (ABSTRACT TRUNCATED)

Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer.

Adenovirus (Ad) vectors are promising for gene therapy of glioma due to their ability to achieve efficient gene transfer upon intratumoral administration. Yet in this context, Ad mediates widespread gene transfer to both tumor and surrounding parenchyma. Ad entry is dependent upon the expression of fiber receptors, such as coxsackie/adenovirus receptor, and alpha(v) integrins on the target cells for binding and internalization, respectively. We hypothesized that the susceptibility of human gliomas to Ad would likely be heterogeneous due to variable expression of these receptors. It was found that established human glioma cell lines exhibited differential susceptibility to Ad-mediated gene transfer, which correlated directly with the level of radiolabeled Ad binding and with the expression of coxsackie/adenovirus receptor but not with the expression of alpha(v) integrins. To circumvent the lack of fiber receptors and to target Ad gene transfer specifically to tumor cells, we used a bispecific antibody conjugate to ablate Ad binding to fiber receptors and retarget binding to the epidermal growth factor receptor (EGFR), a tumor-associated marker negligibly expressed in normal, mitotically quiescent neural tissues. The results demonstrate that EGFR-targeted Ad gene transfer was EGFR specific and independent of fiber-fiber receptor interactions. Furthermore, EGFR targeting significantly enhanced Ad gene delivery to 7 of 12 established glioma cell lines and to 6 of 8 cultured primary gliomas. Interestingly, EGFR-targeted Ad gene transfer did not correlate with EGFR expression across cell lines, suggesting the importance of other factors. This study establishes that fiber receptor expression limits the utility of Ad vectors for gene transfer to glioma cells and suggests that targeting Ad via EGFR may prove valuable for tumor-specific gene transfer to high-grade gliomas. These findings have key relevance in the context of Ad vector-based approaches for glioma gene therapy.

Comparative binding and preclinical localization and therapy studies with radiolabeled human chimeric and murine 17-1A monoclonal antibodies.

Murine MoAb 17-1A is an IgG2a antibody reactive with a gastrointestinal cancer-associated cell surface antigen. Human-mouse chimeric 17-1A MoAbs were constructed in which the murine variable region of 17-1A was joined with human IgG1, IgG2, IgG3, and IgG4 constant regions. Human-mouse IgG1, IgG2, and IgG4 chimeric antibodies were compared with the parental murine antibody and its F(ab')2 fragments for their ability to bind to colon carcinoma cells in vitro, for their blood clearance in normal nude mice, and for their localization and tumor growth inhibition of colon carcinoma xenografts in nude mice. Indirect immunofluorescence experiments with fluorescein-conjugated goat anti-mouse or goat anti-human antibody verified that the substitution of human constant regions in the chimeric MoAbs did not significantly alter the ability of the murine variable region to bind to colon adenocarcinoma cell lines (LS174T, SW948, and C0112). The immunoreactivities of 125I-labeled murine and chimeric 17-1A MoAbs measured in a live cell-binding assay with LS174T, SW948, and C0112 cells revealed that chimeric IgG1, IgG4, and 17-1A F(ab')2 were comparable to murine 17-1A while chimeric IgG2 showed lower binding. The blood half-lives of 125I-labeled murine 17-1A, its F(ab')2 fragments, and chimeric IgG1, IgG2, and IgG4 in normal nude mice determined by serial eye bleeding were 7.5, 0.5, 5.2, 6.9, and 1.9 days, respectively. In biodistribution studies at 4 days after injection of 125I-labeled MoAbs in nude mice bearing LS174T tumors, chimeric IgG1 had the highest tumor concentration of 20.5% injected dose/g with a tumor/blood ratio of 3.2. 131I-labeled murine 17-1A administered in a single injection of 300 microCi or 3 injections of about 300 microCi each to nude mice bearing established LS174T tumors inhibited tumor growth, whereas a comparable amount of unlabeled murine 17-1A did not inhibit tumor growth. 131I-labeled chimeric IgG1 MoAb showed a similar level of tumor growth inhibition. The results of the present study indicate that 17-1A chimeric IgG1 antibody may be the best choice for clinical radioimmunodetection and radioimmunotherapy studies.

Localization and imaging with radioiodine-labeled monoclonal antibodies in a xenogeneic tumor model for human B-cell lymphoma.

Two MoAbs directed towards human B-cell malignancies have been studied in a preclinical animal model to evaluate their potential for in vivo imaging and therapy of B-cell lymphomas. Anti-B1 reacts with virtually all immunoglobulin-bearing malignancies and non-T acute lymphoblastic leukemia. Anti-J5 reacts with the common acute lymphoblastic leukemia antigen found on non-T acute lymphoblastic leukemia and follicular lymphomas. Anti-T1 which recognizes the CD5 antigen on most T-cell leukemias and lymphomas was used as a control antibody. These monoclonal antibodies were radiolabeled with 125I or 131I by the ICl method. Namalwa (B-cell) and MOLT-4 (T-cell) tumors were grown s.c. in irradiated nude mice. The highest tissue concentration of 125I-labeled anti-J5 in Namalwa-bearing mice was in blood and tumor. The tumor/blood ratio ranged from 0.7-1.2, with the highest ratio 4 days after injection. Pharmacokinetic analysis indicated that the t1/2 beta of anti-J5 from blood and other tissues ranged from 40-50 h, while the t1/2 beta for tumor averaged 65 h. The area under the curve of tumor was 2- to 5-fold higher than the area under the curve of liver, kidney, skin, and muscle. The peak tissue levels of 125I-labeled anti-B1 in Namalwa-bearing mice were again in blood and tumor and 6 days following injection more than 5-fold greater activity was found in tumor compared to normal tissues other than blood. The tumor/blood ratio was 1.2 and 0.7 at 4 and 6 days after injection. 125I-labeled anti-B1 showed minimal uptake in antigen-negative MOLT-4 tumors and 125I-labeled anti-T1 showed little uptake in Namalwa tumors. Scintigraphic images were obtained following the injection of 131I-labeled anti-J5 and anti-B1 in nude mice bearing Namalwa tumors. These results indicate that radiolabeled anti-J5 and anti-B1 show promise as diagnostic and possibly therapeutic agents for human B-cell lymphoma, although there may be a limitation to clinical utility due to cross-reactivity with some normal cells.

Enhanced apoptosis with combination C225/radiation treatment serves as the impetus for clinical investigation in head and neck cancers.

PURPOSE: Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous cell carcinomas, and this overexpression is associated with a poor prognosis. Therefore, EGFr has become the target of investigations aimed at disabling the receptor to determine whether this process leads to improved tumor kill with conventional treatment. MATERIALS AND METHODS: C225 is an anti-EGFr monoclonal antibody that inhibits receptor activity by blocking the ligand binding site. A panel of human head and neck squamous cell carcinoma cell lines was used to study the combination of C225 and radiation. RESULTS: It was determined that the combination of C225 (5 microgram/mL) delivered simultaneously with radiation (3 Gy) resulted in a greater decrement in cellular proliferation than either treatment alone. This reduction in proliferation correlated with reduced EGFr tyrosine phosphorylation and a reduction in phosphorylated signal transducer and activator of transcription-3 (STAT-3) protein (known to protect cells from apoptosis). Also, the decrement in proliferation correlated with increased apoptotic events, thereby indirectly linking C225/radiation-induced regulation of STAT-3 protein to apoptosis. CONCLUSION: This preclinical work serves as important support for the ongoing clinical investigation of C225 and radiotherapy for patients with head and neck carcinomas. The initial results of these clinical studies have been promising.

Biodistribution study of 188Re-labeled trisuccin-HuCC49 and trisuccin-HuCC49deltaCh2 conjugates in athymic nude mice bearing intraperitoneal colon cancer xenografts.

The trihydroxamate bifunctional chelating agent (BCA), trisuccin, has been shown to be a potential ligand for radiolabeling of monoclonal antibodies (MAbs) with rhenium radioisotopes, through an indirect postconjugation approach. The use of this trihydroxamate BCA made it possible to prepare stable BCA-MAb conjugates in pure form that could be radiolabeled with carrier-free 188Re. The anti-TAG-72 murine MAb, CC49, and its humanized derivatives are promising agents in the treatment of a number of malignancies with the CH2 domain-deleted MAb (HuCC49deltaCH2), which is of particular interest due to its rapid blood clearance. The biodistribution of 188Re-labeled conjugates of trisuccin with both humanized CC49 (HuCC49) and HuCC49deltaCH2 in athymic nude mice implanted i.p. with LS174T human colon carcinoma was studied. Trisuccin-MAb conjugates were synthesized at different BCA:MAb ratios by the 6-oxoheptanoic acid method using trisuccin hydrazide. The conjugates were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy for the number of incorporated trisuccin molecules. The conjugates were radiolabeled with carrier-free, generator-produced 188Re and purified by gel filtration on Sephadex G-25. Labeling yields and homogeneity of the labeled conjugates were analyzed by high-pressure liquid chromatography and instant TLC. Athymic nude mice were injected i.p. with LS174T human colon carcinoma cells, 7 days prior to injection of the labeled antibodies. 188Re-labeled MAbs were injected i.p., and the mice were sacrificed 24 h postinjection. Matrix-assisted laser desorption/ionization time-of-flight analyses showed stable incorporation of trisuccin into each MAb, with the measured ligand:MAb values positively correlating with the theoretical ratios. Labeling of the conjugates with 188Re proceeded with high yields, producing homogeneous 188Re-MAbs with good stabilities as shown by instant TLC and biodistribution analyses. Biodistribution of the radiolabeled MAbs at 24 h after injection showed median tumor uptake values of 23.5%ID/g and 17.6%ID/g for the 188Re-HuCC49deltaCH2 and 188Re-HuCC49, respectively. The blood clearance of the domain-deleted MAb was faster than that of the intact antibody. The blood values at 24 h after injection were 0.7%ID/g for 188Re-HuCC49deltaCH2 and 3.2%ID/g for 188Re-HuCC49. The results indicate that trisuccin is a promising agent for postconjugation labeling of antibodies with 188Re. Additionally, these results illustrate the potential of 188Re-HuCC49deltaCH2 in radioimmunodiagnosis and radioimmunotherapy of cancer.

Comparison of multiple bolus and continuous injections of 131I-labeled CC49 for therapy in a colon cancer xenograft model.

One of the problems in achieving cures with radioimmunotherapy is that hematological toxicity limits the quantity of radiolabeled monoclonal antibody (MAb) that can be administered. The MAb CC49 binds with high affinity to the TAG-72 antigen expressed in many human adenocarcinomas. We investigated tumor growth inhibition, survival, and tumor and bone marrow dosimetry after multiple bolus injections or continuous infusion of 131I-labeled CC49 MAb in a human colon cancer xenograft model to determine which method of administration results in the highest therapeutic ratio. Groups of athymic nude mice bearing established s.c. LS174T human colon cancer xenografts received three i.p. bolus injections (3X) of 131I-labeled CC49 (3X, days 0, 3, and 7) or were implanted i.p. with mini-osmotic pumps delivering 131I-labeled CC49 over 7 days. The total radionuclide doses administered were broken down into low-dose (< or = 450 microCi), medium-dose (450-800 microCi), and high-dose (> 800 microCi) groups. At the medium-dose level, the bolus-therapy animals did not have a significantly longer survival time but did have a significantly longer time-to-tumor doubling than the pump-therapy animals. The median survival for medium-dose bolus and pump therapy was 157 and 105 days, respectively, and the median time-to-tumor doubling was at least 114 and 77 days, respectively. At the low-dose level, the bolus-therapy animals had a significantly longer survival time but not a significantly longer time-to-tumor doubling than the pump-therapy animals. The median survival for low-dose bolus and pump therapy was 95.5 and 59 days, respectively, and the median time-to-tumor doubling was 73 and 38 days, respectively. The high-bolus dose was toxic. A comparison of the overall survival rate of pump therapy versus bolus therapy, excluding high-dose, resulted in the bolus-therapy animals having a longer survival time and a longer time-to-tumor doubling than the pump-therapy animals. Serial section autoradiography was used to reconstruct tumor activity density distributions over time. Average dose values calculated from total uptake data for 900 microCi administered activity yielded 158 Gy (3X) and 141 Gy (pump). Average three-dimensional doses using the radial histograms to calculate the absorbed fractions were 139 Gy and 123 Gy, respectively. This calculation includes energy loss external to the tumor. With cell proliferation parameters set to single fraction 60Co recurrence results, the effective dose (D(eff)) for local control was 11 Gy and 9 Gy, respectively. Three bolus injections resulted in a more uniform dose rate over a longer period, resulting in a calculated 19% improvement in D(eff) compared with pump administration. Dose to bone marrow was calculated assuming an activity concentration in bone marrow of 0.24 times the concentration in blood and an absorbed fraction of 0.63. For the 900-microCi 131I-labeled CC49 injected activity, pump administration resulted in an 80% higher calculated D(eff) to bone marrow compared with 3X bolus injection. These results demonstrate that 3X bolus injections were clearly superior to pump administration in terms of survival, tumor growth inhibition, tumor absorbed dose, and bone marrow dose.

Selectivity of TAG-72-targeted adenovirus gene transfer to primary ovarian carcinoma cells versus autologous mesothelial cells in vitro.

Efficient gene transfer by recombinant adenovirus (Ad) vectors depends on expression of CAR and alpha(v) integrin on target cells. Because Ad may also infect nearby nontarget cells expressing these receptors, such as peritoneal mesothelial cells after i.p. injection, we hypothesized that targeting Ad gene delivery to a receptor overexpressed on most ovarian carcinoma cells, such as TAG-72, would enhance the selectivity of Ad gene transfer when used in this context. A monoclonal antibody that has been investigated clinically for immunotherapy and immunodetection of ovarian carcinomas, namely CC49, was used to construct a bispecific conjugate with the Fab fragment of a neutralizing anti-knob mAb to target Ad binding via TAG-72. This conjugate facilitated TAG-72-specific, CAR-independent Ad reporter gene transfer to both ovarian cancer cell lines and primary ovarian cancer cells cultured from malignant ascites fluid. Fab-CC49 was very selective for tumor cells, augmenting Ad gene transfer to primary ovarian cancer cells 2- to 28-fold relative to untargeted Ad, while also decreasing gene transfer to autologous cultured mesothelial cells 4- to 9-fold. These data suggest that targeting Ad via TAG-72 may improve the selectivity of Ad gene transfer for ovarian tumors 8- to 252-fold on i.p. vector injection. These results also define the requirements for a candidate target receptor in the rational design of a targeted Ad vector for ultimate clinical utility, one that selectively infects tumor cells and spares normal cells on i.p. injection. Such a vector may increase gene transfer and decrease the toxicity of Ad vectors, which would improve the therapeutic index of cytotoxic gene therapy for ovarian cancer in clinical trials.

Adenoviral-mediated delivery of gastrin-releasing peptide receptor results in specific tumor localization of a bombesin analogue in vivo.

Radioimmunotherapy is hindered by a variety of factors linked to the utilization of monoclonal antibodies. These limitations include restricted tumor penetration as well as low levels of intratumoral antigen expression. To address the latter problem, we used a gene therapy approach to induce tumor cells to express enhanced levels of receptor with high binding affinity for a radiolabeled peptide. In this regard, a radiolabeled bombesin analogue was used in conjunction with a recombinant adenoviral vector encoding the murine gastrin-releasing peptide receptor (mGRPr). A panel of human carcinoma cell lines was infected in vitro with the recombinant adenoviral vector encoding the mGRPr vector to examine the induced binding of a 125I-labeled bombesin peptide. All cell lines examined displayed high levels of induced peptide binding, with approximately 60-80% of the radioactivity bound to the cells, in a live-cell binding assay. The human ovarian carcinoma cell line SKOV3.ip1 was chosen for in vivo analysis of radiolabeled bombesin analogue tumor localization in biodistribution and pharmacokinetic studies in athymic nude mice. Genetic induction of mGRPr in vivo resulted in selective tumor uptake of the radiolabeled peptide and high tumor:blood ratios. The biodistribution results compared favorably to those obtained with 131I-labeled e21 anti-erbB-2 monoclonal antibody in animals bearing i.p. SKOV3.ip1 tumors that endogenously express erbB-2. Thus, a novel method to combine gene transfer and radioimmunotherapy may result in augmented tumor cell targeting of radiopharmaceuticals.

In vivo localization of [(111)In]-DTPA-D-Phe1-octreotide to human ovarian tumor xenografts induced to express the somatostatin receptor subtype 2 using an adenoviral vector.

Adenoviral vectors, encoding genes for cell surface antigens or receptors, have been used to induce their high level expression on tumor cells in vitro and in vivo. These induced antigens and receptors can then be targeted with radiolabeled antibodies or peptides for potential radiotherapeutic applications. The purpose of this study was to determine a dosing schema of an adenoviral vector encoding the human somatostatin receptor subtype 2 (AdCMVhSSTr2) for achieving the highest tumor localization of [(111)In]-DTPA-D-Phe1-octreotide, which binds to this receptor, in a human ovarian cancer model as a prelude to future therapy studies. AdCMVhSSTr2 was produced and used to induce hSSTr2 on A427 human nonsmall cell lung cancer cells and on SKOV3.ipl human ovarian cancer cells in vitro, as demonstrated by competitive binding assays using [125I]-Tyr1-somatostatin and [(111)In]-DTPA-D-Phe1-octreotide. Mice bearing i.p. SKOV3.ip1 tumors administered 1 x 10(9) plaque-forming units of AdCMVhSSTr2 i.p. 5 days after tumor cell inoculation, followed by an i.p. injection of [(111)In]-DTPA-D-Phe1-octreotide 2 days later, showed a range of 15.3-60.4% median injected dose/gram (ID/g) in tumor at 4 h after injection compared with 3.5% ID/g when [125I]-Tyr1-somatostatin was administered and 0.3% ID/g when the negative control peptide [125I]-mIP-bombesin was administered. Mice administered a control adenoviral vector encoding the gastrin-releasing peptide receptor did not have tumor localization of [(111)In]-DTPA-D-Phe1-octreotide (<1.6% ID/g), demonstrating specificity of [(111)In]-DTPA-D-Phe1-octreotide for the AdCMVhSSTr2 induced tumor cells. In another set of experiments, the tumor localization of [(111)In]-DTPA-D-Phe1-octreotide was not different 1, 2, or 4 days after AdCMVhSSTr2 injection (31.8, 37.7, and 40.7% ID/g, respectively; P = 0.88), indicating that multiple injections of radiolabeled peptide can be administered with equivalent uptake over a 4-day period. [(111)In]-DTPA-D-Phe1-octreotide tumor localization in animals administered AdCMVhSSTr2 on consecutive days or 2 days apart was 22.4% ID/g and 53.2% ID/g, respectively (P = 0.009) when [(111)In]-DTPA-D-Phe1-octreotide was given 1 day after the second AdCMVhSSTr2 injection. There was no difference in [(111)In]-DTPA-D-Phe1-octreotide localization after a single AdCMVhSSTr2 injection (40.7% ID/g) or two injections of AdCMVhSSTr2 given 1 (45.9% ID/g) or 2 (53.2% ID/g) days apart, where [(111)In]-DTPA-D-Phe1-octreotide was given in each case 4 days after the first AdCMVhSSTr2 injection (P = 0.65). Therefore, two AdCMVhSSTr2 injections did not increase [(111)In]-DTPA-D-Phe1-octreotide tumor localization compared with one injection, which eliminates concerns about an immune response to a second dose of AdCMVhSSTr2. This will be the basis for a therapeutic protocol with multiple administrations of an octreotide analogue labeled with a therapeutic radioisotope.