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

Vol. 5, 51–60, January 1999 Clinical Cancer Research 51

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

Jason L. Hornick, Leslie A. Khawli, Peisheng Hu, 1.7-fold, respectively. Finally, tumor uptake of the radiola- 125 ϳ Jahangir Sharifi, Chand Khanna, and beled thymidine analogue IUdR also increased 3-fold after pretreatment, indicating that this approach can be Alan L. Epstein2 extended to small molecules such as chemotherapeutic Department of Pathology, University of Southern California School of drugs. Because TNT-3 recognizes a universal nuclear anti- 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, gen accessible in degenerating and necrotic cells within all Bethesda, Maryland 20892 [C. K.] solid tumors, this strategy may be applicable to the majority of human cancers.

Abstract Introduction The efficacy of molecular therapies for human malig- Physiological barriers to the delivery of therapeutic re- nancies is limited by inadequate accumulation within solid agents to solid tumors are a major obstacle to the clinical tumors. Our laboratory has developed a novel approach that success of developing molecular therapies (1). For example, the uses monoclonal antibodies (MAbs) to direct vasoactive pro- limited clinical responses observed in radioimmunotherapy of teins to tumor sites to increase local vascular permeability solid tumors (2) can be attributed in large part to low tumor and, in turn, improve the delivery of therapeutic reagents. localization of radiolabeled MAb3 (1). Although xenograft mod- Previously, we demonstrated that pretreatment with immu- els in nude mice have shown levels of tumor uptake ranging noconjugates containing interleukin-2 (IL-2) enhances spe- from 1–20% ID/g, patient studies have demonstrated exceed- cific tumor uptake of radiolabeled MAbs without affecting ingly low tumor uptake in the range of 0.01% ID/g (3). Thus, an normal tissues. In the present study, we describe a fusion extremely small fraction of antibody delivers radionuclide to protein consisting of a chimeric antinuclear antibody and tumor sites, whereas the majority of the injected dose disperses IL-2 (chTNT-3/IL-2) and illustrate its potential for improv- throughout the body, where it can cause dose-limiting myelo- ing the delivery of both MAbs and drugs. The ability of suppression (4). Recognizing that blood flow and vascular per- pretreatment with chTNT-3/IL-2 to increase specific tumor meability are key parameters controlling the egress of therapeu- uptake of the MAb B72.3 was demonstrated in LS174T tic molecules into tumors (5, 6), our laboratory developed an colon tumor-bearing mice. Tumor accretion of B72.3 in- experimental approach to alter tumor vascular physiology and, creased nearly 3-fold, with no changes in normal tissues. G in turn, increase the delivery of therapeutic reagents to tumors. Abrogation of this effect with N -methyl-l-arginine, a chem- This strategy uses MAbs to direct proteins with vasoactive ical inhibitor of nitric oxide synthase, suggests that rapid properties to tumor sites to increase local vascular permeability generation of nitric oxide in the tumor is responsible for the without affecting normal tissues (7). We previously developed enhanced uptake. To demonstrate that pretreatment with immunoconjugates containing cytokines and other vasoactive chTNT-3/IL-2 can improve the uptake of other clinically molecules and examined their ability to increase tumor uptake relevant MAbs in different tumor models, additional studies of radiolabeled MAbs (8). From these studies, it was determined were performed in both lung and prostate xenograft models. that the immunoconjugates that produced the greatest enhance- Pretreatment with the fusion protein increased specific tu- ment of antibody uptake contained IL-2. mor uptake of the MAb NR-LU-10 in A427 lung tumor- IL-2 is a Mr 15,000 protein secreted by activated T cells bearing mice and enhanced tumor uptake of the MAb CYT- that supports the proliferation and activation of lymphocytes and 351 in LNCaP prostate tumor-bearing mice, 2.1-fold 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 Received 6/29/98; revised 9/15/98; accepted 10/5/98. to increased permeability of blood vessels in the lungs and other The costs of publication of this article were defrayed in part by the organs leading to a toxic side effect known as the capillary leak payment of page charges. This article must therefore be hereby marked syndrome (11–13). In our novel approach, the undesirable prop- 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). 3 The abbreviations used are: MAb, monoclonal antibody; ID/g, injected 2 To whom requests for reprints should be addressed, at the Department dose/g of tissue; IL-2, interleukin 2; muTNT-3, murine TNT-3; of Pathology, University of Southern California School of Medicine, chTNT-3, chimeric TNT-3; chTNT-1, chimeric TNT-1; l-NMA, NG- 2011 Zonal Avenue, HMR 210, Los Angeles, CA 90033. Phone: (323) methyl-l-arginine; 125IUdR, 5-[125I]Iodo-2Ј-deoxyuridine; NOS, nitric 442-1171; Fax: (323) 442-3049. oxide synthase. Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. 52 MAb/IL-2 Fusion Protein Directed against DNA

erty of IL-2 has been harnessed by using MAbs to target IL-2 to LU-10 (IgG2b; Ref. 21), reactive with a Mr 40,000 glycoprotein the tumor site. Our laboratory has demonstrated in animal mod- expressed on many epithelial cell carcinomas, was generously els that administration of immunoconjugates consisting of IL-2 provided by Dr. Don Axworthy of NeoRx Corp. (Seattle, WA). and MAbs directed against various tumor antigens increases The murine MAb CYT-351, also known as 7E11-C5.3 (IgG1;

local tumor vascular permeability and, in turn, enhances tumor Ref. 22), recognizing a Mr 100,000 prostate-specific membrane uptake of radiolabeled MAbs (14, 15). The magnitude of en- glycoprotein (23, 24), was generously provided by CYTOGEN hancement was similar whether the immunoconjugate was di- Corp. (Princeton, NJ). Iodine-125 and iodine-131-labeled MAbs rected against tumor-associated cell surface antigens (14), an were prepared using a modified chloramine T method, as de- extracellular matrix protein in the basement membranes of tu- scribed previously (25). The NS0 murine myeloma cell line was mor vessels (15), or an intracellular antigen accessible in the obtained from Lonza Biologicals. The Raji cell line (derived necrotic regions of solid tumors (8). For this reason, we chose to from an African Burkitt’s lymphoma; Refs. 26 and 27), the develop an antibody/IL-2 fusion protein with specificity for a LS174T human colon adenocarcinoma cell line (28), the A427 nuclear antigen that might serve as a universal targeting agent, human lung adenocarcinoma cell line (29), and the LNCaP owing to its ability to target degenerating cells within all solid human prostatic adenocarcinoma cell line (30) were obtained tumors. from the American Type Culture Collection. In the present study, we describe a fusion protein consisting of chTNT-3 and IL-2 (chTNT-3/IL-2). Because TNT-3 recog- Construction of Expression Vectors nizes DNA exposed in the degenerating and necrotic cells The expression vectors were constructed using standard within solid tumors, it has the potential to target the majority of techniques. The expression vector for the chTNT-3 heavy chain, human malignancies (16). In this study, we examine the ability pEE12/chTNT-3 HC (16), was used as the parent vector. This of chTNT-3/IL-2 to increase the specific tumor uptake of both plasmid contains the cDNA sequence for the human-mouse MAbs and chemotherapeutic drugs in various tumor xenograft chTNT-3 heavy chain, under the control of the cytomegalovirus models in the hope that such a strategy may represent a general major immediate early promoter, and the cDNA sequence for approach to increase the delivery of therapeutic molecules to glutamine synthetase, under the control of the SV40 early pro- solid tumors. moter. To amplify the human IL-2 cDNA from the pBC12/HIV/ IL-2 plasmid template, two primers (5Ј-GGTAAAGCGGCC- Ј Materials and Methods GCAGGAGGTGGTAGCGCACCTACTTCAAGTTCTACA-3 and 5Ј-TCATGCGGCCGCTCAAGTTAGTGTTGAGATGAT- Reagents GCT-3Ј) were used. The PCR fragment was inserted into the The plasmid pBC12/HIV/IL-2 containing human IL-2 NotI site of pEE12/chTNT-3 HC, resulting in the expression cDNA (17) was obtained from the American Type Culture vector 12/chTNT-3/IL-2 encoding a fusion protein consisting of Collection (clone 67618; Manassas, VA). The plasmids the chTNT-3 heavy chain with human IL-2 at its COOH termi- pEE6hCMV-B and pEE12 were purchased with the Glutamine nus. The expression vector for the chTNT-3 light chain, pEE6/ Synthetase Gene Amplification System from Lonza Biologicals chTNT-3 LC, was constructed as described previously (16). (Slough, United Kingdom). Restriction endonucleases, T4 DNA ligase, and other molecular biology reagents were purchased Expression and Purification of Antibody Fusion Protein from New England Biolabs (Beverly, MA) or Boehringer Mann- chTNT-3/IL-2 was expressed in NS0 murine myeloma heim (Indianapolis, IN). Chloramine T, single-stranded DNA cells according to the manufacturer’s protocol (Lonza Biologi- Ј from calf thymus, l-NMA, and ABTS [2,2 -azino-bis(3-ethyl- cals). The highest producing clone was incubated in a 10-liter benzthiazoline-6-sulfonic acid) diammonium salt] were pur- bioreactor, and the fusion protein was purified stepwise from chased from Sigma Chemical Co. (St. Louis, MO). Iodine-125 cell culture medium by protein A affinity and ion-exchange and iodine-131 were obtained as sodium iodide in 0.1N sodium chromatography, as described previously (25). Purity of the hydroxide from DuPont/New England Nuclear (North Billerica, fusion protein was examined by SDS-PAGE according to the 125 MA). IUdR was purchased from Amersham Life Science, method of Laemmli (31). Inc. (Arlington Heights, IL). BALB/c and athymic nude mice were purchased from Harlan Sprague Dawley (Indianapolis, Immunoassays IN). ELISA. Fusion protein-containing supernatants were ini- tially identified by indirect ELISA using microtiter plates coated Antibodies and Cell Lines with single-stranded DNA from calf thymus, as described pre- chTNT-3 (IgG1␬) was constructed and expressed as de- viously (16). For production rate assays, 106 cells were plated in scribed previously (16). chTNT-1 (IgG1␬), of which the vari- 1 ml of selective medium and allowed to incubate for 24 h. able regions of cDNAs were cloned from the murine TNT-1 ELISA was then performed as before. Supernatants were seri- hybridoma (18), was constructed and expressed in the same ally diluted and applied to wells of microtiter plates coated with manner as chTNT-3. The fusion protein chCLL-1/IL-2, consist- goat antihuman IgG (HϩL; CalTag, South San Francisco, CA). ing of the chimeric anti-B-cell MAb CLL-1 with human IL-2 at Dilutions of a control chimeric MAb were used to generate a the C-termini of the chimeric heavy chains, was produced as standard curve using 4-parameter fit by an automated ELISA described previously (19). The murine MAb B72.3 (IgG1; Ref. reader (Bio-Tek Instruments, Inc., Winooski, VT), from which 20), recognizing the tumor-associated glycoprotein TAG-72, concentrations of unknowns were estimated. Rates of produc- was a gift from Celltech Biologicals. The murine MAb NR- tion were compared to identify the highest producing clones.

Raji Cell Competition RIA. The immunoreactivity of specific tumor uptake of radiolabeled MAbs was examined in chTNT-3/IL-2 was also evaluated by a competition RIA for two additional tumor models. Female athymic nude mice, 6 binding to fixed Raji lymphoma cells. For these studies, 2 ϫ 106 weeks of age, received injections of A427 lung adenocarcinoma Raji cells fixed in 2% paraformaldehyde (32) were incubated cells, and male nude mice (6 weeks of age) received injections with 20 ng of 125I-labeled muTNT-3 and serial dilutions of cold of LNCaP prostatic adenocarcinoma cells. The tumors were chTNT-3, chTNT-3/IL-2, or a MAb recognizing a different grown for 10–14 days (A427) or ϳ8 weeks (LNCaP) until they nuclear antigen (chTNT-1). The cells and MAbs were incubated reached 1 cm in diameter. Groups of mice (n ϭ 7) were then for1hatroom temperature with constant mixing. The cells administered i.v. injections of various doses of chTNT-3/IL-2 were then washed twice, and the cell pellet-associated radioac- 2 h before the i.v. injection of 125I-labeled MAb. NR-LU-10 was tivity was measured in a gamma counter. Maximal binding was used with the A427 tumor model, and CYT-351 was used with determined from tubes containing no cold antibodies. the LNCaP tumor model. Animals were sacrificed 5 days after Determination of Avidity. The avidity constant of injection for biodistribution analysis, as described above. To chTNT-3/IL-2 was determined by a fixed cell RIA using the examine whether this pretreatment strategy could be extended to method of Frankel and Gerhard (33). Raji lymphoma cell sus- small molecules such as chemotherapeutic drugs, groups of 6 pensions containing 10 cells/ml were incubated with 10–110 mice (n ϭ 4) received 125IUdR with or without pretreatment 125 ng of I-labeled chTNT-3/IL-2 in 200 ␮l of PBS in duplicate with 15 ␮g of chTNT-3/IL-2. Because of the rapid clearance of for1hatroom temperature with constant mixing. The cells this drug, mice were sacrificed 3 h after injection for biodistri- were then washed three times with PBS containing 1% BSA to bution analysis. All data are presented as medians. Significance remove unbound antibody and counted in a gamma counter. The levels were determined using the Wilcoxon’s rank-sum test. amount of fusion protein bound was determined by the remain- ing cell-bound radioactivity (cpm) in each tube and the specific activity (cpm/ng) of the radiolabeled fusion protein. Scatchard Results analysis was performed to obtain the slope. The equilibrium or Construction, Expression, and Purification of chTNT- ϭ avidity constant Ka was calculated by the equation K 3/IL-2. A PCR fragment containing the human IL-2 cDNA, Ϫ(slope/n), where n is the valence of the antibody fusion protein preceded by a 7 amino acid linker peptide, was inserted into the (2 for IgG). NotI site previously appended immediately downstream of the IL-2 Bioassay. Biological activity of the fusion protein human ␥1 terminal codon, producing a TNT-3 VH/human ␥1/ was determined by a standard IL-2-dependent T-cell prolifera- human IL-2 fusion gene. This resulted in the expression vector tion assay using the murine T cell line CTLL-2. Recombinant 12/chTNT-3 HC/IL-2 encoding a fusion protein consisting of IL-2, obtained from Hoffmann La Roche, Inc. (Nutley, NJ), was the chTNT-3 heavy chain with human IL-2 at its COOH termi- used as a standard. Briefly, serially diluted samples and standard nus. This expression vector was cotransfected with the expres- were incubated with 2 ϫ 104 CTLL-2 cells in triplicate for 20 h sion vector for the chTNT-3 light chain, pEE6/chTNT-3 LC. at 37°C before they were pulsed with [3H]thymidine and har- The fusion protein was expressed in NS0 murine myeloma cells vested 6 h later. Specific activity of the sample was determined using the Glutamine Synthetase Gene Amplification System by regression of the linear portion of a semi-log graph of cpm (Lonza Biologicals). The highest producing transfectant was versus nM IL-2 for the standard. scaled up to a 10-liter bioreactor, and the fusion protein was Pretreatment Studies. Athymic nude mice, 6 weeks of purified stepwise by protein A affinity and ion-exchange chro- age, received injections of a 0.2-ml inoculum containing 2 ϫ matography. The production level of chTNT-3/IL-2 was Ͼ40 107 LS174T human colon adenocarcinoma cells s.c. in the left ␮g/ml. After purification, the chimeric heavy chain fusion pro- thigh. The tumors were grown for 10–14 days until they reached tein was intact, as demonstrated by reducing SDS-PAGE. Two approximately 1 cm in diameter. In the time-dependence study, bands were resolved for chTNT-3/IL-2 at approximately Mr ϭ groups of mice (n 4) were administered i.v. injections of 30 25,000 and Mr 70,000, corresponding to the molecular weights ␮g of chTNT-3/IL-2 at various times before, simultaneously, or of the immunoglobulin light chain and heavy chain plus cyto- subsequently to the i.v. injection of a 0.1-ml inoculum contain- kine, compared with chTNT-3, the heavy chain of which exhib- ␮ ␮ 125 ing 100 Ci/10 g I-labeled B72.3. In the dose-dependence ited an apparent molecular weight of approximately Mr 55,000 study, groups of mice were administered various doses of (Fig. 1). chTNT-3/IL-2 2 h before the i.v. injection of 125I-labeled B72.3. Immunobiochemical Analysis. The immunoreactivity As a control, a group of mice received 15 ␮g of chCLL-1/IL-2. of chTNT-3/IL-2 was assessed by determining binding to fixed To examine the mechanism of vasopermeability enhancement, a Raji lymphoma cells. In a competition RIA, the fusion protein, group of mice received 20 mg/kg l-NMA (34), an inhibitor of chTNT-3, and an isotype-matched control MAb (chTNT-1) NOS, i.p. 30 min before pretreatment with 15 ␮g of chTNT-3/ were evaluated for their ability to inhibit the binding of 125I- IL-2. A control group received l-NMA alone before administra- labeled muTNT-3 to Raji cells (Fig. 2). Because it recognizes a tion of 125I-labeled B72.3. In the preceding groups, animals different nuclear antigen, chTNT-1 was unable to compete with were sacrificed by sodium pentobarbital overdose 72 h after radiolabeled muTNT-3. chTNT-3/IL-2, however, inhibited injection, and tissues were removed, weighed, and measured in binding of 125I-labeled muTNT-3 to a similar extent as a gamma counter. For each mouse, data were expressed as the chTNT-3. Binding studies were then conducted in which 125I- percentage of ID/g and tumor:organ ratio (cpm/g tumor:cpm/g labeled chTNT-3/IL-2 was incubated with fixed Raji cells, and organ). the bound radioactivity was used to calculate the avidity con- The ability of chTNT-3/IL-2 pretreatment to enhance the stant. chTNT-3/IL-2 was found to have a binding constant of

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. 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). The relationship between the dose of fusion protein and tumor uptake of B72.3 was then examined. A dose of 15 ␮g resulted in the greatest increase in tumor uptake (Fig. 3B). With higher doses, the magnitude of tumor uptake began to diminish. 9 Ϫ1 9 Ϫ1 1.6 ϫ 10 M , compared with 1.4 ϫ 10 M for chTNT-3. At the highest dose studied, several normal tissues revealed These studies confirm that chTNT-3/IL-2 maintains the immu- significantly higher MAb uptake (Table 1). Most importantly, noreactivity of chTNT-3 and demonstrate that the cytokine at the lung showed a significant increase in uptake (from a median the COOH terminus of the heavy chain does not interfere with of 0.96% ID/g with 15 ␮g chTNT-3/IL-2 to 1.40% ID/g with 60 binding to the antigenic target under physiological conditions. ␮g; P Յ 0.025). This suggests that toxicity to normal tissues can IL-2 Bioactivity of chTNT-3/IL-2. Biological activity occur with high doses of fusion protein. This inference does not of the IL-2 moiety was determined by examining the ability of explain the increase in blood levels of B72.3 after the highest the fusion protein to support IL-2-dependent T-cell prolifera- dose of fusion protein; the explanation for this increase remains tion. A bioassay with the murine T cell line CTLL-2 was unclear. performed in which chTNT-3/IL-2 was assayed along with Fig. 4 depicts the tissue biodistribution and tumor uptake of chTNT-3 and a recombinant IL-2 standard. Two molecules of 125I-labeled B72.3 under optimal pretreatment conditions. Tu- IL-2/antibody were assumed. On a molar basis, the fusion mor uptake increased significantly from 4.19% ID/g (range, protein displayed roughly 26% of the activity of the IL-2 stand- 3.64–4.61) to 11.18% ID/g (range, 10.32–11.70) after pretreat- ard (data not shown), corresponding to a specific activity of ment with chTNT-3/IL-2 (P Յ 0.025). Under these conditions, ϳ6 ϫ 105 IU/mg chTNT-3/IL-2. As expected, chTNT-3 had no there was no change in radiolabeled MAb uptake in normal IL-2 activity. chCLL-1/IL-2, which was used as a control for the tissues, resulting in significantly higher tumor:normal organ pretreatment studies, had ϳ50% of the activity of the IL-2 ratios (Fig. 4B). On the other hand, pretreatment with the control standard on a molar basis. This corresponds to a specific activity fusion protein chCLL-1/IL-2 (19), which recognizes B cell of ϳ8 ϫ 105 IU/mg (19). malignancies, had no effect on tumor uptake. This demonstrates Pretreatment Studies. The effect of chTNT-3/IL-2 ad- that tumor localization of the fusion protein is necessary for ministration on tumor uptake of the murine MAb B72.3 was enhancing specific uptake of radiolabeled MAb. evaluated in LS174T colon tumor-bearing nude mice. Biodis- The NOS inhibitor l-NMA was administered before pre- tribution analyses were performed 72 h after B72.3 injection. To treatment with chTNT-3/IL-2 to examine the mechanism of determine the relationship between the timing of treatment and increased tumor vascular permeability. l-NMA abrogated the tumor uptake, 30 ␮g of chTNT-3/IL-2 were injected i.v. at effect of fusion protein pretreatment on tumor uptake of B72.3 various times relative to 125I-labeled B72.3. The effect of fusion (Fig. 5). The inhibitor alone, however, did not decrease tumor protein administration on increased tumor uptake was clearly uptake below baseline levels (data not shown). These data time-dependent (Fig. 3A). The highest tumor accretion of B72.3 strongly suggest that nitric oxide generation is responsible for occurred when chTNT-3/IL-2 was injected 1–3 h before the the enhancement of tumor uptake of MAb. administration of B72.3. For this reason, a 2-h interval between The effect of chTNT-3/IL-2 administration on specific pretreatment and radiolabeled MAb injection was used for the tumor uptake of the murine MAb NR-LU-10 was then evaluated remainder of the experiments. in A427 lung tumor-bearing nude mice. Various doses of

Table 1 Median percentage ID/g of the MAb B72.3 administered 2 h after pretreatment with the indicated doses of chTNT-3/IL-2 in LS174T human colon adenocarcinoma-bearing nude mice 72 h after injection Organ 15 ␮g60␮g Pa Blood 1.74 2.78 Յ0.025 Skin 0.74 0.84 Յ0.05 Muscle 0.26 0.41 NSb Bone 0.23 0.35 Յ0.05 Heart 0.47 0.74 Յ0.025 Lung 0.96 1.40 Յ0.025 Liver 0.52 0.74 Յ0.025 Spleen 0.39 0.53 NS Pancreas 0.32 0.76 Յ0.025 Stomach 0.75 0.74 NS Intestine 0.24 0.43 Յ0.025 Kidney 0.49 0.66 Յ0.05 a Ps determined by the Wilcoxon’s rank-sum test. b NS, not significant.

Pretreatment with the fusion protein was next evaluated in a human prostatic adenocarcinoma xenograft model. The relationship between dose of chTNT-3/IL-2 and tumor uptake of 125I-labeled CYT-351 was examined in LNCaP prostate tumor- bearing mice. Mice were again sacrificed 5 days after injection for biodistribution. A dose of 30 ␮g resulted in the greatest increase in tumor uptake (data not shown). As shown in Table 3, tumor uptake increased significantly from 18.59% ID/g (range, 17.29–20.81) to 31.85% ID/g (range, 28.70–34.55) after this pretreatment dose (P Յ 0.001), with no increase in uptake in normal tissues. On the contrary, there was a significant decrease in the levels of radiolabeled MAb in the majority of normal tissues after pretreatment, again resulting in higher tumor:organ ratios. Finally, the effect of chTNT-3/IL-2 pretreatment on tumor uptake of 125IUdR was examined, to assess whether this approach could be applied to small molecules such as chemotherapeutic drugs. IUdR was selected as a representative drug be- Fig. 3 Time- and dose-dependence of chTNT-3/IL-2 pretreatment on cause of the availability of a radioiodinated derivative. Again, tumor uptake of 125I-labeled B72.3 in LS174T colon adenocarcinoma- 15 ␮g of fusion protein were administered 2 h before injection ␮ bearing mice. A, tumor-bearing mice received injections of 30 gof of 125IUdR in LS174T colon tumor-bearing mice. Because 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 the short circulation time of this drug, mice were sacrificed 3 h ,(ء) doses of chTNT-3/IL-2 2 h before administration of B72.3. In each set later for biodistribution analysis. Control tumor uptake was of experiments, mice were sacrificed 72 h after injection for biodistri- 1.44% ID/g (range, 1.28–1.53), increasing significantly to bution analysis. 4.10% ID/g (range, 3.89–4.32) after pretreatment (P Յ 0.025), representing approximately a 3-fold increase in tumor uptake with no effect on normal tissues (Table 4). chTNT-3/IL-2 were administered 2 h before injection of 125I- labeled NR-LU-10. Biodistribution analyses were performed 5 Discussion days after NR-LU-10 injection. The tumor uptake of MAb In this study, a recombinant fusion protein containing the increased with increasing doses of fusion protein (data not chimeric MAb TNT-3 and human IL-2 was generated as a shown). Table 2 depicts the biodistribution of NR-LU-10 after universal pretreatment to enhance the delivery of therapeutic pretreatment with 30 ␮g of chTNT-3/IL-2. Tumor uptake in- molecules to solid tumors. The fusion protein was expressed in creased significantly from 3.09% ID/g (range, 2.83–3.25) to mammalian cells using the Glutamine Synthetase Gene Ampli- 6.37% ID/g (range, 5.75–6.71) after pretreatment with chTNT- fication System so that large-scale production can yield suffi- 3/IL-2 (P Յ 0.001). There was a significant decrease in tissue cient recombinant product for clinical studies (35). Acrylamide levels of radiolabeled MAb in many normal organs after fusion gel electrophoresis demonstrated that the chimeric heavy chain protein pretreatment and, consequently, a significant increase in fusion protein was intact after purification (Fig. 1). As described tumor:organ ratios for all normal tissues (Table 2). previously by our laboratory, for other antibody-cytokine fusion

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 ␮g of chTNT-3/IL-2, followed 2 h later by B72.3. Mice were sacrificed 72 h after injection for biodistribution analysis.

strated in animal models (39–41). 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 persist- ence 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 Fig. 4 Three-day biodistribution of 125I-labeled B72.3 under optimal on developing an approach to improve the delivery of antibodies pretreatment conditions in LS174T colon tumor-bearing mice. Tumor- to tumors that might also be applicable to other therapeutic bearing mice were pretreated with 15 ␮g of chTNT-3/IL-2 or chCLL- molecules. Our laboratory was the first to use immunoconju- 1/IL-2 (negative control) 2 h before the administration of B72.3. A, gates containing vasoactive cytokines to increase vascular per- median tissue uptake of B72.3 measured by the percentage of injected meability (14). Others have shown that the systemic adminis- dose/g. B, median tumor:normal tissue ratios of B72.3. Significant ␥ and P Յ 0.05 ($). tration of tumor necrosis factor (49–51), IFN- (52), and IL-2 (ء) differences are P Յ 0.025 (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 proteins (19, 36), the IL-2 cDNA was inserted downstream of also results in increased uptake in normal tissues including lung, the terminal codon of the chimeric heavy chain, following a liver, and spleen. Hence, the targeted delivery of cytokines to short linker peptide to promote proper folding of the cytokine. tumor sites using MAbs represents a significant advancement of The fusion protein retains the immunoreactivity of the parent this technology. The fusion protein described in the present antibody, as evidenced by competition with 125I-labeled study was designed for enhancing tumor uptake of therapeutic muTNT-3 for binding to fixed Raji Burkitt’s lymphoma cells molecules in a wide variety of human cancers because TNT-3 (Fig. 2). Moreover, chTNT-3/IL-2 maintains the high avidity recognizes a universal nuclear antigen (16) exposed in the constant of chTNT-3. The biological activity of the IL-2 moiety degenerating and necrotic cells present in all solid tumors. was demonstrated by a proliferation assay with a murine IL-2- The ability of chTNT-3/IL-2 to increase tumor uptake of dependent T cell line. both a MAb and a drug (IUdR) was examined in the LS174T As our laboratory and others have demonstrated previ- colon adenocarcinoma xenograft model. Radiolabeled IUdR has ously, MAb/IL-2 fusion proteins are eliminated rapidly from been evaluated in animal tumor models for the diagnosis and normal mice (36–38). The therapeutic potential of MAb/IL-2 therapy of cancers (54) and was used in our studies to determine fusion proteins for eliciting tumor rejection has been demon- whether chemotherapeutic drugs can also be enhanced in tumors

Table 2 Five-day biodistribution of the MAb NR-LU-10 in A427 human lung adenocarcinoma-bearing nude mice after pretreatment with chTNT-3/IL-2 Percentage of ID/g Tumor:organ ratio Organ No pretreatment Pretreatment Pa No pretreatment Pretreatment P Blood 2.32 1.68 Յ0.05 1.40 3.99 Յ0.001 Skin 0.75 0.49 Յ0.05 4.02 13.11 Յ0.001 Muscle 0.17 0.16 NSb 18.17 38.09 Յ0.01 Bone 0.20 0.19 NS 16.56 33.86 Յ0.001 Heart 0.58 0.39 Յ0.025 5.46 17.35 Յ0.001 Lung 0.68 0.61 NS 4.55 11.06 Յ0.001 Liver 0.46 0.33 Յ0.01 6.69 19.02 Յ0.001 Spleen 0.49 0.32 Յ0.025 6.42 20.73 Յ0.001 Pancreas 0.21 0.18 Յ0.001 13.62 35.64 Յ0.001 Stomach 0.39 0.32 NS 8.39 19.89 Յ0.001 Intestine 0.28 0.18 Յ0.005 10.47 35.93 Յ0.001 Kidney 0.64 0.36 Յ0.001 5.11 17.09 Յ0.001 Tumor 3.09 6.37 Յ0.001 a Ps determined by the Wilcoxon’s rank-sum test. b NS, not significant.

Table 3 Five-day biodistribution of the MAb CYT-351 in LNCaP human prostatic adenocarcinoma-bearing nude mice after pretreatment with chTNT-3/IL-2 Percentage of ID/g Tumor:organ ratio Organ No pretreatment Pretreatment Pa No pretreatment Pretreatment P Blood 7.75 6.25 Յ0.005 2.58 5.26 Յ0.001 Skin 1.67 1.55 NSb 11.16 20.26 Յ0.001 Muscle 0.38 0.45 NS 49.54 63.18 NS Bone 1.00 0.49 Յ0.001 20.75 60.25 Յ0.001 Heart 2.30 1.70 Յ0.025 8.32 18.42 Յ0.01 Lung 3.17 2.43 Յ0.005 5.98 11.81 Յ0.001 Liver 1.40 1.27 NS 13.33 26.10 Յ0.001 Spleen 1.40 0.93 NS 14.34 36.00 Յ0.005 Pancreas 0.76 0.82 NS 26.43 40.25 Յ0.025 Stomach 0.99 0.80 NS 21.11 38.74 Յ0.001 Intestine 1.05 0.77 Յ0.025 17.30 40.72 Յ0.001 Kidney 1.78 1.16 Յ0.01 11.31 25.22 Յ0.001 Tumor 18.59 31.85 Յ0.001 a Ps determined by the Wilcoxon’s rank-sum test. b NS, not significant.

in a specific manner. In this model, increased tumor uptake of delivery of macromolecules, this pretreatment strategy has ex- 125I-labeled B72.3 after pretreatment with chTNT-3/IL-2 was citing implications for chemotherapy, as this approach may both time- and dose-dependent (Fig. 3). It seems that normal decrease the systemic toxicity of anticancer drugs while produc- tissue toxicity at the highest pretreatment dose administered ing higher tumor killing. limited the accumulation of B72.3 in the tumor, as levels in The effects of pretreatment with chTNT-3/IL-2 on the normal tissues began to increase. Under optimal conditions, specific tumor uptake of radiolabeled antibodies were examined however, pretreatment with chTNT-3/IL-2 resulted in nearly a in two additional tumor models. The MAb NR-LU-10 recog- 3-fold increase in tumor accretion of both 125I-labeled B72.3 nizes a membrane glycoprotein expressed in many carcinomas (Fig. 4) and 125IUdR (Table 4), with no effect on normal tissues. of epithelial origin. The tumor-targeting ability of this MAb has These results are similar to those observed with chemically been demonstrated previously in colon tumor-bearing mice by produced IL-2 immunoconjugates. The necessity for tumor lo- both biodistribution and imaging studies (55). Moreover, clini- calization of IL-2 was evidenced by the absence of an effect cal studies have illustrated the potential of NR-LU-10 both for when mice were pretreated with the control fusion protein the diagnostic imaging and staging of patients with lung cancer chCLL-1/IL-2 directed against B-cell malignancies. Further- (56) and for the radioimmunotherapy of ovarian cancer (57). In more, as illustrated in Fig. 4, the biodistribution of radiolabeled the present study, the ability of pretreatment with chTNT-3/IL-2 B72.3 in normal tissues was similar with or without the control to enhance the tumor uptake of NR-LU-10 was demonstrated in and experimental fusion protein pretreatment, indicating that the nude mice bearing A427 human lung adenocarcinoma tumors clearance profile of B72.3 was not altered by the administration (Table 2). The tumor targeting ability of the MAb CYT-351 of MAb/IL-2 fusion proteins. In addition to improving the reactive with a prostate-specific membrane antigen has been

Table 4 Three-day biodistribution of 125IUdR in LS174T human colon adenocarcinoma-bearing nude mice after pretreatment with chTNT-3/IL-2 Percentage of ID/g Tumor:organ ratio Organ No pretreatment Pretreatment Pa No pretreatment Pretreatment P Blood 1.64 1.59 NSb 0.89 2.50 Յ0.025 Skin 1.31 1.35 NS 1.10 3.06 Յ0.025 Muscle 0.39 0.40 NS 3.91 10.50 Յ0.025 Bone 0.90 0.82 NS 1.51 5.02 Յ0.025 Heart 0.51 0.52 NS 2.85 7.54 Յ0.025 Lung 1.18 0.92 NS 1.23 4.27 Յ0.025 Liver 0.69 0.76 NS 2.05 5.57 Յ0.025 Spleen 1.18 1.28 NS 1.19 3.15 Յ0.025 Pancreas 0.90 0.80 NS 1.68 5.12 Յ0.025 Stomach 9.61 10.40 NS 0.14 0.39 Յ0.025 Intestine 1.70 1.11 NS 0.84 3.56 Յ0.025 Kidney 1.41 1.19 NS 1.03 3.45 Յ0.025 Tumor 1.44 4.10 Յ0.025 a Ps determined by the Wilcoxon’s rank-sum test. b NS, not significant.

shown previously in the LNCaP nude mouse xenograft model ment represents a strategy with great possibilities for improving (58). Recently, the potential of CYT-351 for the immunoscin- drug delivery. Pretreatment with the fusion protein described in tigraphy of patients with prostate cancer has been demonstrated this study may be applicable to a wide spectrum of human in clinical studies (59, 60). Pretreatment with chTNT-3/IL-2 malignancies because TNT-3 is expected to localize to any significantly increased the specific tumor uptake of CYT-351 in tumor that contains degenerating cells and necrosis. Moreover, LNCaP prostate tumor-bearing mice (Table 3). This pretreat- these studies demonstrate the ability of pretreatment with ment strategy can, thus, be applied to tumors of different histo- chTNT-3/IL-2 to increase tumor uptake of both MAbs and small logical origins. The optimal dose of chTNT-3/IL-2 varied some- molecules such as chemotherapeutic drugs, which should im- what from one tumor model to the next, which might be prove the therapeutic potential of these reagents. attributable to different amounts of fusion protein accumulating at the tumor site. The biodistribution of chTNT-3/IL-2 in each Acknowledgments tumor model was not determined in these experiments, however. We thank Barbara H. Biela and Myra M. Mizokami for assistance The findings described in this report provide evidence for the with the animal studies. 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 pretreat- References ment in the clinical setting for successful application of this 1. Jain, R. K. Delivery of molecular medicine to solid tumors. Science strategy. It is as yet unclear how the much larger vascular (Washington DC), 27: 1079–1080, 1996. volume of patients will affect the window for optimal dose and 2. Wilder, R. B., DeNardo, G. L., and DeNardo, S. J. Radioimmuno- therapy: recent results and future directions. J. Clin. Oncol., 14: 1383– timing observed in the mouse models. 1400, 1996. NOS has been implicated in the capillary leak syndrome 3. Buchsbaum, D. J. Experimental approaches to increase radiolabeled produced by therapy with IL-2. Hypotension observed after antibody localization in tumors. Cancer Res., 55(Suppl. 23): 5729s– administration of IL-2 to dogs decreased after administration of 5732s, 1995. an inhibitor of NOS (34). In addition, NOS inhibitors prevent 4. Vriesendorp, H. M., Quadri, S. M., Andersson, B. S., and Dicke, signs of capillary leak in mice (61, 62). We hypothesized that K. A. Hematologic side effects of radiolabeled immunoglobulin therapy. local generation of nitric oxide was responsible for the increased Exp. Hematol., 24: 1183–1190, 1996. tumor uptake of MAb in our model, especially because the 5. Jain, R. K. Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors. Cancer Res., 50(Suppl. 3): observed vasopermeability effect was rapid in onset and short- 814s–819s, 1990. lived (Fig. 3A). In the present study, administration of l-NMA 6. Sands, H., and Jones, P. L. Physiology of monoclonal antibody completely blocked the transient effect of pretreatment with accretion by tumors. Cancer Treat. Res., 51: 97–122, 1990. chTNT-3/IL-2 (Fig. 5). It has recently been demonstrated that 7. Epstein, A. L. New approaches to improved antibody targeting. In: systemic inhibition of NOS has no effect on vascular perme- R. E. Henkin (ed.), Nuclear Medicine, pp. 516–533. St. Louis: Mosby- ability of the LS174T xenograft (63). In our study, we, likewise, Year Book, 1996. observed no decrease in tumor uptake of radiolabeled MAb after 8. Khawli, L. A., Miller, G. K., and Epstein, A. L. Effect of seven new vasoactive immunoconjugates on the enhancement of monoclonal anti- administration of l-NMA. Hence, it seems that even in those body uptake in tumors. Cancer (Phila.), 73: 824–831, 1994. tumors in which baseline vascular permeability is not responsive 9. Smith, K. A. Lowest dose interleukin-2 immunotherapy. Blood, 81: to NOS inhibition, nitric oxide generation can further increase 1414–1423, 1993. permeability. 10. Oppenheim, M. H., and Lotze, M. T. Interleukin-2: solid-tumor Modifying vascular physiology in the tumor microenviron- therapy. Oncology, 51: 154–169, 1994.

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Jason L. Hornick, Leslie A. Khawli, Peisheng Hu, et al.

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