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Pretreatment with a Monoclonal Antibody/Interleukin-2 Fusion Protein Directed against DNA Enhances the Delivery of Therapeutic Molecules to Solid Tumors¹

Department of Pathology, University of Southern California School of Medicine, Los Angeles, California 90033 [J. L. H., L. A. K., P. H., J. S., A. L. E.] and Pediatric Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 [C. K.]

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
The efficacy of molecular therapies for human malignancies is limited by inadequate accumulation within solid tumors. Our laboratory has developed a novel approach that uses monoclonal antibodies (MAbs) to direct vasoactive proteins to tumor sites to increase local vascular permeability and, in turn, improve the delivery of therapeutic reagents. Previously, we demonstrated that pretreatment with immunoconjugates containing interleukin-2 (IL-2) enhances specific tumor uptake of radiolabeled MAbs without affecting normal tissues. In the present study, we describe a fusion protein consisting of a chimeric antinuclear antibody and IL-2 (chTNT-3/IL-2) and illustrate its potential for improving the delivery of both MAbs and drugs. The ability of pretreatment with chTNT-3/IL-2 to increase specific tumor uptake of the MAb B72.3 was demonstrated in LS174T colon tumor-bearing mice. Tumor accretion of B72.3 increased nearly 3-fold, with no changes in normal tissues. Abrogation of this effect with N^G-methyl-l-arginine, a chemical inhibitor of nitric oxide synthase, suggests that rapid generation of nitric oxide in the tumor is responsible for the enhanced uptake. To demonstrate that pretreatment with chTNT-3/IL-2 can improve the uptake of other clinically relevant MAbs in different tumor models, additional studies were performed in both lung and prostate xenograft models. Pretreatment with the fusion protein increased specific tumor uptake of the MAb NR-LU-10 in A427 lung tumor-bearing mice and enhanced tumor uptake of the MAb CYT-351 in LNCaP prostate tumor-bearing mice, 2.1-fold and 1.7-fold, respectively. Finally, tumor uptake of the radiolabeled thymidine analogue ¹²⁵IUdR also increased 3-fold after pretreatment, indicating that this approach can be extended to small molecules such as chemotherapeutic drugs. Because TNT-3 recognizes a universal nuclear antigen accessible in degenerating and necrotic cells within all solid tumors, this strategy may be applicable to the majority of human cancers.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Physiological barriers to the delivery of therapeutic reagents to solid tumors are a major obstacle to the clinical success of developing molecular therapies (1) . For example, the limited clinical responses observed in radioimmunotherapy of solid tumors (2) can be attributed in large part to low tumor localization of radiolabeled MAb³ (1) . Although xenograft models in nude mice have shown levels of tumor uptake ranging from 1–20% ID/g, patient studies have demonstrated exceedingly low tumor uptake in the range of 0.01% ID/g (3) . Thus, an extremely small fraction of antibody delivers radionuclide to tumor sites, whereas the majority of the injected dose disperses throughout the body, where it can cause dose-limiting myelosuppression (4) . Recognizing that blood flow and vascular permeability are key parameters controlling the egress of therapeutic molecules into tumors (5 , 6) , our laboratory developed an experimental approach to alter tumor vascular physiology and, in turn, increase the delivery of therapeutic reagents to tumors. This strategy uses MAbs to direct proteins with vasoactive properties to tumor sites to increase local vascular permeability without affecting normal tissues (7) . We previously developed immunoconjugates containing cytokines and other vasoactive molecules and examined their ability to increase tumor uptake of radiolabeled MAbs (8) . From these studies, it was determined that the immunoconjugates that produced the greatest enhancement of antibody uptake contained IL-2.

IL-2 is a M_r 15,000 protein secreted by activated T cells that supports the proliferation and activation of lymphocytes and other immune cells (9) . In clinical studies, IL-2 has shown success in the treatment of several human malignancies, in particular melanoma and renal cell carcinoma (10) . It is well established, however, that systemic administration of IL-2 leads to increased permeability of blood vessels in the lungs and other organs leading to a toxic side effect known as the capillary leak syndrome (11, 12, 13) . In our novel approach, the undesirable property of IL-2 has been harnessed by using MAbs to target IL-2 to the tumor site. Our laboratory has demonstrated in animal models that administration of immunoconjugates consisting of IL-2 and MAbs directed against various tumor antigens increases local tumor vascular permeability and, in turn, enhances tumor uptake of radiolabeled MAbs (14 , 15) . The magnitude of enhancement was similar whether the immunoconjugate was directed against tumor-associated cell surface antigens (14) , an extracellular matrix protein in the basement membranes of tumor vessels (15) , or an intracellular antigen accessible in the necrotic regions of solid tumors (8) . For this reason, we chose to develop an antibody/IL-2 fusion protein with specificity for a nuclear antigen that might serve as a universal targeting agent, owing to its ability to target degenerating cells within all solid tumors.

In the present study, we describe a fusion protein consisting of chTNT-3 and IL-2 (chTNT-3/IL-2). Because TNT-3 recognizes DNA exposed in the degenerating and necrotic cells within solid tumors, it has the potential to target the majority of human malignancies (16) . In this study, we examine the ability of chTNT-3/IL-2 to increase the specific tumor uptake of both MAbs and chemotherapeutic drugs in various tumor xenograft models in the hope that such a strategy may represent a general approach to increase the delivery of therapeutic molecules to solid tumors.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Reagents
The plasmid pBC12/HIV/IL-2 containing human IL-2 cDNA (17) was obtained from the American Type Culture Collection (clone 67618; Manassas, VA). The plasmids pEE6hCMV-B and pEE12 were purchased with the Glutamine Synthetase Gene Amplification System from Lonza Biologicals (Slough, United Kingdom). Restriction endonucleases, T4 DNA ligase, and other molecular biology reagents were purchased from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN). Chloramine T, single-stranded DNA from calf thymus, l-NMA, and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt] were purchased from Sigma Chemical Co. (St. Louis, MO). Iodine-125 and iodine-131 were obtained as sodium iodide in 0.1N sodium hydroxide from DuPont/New England Nuclear (North Billerica, MA). ¹²⁵IUdR was purchased from Amersham Life Science, Inc. (Arlington Heights, IL). BALB/c and athymic nude mice were purchased from Harlan Sprague Dawley (Indianapolis, IN).

Antibodies and Cell Lines
chTNT-3 (IgG1) was constructed and expressed as described previously (16) . chTNT-1 (IgG1), of which the variable regions of cDNAs were cloned from the murine TNT-1 hybridoma (18) , was constructed and expressed in the same manner as chTNT-3. The fusion protein chCLL-1/IL-2, consisting of the chimeric anti-B-cell MAb CLL-1 with human IL-2 at the C-termini of the chimeric heavy chains, was produced as described previously (19) . The murine MAb B72.3 (IgG1; Ref. 20 ), recognizing the tumor-associated glycoprotein TAG-72, was a gift from Celltech Biologicals. The murine MAb NR-LU-10 (IgG_2b; Ref. 21 ), reactive with a M_r 40,000 glycoprotein expressed on many epithelial cell carcinomas, was generously provided by Dr. Don Axworthy of NeoRx Corp. (Seattle, WA). The murine MAb CYT-351, also known as 7E11-C5.3 (IgG1; Ref. 22 ), recognizing a M_r 100,000 prostate-specific membrane glycoprotein (23 , 24) , was generously provided by CYTOGEN Corp. (Princeton, NJ). Iodine-125 and iodine-131-labeled MAbs were prepared using a modified chloramine T method, as described previously (25) . The NS0 murine myeloma cell line was obtained from Lonza Biologicals. The Raji cell line (derived from an African Burkitt’s lymphoma; Refs. 26 and 27 ), the LS174T human colon adenocarcinoma cell line (28) , the A427 human lung adenocarcinoma cell line (29) , and the LNCaP human prostatic adenocarcinoma cell line (30) were obtained from the American Type Culture Collection.

Construction of Expression Vectors
The expression vectors were constructed using standard techniques. The expression vector for the chTNT-3 heavy chain, pEE12/chTNT-3 HC (16) , was used as the parent vector. This plasmid contains the cDNA sequence for the human-mouse chTNT-3 heavy chain, under the control of the cytomegalovirus major immediate early promoter, and the cDNA sequence for glutamine synthetase, under the control of the SV40 early promoter. To amplify the human IL-2 cDNA from the pBC12/HIV/IL-2 plasmid template, two primers (5'-GGTAAAGCGGCCGCAGGAGGTGGTAGCGCACCTACTTCAAGTTCTACA-3' and 5'-TCATGCGGCCGCTCAAGTTAGTGTTGAGATGATGCT-3') were used. The PCR fragment was inserted into the NotI site of pEE12/chTNT-3 HC, resulting in the expression vector 12/chTNT-3/IL-2 encoding a fusion protein consisting of the chTNT-3 heavy chain with human IL-2 at its COOH terminus. The expression vector for the chTNT-3 light chain, pEE6/chTNT-3 LC, was constructed as described previously (16) .

Expression and Purification of Antibody Fusion Protein
chTNT-3/IL-2 was expressed in NS0 murine myeloma cells according to the manufacturer’s protocol (Lonza Biologicals). The highest producing clone was incubated in a 10-liter bioreactor, and the fusion protein was purified stepwise from cell culture medium by protein A affinity and ion-exchange chromatography, as described previously (25) . Purity of the fusion protein was examined by SDS-PAGE according to the method of Laemmli (31) .

Immunoassays
ELISA.
Fusion protein-containing supernatants were initially identified by indirect ELISA using microtiter plates coated with single-stranded DNA from calf thymus, as described previously (16) . For production rate assays, 10⁶ cells were plated in 1 ml of selective medium and allowed to incubate for 24 h. ELISA was then performed as before. Supernatants were serially diluted and applied to wells of microtiter plates coated with goat antihuman IgG (H+L; CalTag, South San Francisco, CA). Dilutions of a control chimeric MAb were used to generate a standard curve using 4-parameter fit by an automated ELISA reader (Bio-Tek Instruments, Inc., Winooski, VT), from which concentrations of unknowns were estimated. Rates of production were compared to identify the highest producing clones.

Raji Cell Competition RIA.
The immunoreactivity of chTNT-3/IL-2 was also evaluated by a competition RIA for binding to fixed Raji lymphoma cells. For these studies, 2 x 10⁶ Raji cells fixed in 2% paraformaldehyde (32) were incubated with 20 ng of ¹²⁵I-labeled muTNT-3 and serial dilutions of cold chTNT-3, chTNT-3/IL-2, or a MAb recognizing a different nuclear antigen (chTNT-1). The cells and MAbs were incubated for 1 h at room temperature with constant mixing. The cells were then washed twice, and the cell pellet-associated radioactivity was measured in a gamma counter. Maximal binding was determined from tubes containing no cold antibodies.

Determination of Avidity.
The avidity constant of chTNT-3/IL-2 was determined by a fixed cell RIA using the method of Frankel and Gerhard (33) . Raji lymphoma cell suspensions containing 10⁶ cells/ml were incubated with 10–110 ng of ¹²⁵I-labeled chTNT-3/IL-2 in 200 µl of PBS in duplicate for 1 h at room temperature with constant mixing. The cells were then washed three times with PBS containing 1% BSA to remove unbound antibody and counted in a gamma counter. The amount of fusion protein bound was determined by the remaining cell-bound radioactivity (cpm) in each tube and the specific activity (cpm/ng) of the radiolabeled fusion protein. Scatchard analysis was performed to obtain the slope. The equilibrium or avidity constant K_a was calculated by the equation K = -(slope/n), where n is the valence of the antibody fusion protein (2 for IgG).

IL-2 Bioassay.
Biological activity of the fusion protein was determined by a standard IL-2-dependent T-cell proliferation assay using the murine T cell line CTLL-2. Recombinant IL-2, obtained from Hoffmann La Roche, Inc. (Nutley, NJ), was used as a standard. Briefly, serially diluted samples and standard were incubated with 2 x 10⁴ CTLL-2 cells in triplicate for 20 h at 37°C before they were pulsed with [³H]thymidine and harvested 6 h later. Specific activity of the sample was determined by regression of the linear portion of a semi-log graph of cpm versus nM IL-2 for the standard.

Pretreatment Studies.
Athymic nude mice, 6 weeks of age, received injections of a 0.2-ml inoculum containing 2 x 10⁷ LS174T human colon adenocarcinoma cells s.c. in the left thigh. The tumors were grown for 10–14 days until they reached approximately 1 cm in diameter. In the time-dependence study, groups of mice (n = 4) were administered i.v. injections of 30 µg of chTNT-3/IL-2 at various times before, simultaneously, or subsequently to the i.v. injection of a 0.1-ml inoculum containing 100 µCi/10 µg ¹²⁵I-labeled B72.3. In the dose-dependence study, groups of mice were administered various doses of chTNT-3/IL-2 2 h before the i.v. injection of ¹²⁵I-labeled B72.3. As a control, a group of mice received 15 µg of chCLL-1/IL-2. To examine the mechanism of vasopermeability enhancement, a group of mice received 20 mg/kg l-NMA (34) , an inhibitor of NOS, i.p. 30 min before pretreatment with 15 µg of chTNT-3/IL-2. A control group received l-NMA alone before administration of ¹²⁵I-labeled B72.3. In the preceding groups, animals were sacrificed by sodium pentobarbital overdose 72 h after injection, and tissues were removed, weighed, and measured in a gamma counter. For each mouse, data were expressed as the percentage of ID/g and tumor:organ ratio (cpm/g tumor:cpm/g organ).

The ability of chTNT-3/IL-2 pretreatment to enhance the specific tumor uptake of radiolabeled MAbs was examined in two additional tumor models. Female athymic nude mice, 6 weeks of age, received injections of A427 lung adenocarcinoma cells, and male nude mice (6 weeks of age) received injections of LNCaP prostatic adenocarcinoma cells. The tumors were grown for 10–14 days (A427) or 8 weeks (LNCaP) until they reached 1 cm in diameter. Groups of mice (n = 7) were then administered i.v. injections of various doses of chTNT-3/IL-2 2 h before the i.v. injection of ¹²⁵I-labeled MAb. NR-LU-10 was used with the A427 tumor model, and CYT-351 was used with the LNCaP tumor model. Animals were sacrificed 5 days after injection for biodistribution analysis, as described above. To examine whether this pretreatment strategy could be extended to small molecules such as chemotherapeutic drugs, groups of mice (n = 4) received ¹²⁵IUdR with or without pretreatment with 15 µg of chTNT-3/IL-2. Because of the rapid clearance of this drug, mice were sacrificed 3 h after injection for biodistribution analysis. All data are presented as medians. Significance levels were determined using the Wilcoxon’s rank-sum test.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Construction, Expression, and Purification of chTNT-3/IL-2.
A PCR fragment containing the human IL-2 cDNA, preceded by a 7 amino acid linker peptide, was inserted into the NotI site previously appended immediately downstream of the human 1 terminal codon, producing a TNT-3 VH/human 1/human IL-2 fusion gene. This resulted in the expression vector 12/chTNT-3 HC/IL-2 encoding a fusion protein consisting of the chTNT-3 heavy chain with human IL-2 at its COOH terminus. This expression vector was cotransfected with the expression vector for the chTNT-3 light chain, pEE6/chTNT-3 LC. The fusion protein was expressed in NS0 murine myeloma cells using the Glutamine Synthetase Gene Amplification System (Lonza Biologicals). The highest producing transfectant was scaled up to a 10-liter bioreactor, and the fusion protein was purified stepwise by protein A affinity and ion-exchange chromatography. The production level of chTNT-3/IL-2 was >40 µg/ml. After purification, the chimeric heavy chain fusion protein was intact, as demonstrated by reducing SDS-PAGE. Two bands were resolved for chTNT-3/IL-2 at approximately M_r 25,000 and M_r 70,000, corresponding to the molecular weights of the immunoglobulin light chain and heavy chain plus cytokine, compared with chTNT-3, the heavy chain of which exhibited an apparent molecular weight of approximately M_r 55,000 (Fig. 1) .

View larger version (96K): [in this window] [in a new window]   Fig. 1. Electrophoretic identification of chTNT-3/IL-2. Coomassie Blue-stained 10% acrylamide reducing gel of purified chTNT-3 (Lane 2) and chTNT-3/IL-2 (Lane 3). Lane 1, molecular weight standards (kDa).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Competitive binding RIA with chTNT-3/IL-2. Purified fusion protein was assayed for its ability to inhibit the binding of 125I-labeled muTNT-3 to fixed Raji human lymphoma cells. chTNT-3 and chTNT-1 served as positive and negative controls, respectively.

Pretreatment Studies.
The effect of chTNT-3/IL-2 administration on tumor uptake of the murine MAb B72.3 was evaluated in LS174T colon tumor-bearing nude mice. Biodistribution analyses were performed 72 h after B72.3 injection. To determine the relationship between the timing of treatment and tumor uptake, 30 µg of chTNT-3/IL-2 were injected i.v. at various times relative to ¹²⁵I-labeled B72.3. The effect of fusion protein administration on increased tumor uptake was clearly time-dependent (Fig. 3A) . The highest tumor accretion of B72.3 occurred when chTNT-3/IL-2 was injected 1–3 h before the administration of B72.3. For this reason, a 2-h interval between pretreatment and radiolabeled MAb injection was used for the remainder of the experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Time- and dose-dependence of chTNT-3/IL-2 pretreatment on tumor uptake of 125I-labeled B72.3 in LS174T colon adenocarcinoma-bearing mice. A, tumor-bearing mice received injections of 30 µg of chTNT-3/IL-2 at various times relative to the administration of B72.3 (*), as indicated. B, tumor-bearing mice received injections of various doses of chTNT-3/IL-2 2 h before administration of B72.3. In each set of experiments, mice were sacrificed 72 h after injection for biodistribution analysis.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Three-day biodistribution of 125I-labeled B72.3 under optimal pretreatment conditions in LS174T colon tumor-bearing mice. Tumor-bearing mice were pretreated with 15 µg of chTNT-3/IL-2 or chCLL-1/IL-2 (negative control) 2 h before the administration of B72.3. A, median tissue uptake of B72.3 measured by the percentage of injected dose/g. B, median tumor:normal tissue ratios of B72.3. Significant differences are P 0.025 (*) and P 0.05 ($).

View larger version (29K): [in this window] [in a new window]   Fig. 5. Effect of l-NMA on tumor uptake of 125I-labeled B72.3 after chTNT-3/IL-2 pretreatment in LS174T colon tumor-bearing nude mice. Tumor-bearing mice were administered 20 mg/kg l-NMA 30 min before pretreatment with 15 µg of chTNT-3/IL-2, followed 2 h later by B72.3. Mice were sacrificed 72 h after injection for biodistribution analysis.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

As our laboratory and others have demonstrated previously, MAb/IL-2 fusion proteins are eliminated rapidly from normal mice (36, 37, 38) . The therapeutic potential of MAb/IL-2 fusion proteins for eliciting tumor rejection has been demonstrated in animal models (39, 40, 41) . The rapid clearance of these fusion proteins may prove beneficial in the clinical setting, where potentially injurious exposure of healthy tissues to the high doses of IL-2 (13 , 42 , 43) necessary to evoke cellular immune responses against solid tumors (10) may be minimized as the antibody concentrates the cytokine at the tumor site (44) . It will, of course, be necessary to evaluate the toxicity of MAb/IL-2 fusion proteins in patients because the serum persistence of the fusion protein compared with free recombinant IL-2 (45) may still result in toxicities.

The efficacy of MAbs in the radioimmunotherapy of human malignancies is limited by insufficient accumulation within solid tumors (46) . Investigators have shown that IFNs can enhance the expression of tumor-associated antigens leading to increased tumor uptake of MAbs (47 , 48) . This approach, however, is limited to MAbs directed against tumor antigens that can be up-regulated by such treatment. We have focused our efforts on developing an approach to improve the delivery of antibodies to tumors that might also be applicable to other therapeutic molecules. Our laboratory was the first to use immunoconjugates containing vasoactive cytokines to increase vascular permeability (14) . Others have shown that the systemic administration of tumor necrosis factor (49, 50, 51) , IFN- (52) , and IL-2 (14 , 53) increases tumor uptake of radiolabeled MAbs in mouse models, but as demonstrated in these studies and in those from our laboratory (14) , pretreatment with free vasoactive cytokines also results in increased uptake in normal tissues including lung, liver, and spleen. Hence, the targeted delivery of cytokines to tumor sites using MAbs represents a significant advancement of this technology. The fusion protein described in the present study was designed for enhancing tumor uptake of therapeutic molecules in a wide variety of human cancers because TNT-3 recognizes a universal nuclear antigen (16) exposed in the degenerating and necrotic cells present in all solid tumors.

The ability of chTNT-3/IL-2 to increase tumor uptake of both a MAb and a drug (IUdR) was examined in the LS174T colon adenocarcinoma xenograft model. Radiolabeled IUdR has been evaluated in animal tumor models for the diagnosis and therapy of cancers (54) and was used in our studies to determine whether chemotherapeutic drugs can also be enhanced in tumors in a specific manner. In this model, increased tumor uptake of ¹²⁵I-labeled B72.3 after pretreatment with chTNT-3/IL-2 was both time- and dose-dependent (Fig. 3) . It seems that normal tissue toxicity at the highest pretreatment dose administered limited the accumulation of B72.3 in the tumor, as levels in normal tissues began to increase. Under optimal conditions, however, pretreatment with chTNT-3/IL-2 resulted in nearly a 3-fold increase in tumor accretion of both ¹²⁵I-labeled B72.3 (Fig. 4) and ¹²⁵IUdR (Table 4) , with no effect on normal tissues. These results are similar to those observed with chemically produced IL-2 immunoconjugates. The necessity for tumor localization of IL-2 was evidenced by the absence of an effect when mice were pretreated with the control fusion protein chCLL-1/IL-2 directed against B-cell malignancies. Furthermore, as illustrated in Fig. 4 , the biodistribution of radiolabeled B72.3 in normal tissues was similar with or without the control and experimental fusion protein pretreatment, indicating that the clearance profile of B72.3 was not altered by the administration of MAb/IL-2 fusion proteins. In addition to improving the delivery of macromolecules, this pretreatment strategy has exciting implications for chemotherapy, as this approach may decrease the systemic toxicity of anticancer drugs while producing higher tumor killing.

The effects of pretreatment with chTNT-3/IL-2 on the specific tumor uptake of radiolabeled antibodies were examined in two additional tumor models. The MAb NR-LU-10 recognizes a membrane glycoprotein expressed in many carcinomas of epithelial origin. The tumor-targeting ability of this MAb has been demonstrated previously in colon tumor-bearing mice by both biodistribution and imaging studies (55) . Moreover, clinical studies have illustrated the potential of NR-LU-10 both for the diagnostic imaging and staging of patients with lung cancer (56) and for the radioimmunotherapy of ovarian cancer (57) . In the present study, the ability of pretreatment with chTNT-3/IL-2 to enhance the tumor uptake of NR-LU-10 was demonstrated in nude mice bearing A427 human lung adenocarcinoma tumors (Table 2) . The tumor targeting ability of the MAb CYT-351 reactive with a prostate-specific membrane antigen has been shown previously in the LNCaP nude mouse xenograft model (58) . Recently, the potential of CYT-351 for the immunoscintigraphy of patients with prostate cancer has been demonstrated in clinical studies (59 , 60) . Pretreatment with chTNT-3/IL-2 significantly increased the specific tumor uptake of CYT-351 in LNCaP prostate tumor-bearing mice (Table 3) . This pretreatment strategy can, thus, be applied to tumors of different histological origins. The optimal dose of chTNT-3/IL-2 varied somewhat from one tumor model to the next, which might be attributable to different amounts of fusion protein accumulating at the tumor site. The biodistribution of chTNT-3/IL-2 in each tumor model was not determined in these experiments, however. The findings described in this report provide evidence for the potential of this approach as a universal pretreatment to enhance the delivery of therapeutic molecules to solid tumors. It seems that it will be necessary to optimize the conditions of pretreatment in the clinical setting for successful application of this strategy. It is as yet unclear how the much larger vascular volume of patients will affect the window for optimal dose and timing observed in the mouse models.

NOS has been implicated in the capillary leak syndrome produced by therapy with IL-2. Hypotension observed after administration of IL-2 to dogs decreased after administration of an inhibitor of NOS (34) . In addition, NOS inhibitors prevent signs of capillary leak in mice (61 , 62) . We hypothesized that local generation of nitric oxide was responsible for the increased tumor uptake of MAb in our model, especially because the observed vasopermeability effect was rapid in onset and short-lived (Fig. 3A) . In the present study, administration of l-NMA completely blocked the transient effect of pretreatment with chTNT-3/IL-2 (Fig. 5) . It has recently been demonstrated that systemic inhibition of NOS has no effect on vascular permeability of the LS174T xenograft (63) . In our study, we, likewise, observed no decrease in tumor uptake of radiolabeled MAb after administration of l-NMA. Hence, it seems that even in those tumors in which baseline vascular permeability is not responsive to NOS inhibition, nitric oxide generation can further increase permeability.

Modifying vascular physiology in the tumor microenvironment represents a strategy with great possibilities for improving drug delivery. Pretreatment with the fusion protein described in this study may be applicable to a wide spectrum of human malignancies because TNT-3 is expected to localize to any tumor that contains degenerating cells and necrosis. Moreover, these studies demonstrate the ability of pretreatment with chTNT-3/IL-2 to increase tumor uptake of both MAbs and small molecules such as chemotherapeutic drugs, which should improve the therapeutic potential of these reagents.

	ACKNOWLEDGMENTS

 
We thank Barbara H. Biela and Myra M. Mizokami for assistance with the animal studies.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by Cancer Therapeutics, Inc. (Los Angeles, CA), Techniclone Corp. (Tustin, CA), and Brilliance Pharmaceuticals (Shanghai, China).

² To whom requests for reprints should be addressed, at the Department of Pathology, University of Southern California School of Medicine, 2011 Zonal Avenue, HMR 210, Los Angeles, CA 90033. Phone: (323) 442-1171; Fax: (323) 442-3049.

³ The abbreviations used are: MAb, monoclonal antibody; ID/g, injected dose/g of tissue; IL-2, interleukin 2; muTNT-3, murine TNT-3; chTNT-3, chimeric TNT-3; chTNT-1, chimeric TNT-1; l-NMA, N^G-methyl-l-arginine; ¹²⁵IUdR, 5-[¹²⁵I]Iodo-2'-deoxyuridine; NOS, nitric oxide synthase.

Received for publication 6/29/98. Revision received 9/15/98. Accepted for publication 10/ 5/98.

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TOP ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

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