Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL 2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.

Current understanding as to how tumors escape immune surveillance involves T cell anergy as a possible mechanism in models of tumorigenesis. Many tumors express antigenic determinants that are tumor specific or are at least more prevalent in neoplastic tissue (1). Rejection of these tumors is mediated predominately by T lymphocytes (2, 3), and the primary T cell activation signal is delivered via interaction with the TCR of the specific antigenic peptide in association with MHC I or II (4). Normally, activation involves members of the B7 family of costimulatory molecules, as the primary Ag signal is not sufficient to elicit the activation response. Anergic T cells, while capable of recognizing the antigenic epitope(s) on tumor cells, fail to eliminate these cells and consequently do not prevent tumor growth.

The first identified member of the B7 costimulatory family of molecules was B7-1 (CD80). Molecular characterization of B7-1 has shown that the molecule is a 44- to 54-kDa member of the Ig superfamily and is the ligand for CD28 and CTLA-4 counter-receptors on T cells (5, 6, 7, 8). B7-1 was first described as an activated B cell marker (9, 10); however, B7-1 expression has since been localized to other APC, including dendritic cells, monocytes, and macrophages. The interaction of B7-1 with CD28 is vital for the amplification and generation of signals necessary for Ag-specific T cell responses and effector functions (11, 12, 13, 14). B7-1 and CD28 interactions have been shown to contribute to Th cell activation and function. In addition, B7-1 has been implicated as a necessary requirement for the generation of CD8+ CTL in the absence of help from CD4+ T cells (15, 16). Inhibiting the interaction of B7-1 with CD28 while allowing Ag-specific interaction can result in T cell anergy, probably by the lack of suitable autocrine growth factor, IL-2, production (as reviewed in 17 . Anergic tumor-infiltrating lymphocytes (TILs)3 have been demonstrated in some tumors. The use of rIL-2 in vitro to overcome anergy has shown that TILs can recognize tumor-specific Ags and be activated by appropriate signals to develop cytotoxicity (18).

Recently, a number of studies have demonstrated potential for the use of the B7 family of costimulators in tumor immunotherapy. The expression of B7 family members in murine tumor models has been shown to activate CD8+ T cells and or CD4+ T cells against the respective tumor cells (6, 16, 19, 20, 21, 22, 23, 24, 25, 26, 27). Moreover, administration of IL-2 has been shown to promote antitumor immunity, presumably by alleviating the anergic block seen in T cells in some tumor models and thereby preventing the onset of anergy (28, 29). We have previously shown (30) that an adenovirus vector expressing human IL-2 administered intratumorally results in approximately 45% regression in a murine adenocarcinoma model. The potential for additive or synergistic effects from the expression of both B7-1 and IL-2 prompted us to investigate the antitumor effects of intratumoral injection of a single adenoviral vector constructed to express both B7-1 and IL-2 from the same cell.

In this study we demonstrate that intratumoral injection of an adenovirus (Ad) vector constructed to express murine B7-1 (Ad5 mB7-1) results in 38% complete regression of tumors, while an Ad vector expressing IL-2 (Ad CAIL-2) (30) demonstrated complete regression in 42% of tumor-bearing mice. In contrast, intratumoral administration of a dicistronic vector expressing both B7-1 and IL-2 (Ad5 mB7-1/hIL-2) resulted in complete regression in 100% of tumor-bearing mice. Cured mice were shown to have generated systemic immunity to a subsequent challenge with fresh tumor cells, and tumor-specific cytotoxic T lymphocyte activity could be detected.

Six- to eight-week-old FVB/n mice were purchased from Taconic Laboratories (Germantown, NY) and housed in a pathogen-free facility until use.

The cell lines used include the following: A549 (American Type Culture Collection, Rockville, MD; CCL-185); WM35, radial phase human melanoma (31); MRC5, human fibroblast cell line (American Type Culture Collection, CCL-171); PyMT, primary polyoma middle T Ag-transformed murine cells obtained from explanted tumors; PTO516, FVB/n kidney-derived cells, 516 MT3 cells derived from PTO516 and stably transformed to express polyoma middle T Ag; 293, human embryonic kidney cells transformed with adenoviral E1 sequences (32); and 293N3S, contact-independent 293 derivative (33). All cell culture reagents were purchased from Life Technologies (Grand Island, NY).

Total RNA was isolated from FVB/n splenocytes using the reagent Trizol (Life Technologies). RT-PCR was performed using the First Strand cDNA Synthesis Kit (Life Technologies). Briefly, cDNA was synthesized using oligo(dT) as the primer. PCR was performed using Vent DNA polymerase (New England Biolabs, Beverley, MA) and the following parameters: denaturation at 94°C for 1 min, annealing 55°C for 30 s, and extension at 72°C for 1 min. This was performed using the following sense and antisense primers designed to anneal to the 5′- and 3′-ends of the B7 cDNA sequence as deposited in GenBank. The sense primer 5′-AAGATCTCTCCATTGGCTCTAGATTCCTGGC-3′ and the antisense primer 5′-GAAGATCTGATTGTACCTCATGAGCCACATAATA-3′ were designed to include BglII restriction sites (underlined). The amplified fragment of 1023 bp was directly ligated into the EcoRV-digested pDK6 shuttle plasmid (34) to create pDK6-mB7-1 (Fig. 1). The shuttle plasmid pDK6-mB7-1 was amplified and purified by alkaline lysis and cesium chloride gradient centrifugation. Purified plasmid was then combined with the rescue plasmid pBHG10 (35) and cotransfected into 293 cell to produce Ad5 mB7-1.

FIGURE 1.

A schematic diagram of the construction of Ad5 mB7-1 and Ad5 mB7-1/hIL-2. For a more detailed vector construction description, see Materials and Methods.

FIGURE 1.

A schematic diagram of the construction of Ad5 mB7-1 and Ad5 mB7-1/hIL-2. For a more detailed vector construction description, see Materials and Methods.

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The shuttle plasmid containing mB7-1 (pDK6 mB7-1) was sequenced, amplified, and then subjected to the following manipulation to generate a dicistronic construct expressing mB7-1 and hIL-2. The human IL-2 open reading frame was amplified using the following 5′-phosphorylated primers, 5′-TACAGGATGCAACTCCTGTCTTGC-3′ (sense) and 5′-CTAATTATCAAGTCAGTGTTGA-3′ (antisense). It should be noted that the sense primer is constructed such that the underlined codon (TAC) is the first amino acid immediately after the ATG start codon. The PCR product of 469 bp was then blunt end ligated into the pCITE 2a plasmid (Novagen, Milwaukee, WI), which carries the encephalomyocarditis virus internal ribosomal entry site (IRES). This plasmid was digested with NcoI (recognition site CCATGG) and subsequently blunt ended (blunting was performed with Klenow polymerase large fragment from New England Biolabs) to produce an ATG start codon before ligation with the IL-2 PCR product. Ligation of the IL-2 PCR product with the pCITE 2a plasmid resulted in pCITE—hIL2, which carries the hIL-2 open reading frame driven by the IRES sequence.

The pCITE–hIL-2 plasmid was used as a template for a second round of PCR. Using the following phosphorylated primer, 5′-TTCCGGTTATTTTCCACCATATTG-3′ (IRES sense), and the original hIL-2 antisense primer, a PCR product of 979 bp was amplified corresponding to the IRES/hIL-2 hybrid molecule. This PCR product was blunt end ligated into pDK6 mB7-1 digested with SalI (3′ to the stop codon of mB7-1) to produce pDK6 mB7-1/IRES/hIL-2 (Fig. 1). This plasmid was then processed in an identical manner to pDK6 mB7-1 (see Ad5 mB7-1) and cotransfected with pBHG10 to generate Ad5 mB7-1/hIL-2. Both Ad5 mB7-1 and Ad5 mB7-1/hIL-2 were screened using Southern and Northern techniques to characterize the presence of inserted DNA or the production of monocistronic (Ad5 mB7-1) or dicistronic mRNAs (Ad5 mB7-1/hIL-2). In the case of Ad5 mB7-1/hIL-2 Western analysis was performed to detect hIL-2 production. All cloning was confirmed by sequencing.

A description of these viruses was provided by Addison (30).

Flow cytometric analysis was performed on MRC5, A549, WM35, and PyMT cells to characterize mB7-1 expression from both mB7-1-expressing vectors. Cells were infected for 48 h (MRC5, A549, and WM35) before analysis, and PyMT cells were infected and analyzed over 1 to 3 days. All groups were harvested at specific times, washed in PBS, and incubated with anti-mB7-1 Ab. The PyMT group was also incubated with anti-MHC I or II and anti-B7-2 Abs before flow cytometric analysis. Analysis was performed using a flow cytometer (Becton Dickinson, Mountain View, CA).

MRC5, A549, and WM35 cells were infected at a multiplicity of infection (MOI) of 10 plaque-forming units/cell of either Ad5 CAIL-2 or Ad5 mB7-1/hIL-2. Infected cultures were incubated for 4 days in the case of MRC5 and WM35, and for 5 days in the case of A549 cells. At 24-h intervals, 200-μl aliquots were removed and stored at −70°C for quantification. Secreted hIL-2 was quantitated using the hIL-2 DuoSet Kit (Genzyme Diagnostics, Cambridge, MA).

A transgenic mouse strain (FVB/n) expressing the polyoma middle T (PyMT) Ag under the control of the mouse mammary tumor virus long terminal repeat was the source of the tumor cells used in this study (36). Expression of PyMT Ag results in spontaneous transformation of the mammary epithelium by 8 to 10 wk of age. Tumors were excised from transgenic mice and subjected to enzymatic digestion to generate a single cell suspension (30). The single cell suspension was washed with PBS, and aliquots of 106 tumor cells were injected s.c. into the right hind flank of normal syngeneic FVB/n mice. Palpable tumors (normally 50–75 mm3) arise in these recipients 21 days after initial tumor cell injection, at which time appropriate amounts of virus were injected in a volume of 40 μl. After injection of adenoviral vectors, tumors were monitored weekly using calipers. The volume of the tumor was calculated from the longest diameter and average width, assuming a prolate spheroid. Mice with tumors not responding to vector treatment were killed when the longest diameter exceeded 20 mm. Regressed mice were left for approximately 3 mo and then challenged with 1 × 106 freshly isolated PyMT tumor cells on the left hind flank.

Splenocytes (effectors) were obtained from mice whose tumors had regressed as a result of Ad5 mB7-1, Ad CAIL-2, or Ad5 mB7-1/hIL-2 treatment and cocultured with 516 MT3 cells (stimulators) at a concentration of 1.2 × 105 516 MT3 to 1.2 × 107 splenocytes for 5 days in 12-well dishes. Serial dilutions of the effector cells were incubated in a V-bottom 96-well plate with 5 × 103 516 MT3 or PTO516 target cells. Target cells (106) were labeled with 100 μCi of 51Cr sodium salt for 2 h before coculture with the effector cells. Cells were cocultured for 5 to 6 h, at which time 80 μl of supernatant was removed for counting. The percent specific lysis was calculated as follows: 100 × (experimental cpm − spontaneous cpm)/(maximal cpm − spontaneous cpm).

Recombinant adenoviral vectors that express murine B7-1 (Ad5 mB7-1) or both mB7-1 and human IL-2 (Ad5 mB7-1/hIL-2) were constructed as outlined in Figure 1. Expression cassettes for both mB7-1 or mB7-1/hIL-2 were inserted into the E1-deleted region of the human adenovirus type 5 genome. For both constructs, transgenes were flanked by the murine CMV immediate early promoter (mCMV) and an SV40 polyadenylation signal (SV40). In the expression cassette for Ad5 mB7-1/hIL-2, the encephalomyocarditis virus IRES (37) was placed between the mB7-1- and the hIL-2-coding sequences, resulting in a dicistronic DNA fragment. The IRES functions as an internal ribosome initiation site for translation of hIL-2 in the resulting dicistronic mRNA.

Expression of the mB7-1 gene and hIL-2 gene (Ad5 mB7-1 and Ad5 mB7-1/hIL-2) was confirmed in human and murine cell lines infected with each recombinant adenoviral vector. We initially decided to characterize freshly isolated PyMT cells for the endogenous expression of crucial immunologic molecules. Single cell suspensions of PyMT tumor cells were checked by flow cytometry for surface expression of mB7-1, mB7-2, MHC I and II (Fig. 2, A–D). No mB7-1 expression could be detected on uninfected PyMT tumor cells (Fig. 2,A). Since no detectable mB7-1 protein could be demonstrated on PyMT cells, we proceeded to determine whether the Ad5 mB7-1 and Ad5 mB7-1/hIL-2 vectors could change the PyMT B7-negative phenotype to an mB7-1-positive phenotype. Figure 2, E and F, clearly demonstrates that mB7-1 protein was produced and integrated into the cell membranes of PyMT tumor cells infected with either Ad5 mB7-1 or Ad5 mB7-1/hIL-2. This result confirms that the expression of mB7-1 on PyMT tumor cells infected with both Ad5 mB7-1 or Ad5 mB7-1/hIL-2 vectors is due to the presence of the mB7-1 transgene (Fig. 2, E and F). WM35, A549, and MRC5 cells could also be converted from a mB7-1-negative phenotype to a positive phenotype after infection with the mB7-1-expressing vectors (data not shown).

FIGURE 2.

Flow cytometric data demonstrating the expression of important immunoregulatory molecules on PyMT tumor cells (B7-1, B7-2, MHC I, and MHC II; A–D). Flow cytometric analysis demonstrating vector-derived mB7-1 transgene expression from both Ad5 mB7-1/hIL-2 (E) and Ad5 mB7-1 (F) on PyMT tumor cells. PyMT cells were infected with both Ad5 mB7-1 and Ad5 mB7-1/hIL-2 in vitro at an MOI of 10 for 3 days. Single cell suspensions were then subjected to flow cytometry using an anti-mB7-1 Ab.

FIGURE 2.

Flow cytometric data demonstrating the expression of important immunoregulatory molecules on PyMT tumor cells (B7-1, B7-2, MHC I, and MHC II; A–D). Flow cytometric analysis demonstrating vector-derived mB7-1 transgene expression from both Ad5 mB7-1/hIL-2 (E) and Ad5 mB7-1 (F) on PyMT tumor cells. PyMT cells were infected with both Ad5 mB7-1 and Ad5 mB7-1/hIL-2 in vitro at an MOI of 10 for 3 days. Single cell suspensions were then subjected to flow cytometry using an anti-mB7-1 Ab.

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The expression of hIL-2 from Ad5 mB7-1/hIL-2 was characterized by Western blot analysis. Supernatants from 293 cell cultures infected with Ad5 mB7-1/hIL-2 and, for comparison, Ad CAIL-2 were size fractionated by SDS-PAGE and Western blotted with an anti-hIL-2 Ab, demonstrating the presence of two polypeptides of 15 and 17 kDa (data not shown). IL-2 from the dicistronic Ad5 mB7-1/hIL-2 and the monocistronic IL-2 control vector Ad CAIL-2 was quantified by ELISA (Fig. 3, A–C). IL-2 levels derived from the Ad5 mB7-1/hIL-2 vector were between 9 and 50 ng/ml compared with hIL-2 levels derived from Ad CAIL-2, which were between 50 and 625 ng/ml for infections with the same MOI. The expression observed per 1 × 106 cells with Ad5 mB7-1/hIL-2 was approximately 13-fold lower than that with Ad CAIL-2 (Fig. 3, A–C).

FIGURE 3.

The production of hIL-2 from Ad5 mB7-1/hIL-2- and Ad CAIL-2-infected A549 (A), MRC5 (B), and WM35 (C). Cells were infected with adenoviral vectors at an MOI of 10 for both Ad CAIL-2 and Ad5 mB7-1/hIL-2. Aliquots were removed, and supernatants were analyzed by ELISA for hIL-2.

FIGURE 3.

The production of hIL-2 from Ad5 mB7-1/hIL-2- and Ad CAIL-2-infected A549 (A), MRC5 (B), and WM35 (C). Cells were infected with adenoviral vectors at an MOI of 10 for both Ad CAIL-2 and Ad5 mB7-1/hIL-2. Aliquots were removed, and supernatants were analyzed by ELISA for hIL-2.

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We have previously shown that intratumoral injection of adenoviral vectors expressing IL-2 or IL-12 to polyoma middle T tumor-bearing mice cause regression at a dose of 5 × 108 plaque-forming units (30, 38). We used this same dose for Ad5 mB7-1, Ad5 mB7-1/hIL-2, and Ad CAIL-2 and compared the effects to those seen with the E1-deleted control vector, Ad5 dl70-3. Administration of control virus did not modify the progression of the polyoma middle T tumor growths in any of the control groups described here or previously (30, 38). Conversely, treatment with Ad5 mB7-1 resulted in 38% (9 of 24 tumors) total regression of established tumors (Table I), and all the remaining 62% of Ad5 mB7-1-treated tumors showed a pronounced growth retardation, as shown in Table I. Ad CAIL-2-treated tumors demonstrated different growth or regression kinetics compared with Ad5 mB7-1. While 42% (10 of 24 tumors) of the Ad CAIL-2-treated mice demonstrated complete regression (Table I), 37% (9 of 24 tumors) demonstrated a partial growth delay (19 of 24 tumors, or 79% overall responded), and 21% (5 of 24 tumors) showed no response to treatment. In contrast, administration of Ad5 mB7-1/hIL-2 caused complete regression of all (100%; n = 38) tumors treated, suggesting at least additive, if not synergistic, effects between mB7-1 and hIL-2 expressed from the same cell (Table I). Ad5 dl70-3-treated tumors demonstrated no growth alteration to the vector; as a result, these mice were usually killed between days 30 and 35 post-tumor cell injection. All mice exhibiting complete regression, regardless of which vector was used, were tumor free for at least 110 days postinjection.

Table I.

Growth response of PyMT tumors in response to intratumoral injection of Ad5 mB7-1, Ad CAIL-2, and Ad5 mB7-1/hIL-2

VirusResponseTotal
Expt. 1Expt. 2Expt. 3Expt. 4Expt. 5Expt. 6
Ad5 mB7-1        
Nonea 0/4 0/4 0/4 0/4 0/4 0/4 0/24 (0%) 
Partial 3/4 2/4 3/4 1/4 3/4 3/4 15/24 (62.5%) 
Complete 1/4 2/4 1/4 3/4 1/4 1/4 9/24 (37.5%) 
Ad CAIL-2        
None 0/4 2/4 0/4 1/4 1/4 1/4 5/24 (21%) 
Partial 2/4 1/4 3/4 0/4 1/4 2/4 9/24 (37%) 
Complete 2/4 1/4 1/4 3/4 2/4 1/4 10/24 (42%) 
Ad5 mB7-1/hIL-2        
None 0/4 0/4 0/5 0/4 0/3 0/18 0/38 (0%) 
Partial 0/4 0/4 0/5 0/4 0/3 0/18 0/38 (0%) 
Complete 4/4 4/4 5/5 4/4 3/3 18/18 38/38 (100%) 
VirusResponseTotal
Expt. 1Expt. 2Expt. 3Expt. 4Expt. 5Expt. 6
Ad5 mB7-1        
Nonea 0/4 0/4 0/4 0/4 0/4 0/4 0/24 (0%) 
Partial 3/4 2/4 3/4 1/4 3/4 3/4 15/24 (62.5%) 
Complete 1/4 2/4 1/4 3/4 1/4 1/4 9/24 (37.5%) 
Ad CAIL-2        
None 0/4 2/4 0/4 1/4 1/4 1/4 5/24 (21%) 
Partial 2/4 1/4 3/4 0/4 1/4 2/4 9/24 (37%) 
Complete 2/4 1/4 1/4 3/4 2/4 1/4 10/24 (42%) 
Ad5 mB7-1/hIL-2        
None 0/4 0/4 0/5 0/4 0/3 0/18 0/38 (0%) 
Partial 0/4 0/4 0/5 0/4 0/3 0/18 0/38 (0%) 
Complete 4/4 4/4 5/5 4/4 3/3 18/18 38/38 (100%) 
a

None = no response to vector treatment; partial = growth retardation to treatment in comparison to control; complete = complete regression.

Mice that had undergone complete regression were challenged on the left hind flank with 1 × 106 freshly isolated polyoma middle T cells on day 110 after the initial vector administration (see Materials and Methods). None of the challenged mice developed tumors, and all remained tumor free for an additional 120 days after challenge (Table II). To ensure the tumor-forming capacity of these cells, untreated syngeneic mice were included to observe the kinetics of tumor growth (Table II).

Table II.

Protective immunity in mice completely regressed with Ad5 mB7-1, Ad CAIL-2, and Ad5 mB7-1/hIL-2a

VirusResponseTotal
Expt. 1Expt. 2Expt. 3
Ad5 mB7-1     
Protectionb 1/1 2/2 1/1 4/4 (100%) 
No protection 0/1 0/2 0/1 0/4 
Ad CAIL-2     
Protection 2/2 1/1 1/1 4/4 (100%) 
No protection 0/2 0/1 0/1 0/4 
Ad5 mB7-1/hIL-2     
Protection 4/4 4/4 5/5 13/13 (100%) 
No protection 0/4 0/4 0/5 0/13 
VirusResponseTotal
Expt. 1Expt. 2Expt. 3
Ad5 mB7-1     
Protectionb 1/1 2/2 1/1 4/4 (100%) 
No protection 0/1 0/2 0/1 0/4 
Ad CAIL-2     
Protection 2/2 1/1 1/1 4/4 (100%) 
No protection 0/2 0/1 0/1 0/4 
Ad5 mB7-1/hIL-2     
Protection 4/4 4/4 5/5 13/13 (100%) 
No protection 0/4 0/4 0/5 0/13 
a

All mice were challenged with freshly isolated PyMT tumor cells 110 days post-initial tumor injection.

b

Protection = no tumor growth for 120 days after challenge; no protection = tumor growth after challenge.

Two cured mice from each of the following treatments were killed, and their spleens were removed 120 days after challenge. Splenocytes prepared from Ad5 mB7-1-treated mice (120 days after challenge) demonstrated a 13 to 23% specific lysis of 516 MT3 cells at an E:T cell ratio of 3.3:1 (Fig. 4). Lysis of PT0516 (no polyoma middle T Ag) was also observed, however at a much lower percentage. In contrast, splenocytes from Ad CAIL-2-treated mice (120 days after challenge) demonstrated 57 to 68% specific lysis of 516 MT3 cells and undetectable levels of lysis on PT0516 cells (at E:T cell ratios of 3.3:1; Fig. 4). Similarly, splenocytes from Ad5 mB7-1/hIL-2-treated mice (120 days after challenge) demonstrated 65 to 73% lysis of 516 MT3 targets and undetectable levels of lysis on PT0516 cells (at E:T cell ratios of 3.3:1; Fig. 4). When splenocytes from control mice were used, target cell killing was similar to or less than that seen for PyMT-negative targets at all effector cell ratios. This suggests the presence of significant numbers of effector cells capable of killing PyMT-transformed cells.

FIGURE 4.

Cytotoxic T lymphocyte activity in totally regressed mice induced by Ad5 mB7-1 (A), Ad5 mB7-1/hIL-2 (B), and Ad CAIL-2 (C). Solid squares represent 516 MT3 targets (516 MT3 cells express polyoma middle T Ag); open squares demonstrate PTO516 targets (PTO516 cells do not express polyoma middle T Ag). Splenocytes were cocultured with 516 MT3 cells for 5 to 7 days and then checked for CTL activity as outlined in Materials and Methods.

FIGURE 4.

Cytotoxic T lymphocyte activity in totally regressed mice induced by Ad5 mB7-1 (A), Ad5 mB7-1/hIL-2 (B), and Ad CAIL-2 (C). Solid squares represent 516 MT3 targets (516 MT3 cells express polyoma middle T Ag); open squares demonstrate PTO516 targets (PTO516 cells do not express polyoma middle T Ag). Splenocytes were cocultured with 516 MT3 cells for 5 to 7 days and then checked for CTL activity as outlined in Materials and Methods.

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Immunosurveillance by potential effector cells of the immune system is, in large part, responsible for the rejection of neoplastic cells. However, in individuals with seemingly intact immune systems, neoplastic growths arise and are not rejected. This ability of some neoplastic growths to evade the immune system suggests that potentially antigenic tumors elicit specific effects that act to suppress the immune system. Conversely, tumors that do not express associated Ags are cryptic and hence offer no target(s) for immune rejection. Thus, understanding the mechanisms of tumor-mediated immune suppression or the parameters encompassing the efficient activation of immune effector functions will allow the development of new strategies in tumor immunotherapy.

Recent evidence has demonstrated the ability of certain cytokines to promote tumor rejection, and since the initial IL-2 study by Rosenberg et al. (39), cytokines such as IL-2, IL-4, IL-7, and IL-12 have been studied to assess their potential for mediating tumor rejection in various immunotherapy protocols. Most of these therapies have been limited by the appearance of toxic effects elicited by certain cytokines or the inability of the cytokines under investigation to induce efficient antitumor effector activity (40, 41, 42, 43). TILs and lymphokine-activated killer cells have also been used with only limited success (41, 42, 44). Evidence suggests that genetically modified tumors cells expressing IL-2 or IL-4 either by transfection or retroviral integration can abrogate the ability of tumors to grow (45, 46, 47, 48). We have recently used adenoviral vectors to deliver cytokines such as IL-2, IL-4, and IL-12 intratumorally (30, 38, 49) and have shown that the transient expression of IL-2, IL-4, and IL-12 could augment the recognition of the tumor by the immune system and result in complete regression in a significant fraction of the tumors treated.

In contrast to the use of cytokine mediators, other tumor immunotherapy regimens have focused on the B7 family of costimulatory molecules. B7 family members are required for the transduction of signals that promote T cell activation via B7 ligation with T cell-derived CD28 in the presence of MHC I or II Ag/TCR interactions (50, 51, 52, 53). Transfection of tumor cells with B7-1 has been extensively used and demonstrated to result in only moderate effects on tumor regression (21, 24, 26, 54, 55). More pertinent to this work are the observations of a number of groups that show that B7-1 can be used to augment the activity of IL-12, IL-2, and IL-7 in vivo (56, 57, 58, 59, 60) by positively effecting tumor regression, possibly through interaction with adhesion molecules such as intercellular adhesion molecule-1 (61). Taken together these studies demonstrate that B7-modified tumor cells are capable of delivering, in conjunction with MHC I or II, Ag-specific activation signals to T cells. By far one of the more beneficial effects of B7 enhancement of tumorigenicity is the ability of B7 to directly activate naive CD8+ CTL in the absence of CD4+ help (62).

It is evident from the current literature that the use of cytokines or costimulatory molecules in isolation to modulate the immune response against established tumors is of limited efficacy. The biologic evidence for the activity of IL-2 and B7-1 on tumor rejection prompted us to examine augmentation of the immune response by supplying both B7-1 and IL-2 to the same cell in vivo, reasoning that this might enhance the ability of the immune system to recognize and react against established tumors.

We have demonstrated the efficiency of Ad vectors (Ad5 mB7-1 and Ad5 mB7-1/hIL-2) constructed to express the costimulatory molecule B7-1 (murine) at converting B7-1-negative PyMT tumor cells to a B7-1-positive phenotype (Fig. 2, A, E, and F). We have also demonstrated the ability of the double recombinant vector (Ad5 mB7-1/hIL-2) to produce hIL-2 (Fig. 3); however, hIL-2 production was at a level 1 log lower than that observed for the single vector expressing hIL-2 (Ad CAIL-2; Fig. 3) that we had previously used in this model (30).

Comparison of the effects of intratumoral injection of Ad5 mB7-1 or Ad CAIL-2 vs Ad5 mB7-1/hIL-2 demonstrated that the combination vector was much more effective at inducing complete regression in the PyMT model than either of the vectors expressing mB7-1 or IL-2 alone. Intratumoral administration of Ad5 mB7-1 demonstrated 37.5% total regression (9 of 24 mice; Table I), with the other 62.5% (15 of 24 mice) showing a partial response characterized by a drastic reduction in tumor volume but subsequent relapse into a rapid growth phase (all 24 mice responded). Similar to our previous data (30), Ad CAIL-2 injection resulted in 10 of 24 (or 42%) of the animals demonstrating complete regression and 37% exhibiting partial reduction in tumor volume or a growth delay (19 of 24 mice responded), with an overall response of 79%. In contrast, Ad5 mB7-1/hIL-2 treated tumors resulted in complete regression of all tumors treated (100%). Another important observation demonstrated by Ad5 mB7-1/hIL-2 is the shorter time taken for PyMT tumors to regress completely. Consistently, all tumors regressed within 14 days of vector administration. On the other hand, tumors undergoing complete regression by treatment with either Ad5 mB7-1 or Ad CAIL-2 took 19 to 27 days to completely regress.

To determine the ability of all vectors to induce protection from freshly isolated PyMT tumor cells, we challenged all completely regressed mice (from Expt. 1, 2, and 3; Table I) with freshly isolated PyMT tumor cells 110 days after primary tumor injection. Tumor cell challenge was always administered on the opposite hind flank to the site of the regressed tumor. All mice that exhibited complete regression were found to be protected against challenge, demonstrating that all vectors were capable of generating long lasting systemic immunity (Table II).

To determine a possible mechanism and the specificity of the effector function generated during treatment with the vectors, we examined the CTL activity in the spleen of each completely regressed animal (Fig. 4, A–C). Ad5 mB7-1-treated animals demonstrated 13 to 23% specific lysis on 516 MT3 polyoma middle T-expressing cell line at an E:T cell ratio of 3.3:1. This is low, but, nevertheless, significant, since the same spleen cells resulted in only 1% lysis of the parental line PTO516 that does not express polyoma middle T (Fig. 4,A). Conversion of the PyMT tumor from a B7-1 to a B7-1+ phenotype could enhance the ability of NK cells to target the tumor cells via a mechanism similar to that observed by Yeh et al. (63) and Chambers et al. (64). Therefore, B7-1+ PyMT tumor cells could enhance not only MHC-restricted effector cells, but also non-MHC restricted NK cells. The conversion of PyMT cells from a B7-1 to a B7-1+ population potentially provides an environment for the activation of at least two effector cell populations (NK and CD4+/CD8+), resulting in the 100% response observed for tumors treated with Ad5 mB7-1. Ad5 mB7-1/hIL-2 treatment results in high PyMT-specific CTL activity (65 to 73% lysis on 516 MT3), as opposed to undetectable lysis on nonspecific targets at an E:T cell ratio of 3.3:1 (Fig. 4,B), similar to that seen in Ad CAIL-2-treated mice that underwent total regression (Fig. 4 C). In contrast to the single vectors, Ad5 mB7-1/hIL-2 encompasses both effects mentioned above for Ad5 mB7-1 and Ad CAIL-2 into a single system capable of up-regulating IL-2R levels and providing the autocrine activity of IL-2 directly at the site of CD28 (T cell): mB7-1 (PyMT tumor cells) ligation. mB7-1 ligation with CD28 may act to provide the missing signal(s) necessary to reverse anergy and allow for a greater proliferative response of effector cells. We know that the hIL-2 levels produced by Ad5 mB7-1/hIL-2 are 13-fold lower than those observed for Ad CAIL-2. Hence, we propose that hIL-2 and mB7-1 act synergistically to overcome anergy by providing a microenvironment maximally conducive to effector function and proliferation.

The findings of this study demonstrate the effectiveness of augmenting the immune response against tumors with adenoviral vectors expressing mB7-1 in combination with hIL-2 (Ad5 mB7-1/hIL-2). Future analyses will determine the effector population activated by the double construct and the effectiveness of this construct in the treatment of other established murine tumors.

We thank D. Chong and X. Feng for their technical expertise. We also thank D. Snider and H. Liang for the FACS analysis and N. Sienna for critical reading of the manuscript.

1

This work was supported by grants from the National Cancer Institute of Canada, the Medical Research Council of Canada, Baxter Healthcare, London Life Insurance, a Ph.D. studentship from the Medical Research Council of Canada (to P.C.R.E.), a fellowship from the Medical Research Council of Canada (to J.L.B.), and a Terry Fox Research Scientist Award from the National Cancer Institute of Canada (to F.L.G.).

3

Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; Ad, adenovirus; hIL, human IL; IRES, internal ribosome entry site; MOI, multiplicity of infection.

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