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© 2002 American Society for Clinical Oncology Phase I Trial of Adoptive Immunotherapy With Cytolytic T Lymphocytes Immunized Against a Tyrosinase EpitopeByFrom the Center for Biological Therapy and Melanoma Research, University of California San Diego School of Medicine, San Diego, CA. Address reprint requests to Malcolm S. Mitchell, MD, Hudson-Webber Cancer Research Center, Rm 740.2, Karmanos Cancer Institute, 110 East Warren Ave, Detroit, MI 48201; email: mitchell@ karmanos.org.
PURPOSE: To study distribution and toxicity of cytolytic T lymphocytes (CTLs) against a single melanoma epitope. PATIENTS AND METHODS: CD8+ T cells obtained by leukapheresis from 10 patients with disseminated HLA-A2.1+, tyrosinase-positive melanomas were immunized in vitro against tyrosinase369-377 (YMNGTMSQV). Drosophila cells transduced with HLA-A2.1, CD80, and CD54 (intracellular adhesion molecule-1) were used for priming, followed by two rounds of immunization with mononuclear cells as antigen-presenting cells. 1 x 108 CTL were infused intravenously (IV) on day 1. CTL frequency was measured by limiting dilutions in five patients. 111In labeling and scintigraphy measured distribution of CTL in next five. Five days later, 1 x 108 CTLs were infused on 4 successive days to both groups. Immunohistology of response was judged by biopsies. RESULTS: Infusions were nontoxic. CTLs were undetectable in the blood, going to lungs within 5 minutes. At 4, 24, and 72 hours, they were found in liver and spleen. Lesions were visualized by scintiscans in one responding patient where two subcutaneous nodules were noted at 4 and 24 hours. A second patient had a partial response and remains alive with disease 2 years later. CD8+ T cells were found in lesions of responders, associated with the presence of HLA-A2 molecules and tyrosinase. Two nonresponders without tyrosinase and HLA-A2 molecules had a paucity of CD8+ T cells in their lesions. Whether the CD8+ T cells in lesions of responders were those we had reinfused is uncertain. CONCLUSION: CTLs immunized against a single melanoma epitope were nontoxic but did not specifically localize to tumor sites. Nevertheless, two patients had disease regression. Additional therapeutic studies with specifically immunized CTL seem justified.
SPECIFIC IMMUNOTHERAPY OF cancer implies the use of antibodies, cancer vaccines, or T cells directed against tumor-associated antigens.1,2 The number of cancer cells and the subversion of the immune response, by such cytokines as interleukin (IL)-10 and transforming growth factor–beta (TGF-ß) made or induced by the tumor, have thwarted most efforts at curing metastatic disease by immunologic means, particularly with cancer vaccines alone.3 It is possible that clinical results might be improved if a complementary form of specific immunotherapy, such as cytolytic T lymphocytes (CTLs), were administered to reduce the tumor cell burden. Among the first attempts at adoptive cellular immunotherapy were activated natural killer cells (also known as lymphokine-activated killer [LAK] cells) administered with high doses of IL-2.4 This suffered from the nonspecificity of the LAK cells, the failure of those cells to penetrate the tumor, and the toxicity of the high-dose IL-2. The introduction of tumor-infiltrating lymphocytes immunized against melanoma cells, also given with high doses of IL-2,5 was an effort to improve the specificity of the transferred cells.
The rapid increase in the understanding of tumor-associated antigens and specific epitopes recognized by cytolytic T lymphocytes has made it possible to immunize CTL in vitro against shared epitopes of a particular histotype of tumor. Tyrosinase is an important shared antigen in melanoma, which contains several immunogenic peptides recognized by CTL in the context of HLA-A2, HLA-A24, or HLA-B44.6-8 Tyrosinase is an enzyme involved in the conversion of phenylalanine to tyrosine, intermediate steps in the production of melanin, and is found in more than 90% of melanomas.9 Tyrosinase369-377 (YMNGTMSQV) (tyrosine-methionine-asparagine-glycine-threonine-methionine-serine-glutamine-valine) is an epitope of tyrosinase restricted by HLA-A2.1, an HLA class I molecule found in approximately 40% of white people. A posttranscriptional variant TMDGTMSQV (asparagine371 We studied a strategy in which CTL obtained by leukapheresis from patients with melanoma were immunized in vitro against tyrosinase369-377 nonamer (hereafter referred to, for simplicity, as tyrosinase epitope) and then reinfused into the patient in large numbers as specific adoptive immunotherapy. We used Drosophila cells transduced with human HLA class I and accessory molecules as antigen-presenting cells (APCs) to obtain efficient de novo immunization. In this phase I study, we have focused on the toxicity and distribution of the CTL, administered alone without cytokines, and performed biopsies to determine the presence of CD8+ and CD4+ T cells and CD16+ NK cells within tumor masses before and after the infusions. Despite 111In scans indicating limited distribution of the T cells, evidence for a clinical response of tumor masses was obtained in two of the patients, one of whom had a PR and remains alive more than two years later.
In Vitro Immunization With Drosophila Cells as APCs Drosophila melanogaster cells were used as APCs.11,12 These cells are efficient vehicles for the presentation of peptides in the context of HLA class I, especially for de novo immunization of CD8+ CTL. The Schneider S2 Drosophila cell line (American Type Culture Collection CRL 10974, Rockville, MD) was transduced with HLA-A2.1, CD80 (B7-1) and CD54 (intracellular adhesion molecule-1) with a pRmHa-3 plasmid vector. Drosophila cells were grown in Schneider’s medium (106 cells/mL) with 10% fetal bovine serum and CuSO4 at 27°C, the optimal temperature for these insect cells. They were harvested, washed, and resuspended in X-press medium (Bio Whittaker, Walkersville, MD) containing 100 µg/mL of the tyrosinase epitope. CD8+ T cells were obtained from peripheral blood mononuclear cells (PBMCs) by positive selection with a novel anti-CD8 monoclonal antibody (mAb) (LeTurcq et al, manuscript in preparation), captured with a sheep anti-mouse magnetic bead (Dynal, Lake Success, NY).13 After incubation at 27°C with the tyrosinase epitope for 3 hours, the presence of tyrosinase on the S2 Drosophila cells was detected by a binding competition assay.14 Hepatitis B virus core peptide 18-27 (FLPSDFFPSV) (HBVc) was radiolabeled with 125I as a standard and incubated with the Drosophila cells with attached unlabeled tyrosinase epitope. The cells were then layered on fetal bovine serum and centrifuged to separate free and cell-bound peptide. Binding inhibition was calculated as 100 x (1 - binding of HBVc in the absence of unlabeled tyrosinase/binding of HBVc in the presence of unlabeled peptide). This inhibition of binding of the standard peptide indicated that the tyrosinase epitope was bound to HLA-A2 molecules on the Drosophila cells. The Drosophila cells were then incubated with the CD8+ T cells at 37°C at a ratio of 1:10 in RPMI 1640 medium containing 10% autologous serum. Two days later, 20 IU of IL-2 and 30 IU of IL-7 were added to the growth medium. Incubation was continued for 1 week, after when the Drosophila cells were replaced with autologous irradiated PBMCs (30 Gy) and tyrosinase peptide. This was repeated for one additional round of stimulation, after when the CD8+ T cells were tested for cytotoxicity by a 4-hour 51Cr release assay. The final preparation contained at least 92% CD8+ T cells, with 4% or less CD16+ (natural killer) cells and 4% or less CD4+ T cells.
In Vitro Feasibility Study of Healthy Individuals and Patients With Melanoma
The number of CTLs obtained was in the range of 0.5 to 1 x 109 cells in 3 to 4 weeks (three to four stimulations with tyrosinase epitope). These results showed that a clinical trial of reinfusion with our projected dose of CTL was feasible within 1 month after CD8+ T cells were obtained by leukapheresis. We felt that this was not an inordinately long period to require a patient with melanoma to wait for therapy after discontinuing his or her previous treatment.
Obtaining T Cells From Patients and Reinfusing Them After Immunization In Vitro No Drosophila cells remained in the CTL preparation after the immunization procedure. Drosophila cells are viable at 27°C but are nonviable at 37°C. Furthermore, two rounds of immunization with changes of medium each time were performed subsequent to the initial immunization with the fly cells. Finally, the polymerase chain reaction was used to try to detect residual Drosophila DNA in the final preparation of CTL before reinfusion. Drosophila DNA was uniformly absent by this sensitive method. For infusion into the patient, the CTLs were resuspended in 200 mL of 0.9% saline with 5% human serum albumin in a transfer pack (Baxter [McGaw Park, IL] catalog no. 4R-2014 plastic blood cell infusion bag) and were administered intravenously over a period of 1 hour on the stem-cell transplantation unit. Experienced nurses took vital signs every 15 minutes and monitored the patients for signs of toxicity or immediate hypersensitivity reactions.
Viability of 111In-Labeled CTLs
Phase I Trial The purpose of the study was to determine the toxicity and distribution of reinfused autologous CTLs generated in vitro against the tyrosinase epitope. Ten eligible patients with metastatic melanoma were treated and were included in the study. All had received and were considered resistant to standard therapy, such as surgery, radiation therapy, and chemotherapy. The most crucial criteria for inclusion were the following: (1) histologically confirmed melanoma, clinical stage IV (metastatic); (2) presence of tyrosinase in a biopsy specimen of the tumor, as determined by immunohistochemistry with mAb T311 to recombinant tyrosinase protein expressed in Escherichia coli; and (3) HLA-A2.1 positivity. For this last parameter, lymphocytes from the patients were first screened with mAb BB7.2 (ATCC HB-82) for HLA-A2 positivity. Lymphocytes that were positive by the mAb were additionally subtyped by the polymerase chain reaction with the Dynal HLA-A2-SSP kit (catalog no. 571.01). Other requirements for inclusion were age of 18 years or older, not being pregnant, Karnofsky performance status of at least 70%, normal leukocyte count (  3,000/µL) and normal platelet count ( 100,000/µL), and signed informed consent. These studies were performed after review by the human investigations committees at the University of California, San Diego and in accord with an assurance filed with and approved by the Department of Health and Human Services. The protocol was reviewed as well by the Food and Drug Administration as part of an investigator-sponsored IND application. Ineligibility criteria included medical or psychologic impediment to probable compliance with the protocol, severe renal, cardiac or other dysfunction, uncontrolled brain metastases, and human immunodeficiency virus positivity. CTLs were infused in a volume of 200 mL of 0.9% saline with 5% human serum albumin over a period of 60 minutes. The first five patients were studied to see whether and for how long during the peri-infusion period we could detect the presence in the blood of CTLs against the tyrosinase epitope. Because we did not know whether a dose of CTLs at any level might be toxic, the first patient was given a test dose of 1 x 107 CTLs. A full dose of 1 x 108 cells was to be given 1 day later. Because of an adverse event in this first patient unrelated to the CTL infusion, which we will shortly describe, the full dose was delayed until 2 days later. All other patients received 1 x 108 lymphocytes on day 1, 7 days after the last restimulation of the CTL in vitro. The presence of CTL in the peripheral blood was assayed by limiting dilutions immediately before and after the infusion and at 30 minutes. Five days later, 12 days after the last restimulation of the CTL in vitro, the patients received 1 x 108 CTL per day for a maximum of 4 consecutive days (days 6 through 9), depending on availability of the CTLs. Our plan was to treat all patients with a total of 5 x 108 CTLs in five daily doses; all but two patients received the projected dose. The projected fifth dose for patient no. 5 failed to meet safety release criteria, whereas patient no. 8 had insufficient cells for a fifth infusion. The second five patients were given 1 x 108 111In-labeled CTLs15 and had scintiscans immediately afterward and at 4, 24, 48, and 72 hours after the single infusion on day 1. The 72-hour half-life of 111In precluded study beyond that time. Patient no. 9 was studied after the infusion on day 6 because of technical problems on day 1, receiving other unlabeled infusions on days 7 through 9 during the period of study of the 111In-labeled cells. Toxicity was assessed in part through repeated complete blood counts and blood chemistries, performed weekly for 2 weeks after the first infusion. Patients who had at least three subcutaneous lesions were requested to permit a biopsy before treatment and again 2 to 4 weeks after the conclusion of the reinfusion. The remaining lesions were used to monitor response. Immunostaining for the presence of the following molecules was performed on a portion of the biopsy snap-frozen and stored at -80°C: HLA class I molecules (with mAb w6/32), HLA-A2 (with mAb BB7.2), tyrosinase (with a rabbit polyclonal antibody against tyrosinase), CD4+, CD8+ T cells, and CD16+ cells, with "Leu" series mAbs from Becton Dickinson (San Jose, CA). Table 1 summarizes the clinical data on the patients in the study.
Toxicity of Reinfused CTLs No immediate or delayed hypersensitivity to the CTLs was noted. No patient experienced fever, chills, or back pain. Two patients, patient no. 5 (a responder) and patient no. 10 (a nonresponder) felt pain in the region where their tumor was located briefly after the fourth infusion. No abnormality of blood chemistries or complete blood count was found in the 2-week period of study beginning after the first infusion.
Attempt to Detect CTL in the Blood After Reinfusion We were unable to detect an increase in the frequency of circulating CTL directed against tyrosinase by this method. The frequency before and within 5 minutes after the infusion was less than 1:50,000, which was our lower limit of detection. Limiting dilutions analyses have been used successfully for over a decade in our laboratory to quantitate CTL against melanoma and breast cancer antigens in other clinical trials.16
Distribution of the CTLs After Reinfusion Within 5 minutes of administration, CTLs were found in both lungs (Fig 2A), reinforcing the limiting dilutions data that they had rapidly left the circulation. At 4 hours, they were still in the lungs, although there was some movement to the liver and spleen at that time (Fig 2B). At 24 hours, CTLs were detected in the liver and spleen in all five patients, with most cells having left the lungs (Fig 2C). They seemed to remain in the liver and spleen thereafter (Figs 2D and 2E). There was little or no localization in most patients to the sites of the tumor. A noteworthy exception was patient no. 9 (Figs 2C, 2D, and 2E), whose subcutaneous nodules on the upper arm were identified on scans taken at 4 and 24 hours.
Clinical Responses Patient no. 5 had clear evidence of a response to the treatment. Despite having received only four of the projected five doses, a total of 4 x 108 CTLs, he had rapid and persistent disappearance of numerous small pretracheal and prevascular lymph nodes, coalescence and shrinkage of two contiguous 2.3-cm diameter left lateral subcutaneous thigh nodules to 2.2 cm in total diameter, and shrinkage of large masses in the left external iliac-femoral region and the left anterior thigh (region of the anterior tensor fascia lata). The left anterior thigh mass regressed from 3.5 x 2.5 to 2.8 x 2.1 cm and became flat, whereas the 4.5 x 3.5 cm iliac mass also became flat to palpation and 3.0 x 2.5 cm by physical examination. The anterior thigh mass continued to regress for several months after the initial shrinkage. The first evidence of response was found by physical examination 2 weeks after completion of the infusions and persisted for 7 months. By computed tomography (CT) scans, one large lesion began to respond only 4 months after treatment and continued thereafter. This patient’s CT scans of the chest and pelvis before and after treatment are shown in Fig 3. Displayed are the scans pretreatment on March 18, 1998, on May 20, 1998, when a complete response of four pretracheal nodes was apparent (see arrows), on July 22, 1998, when the large iliac mass was beginning to respond, and on October 16, 1998, when shrinkage of the iliac lesion was considerable. Before therapy it was impossible to biopsy any of his lymph node or subcutaneous masses, because none could be excised completely without major surgery, and we were concerned about the possibility of excessive bleeding and poor healing of an incisional biopsy. Biopsy after treatment of the inguinal mass was obtained. The patient had an indolent course after his treatment with the CTL, failing to respond to several chemotherapy regimens. He has remained alive without progression at treated sites for longer than 2 years after treatment, but brain metastases present before therapy recurred on August 1, 2000, more than 3 years after receiving radiation therapy.
Patient no. 9 also had clinical regression of some of his disease, mainly subcutaneous nodules. He had a disappearance of a 1.3 x 1.3-cm subcutaneous nodule in the right biceps region, shrinkage of a midback lesion from 1.5 x 1.5 to 1.0 x 1.0 cm, and flattening and shrinkage of an upper arm lesion from 1.6 x 1.2 to 1.3 x 1.1 cm. However, lesions in the liver increased significantly by CT scan, despite the migration of the CTL to the liver by scintiscans. His response overall was considered a mixed response. Patient no. 1 had an unusual course after treatment with the test dose of 1 x 107 cells. Approximately 9 hours after the end of the infusion, she experienced shaking chills and fever and was seen in the emergency room. Blood cultures confirmed the presence of Staphylococcus aureus sepsis, with a probable source in her infusion catheter, which contained the same organism. She survived the sepsis only to die three weeks later of disseminated melanoma. Her lesions shrank within 24 hours of the treatment with the small dose of CTLs, and a biopsy showed only necrotic material. The immediate shrinkage was probably attributable to the effects of the sepsis on leukocytes, with consequent release of tumor necrosis factor alpha and other antitumor cytokines into the circulation. The other patients in the study had progressive disease.
Immunohistology of Lesions Patient no. 5 had inguinal and thigh lesions that were considered too large to biopsy before treatment. However, after the sites regressed with treatment, two biopsies were performed 3 months apart. On his first posttreatment biopsy on May 20, 1998, when regression of these lesions had been noted, tyrosinase, HLA class I, and, specifically, HLA-A2 molecules were present, and there were CD8+, CD4+, and CD16+ T cells present in the nodule (Fig 4). There was an increase in the lymphocytes of all three phenotypes when the region was again biopsied on August 10, 1998 and a persistence of the tyrosinase and HLA-A2 molecules (not shown).
Patient no. 9 had a pretreatment biopsy of deltoid region mass that proved to be an angiolipoma rather than a melanoma nodule. However, in a melanoma nodule in the right upper arm that regressed after treatment, tyrosinase, HLA class I and HLA-A2 molecules, and CD8+ and CD4+ T cells were found in abundance. CD16+ cells were also present. (Fig 5). Of course, whether the CD8+ T cells were the ones administered and were cytotoxic in situ could not be determined.
Biopsies of patient no. 2, a nonresponder, were significantly different from the previous two responding patients. His CTLs after in vitro immunization were strongly reactive to tyrosinase-containing melanomas, and his original metastatic specimen had tyrosinase. Yet biopsies before and after treatment with CTL showed tyrosinase largely absent. HLA class I molecules were present, but HLA-A2 was patchily distributed and of low intensity on staining. Associated with these findings, both CD8+ and CD4+ T cells were scarce in biopsies before and after treatment. CD16+ cells were found in approximately equal numbers in lesions biopsied before and after treatment (Fig 6).
Patient no. 1, who had rapid softening and shrinkage of her tumor masses within 24 hours after an episode of staphylococcal sepsis, also had little or no tyrosinase, HLA-A2 staining, and few CD4+ or CD8+ T cells in a biopsied nodule before treatment. A biopsy taken at 24 hours showed only necrotic material and was uninterpretable for cells. Similarly, autopsy specimens 3 weeks after treatment were too autolyzed to permit accurate determination of infiltrating cells.
This phase I trial was designed to explore the toxicity and distribution of human CTL immunized against a specific melanoma epitope, tyrosinase369-377 (YMNGTMSQV), and administered intravenously to melanoma patients. Tyrosinase-specific CTLs generated in 3 weeks did not cause significant toxicity in the patients on reinfusion, except for pain at the site of tumor in two patients after the fourth infusion. By scintigraphy, labeled CTL went to the lungs and emigrated to the liver and spleen within 24 hours but did not localize to sites of tumor except in one patient. That patient had a regression of the visualized subcutaneous nodules but not of (nonvisualized) liver lesions. A second remission occurred in a patient who did not have a scintiscan. Immunohistology showed an association between the presence of CTL at sites of tumor and positive staining for tyrosinase and HLA-A2, but it was impossible to determine whether the CTL were those that had been reinfused and to conclude that the CTL had caused the response. CD4+ T cells were also present in abundance in the same lesions. A unique aspect to this study was that Drosophila cells were used as APCs. Drosophila cells are blank-slate APCs, which can be transduced with a variety of molecules and subsequently loaded with peptides.11,12 Because Drosophila cells do not present intrinsic peptides, unlike autologous dendritic cells, the added peptides do not have to compete with endogenous peptides for binding pockets on HLA molecules. It is therefore possible to display many copies of a peptide on the surface of the fly cells. Leturcq et al confirmed previous data from the same laboratory11 that Drosophila cells transduced with (human) HLA-A2, CD80 (B7-1), and CD54 (intracellular adhesion molecule-1) are effective at presenting peptides to naive T cells (Leturcq et al, manuscript in preparation). Naive T cells were immunized de novo against the tyrosinase epitope over the course of 4 weeks, with only the first round of immunization requiring presentation by Drosophila. Autologous irradiated adherent PBMCs were as good as Drosophila for subsequent rounds. This is similar to observations that dendritic cells are best at de novo priming of T cells but no better than, and perhaps inferior to, macrophages and lymphoblastoid cells for secondary stimulation Whether dendritic cells or Drosophila is superior for primary immunization in vitro remains to be determined by direct comparison. Whether the infused CD8+ T cells caused the regressions observed could not be determined, but there was a clear temporal relationship between the two. The mechanism by which they might have led to regression is likewise a matter for conjecture. CD8+ T cells were present in biopsy specimens from patients with clinical responses and were absent in tumor specimens lacking HLA class I molecules. If CTLs reached a site of metastasis as in patient no. 9 (from his positive scintiscan), they apparently remained there only when the target epitope was presented on the expressed HLA class I molecules. It is interesting that the patients who responded had HLA-A2 and tyrosinase on their tumor cells, whereas two patients who did not respond lacked both of these elements. For entry into the study, we required that both of these molecules be present in the original tumor or its first metastasis. The markers had clearly been lost in patients at some time before the treatment was begun. Obviously it is insufficient to have potent CTLs in the vicinity of a tumor if the latter cannot be immunologically recognized by the T-cell receptors. This situation might be remedied by administration of interferon alfa or interferon gamma, which are capable of upregulating, respectively, HLA class I expression or both HLA class I and class II molecules. This important element of immunotherapy, which we have called cytomodulatory,18,19 might be an important adjunct to many forms of specific immunotherapy to ensure optimal targeting of the tumor cells. Biopsies on two occasions of responding lesions of patient no. 5 showed an abundance of infiltrating CD8+ T cells but also CD4+ T cells, which were clearly endogenous. Whether these CD4+ T cells were antigen-specific is unknown. In other studies, we found that the immunohistology of biopsies obtained from regressing lesions of patients treated by active specific immunotherapy with an allogeneic melanoma lysate vaccine revealed both CD8+ and CD4+ T cells and macrophages in the lesions.20 This suggested that cytokines liberated at the site of T-cell infiltration, attracting macrophages and perhaps modifying tumor vascular endothelium, may be an important function of the T cells, apart from direct cytolysis of tumor cells. Long-term immunologic memory mediated by tumor antigen–reactive CD4+ T cells, which can be elicited by specific active immunotherapy (cancer vaccines), may be an important element presently missing from adoptive immunotherapy regimens that rely solely on the infusion of CTLs. The CTLs did not specifically localize to tumor sites after intravenous infusion, with a distribution suggesting trapping in the reticuloendothelium of the lungs, liver, and spleen. It was encouraging, however, that a definite pattern of lymphocyte traffic emerged, with rapid initial migration to the lungs, followed at 24 hours by movement out of the lungs to the liver and spleen. After 72 hours, there may have been true localization to a site of tumor, at least in patient no. 9. That patient alone showed what might have been localization to subcutaneous nodules of the shoulder. There was a local response in those sites in that patient but overall progression of disease in the liver. The CTLs were probably not at the sites of tumor metastases in the liver but instead trapped within the Kupffer cells or otherwise sequestered within that organ. Maneuvers designed to maintain the motility of the reinfused lymphocytes after in vitro immunization to avoid sequestration might therefore be considered in additional trials. By design, CTLs were given here without a T-cell growth factor, such as IL-2, to define the toxicity and distribution of the T cells alone as a baseline. The CTLs had a short lifespan in the bloodstream, as judged by our inability to detect them by a limiting dilution analysis that we have used successfully in several other studies and their rapid movement into the lungs within 5 minutes. Inclusion of a T-cell growth factor, such as IL-2, to promote proliferation and prolong the survival of the CTL in vivo seems an obvious next step. For similar reasons, we did not immunize and reinfuse CD4+ helper T cells together with the CD8+ CTL here. However, we are aware that the additional presence of T helper cells is important for CD8+ T cells to be optimally effective after adoptive transfer. Riddell and Greenberg21 found that CD4+ T cells caused CD8+ T cells directed against CMV antigens either to decline more slowly over a 12-week period or, alternatively, increase in frequency after an initial small decline. Coincident with the latter changes was a gradual rise in the CD4+ T cells in the circulation, as measured by 3H-TdR incorporation in response to CMV in vitro. For phase II trials, the addition of specifically immunized CD4+ T cells to the infusion may well be of therapeutic value. Whether they will be necessary in addition to the nonspecific help provided by cytokines such as IL-2 remains to be seen. No toxicity of the CTL was observed, even though the cells had been removed by leukapheresis, cultivated for 3 weeks in vitro, and then returned to the patient. No fever, chills, hives, or delayed hypersensitivity reactions were noted in nine of the 10 patients. The one severe adverse event associated with the infusions, staphylococcal sepsis, was traceable to a contaminated infusion catheter that had been accessed and manipulated several times during that day. T cells and medium were found to be free of bacterial contamination. The patient recovered from the episode after antibiotics and vasopressors were administered. The optimal number of CTLs for therapy remains to be determined. We infused 5 x 108 CD8+ T cells, because we had some concern about possible toxicity caused by infusing large numbers of highly immunized CTLs. The number of cells may have been suboptimal, because we estimated from cloning that 10% to 30% of the infused CTLs were specific for the tyrosinase epitope. Thus, a maximum of 1.5 x 108 tyrosinase-specific CTLs were infused. At least 1 log10 more CTL than the 1 x 109 we started with can be obtained by continuing the culture for several more weeks or obtaining a larger number of leukocytes at leukapheresis. A single tyrosinase epitope was used as a target in these studies solely as a prototype. We recognize that many other epitopes, both HLA-A2 restricted and those restricted by other common HLA class I alleles, should be included for broader immunization. Nevertheless, our results have encouraged us to consider phase II trials incorporating some of these elements in an attempt to improve on the clinical activity of what seems to be a nontoxic and potentially useful therapy.
Supported by a contract with the R.W. Johnson Pharmaceutical Research Institute. Performed under Bureau of Biologics Investigational New Drug Application no. 6875, Bureau of Biologics, United States Food and Drug Administration (M.S.M., principal investigator).
1. Mitchell MS: Principles of tumor immunology and their application to the biomodulation of cancer, in Calabresi P, Schein PS (eds): Medical Oncology ( ed 2 ). New York, NY, McGraw-Hill, 1993, pp 323-344 2. Quan WDY, Mitchell MS: Principles of biologic therapy, in Haskell CM (ed): Cancer Treatment ( ed 4 ). Philadelphia, PA, WB Saunders, 1995, pp 57-69 3. Hersey P: Impediments to successful immunotherapy. Pharmacol Ther 81: 111-119, 1999[CrossRef][Medline] 4. Rosenberg SA, Lotze MT, Muul LM, et al: Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 313: 1485-1492, 1985[Abstract]
5.
Rosenberg SA, Lotze MT, Yang JC, et al: Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer. J Natl Cancer Inst 85: 622-632, 1993 6. Kang X, Kawakami Y, el Gamil M, et al: Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes. J Immunol 155: 1343-1348, 1995[Abstract]
7.
Robbins PF, el Gamil M, Kawakami Y, et al: Recogni-tion of tyrosinase by tumor-infiltrating lymphocytes from a patient responding to immunotherapy. Cancer Res 54: 3124-3126, 1994
8.
Brichard V, Van Pel A, Wolfel T, et al: The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 178: 489-495, 1993 9. Fetsch PA, Riker AI, Marincola FM, et al: Tyrosinase immunoreactivity in fine-needle aspiration samples of metastatic malignant melanoma. Cancer 90: 252-257, 2000[CrossRef][Medline] 10. Skipper JC, Hendrickson RC, Gulden PH, et al. An HLA-A2-restricted: tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J Exp Med 183:527-534, 1996
11.
Cai Z, Brunmark A, Jackson MR, et al: Transfected drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells. Proc Natl Acad Sci U S A 93: 14736-14741, 1996 12. Sun S, Cai Z, Langlade-Demoyen P, et al: Dual function of Drosophila cells as APCs for naive CD8+ T cells: Implications for tumor immunotherapy. Immunity 4: 555-564, 1996[CrossRef][Medline] 13. Luxembourg AT, Borrow B, Teyton L, et al: Biomagnetic isolation of antigen-specific CD8+ T cells usable in immunotherapy. Nat Biotechnol 16: 281-285, 1998[CrossRef][Medline] 14. del Guercio MF, Sidney J, Hermanson G, et al: Binding of a peptide antigen to multiple HLA alleles allows definition of an A2-like supertype. J Immunol 154: 685-693, 1995[Abstract] 15. Fisher B, Packard BS, Read EJ, et al: Tumor localization of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma. J Clin Oncol 7: 250-261, 1989[Abstract]
16.
Mitchell MS, Kan-Mitchell J, Kempf RA, et al: Active specific immunotherapy of melanoma: Phase I trial of allogeneic melanoma lysates and a novel adjuvant. Cancer Res 48: 5883-5893, 1988 17. Fazekas DS: The evaluation of limiting dilutions assays. J Immunol Methods 49: R11–R23, 1982[CrossRef][Medline] 18. Mitchell MS: Biomodulation: A classification and overview, in Reif AE, Mitchell MS (eds): Immunity to Cancer. Orlando, FL, Academic Press, 1985, pp 401-411. 19. Mitchell MS: Biological Approaches to Cancer Treatment: Biomodulation. New York, NY, McGraw-Hill, 1992 20. Mitchell MS, Harel W, Kan-Mitchell J, et al: Active specific immunotherapy of melanoma with allogeneic cell lysates: Rationale, results and possible mechanisms of action. Ann N Y Acad Sci 690,153-166, 1993 21. Riddell SR, Greenberg PD: Principles for adoptive T cell therapy of human viral diseases. Annu Rev Immunol 13: 545-586, 1995[CrossRef][Medline] Submitted December 8, 2001; accepted October 12, 2001.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
aspartic acid) has also been identified.
