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The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations.
In phase I studies, 54 patients received escalating doses (between 107 and 1011 plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed.
Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients.
High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens.
The cloning and characterization of the genes encoding melanoma-associated antigens recognized by human T cells have opened new possibilities for the development of active immunization strategies for the treatment of patients with metastatic melanoma (1,2). Two immuno-dominant antigens, MART-1/MelanA and gp100, were recognized by the majority of tumor-infiltrating lymphocytes (TILs) obtained from HLA-A2-positive patients with metastatic melanoma (3-6). In prior studies (7-9), we have reported the initial results of immunization of patients with melanoma with immunodominant peptides obtained from the MART-1 or gp100 proteins incorporated in incomplete Freund's adjuvant (IFA) and have demonstrated that antitumor precursor cells are generated in the peripheral blood of immunized patients when comparing preimmunization and postimmunization samples. These studies suggested that improved response rates were seen when peptide immunization was followed by the administration of interleukin 2 (IL-2) (9).
In murine models, immunization with recombinant adenoviruses, vaccinia viruses, and fowlpox viruses encoding model tumor antigens generated antitumor responses that were capable of significantly reducing the number of established pulmonary micrometastases (10,11). These preclinical studies have stimulated efforts to develop immunization strategies against tumor-associated antigens in humans using recombinant viruses.
Adenoviruses are attractive candidates for use in the development of human vaccines and for human gene therapy because the adenovirus genome can be readily manipulated by recombinant DNA techniques and inserts of foreign genes are stably integrated [reviewed in (12,13)]. The incorporation of large DNA fragments into adenovirus requires the deletion of wild-type viral DNA sequences. Most commonly, DNA sequences from the E1, E3, or E4 regions are deleted, which results in a virus deficient in viral replication.
Administration of adenoviruses has been shown to be safe, and vaccines consisting of unattenuated adenovirus have been administered to millions of military recruits over the past several decades (14,15).
Recombinant adenoviruses have been used as vectors for gene therapy in patients with a variety of diseases (16-24) or as vaccines to raise cellular or antibody reactivity against infectious agents (12,13,25-27). In our own preclinical study (10), we demonstrated that immunization with a recombinant adenovirus expressing the model tumor antigen, β-galactosidase, could produce specific cytolytic T cells and could reduce established metastases in tumor-bearing mice that could be enhanced by the concomitant administration of IL-2.
Thus, recombinant adenoviruses were generated expressing the MART-1 and gp100 melanoma-associated antigens and the characteristics of these viruses were determined (28). We have now conducted phase I clinical trials in patients with metastatic melanoma who received active immunization with multiple doses of these recombinant adenoviruses. The immunologic, therapeutic, and safety aspects of these immunizations in humans constitute the subject of this report.
Human melanoma cell lines 888-mel, 1173-mel, 624-mel, and 1300-mel were established in our laboratory and were maintained in RPMI-1640 medium containing 10% fetal calf serum. The human embryonic kidney cell line 293, the human breast carcinoma cell line MDA-231, and the melanoma cell line SK23 were purchased from the American Type Culture Collection (Manassas, VA). The 293 and MDA-231 cells were cultured in Iscove's medium supplemented with 10% fetal calf serum. The T2 cell line is an HLA-A2-positive, TAP-deficient T-B cell hybrid and was maintained in continuous culture in medium consisting of RPMI-1640 supplemented with 10% fetal calf serum.
Type II adenoviruses encoding either the MART-1 or gp100 genes were constructed as previously described (28). In brief, to construct a plasmid encoding the MART-1 antigen, a 2.0-kilobase (kb) NdeI/XbaI fragment containing the MART-1 complementary DNA (cDNA) was inserted into the NdeI/AvrII-digested parental plasmid pAd2CMV. The pAd2CMV is a pBR322-based plasmid containing a 7.6-kb insert derived from the 5′ region of Ad2 (1–355,3328–10685) with a complete E1A and a partial E1B deletion (356–3327). A synthetic bovine growth hormone polyadenylation signal is also present. In the resulting plasmid pAd2CMV-MART-1, the MART-1 gene was under the control of the cytomegalovirus immediate early promoter. Another plasmid, pAd2CMV-gp100, was constructed in a similar fashion by ligation of the 2.0-kb SpeI/XbaI fragment containing gp100 cDNA from pCR2-gp100 to SpeI/AvrII-digested pAd2CMV.
To generate the recombinant adenovirus vectors, XbaI-digested pAd2CMV-MART-1 or pAd2CMV-gp100 DNA and the PshA1-digested wild-type Ad2 DNA were co-transfected into the adenovirus packaging cell line 293 by calcium phosphate precipitation (Promega Corp., Madison, WI). Viral plaques were isolated and expanded and the recombinant viruses were identified by restriction digestion and polymerase chain reaction (PCR) analysis. After multiple rounds of plaque purification, the adenovirus plaques were sent to Genzyme Corporation (Framingham, MA) for production of large quantities of adenovirus under good laboratory practice conditions. The final adenoviral stocks contained up to 2.75 × 1011 infectious units (IU) per mL. In a HeLa/A549 assay, there were no replication competent adenoviruses detected in up to 109 IU. Southern blot analyses for adeno-associated virus were negative.
The recombinant adenoviruses were evaluated in vitro by infecting HLA-A2-positive cell lines that were negative for MART-1 or gp100 expression and then testing for recognition by TILs that recognized MART-1 (TIL 1235) or gp100 (TIL 1200) immuno-dominant peptides (3,29,30). The A375 melanoma line and the MDA-231 breast cancer line are HLA-A2-positive lines that do not express either MART-1 or gp100. Both lines expressed the gp100 antigen as detected by recognition by TIL 1200 when infected with adenovirus gp100 but not with adenovirus MART-1. Similarly, both lines were recognized by TIL 1235 when infected with adenovirus MART-1 and not with adenovirus gp100. Fluorescence-activated cell-scanning assays also demonstrated that cell lines infected with either adenovirus MART-1 or adenovirus gp100 expressed only the appropriate antigen (data not shown).
The clinical protocol used for the treatment of these patients was approved by the Investigational Review Board of the National Cancer Institute and by the U.S. Food and Drug Administration. All patients had histologically confirmed metastatic melanoma and underwent a complete clinical evaluation, including measurements and radiologic examination of all evaluable tumor sites. HLA typing was performed using high-resolution, nested-sequence, PCR subtyping. No patient had received any treatment in the prior month or any immunosuppressive drugs including steroids. Cohorts of at least three patients each received escalating doses of recombinant adenovirus into either the subcutaneous tissue of the anterior thigh or intramuscularly into either the gluteus maximus or deltoid muscles. Injection sites were alternated with each injection and injections were given every 4 weeks. All patients who received adenovirus immunization are included in this report.
Thirty-six patients were treated with adenovirus MART-1 and 18 patients were treated with adenovirus gp100. Seventy percent of the patients were between the ages of 31 and 60 years and all patients but one had an Eastern Cooperative Oncology Group performance status of 0 or 1. All patients had undergone surgical excision of their primary melanoma, 41% had received systemic chemotherapy, and 22% and 28% had also been treated with radiation therapy or hormonal therapy, respectively. Seventy-six percent of the patients had received two or more treatments for their melanoma before entering this trial.
The dose escalation for administration of the adenovirus MART-1 and adenovirus gp100 in cohorts of three or more patients is shown in Table 1. The first trial to be performed was with adenovirus MART-1 and patients began their treatment with 107 plaque-forming units (pfu) given subcutaneously per injection. Subcutaneous injections of up to 3 × 1010 pfu adenovirus MART-1 were given. Intramuscular injections were begun at 109 pfu and escalated to 1011 pfu. The adenovirus gp100 protocols started after the safety of administration of up to 1010 pfu of adenovirus MART-1 had been demonstrated and thus the escalation of the adenovirus gp100 began at the 109-pfu dose given intramuscularly. As seen in Table 1, cohorts of patients were also treated with between 109 and 1011 pfu of adenovirus MART-1 or gp100 in conjunction with IL-2 starting the day following the adenoviral injection. All patients but two received at least two adenoviral injections (fewer than two injections were given only if rapid disease progression precluded further patient treatment).
IL-2 (supplied by Chiron Therapeutics, Emery-ville, CA) was administered at a dose of 720 000 IU/kg intravenously over 15 minutes starting 1 day after the adenoviral injection. Patients received IL-2 every 8 hours until grade 3 or 4 toxicity was reached that could not be easily reversed by standard supportive measures. IL-2 was routinely administered on a general surgical ward, although some patients were transferred to an intensive care unit for monitoring of the administration of vasopressors. All patients receiving IL-2 also received concomitant medications, including acetaminophen (650 mg every 4 hours), indomethacin (50 mg every 8 hours), and ranitidine (150 mg every 12 hours), to prevent some of the side effects associated with IL-2 administration.
Patients underwent leukapheresis prior to receiving adenoviral immunization and approximately 4 weeks after each adenoviral immunization. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque separation and were cryo-preserved in dimethyl sulfoxide (DMSO) at 108 cells per vial and stored at −180 °C.
The MART-1:27–35 peptide, the three gp100 peptides (gp100:154–162, gp100:209–217, and gp100:280–288), and the flu M1:58–66 peptide from the influenza matrix protein were synthesized with the use of solid-phase techniques. The MART-1 peptide was dissolved in 100% DMSO and stored at −70°C. The gp100 peptides and the flu peptide were soluble in aqueous solution.
PBMCs from patients who were cryopreserved before and after immunization were tested for the presence of antitumor T-cell precursors using a peptide sensitization assay based on the ability of circulating precursors to generate antipeptide and antitumor reactivity following in vitro sensitization with immunodominant peptides derived from either the MART-1 or gp100 molecules (8,9). In brief, cryo-preserved PBMCs were thawed and cultured at 6 × 106 PBMC per 2-mL well in 24-well plates (Corning Costar Corp., Cambridge, MA) in 2 mL of Iscove's medium (Biofluids, Rockville, MD) plus 0.3% 1-glutamine, 100 U/mL penicillin, 10% heat-inactivated human AB serum (Biofluids), and 25 mM HEPES buffer (Biofluids). On the day of initiation of the culture, 1–10 μg/mL peptide was added and IL-2 at 300 IU/mL (Chiron Therapeutics) was added 48 and 96 hours after each stimulation. At weekly intervals, cells were harvested and aliquots of cells were either tested for immunologic reactivity or cryopreserved for later analysis. The remaining cells were recultured at 106 cells per 2-mL well and restimulated with the use of 2.5–10 × 106 irradiated (3000 rads) autologous preimmunization PBMCs per well that had been previously incubated for 1–2 hours at 37 °C in 1–10 mg/mL peptide. Cultures were restimulated weekly in vitro for 3–4 weeks with weekly testing or cryopreservation of samples.
To test for immunologic reactivity, interferon gamma release assays were performed as previously described (8,9). In brief, 105 responder cells were incubated in 0.2 mL either alone, with 105 A2+ or A2− tumor cells, or with 105 T2 cells that had been pulsed with relevant or irrelevant peptides. After 24 hours of incubation, the supernatants were harvested and tested for interferon gamma release utilizing an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., Minneapolis, MN).
Immunoglobulin G (IgG) antibodies and neutralizing antibodies reactive with adenovirus type 2 were measured in sera obtained from patients before and after adenoviral immunization. Neutralizing antibodies were assayed by their ability to block infection and lysis of 293 cells by the adenovirus. Sera were inactivated at 56 °C for 30 minutes and serial twofold dilutions were added to 96-well flat-bottom plates in medium containing 10% fetal calf serum. Adenovirus (3 × 105 IU/mL) was added, and after 1-hour incubation at 37 °C , 3 × 104 293 cells were added to each well and the wells were incubated at 37 °C in 5% CO2 for 72–96 hours. Control wells included 293 cells alone or plus untreated virus or virus incubated with serial dilutions of sera. The assays were read when the 293 cells incubated alone were 80%–100% confluent and the wells containing untreated virus plus 293 cells showed complete lysis of the cell monolayer. The neutralization titer for a given sample was the highest dilution that showed detectable protection from cytopathic effects. IgG titers were tested in an ELISA assay using adenovirus type 2 as the coating reagent and an anti-human IgG conjugated to horseradish peroxidase as a detector. The results were expressed as the greatest dilution providing an increase over baseline.
One patient had mild hepatic transaminase elevations after receiving the first adenovirus gp100 injection alone at 109 pfu that returned to normal within 4 weeks and did not recur after the second injection. Except for mild and transient erythema at the injection site, there were no other significant toxic effects associated with the injection of either adenovirus MART-1 or adenovirus gp100, even at 1011 pfu per injection. The administration of high-dose bolus IL-2 was associated with the expected side effects, although they were transient and reversed to normal within several days after stopping IL-2 administration. There were no treatment-related deaths in this protocol.
Of the 16 patients receiving the escalating doses of adenovirus MART-1 without IL-2, there was one complete response, ongoing at 31 months, in a patient with biopsy-proven progressive melanoma in the mediastinum and breast who received four sequential doses of 108 pfu subcutaneously (Table 1; Fig. 1). Of the 20 patients who received adenovirus MART-1 plus high-dose IL-2, there were two complete responses and two partial responses.
There were no responses among the six patients who received the adenovirus gp100 alone and one complete response of extensive pulmonary metastatic disease in a patient who received adenovirus gp100 plus high-dose IL-2 (Table 1). The overall clinical response rate in patients receiving adenovirus in conjunction with high-dose IL-2 was 16% and was similar to that expected from the use of high-dose IL-2 alone.
To determine the HLA-A2-restricted-immunologic reactivity of circulating peripheral blood lymphocytes against the MART-1 or gp100 peptides, in vitro sensitizations were conducted comparing cells obtained before and after adenoviral immunization. Up to four weekly in vitro stimulations of PBMCs were performed either against the MART-1 peptide or against multiple gp100 peptides. In vitro sensitizations against influenza peptide were sometimes performed and with rare exceptions resulted in antiflu reactivity after just one or two in vitro stimulations.
A summary of the first cytokine-release assay performed on the patients who received adenovirus MART-1 is shown in Table 2. Many patients were tested more than once with similar results. Adequate numbers of cells were not available in seven patients, and four of the patients were not suitable for testing in this assay because they were not HLA-A2 positive. In PBMCs from 22 patients tested prior to receiving adenovirus immunization, 12 patients exhibited significant specific reactivity to T2 cells pulsed with the MART-1 peptide following three in vitro sensitizations. All patients except one (No. 6) also exhibited specific reactivity to the MART-1 peptide after immunization and only three patients exhibited more than a twofold increase in reactivity following adenovirus immunization. Of the 10 patients tested who did not exhibit reactivity to the MART-1 peptide prior to immunization, two (Nos. 5 and 12) converted to positive following immunization. Thus, five of 23 patients showed evidence of immunization to the MART-1: 27–25 peptide after receiving recombinant adenovirus encoding the MART-1 antigen. Similar results were obtained in studies of the 16 patients receiving the adenovirus encoding gp100. No consistent evidence for HLA-A2-restricted reactivity to the three gp100 epitopes tested was seen in PBMCs derived from these patients (data not shown). There was no relationship between immunologic reactivity and the dose of adenovirus administered nor was reactivity greater in the clinically responding patients, although the number of responding patients was too small to draw definitive conclusions. There was no association of immune response depending on whether virus was given subcutaneously or intramuscularly, whether patients received concomitant administration of IL-2, or whether patients had high titers of neutralizing antibody against adenovirus (see next section).
The pretreatment levels of antiadenoviral IgG or neutralizing antibodies are shown in Fig. 2, A. Only one of the sera from the 53 patients tested had a preimmunization titer of IgG antibody less than 1000 with the majority of patients having IgG titers of 8000 or greater. When measuring the ability to neutralize infectious adenovirus, six of 54 pretreatment sera had titers of less than 100, with the majority having neutralizing titers of 400 or greater. The increase in IgG as well as neutralizing antibody following one or two adenoviral immunizations is shown in Fig. 2, B. Thirty-four of the 45 sera tested both before and after immunization showed an increase in titer of IgG following immunization, and patients receiving concomitant IL-2 had a higher incidence of elevated antiadenoviral IgG titers (24 of 26) compared with patients who did not receive IL-2 (10 of 19; P = .004). Most of the increase in antiadenoviral antibody occurred after the first immunization, with only a few additional patients exhibiting additional increase in titers following a second immunization (Fig. 2, B). There was no association between the levels of IgG and neutralizing antibody.
Recombinant adenoviruses have several advantages for use in the active immunization of patients with cancer [reviewed in (12,13)]. Genes encoding tumor antigens can be readily inserted into replication-incompetent adenoviruses that can be safely administered in large doses to humans. Adenoviruses have a broad range of infectivity of various cell types and have been popular for use in gene therapy (16-24). Vaccines based on recombinant viruses have been widely used in multiple animal species (12,13,25-27). In murine tumor models, immunization with recombinant adenovirus-encoding model tumor-associated antigens has resulted in successful prevention as well as treatment of metastatic disease. Significant reduction of established pulmonary metastases occurred when 108 pfu of recombinant adenovirus expressing the model β-galactosidase tumor antigen was administered to mice bearing pulmonary micrometastases (10,31,32). The administration of exogenous IL-2 significantly augmented this antitumor effect.
In recent years, multiple tumor-associated antigens present in human melanomas have been identified and characterized [reviewed in (1,2,33)]. TILs obtained from HLA-A2-positive individuals predominantly recognize the MART-1/MelanA and gp100 nonmutated, melanoma–melanocyte differentiation antigens expressed by the great majority of melanomas and these melanoma–melanocyte differentiation antigens are also expressed on melanocytes but not other normal tissues or other tumors (3-5,30). The adoptive transfer of TILs that recognize MART-1/MelanA and gp100 has been associated with regression of cancer in patients with metastatic melanoma (34,35).
To evaluate the therapeutic potential of immunization with recombinant adenoviruses expressing tumor antigens, replication-incompetent adenoviruses were constructed that encoded either the MART-1 or gp100 antigens in deleted E1A and E1B portions of the viral genome (28). The current study has evaluated the safety, immunologic, and potential therapeutic aspects of the immunization of patients with metastatic melanoma using these recombinant adenoviruses.
The patients in this study all had metastatic melanoma, many had been heavily pretreated, and all had progressive disease at the time of entrance in this phase I study. The administration of recombinant adenoviruses at doses up to 1011 pfu per injection was well tolerated, with no significant side effects associated with up to four sequential doses given at monthly intervals. One of 16 patients who received recombinant adenovirus MART-1 alone achieved an objective response (Table 1; Fig. 1), which is ongoing at 31 months. The objective response rate in patients who received recombinant adenovirus in conjunction with high-dose IL-2 was 16% and was similar to the 17% objective response rate seen in 134 consecutive patients with metastatic melanoma treated by us with this regimen of high-dose bolus IL-2 alone in prior studies (36). Thus, except for the single responding patient to adenovirus MART-1 alone, there was no evidence that the adenoviral immunizations enhanced the antitumor effects seen with IL-2 administration.
In previous studies using the same in vitro assay used here, we had demonstrated that the administration of an immunodominant MART-1 peptide (MART-1:27–35) in IFA or the administration of several gp100 peptides (gp100:209–217 or gp100:280–288) in IFA could increase the immune precursors present in immunized patients reactive against peptide and tumor (7-9) and that clinical responses could be enhanced by IL-2 administration (9). Because patients with melanoma have pre-existing immune reactivity against the MART-1 and gp100 antigens, it was necessary to compare preimmunization and postimmunization samples (7-9,37,38). In patients receiving multiple injections of 0.1–10 mg of the immunodominant MART-1 peptide in IFA, 15 of 16 postvaccination PBMCs demonstrated at least a threefold increase in immune reactivity compared with prevaccination PBMCs (7). Similarly, immunization with the two gp100-immunodominant peptides (gp100:209–217 and gp100:280–288) could successfully immunize patients as well (8). Modification of the gp100:209–217 peptide by substituting anchor amino acids to increase binding to HLA-A2 substantially increased its immunogenicity. Administration of this modified peptide plus IL-2 resulted in a 42% objective response rate in patients with metastatic melanoma (9).
In the present study, patients receiving adenovirus MART-1 or adenovirus gp100 showed only sporadic evidence of HLA-A2-restricted reactivity to either the MART-1:27–35 peptide or the gp100:154–162, 209–217, or 280–288 peptides. Given that the adenoviral vectors encode the entire MART-1 or gp100 proteins, one cannot rule out the possibility that reactivity was generated to peptides other than the immunodominant peptides examined here, that peptide reactivity was manifested via a histocompatibility restriction other than HLA-A2, or that assays measuring lymphocyte properties other than specific γ-interferon secretion may have given other results. Thus, only five of the 22 patients tested before and after immunization exhibited some evidence of immunization as a result of the adenoviral administration. It was not possible to assess the antipeptide reactivity in PBMCs derived from 11 of the 34 non-HLA-A2 patients who completed treatment with the Ad2/MART-1 virus, two of whom showed objective clinical response. Therefore, there are insufficient data to permit one to conclude that there was antipeptide T-cell reactivity in PBMCs in those patients who showed objective clinical responses following administration of Ad2/MART-1 virus either with or without IL-2. Similarly, there was little evidence of immunization against the gp100 epitopes in patients receiving adenovirus gp100.
To understand the relative lack of immunizing and therapeutic activity of the recombinant adenoviruses, studies were performed of the antiadenoviral IgG and neutralizing antibodies circulating in the sera of patients prior to immunization. Fifty-two of 53 patients tested had a preimmunization titer of antiadenoviral IgG antibody of 1000 or greater and 48 of 54 pretreatment sera had neutralizing titers of 100 or greater. These high titers of IgG and neutralizing antibodies might be expected to significantly impair the immunizing potential of recombinant adenovirus, although little is known about the level or impact of neutralizing antibodies at a subcutaneous or intramuscular injection site. Neutralizing antibodies can diminish but often not eliminate the efficiency of gene transfer (38-41). Furthermore, it should be emphasized that the patient exhibiting a complete tumor regression following administration of adenovirus-MART-1 alone had a serum-neutralizing titer of 400.
More than 75% of patients showed a significant increase in IgG or neutralizing antibody titers after undergoing two immunizations with adenoviral vectors. Thus, the ability to give repeated injections of adenovirus is limited. Reactivity to adenoviral proteins has been seen by others. In a study (42) of potential gene therapy patients, 57% of adult humans exhibited neutralizing antibodies to adenovirus type 5. In several models, both humoral and cellular responses against adenoviral antigens prevented successful immunization with recombinant adenovirus and could, in fact, lead to destruction of cells expressing the adenoviral genes (43-46). Thus, the immune response to adenoviral proteins may be a significant limitation of the ability of recombinant adenoviruses to successfully immunize experimental animals as well as humans. These studies emphasize the need to develop recombinant adenoviruses that are not susceptible to pre-existing immune reactions in patients and a variety of approaches are being explored (47).
Editor's note: B. Roberts is a senior investigator at Genzyme Corporation and is presently involved in the reported research that is the subject of a cooperative research and development agreement between the National Cancer Institute and Genzyme Corporation.