Cellular and humoral immune responses to adenovirus and ... - Nature

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Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumoral injection of an adenovirus vector expressing wild-type p53 (Ad-p53) Nancy Yen,1 Constantin G. Ioannides,2 Kai Xu,1 Stephen G. Swisher,1 David D. Lawrence,3 Bonnie L. Kemp,4 Adel K. El-Naggar,4 Richard J. Cristiano,1 Bingliang Fang,1 Bonnie S. Glisson,5 Waun K. Hong,5 Fadlo R. Khuri,5 Jonathan M. Kurie,5 J. Jack Lee,6 Jin S. Lee,5 James A. Merritt,7 Tapas Mukhopadhyay,1 Jonathan C. Nesbitt,1 Dao Nguyen,1 Roman Perez-Soler,5 Katherine M. W. Pisters,5 Joe B. Putnam Jr.,1 David S. Schrump,1 Dong M. Shin,5 Garrett L. Walsh,1 and Jack A. Roth1 1

Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, University of Texas MD Anderson Cancer Center, Houston, Texas 77030; Departments of 2Gynecologic Oncology, 3 Diagnostic Imaging, and 4Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030; Departments of 5Thoracic/Head and Neck Medical Oncology and 6Biomathematics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030; and 7Introgen Therapeutics, Houston, Texas 77054. The immune responses of 10 patients with advanced non-small cell lung cancer receiving monthly intratumoral injections of a recombinant adenovirus containing human wild-type p53 (Ad-p53) to adenovirus and transgene antigens were studied. The predominate cellular and humoral immune responses as measured by lymphocyte proliferation and neutralizing antibody (Ab) formation were to adenovirus serotype 5 vector antigens, with increased responses in posttreatment samples. Consistent alterations in posttreatment cellular and humoral immune responses to p53 epitopes were not observed, and cytotoxic Abs to human lung cancer cells were not generated. Patients in this study had evidence of an antitumoral effect of this treatment with prolonged tumor stability or regression; however, neither Abs to p53 protein nor increased lymphocyte proliferative responses to wild-type or mutant p53 peptides have been consistently detected. Cancer Gene Therapy (2000) 7, 530 –536

Key words: Adenovirus vector; p53; lung cancer; lymphocyte proliferation; anti-p53 antibodies.

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tudies have shown that regional administration of viral vectors expressing wild-type (wt) p53 in orthotopic tumor models prevents the growth of tumors with mutant p53 and mediates regression of large established tumors. These data provide the rationale for a clinical protocol to replace a defective p53 gene with a normal p53 gene expressed by a recombinant adenovirus injected intratumorally (i.t.). Adenoviral vectors are currently the most efficient vectors for gene transfer. However, this type of vector is immunogenic, which is thought to limit its ability to be

Received February 5, 1999; accepted July 16, 1999. Address correspondence and reprint requests to Dr. Jack A. Roth, Department of Thoracic and Cardiovascular Surgery, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Box 109, Houston, TX 77030.

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administered repetitively. Recently, i.t. injection of a retroviral vector expressing the wt p53 tumor-suppressor gene was shown to mediate tumor regression in patients by induction of apoptosis.1 We have subsequently evaluated the safety and gene transfer efficacy of monthly i.t. injections of a recombinant adenovirus containing wt p53 (Ad-p53) in patients with advanced non-small cell lung cancer (NSCLC) and head and neck cancer who failed conventional treatments.2,3 Adenovirus-mediated transfer and expression of the marker gene lacZ, which codes for ␤-galactosidase (␤-gal), into lung cancers by direct injection has been reported.4 Adenoviral vectors may elicit immune responses to both the vector and the transgene.5,6 The immune response to i.t. injections of adenoviral vector expressing wt p53 (Ad-p53) in humans is unknown; therefore, we report an analysis of humoral and cell-mediated immune responses after administration of this vector in 10 patients treated in the clinical trial.

Cancer Gene Therapy, Vol 7, No 4, 2000: pp 530 –536

YEN, IOANNIDES, XU, ET AL: IMMUNE RESPONSES AFTER Ad-p53 TREATMENT

MATERIALS AND METHODS

Protocol approval This protocol was approved by the Biosafety and Surveillance Committees of the MD Anderson Cancer Center, by the Recombinant DNA Advisory Committee of the National Institutes of Health, and by the United States Food and Drug Administration.7

Gene transfer vector The construction and generation of Ad-p53 has been reported previously.5 Briefly, an E1-deleted replication-defective recombinant adenovirus was constructed using a modified type 5 adenovirus genome. The cytomegalovirus promoter was used to drive transcription of the human wt p53 gene. Ad-p53 was propagated in 293 cells.

Eligibility criteria and treatment protocol Patients with histological proof of NSCLC were enrolled in the trial. Patients had unresectable tumors and were either unable to receive primary external beam radiation therapy or had a recurrence after such therapy. Patients were also eligible if they did not respond to or relapsed after chemotherapy. Patients had either endobronchial tumor accessible by the bronchoscope with some clinical evidence of bronchial obstruction, advanced local-regional cancer that was unresectable, or isolated metastases whose regression or stabilization would offer potential benefit to the patient. The patients signed an informed consent document indicating that they were aware of the investigational nature of this study, in keeping with the policies of the MD Anderson Cancer Center. Patients with overexpression of the p53 protein according to the criteria of Nishio et al8 were eligible for the protocol. Mutations in the p53 gene were identified by DNA sequencing of a tumor biopsy as described previously.9 All mutations were confirmed by sequencing of a second independent polymerase chain reaction. Patients were not treated on protocol until 4 weeks after completion of systemic or local therapy. The preclinical safety studies and treatment protocol have been described previously.10,11 Ad-p53 was administered by needle injection directly into the tumor percutaneously with computed tomographic guidance. For lesions of ⱖ4 cm in the largest diameter, the final volume given was 10 mL; for lesions with a diameter of ⬍4 cm, the final volume given was 3 mL. The entire volume was injected at a single site. Patients were treated monthly for up to six injections. Evidence for gene transfer and transgene expression for this vector has been described previously.2 Five of the patients received cisplatin (80 mg/m2 intravenously) 3 days before the administration of Ad-p53 at each course. Response to therapy was assessed by chest radiograph or computed tomography (CT) scans before each course of treatment using standard criteria. Responses were confirmed by two evaluations taken 4 weeks apart. Patients were evaluable for response if they had received at least one course of therapy followed by an appropriate radiograph to document response. Response criteria were defined as follows: Complete response, disappearance of all clinical evidence of tumor by physical examination, radiography, and CT (or magnetic resonance imaging) scans for a minimum of 4 weeks; partial response, a ⱖ50% decrease in the sum of the products of the perpendicular diameters of measurable lesions for a minimum of 4 weeks, with no simultaneous increase of ⱖ25% in the size of any lesion and no appearance of any new lesion; stabilization, any variation of the indicator lesion not meeting the

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criteria of a complete or partial response or progression; progressive disease, an increase of ⱖ25% in the size of a bidimensionally or unidimensionally measurable lesion, a clinically significant increase in the size of nonmeasurable disease, or the appearance of any unequivocal new lesion.

Assay for neutralizing antibody (Ab) Serially diluted patient sera were mixed with 200 plaqueforming units (PFU) of Ad-LacZ adenovirus type 5 vector, which expresses ␤-gal, and added to 293 cell monolayers as described previously.12 The mixture dilution, which reduced plaque formation by 50% as estimated from the dilution plot, was considered to be the anti-adenovirus type 5 neutralizing Ab titer. A mouse anti-Ad5 antiserum and negative control sera were tested with each assay.

Assay for anti-p53 Abs The assay for anti-p53 Abs was performed using an anti-p53 enzyme-linked immunosorbent assay kit (PharmaCell, Paris, France). Two positive control sera containing relatively high and low levels of anti-p53 Ab and negative control sera were tested with each assay. The enzyme-linked immunosorbent assay procedure using microtiter plates coated with human p53 protein was performed according to the manufacturer’s instructions. All sera from a patient were tested at the same time in duplicate.

Ab-dependent complement-mediated cytotoxicity The complement-mediated serum cytotoxicity against the human NSCLC cell lines H1299 (large cell), H460 (large cell), and H322 (adenocarcinoma, all gifts of A. Gazdar and J. Minna, Dallas, Tex) unmodified or transduced with DL312 (E1-deleted serotype 5 adenovirus) was measured using an assay for the release of lactate dehydrogenase (LDH) by killed cells (Promega, Madison, Wis). Cells were seeded in microtiter plates at 106 cells/well; next, 10 ␮L of heat-inactivated sera was added to each well and incubated overnight. A 1/16 dilution of rabbit complement was added to each well, and LDH absorbance was measured after 4 hours at 37°C. A doubling of this value was considered to represent the presence of cytotoxic Abs. Experimental controls included a complete lysis control, complement only, a serum that was negative for cytotoxic activity, an anti-B-cell Ab (Accurate Chemical and Scientific, Westbury, NY) reacted against the KR-12 human hybrid myeloma cell line (American Type Culture Collection, Manassas, Va) as a positive control, and preimmune and immune sera raised in rabbits against the H1299 cells for lysability of H1299 cells.

Lymphocyte proliferation assay The p53 peptides tested were selected if they contained the general binding motif for human class major histocompatibility complex (MHC) class II antigens (Ags) as well as the anchors for a number of MHC class II Ags: human histocompatibility leukocyte Ag (HLA)-DR1, HLA-DR3, HLA-DR4, HLADR11, and HLA-DQ7, the sum of whose allelic frequencies covers between 75–100% of the American population as described previously.13 The general peptide binding motif for various human MHC class II molecules consists of a position 1 (P1) anchor (i.e., an aromatic (F, Y, or W) or large (L, I, V, or M) aliphatic residue) when none of the other residues were present in the first three to five amino acids close to the N-terminus and other major but fewer essential anchors at P4,

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Table 1. Wt p53 and Mutant p53 Peptides Used in this Study (Listed by Patient) Patient

Wt p53 sequence

Mutant p53 sequence

C1 D1 A2 S2 E2 R4 E3 S3 M2 C5

GNLRVEYLDDRNTF* RPILTIITLEDSSG RPILTIITLEDSSG VQLWVDSTPPPGTR PQHLIRVEGNLRVEY NSFEVRVCACPGR AIYKQSQHMTEV PEVGSDCTTIHYNY CTYSPALNKMFCQL GNLRVEYLDDRNTF

GNLRVSIWMTETLF* RPILAIITLEDSSG RPILTIITREDSSG VQLWVDSTPPPGAR PQHLNRVEGNLRVEY NSFEVRVGACPGR AIYEQSQHMTEV PEVGSDCTAIHYNY CTYSPALIKMFCQL GNLRVECLDDRNTF

*Patient C1 has one base change in the E codon (GGA3 GAG) follow by an entire sequence change compared with the wt p53. The sequence was chosen to align V3 as the primary anchor for HLA-DR and F12 as the C-terminal anchor.

P5, P7, and P9 counting from the P1 anchor. Because many peptides are capable of binding to many different MHC class II molecules and because their sequences contain overlapping binding motifs for MHC class II molecules, the peptides synthesized contained at least two of three anchors for each HLA-DR Ag when possible, as well as the main P1 anchors for most class II alleles. An additional criterion used for selection was the positioning of the mutated residue in the chosen sequence between the P1-P9 anchor. This was made to ensure that the mutated residue is present in the central area, which is more likely to interact with T-cell receptor than are the flanking sequences. Peptides that were 13–16 residues long were prepared by the Synthetic Antigen Laboratory of MD Anderson Cancer Center using a solid phase method. Their identity was determined by amino acid analysis. Their purity was 93–97% as determined by high performance liquid chromatography. Peptides were dissolved in phosphate-buffered saline or in phosphate-buffered saline in dimethylsulfoxide, aliquoted at 1– 4 mg/mL, and stored frozen at ⫺20°C until use. The peptide sequences used for each patient are shown in Table 1. Peripheral blood lymphocytes were separated on Ficoll-

Hypaque gradients. Both wt and mutant p53 peptides were synthesized based on sequence analysis of the pretreatment tumor biopsy. Peptides ranged in size from 13 mer to 16 mer. DL312 at a multiplicity of infection of 20 was used as the source of adenoviral Ags. Phytohemagglutinin (PHA) at a concentration of 20 ␮g/mL was used as a positive control for lymphocyte proliferation, and medium only was the negative control. The wt or mutant p53 peptides were added to 106 lymphocytes at a concentration of 25 ␮g/mL and cultured for both 5 and 6 days. Preliminary experiments showed that 106 lymphocytes stimulated in bulk culture and then plated at 105 lymphocytes/well was the threshold number of lymphocytes for detecting a proliferative response to p53 peptides. At the end of day 5 or day 6, lymphocytes were seeded at 105 cells/well in triplicate in microtiter plates, labeled with 2 ␮Ci of [3H]thymidine per well, and harvested onto filters after 16 hours for counting. Stimulation indices were calculated by dividing the mean value of the stimulated culture counts per minute by the mean value for the cultures incubated in media alone. Mean stimulation indices ⱖ2 were always statistically significant compared with untreated lymphocyte cultures by the MannWhitney test (P ⬍ .05) using StatMost software (DataMost, Salt Lake City, Utah). Additional comparisons were made using the two-sided Wilcoxon signed-rank test.

RESULTS Pre- and posttreatment lymphocyte and serum samples adequate for testing in quantity and, for lymphocytes, viability were available from 10 lung cancer patients enrolled in the phase I clinical trial of i.t. injection of Ad-p53. The demographic and clinical characteristics of the patients are shown in Table 2. The doses of Ad-p53 administered by i.t. injections ranged from 108 PFU to 1011 PFU, with patients receiving from two to five injections at 1-month intervals.

Anti-adenovirus neutralizing Abs All patients had detectable pretreatment Ab titers ranging from 1:10 to 1:3000, with eight of the patients having

Table 2. Characteristics of Patients and Tumors and Responses to Treatment with Ad-p53

Patient C1 D1 A2 S2 E2 R4 E3 S3 M2 C5

Dose (PFU) 108 108 109 109 3 ⫻ 109 1010 1010 1010 1011 1011

Age Cisplatin (years) Gender Yes Yes No Yes No No No Yes No Yes

64 70 78 69 72 68 55 72 62 68

Male Male Male Male Female Female Male Female Female Male

Prior Prior chemotherapy Prior Histology* surgery courses† radiation‡ Squamous Adeno Adeno Squamous Adeno Large cell Squamous Large cell Squamous Squamous

No Yes Yes No Yes No No No No Yes

5 4 2 12 0 0 3 4 2 1

6600 5400 6000 None 8000 None 6000 6380 6300 6660

Injection site§ Chest wall Lung Liver Adrenal Lung Lung Lung Liver Lung Chest wall

Baseline Number measurement of (cm) courses Response 3.5 ⫻ 9 1.5 ⫻ 2 4⫻5 3.5 ⫻ 5 6.5 ⫻ 7 5⫻5 5⫻5 6 ⫻ 6.5 5 ⫻ 6.5 6⫻3

5 2 3 3 2 2 5 2 3 3

Stable PR¶ Stable Stable Stable Stable Stable Stable Stable Stable

*All tumors histologically confirmed pretreatment to be viable NSCLC: Squamous, squamous cell carcinoma; Adeno, adenocarcinoma, Large cell, large cell carcinoma; Sarcomatoid, sarcomatoid subset of NSCLC. †Number of chemotherapy courses given at least 3 months before Ad-p53 treatment. ‡Centigray (cGy) of external beam radiation therapy given at least 3 months before Ad-p53 therapy. §Location of indicator lesion injected with Ad-p53. ¶PR, partial response. Did not have required follow-up CT scan to confirm response.



Table 3. Pretreatment and Highest Posttreatment Serum AntiAdenovirus Neutralizing Ab Titers Patient

Pretreatment titer

Highest posttreatment titer

1:300 1:200 1:10 1:30 1:3,000 1:1,000 1:60 1:30 1:100 1:60

1:40,000 1:2,000 1:2,000 1:20,000 1:200,000 1:20,000 1:50,000 1:3,000 1:20,000 1:4,000

C1 D1 A2 S2 E2 R4 E3 S3 M2 C5

initial titers of ⱕ1:300 (Table 3). All patients had an increase in Ab titer after treatment, with titers ranging from 1:2,000 to 1:200,000 in the patient with the highest pretreatment titer, representing 10- to 833-fold increases with the median and mean increases being 117-fold and 230-fold, respectively. The time course for the titers of the Ab during the course of treatment is shown in Figure 1. There was no correlation between the dose of the vector and the fold increase in the Ab titer.

Anti-p53 Abs Only 1 of the 10 patients (E3) had detectable anti-p53 Ab levels before treatment with Ad-p53. This patient had an initial titer of 1:50,000, which increased to 1:750,000 after Ad-p53 injection and remained at this level for four consecutive monthly treatments. The other patients remained negative for anti-p53 Abs throughout their treatment.

Serum cytotoxicity against human lung cancer cells alone and transduced with DL312 Pre- and posttreatment sera were tested for the presence of cytotoxic Abs against a human NSCLC cell line (H1299) with and without transduction by the adenoviral vector DL312, which has the adenoviral backbone used

Figure 1. Time course of neutralizing anti-adenoviral Ab formation. Neutralizing Abs to adenoviral Ags were measured immediately before the injection of vector during the treatment courses shown.


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for Ad-p53 but does not contain a transgene. Both an anti-B-cell Ab and sera from a rabbit immunized with H1299 cells generated a cytotoxic response against a B-cell lymphoblastoid cell line and H1299 cells transduced with DL312, respectively, in the presence of rabbit complement (Fig 2). However, none of the pre- or posttreatment sera from patients showed any cytotoxic activity to H460 cells alone or H460 cells transduced with DL312 exceeding that occurring with complement alone or with test sera in the absence of complement. Similar results were seen for sera incubated with H322 or H1299 human lung cancer cell lines (data not shown).

Lymphocyte proliferation to PHA, adenoviral Ags, and mutant and wt p53 peptides All patients had pretreatment lymphocyte proliferative responses to PHA that were ⱖ4-fold the untreated control lymphocyte cultures, indicating that the lymphocytes were capable of proliferative responses before treatment. After treatment, four patients had a ⱖ2-fold increase in the proliferative responses to PHA, whereas one patient had a ⬎2-fold decrease. A total of 3 of the 10 patients had evidence of preexisting cellular immunity to adenovirus serotype 5 Ags, with lymphocyte proliferative responses ⱖ2-fold over unstimulated control lymphocyte cultures to DL312 (Table 4). After treatment, six patients had proliferative responses to DL312, with three patients becoming positive, one patient increasing his response by 2-fold, two patients remaining positive, and one patient becoming negative. However, overall the changes before and after treatment were not statistically significant according to the Wilcoxon signed-rank test (P ⫽ .11). Lymphocyte proliferation to peptides representing wt and mutant p53 epitopes was assessed. Two patients showed significant proliferative responses to wt p53 epitopes in pretreatment lymphocyte cultures. However, none of the posttreatment cultures showed significant lymphocyte proliferation to wt epitopes. Pretreatment lymphocyte cultures from three patients showed proliferative activity to mutant epitopes. These patients showed no proliferative activity to the wt peptide. Two of these patients lost the proliferative response after treatment, whereas two patients who had not shown pretreatment proliferative responses became responsive to the mutant peptide. One patient with prolonged stable disease during treatment had an increased proliferative response to mutant peptide. However, the one responding patient in this group showed no proliferative response pre- or posttreatment to mutant peptide, whereas a second patient with prolonged stable disease had a decrease in lymphocyte proliferation to mutant peptide. The administration of cisplatin did not alter the number of patients showing proliferative responses compared with those receiving Ad-p53 alone. Overall, the changes before and after treatment were not statistically significant for either the wt or mutant p53 peptides according to the Wilcoxon signed-rank test (P ⫽ .41 and P ⫽ 1.00, respectively).

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DISCUSSION

Figure 2. Ab-dependent complement-mediated cytotoxicity. Complement-mediated serum cytotoxicity against the human NSCLC cell line H1299 unmodified or transduced with DL312 was measured using an assay for the release of LDH by killed cells. Cells were seeded in microtiter plates at 106 cells/well; next, 10 ␮L of heatinactivated sera was added to each well and incubated overnight followed by the addition of rabbit complement. C# Serum, serum from an Ad-p53 treatment course #, with C1 being the first course of treatment; 9809 A, preimmunization rabbit sera. 9809 C, rabbit sera 1 month after immunization with H1299 cells. Experimental controls included a complete lysis control, complement only, a serum negative for cytotoxic activity, an anti-B-cell Ab reacted against the KR-12 human hybrid myeloma cell line as a positive control, and preimmune and immune sera raised in rabbits against the H1299 cells transduced with DL312 (anti-H1299 sera) for lysability of H1299 cells. (⫾ SD).

Recently, it has been shown that i.t. injection of a replication-defective retrovirus expressing wt p53 tumorsuppressor gene can mediate tumor regression and is not associated with toxicity.1 Thus i.t. injections of tumorsuppressor gene expression vectors is a feasible strategy for enhanced local-regional tumor control. An adenovirus vector expressing wt p53 mediates increased apoptosis and regression of established human tumors in nu/nu mice.14,15 These results provided the rationale for the present protocol.11 Transient expression of wt p53 is sufficient to mediate apoptosis in cancer cells, but repeated administration of the vector appears desirable.16,17 Therefore, characterization of the humoral and cellular immune responses to the vector and transgene are important. The regression of tumors despite the lack of complete transduction of all tumor cells has suggested possible cytotoxic effects of transduced cells on nontransduced cells (sometimes referred to as a bystander effect). Immunological responses to the tumor induced by the vector or transgene could be a possible explanation for this. The first-generation recombinant adenoviruses used in this study, which are produced by homologous recombination of the transgene into the E1 region of the viral genome, are efficient in transducing nondividing cells and can be produced in high titers. Studies have shown that an immune response develops to adenoviral or transgene proteins. Some animal studies and clinical trials have shown a loss of transgene expression within 2–3 weeks of administration, as well as an inability to achieve transgene expression after repeated administrations into normal tissue.18,19 Both humoral and cellular immune responses have been detected. Subsequent studies have shown that cytotoxic T lymphocytes are generated that are targeted to viral vector proteins expressed by the genetically modified cell.19 Destruction of these cells by cytotoxic T lymphocytes could therefore account for the short duration of transgene expression. Investigators also observed that subsequent administration of adenovirus vectors after the initial dose often failed to mediate transgene expression. The appearance of antiviral neutralizing Abs correlated with the inability to achieve repeated transgene expression with readministration.20,21 Anti-adenovirus Abs have been detected in cystic fibrosis patients injected with an adenovirus vector expressing the cystic fibrosis transmembrane conductance regulator.18,22 In patients undergoing i.t. injection of lung cancers with an adenovirus expressing ␤-gal, humoral and cell-mediated immune responses to the transgene as well as adenoviral Ags were observed.5 However, despite preexisting immune responses to adenovirus, transgene expression occurred. Abs against fiber and penton base viral capsid proteins appear to be necessary for neutralizing activity.6 Despite the appearance of anti-adenovirus Abs, subsequent i.t. injections of Ad-p53 did not cause major toxicity. Furthermore, in the present trial, despite the appearance of high titers of anti-adenovirus Abs, con-


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Table 4. Lymphocyte Stimulation Indices to DL312, Wt p53 Peptides, and Mutant p53 Peptides [PHA] Patient C1 D1 A2 S2 E2 R4 E3 S3 M2 C5

DL312

Wt p53 peptide

Mutant p53 peptide

Pre

Post

Pre

Post

Pre

Post

Pre

Post

3.49 14.35 7.60 21.91 28.44 104.70 11.14 47.59 14.54 5.87

4.77 3.92 18.31 157.10 7.07 102.80 29.38 28.42 11.81 5.77

1.02 1.39 1.23 1.63 2.16 1.68 3.16 1.03 1.56 2.01

3.23 0.46 1.99 1.40 3.63 2.04 2.44 1.32 3.48 4.48

1.73 1.37 4.04 0.99 1.60 1.32 1.03 2.29 1.23 1.36

1.85 0.93 0.73 1.13 1.62 0.90 1.17 1.45 1.31 1.33

1.36 1.14 6.15 0.96 1.87 1.73 2.81 1.29 1.47 2.01

2.79 0.83 1.15 2.26 1.21 1.81 2.17 1.97 1.91 1.51

Indices were calculated by dividing the mean value of the stimulated culture counts per minute by the mean value for the cultures incubated in media alone. Pretreatment and 1-month posttreatment lymphocyte samples were tested. The results shown are from day 5 cultures, with similar results seen for the day 6 cultures. Each value represents the mean of three replicates. Indices ⱖ2 are statistically significant compared with untreated lymphocyte cultures (Mann-Whitney test, P ⬍ .05).

tinued gene expression was documented with subsequent injections.2 Immunological protection against subsequent tumor challenge after i.t. injection of Ad-p53 and Ad-p53 plus IL-2 has been described for syngeneic mouse tumors.23 Although we did not test specifically for cytotoxic lymphocytes recognizing adenovirus Ags, it is unlikely that these would mediate tumor regression or protective responses. Only a fraction of the tumor is transduced and thus would express adenovirus Ags. This of course would not explain protection against subsequent growth of nontransduced tumors. The predominate immune response noted in this trial is directed against adenoviral Ags. Increases in anti-adenoviral Abs and lymphocyte proliferative responses to adenovirus were noted for some patients after injection of the Ad-p53 vector. It does not appear that consistent immune responses against a wt or mutant p53, as measured by the assays used in this study, are induced after intralesional injection of the Ad-p53 vector. Only one patient had preexisting Abs to p53, and these did increase in titer during treatment. However, none of the other patients had induction of anti-p53 Abs during treatment. Lymphocyte proliferative responses to wt and mutant peptides were inconsistent. In two patients with pretreatment proliferative responses to wt peptide, decreases were noted with treatment. For the mutant peptides, two patients showed an increase in lymphocyte proliferative responses, two showed a decrease, and one remained elevated. Patients with advanced lung cancer are known to have suppressed cell-mediated immune responses.24 Perhaps some of the unresponsiveness or decrease in responsiveness noted was due to progression of the patient’s cancer. However, all patient lymphocytes had proliferative capacity as shown by the proliferation of pre- and posttreatment lymphocytes to PHA. Alternatively, there could have been an induction of tolerance through repetitive administration. Although patient-specific lymphocyte proliferation could not be shown for each p53 peptide, it is clear that proliferative responses can be elicited for this class of peptides. This does not eliminate


the possibility that Ad-p53 expression may elicit an enhanced immune response to other tumor-associated Ags. The addition of Ad-p53 to IL-2 treatment resulted in an immune response to a mouse breast cancer. When an adenovirus vector expressing IL-2 was combined with Ad-p53 for i.t. injection, tumor regression was observed and mice were protected against a subsequent challenge of the syngeneic tumor.23 However, if this is occurring, it is not reflected in the Abs being generated that recognize tumor-associated Ags on lung cancer cells, as indicated by the results of the Ab-mediated cytotoxicity assay. In conclusion, in this study some patients receiving an i.t. injection of Ad-p53 develop anti-adenovirus Abs and lymphocyte proliferative responses to adenoviral Ags. Neither Abs nor increased lymphocyte proliferative responses to wt or mutant p53 peptides have been detected consistently. ACKNOWLEDGMENTS This study was supported in part by Grant R01 CA45187 from the National Cancer Institute and the National Institutes of Health, by Grant P50-CA70907 from the Specialized Program of Research Excellence in Lung Cancer, by gifts to the Division of Surgery and Anesthesiology from Tenneco and Exxon for the Core Laboratory Facility, by University of Texas MD Anderson Cancer Center Support Core Grant CA16672, and by a sponsored research agreement with Introgen Therapeutics, Inc.

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