Materials and methods

Selection of patients

A total of 15 patients with newly diagnosed or relapsed metastatic NSCLC were treated. The patients had already failed chemotherapy, radiotherapy, surgery or a combination of all. Eligibility criteria were age >18 years, ECOG performance status 0–2, measurable disease, and signed informed consent. Patients with brain metastasis were included if these were already treated. Patients were not eligible for study if they were receiving chemotherapy, radiation therapy or a biologic modifying agent. All patients were treated in the outpatient clinic at Sylvester Comprehensive Cancer Center/University of Miami. A complete history and physical examination was performed, including weight and vital signs, with performance status assessed by ECOG criteria. The following tests were performed prior to enrollment: complete blood count; platelet count; chemistries (uric acid, calcium, phosphorus, transaminases including SGOT and SGPT, alkaline phosphatase, LDH, total and direct bilirubin, BUN, creatinine, albumin, total protein, electrolytes, and glucose); and EKG. HLA typing was obtained. Tumor measurements were obtained from the results of radiographic studies, including CT scans of relevant sites.

Vaccine cell line and genetic modification

A human lung adenocarcinoma cell line was established in 1994 by Dr N Savaraj (Department of Medicine, University of Miami) from a biopsy of a lung cancer patient, designated as AD#100. The patient was a 74-year-old white male who presented in 1993 with initial symptoms of pelvic pain from bone erosion of the iliac crest due to metastatic pulmonary adenocarcinoma. Cancer cells for culture were obtained by bone marrow aspiration from the area of pelvic bone destruction. The patient was treated with radiation therapy to the pelvis, but expired 1 month after diagnosis. The cell line derived from this patient has been kept in culture in standard medium (described below) and is free of contamination by mycoplasma, virus or other adventitious agents. The cell line is homogeneous, adherent to plastic, and grows with a rate of division of approximately 26 h.

Genetic modification

AD#100 was transfected with plasmid cDNA, pBMG-Neo-B7.1 and pBMG-His-HLA A2 or with B45-Neo-CM-A1-B7.1.²³ Transfected cells were selected with G418 and Histidinol. Verification of correct sequences was based on restriction analysis and the expression of the relevant gene products, namely G418 or histidinol resistance for the vector sequence, HLA A1, A2, and B7.1 expression for the transfected cDNA. The cells were irradiated with 1200 Gy and stored frozen in 10% DMSO in aliquots of 5 10⁷ cells until use. Upon replating in tissue culture, the cells appeared viable for about 14 days, but were unable to form colonies, indicating their inability to replicate (data not shown). They were, therefore, considered safe for use as vaccine cells. The minimum requirement for their use as vaccine was the coexpression of HLA A1 or A2 plus B7.1 on at least 70% of the cells as shown in Figure 1a for representative batches of vaccine cells. The untransfected AD100 line was negative by FACS for staining with anti HLA A1 or A2 or B7.1 (data not shown).

Figure 1.

(a) Quality control of vaccine cells. Representative samples of vaccine cells coexpressing B7.1 (CD80) and HLA A1 (left panel) or HLA A2 (right panel) analyzed by flow cytometry. The percentage of double-positive cells is indicated. CD80 and the HLA A allele must be coexpressed on 70% or more of the cells to qualify for immunization. (b) Patient CD8 cells purified for ELI-spot assays. Flow cytometry of a representative sample of patient CD8 (right panel) cells purified by negative selection and used for ELI-spot analysis; the purity of cells is given in %. Left panel shows isotype control.

Full figure and legend (126K)

Treatment

Immunizations

Intracutaneous injections were given at multiple body sites to reduce the extent of local skin reactions. On a given vaccination day, the patient received the total dose of 5 10⁷ irradiated cells (12,000 rad) divided into two to five aliquots for administration as two to five intradermal injections of each aliquot in an extremity, spaced at least 5 cm at needle entry from the nearest neighboring injection. A total of nine immunizations (4.5 10⁸ cells) were given over the course of therapy, one every 2 weeks, provided that no tumor progression occurred under therapy (Table 1). On subsequent vaccinations, the injection sites were rotated to different limbs in a clockwise manner. One course of vaccination comprised three biweekly injections. Patients with evidence of stable disease (SD) or responding NSCLC by imaging evaluation (CT scans) and none to moderate toxicity (grade 2) were treated with an additional course at the same dose. The second course of injections started 2 weeks after the third vaccination that completed the first course. In the absence of tumor progression by CT scans and with no severe or life-threatening toxicity (grade >3), a third course at the same dose of therapy was given, starting 2 weeks after the third vaccination of the second course of therapy. Clinical and immunologic evaluation by blood tests before and after each course was performed. Patients were followed clinically weekly during the study, including monitoring blood counts and basic chemistries (Table 1).

Table 1 -  Study design.

Full table

Immunological testing

The tests included were skin tests (DTH) and ELI-spot assays for Ifn-. Immune responses mediated by CD4 cells were examined by DTH-reaction following intradermal injection of 10⁵ A1, A2 or untransfected AD100-B7 vaccine cells; however, DTH responses were not reliable indicators for immunity and are not further reported. Purified CD8 cells were obtained from patients prior to and after each course of three immunizations. CD8 cells were enriched by negative depletion with anti-CD56 and anti-CD4 and other antibodies using the Spin-sep prep from Stem Cell Technologies (Vancouver, Canada). Purity was better than 80% (Fig 1b) the primary contaminating cells being B cells (not shown). CD8 cells were frozen in 10% DMSO and 20% FCS containing medium for analysis until all vaccinations of a study patient were completed. Analysis for preimmune and postvaccination ELI-spot frequency was carried out on the same day in the same microtiter plate. Assays were performed in quadruplicate, stimulating 2 10⁴ purified patient CD8 cells with, respectively, 10³ A1 or A2 transfected or untransfected AD100, with K562 or with media only (none in Fig 2) for 3 days and determining the frequency of Ifn--producing cells by ELI-spot. Immune assays were performed prior to immunization and after 3, 6, and 9 immunizations.

Figure 2.

Immunization of advanced lung tumor patients generates strong CD8 response. The frequency of Ifn--spot-forming CD8 cells obtained from lung tumor patients is plotted against the time on study in weeks. Immunizations were given every 2 weeks, zero representing the preimmunization status. A total of 20,000 purified CD8 cells were used for ELI-spot assays. (a) Frequency of spot-forming CD8 cells from HLA A1- and A2-positive patients challenged with HLA A1- or A2-transfected (matched) AD100 tumor cells at a ratio of 20:1=CD8:AD100. (b) Frequency of spot-forming CD8 cells from HLA A1-positive patients challenged with A2-AD100 or HLA A2-CD8 cells were challenged with A1-AD100 (mismatched). (c) Frequency of spot-forming CD8 cells from non-HLA A1 or A2 patients cells challenged with A1 and A2 transfected AD100 (unmatched). (d) Frequency of spot-forming CD8 cells from all patients challenged with untransfected w.t. AD100. (e) Frequency of spot-forming CD8 cells from all patients challenged with K562. (f) Mean frequency of spot-forming CD8 cells from all patients challenged with any of the AD100 w.t. or transfected cells. (g) CD8 spot-forming response of individual, clinically responding patients. The mean number of spots after restimulation with AD100 w.t., AD100-A1, AD100-A2, K562 or nothing in quadruplicate wells is plotted against time after study entry. Arrows indicate the time of last immunization. Patient 1004, 1007, 1010 contain follow-up data analyzed at the points indicated after completion of nine immunizations (18 weeks). HLA type of each patient is indicated in brackets.

Full figure and legend (173K)

Results

Specific CD8 T-cell response of advanced lung cancer patients to whole-cell immunization

Patients with advanced NSCLC stage IIIB/IV were HLA typed. HLA A1-positive patients received the AD-A1-B7 vaccine; HLA A2-positive patients received the AD-A2-B7 vaccine and patients who were neither HLA A1- nor A2-positive received either the AD-A1-B7 or AD-A2-B7 vaccine. In order to determine the cell-mediated response, we were primarily interested in the response of CD8 cells because they are believed to be critically important for tumor immunity. The CD4 response is required for memory formation of CD8 cells. Since lung tumor antigens have not yet been defined, evaluation of the CD4 response is complicated by the absence of MHC class II on the vaccine cells. We, therefore, focused on the CD8 response and will address the CD4 response in future studies. The frequency of Ifn- secreting CD8 cells was determined by ELI-spot after restimulation of purified patient-CD8 cells in vitro with HLA A1- or A2-transfected or untransfected AD100. Controls included stimulation with K562 and incubation of CD8 cells without stimulator cells. Since the vaccine cells are allogeneic, an alloantigen response is expected to also recognize untransfected (wild type, w.t.) AD100 cells, while an HLA A1-positive patient may respond preferentially to challenge with A1-transfected AD100 and an A2 patient to the A2-transfected AD100.

ELI-spot responses of immunized tumor patients are presented as HLA-matched responses (Fig 2a) representing the number of Ifn- secreting CD8 cells obtained from HLA A1 or A2 patients challenged in vitro for 3 days with HLA A1- or A2-transfected AD100 cells, respectively. HLA-mismatched responses indicate the number of spots formed when CD8 cells from A1 or A2 patients were challenged with A2 or A1 transfected AD100, respectively (Fig 2b). The matched response increased 15-fold, from 64 (standard error of the mean, SEM) Ifn--secreting, preimmune CD8 cells (per 20,000) to maximal 9035 (SEM) Ifn--secreting cells after six immunizations and remained at this level during the next three immunizations. The mismatched response increased 5.7-fold, from 2418 to 14242 maximal. The differences between matched and mismatched response is not statistically significant. Included in this group of nine patients is the one patient who showed no response (0 spots) before or after three immunizations, at which time the tumor progressed and the patient was taken off trial.

The remaining five patients were negative for HLA A1 or A2; their CD8 response to challenge with A1- or A2-transfected AD100 is shown as unmatched response in Figure 2c. The frequency of Ifn--secreting CD8 cells increased 21-fold from 4.81.8 preimmune to 10524 after three immunizations and stayed constant throughout the trial. This increase in frequency is similar to that of all patients' CD8 cells when challenged with the untransfected w.t. AD100 (Fig 2d). Finally, the specificity of the response is evident from the absence of an increase of the response to K562 (Fig 2e) or of unchallenged CD8 cells (data not shown). The CD8 response to K562 and to AD100 in its w.t. form or after genetic modification is significantly different at each time point after vaccination (Fig 2f). The inability of the CD8 cells to react to K562 rules out that ELI-spots are due to natural killer (NK) cells or to promiscuous CTL, suggesting that the CD8 cells are specific for the NSCLC vaccine cells.

Clinical response is associated with strong CD8 response

The CD8 response listed in Table 2 reports the response to the matched vaccine for A1- or A2-positive patients; for non A1, A2 patients it is the response to AD100-A2. One of the 15 patients could not be analyzed due to renal failure unrelated to the trial before completing the first course of immunization. Of the 15 patients treated, five patients had clinical responses: one partial response (PR) and four patients with stable disease (SD). Four of these patients with clinical responses (PR+3SD) are still alive with stabilization of their diseases without further therapy for 31, 28, 25, and 12 months. They are being followed and re-evaluated with CT scans every 3 months. The patient who died, originally had SD for 5 months, then progressed and died 15 months later in spite of several courses of palliative chemotherapy. In contrast, nine of the other 10 patients who did not respond to the vaccination are deceased except one patient who achieved SD after therapy with Iressa. Table 2 summarizes the data for all patients, including pretrial treatment, clinical response to immunization and immune response. Patients who had progressive disease while under treatment went off study as indicated in Table 2.

Table 2 - Clinical and Immunological response.

Full table

Five patients had a clinical response and the frequency of Ifn-spot-forming CD8 cells increased upon successive immunization as measured by challenge ex vivo with transfected or untransfected AD100, while the reactivity to K562 remained low and unchanged (Fig 2e). In three of the clinically responding patients (Fig 2; 1004, 1007, 1010), blood samples were obtained after completion of the 18-week treatment period at 35–75 weeks post-trial entry and showed still a considerable titer of CD8 cells responding to AD100 (Fig 2g). Indeed, in two of the patients (1004, 1007), the titer increased further even after immunization was ended at 18 weeks.

The median survival time of all patients at the time of analysis was 18 months, exceeding the expected median survival time of less than 1 year for this group of patients (Fig 3). The 90% confidence intervals (CIS) are shown in Table 3. Analysis of survival by MHC matching and by clinical reponse revealed that HLA-unmatched patients showed a survival advantage that with P=.07 was not statistically significant, while clinical responders had a significant (P=.008) survival advantage when compared to nonresponders.

Figure 3.

Survival analysis of treated patients. Left panel: Kaplan–Meier plot of all patients, including one patient who received only one injection and then went off study. Surviving patients are indicated with tick marks. Median survival: 18 months (90% CI, 7.3 months to indeterminate). Center panel: Comparison of HLA A matched versus unmatched patients; median survival of matched group: 13 months; unmatched: indeterminate, but >29 months. The difference is not statistically significant, P=.073. Right panel: Comparison of responding and nonresponding patients; median survival of nonresponders: 10.5 months; nonresponders: indeterminate but >27 months. The difference is statistically significant, P=.0087.

Full figure and legend (117K)

Table 3 - Patient survival.

Full table

Immunization is safe and nontoxic

None of the 15 patients entered into the trial experienced any treatment-related serious adverse events, defined as deaths or events requiring hospitalization. Treatment-related side effects consisted of local erythema and swelling that resolved in 3–4 days. One patient complained about transient arthralgias that may have been treatment related. One patient died within 30 days of the last immunization due to pulmonary failure; one patient who had previous episodes of pericarditis experienced pericardial effusion during the last course of immunization, requiring a pericardial window. No tumor cells were detected in the fluid; the patient responded to immunization and is still in SD. As mentioned above, one patient had renal failure before completion of one course of immunization. None of these events was deemed likely to be treatment related by an independent safety monitoring board.

Discussion

Rejection of tumors by the immune response is rarely seen in advanced disease and is not a sensitive indicator for the ability of a vaccine to generate immunity. Therefore, the evaluation of active immunotherapy in cancer patients requires the development of an assay that measures the generation of tumor immunity especially in patients with advanced stage disease that may not show a clinical response. A second reason to study immune responses in patients with lung cancer stems from the consensus opinion that lung cancers are nonimmunogenic, and may even be immunosuppressive. It may be questioned, therefore, whether it is possible to generate immune responses in the environment of advanced metastatic lung cancer. The skepticism underlying this question is underlined by recent studies demonstrating the presence of T-regulatory, CD4+/CD25+/CTLA4+ cells residing as tumor infiltrating cells in stage I/II NSCLCs removed by surgery.¹ T-regulatory cells, including those isolated from lung tumor specimens, are capable of suppressing autologous T-cell responses upon activation with anti-CD3. This finding raised the possibility that T-regulatory cells may also abrogate the effect of vaccines in cancer patients.

To achieve effective immunization of lung cancer patients, we developed a whole-cell vaccine, generated from a cell line, AD100, isolated and established in our facilities from a patient with NSCLC. A whole-cell vaccine offers the advantage of containing all potential antigens, thereby allowing an immune response against multiple antigenic epitopes. Since the cell line is derived from a different patient, it is allogeneic. To generate an effective, CD8-mediated immune response, antigenic epitopes must be presented in the context of the patient's MHC. This can be achieved by crosspresentation by the recipient's antigen presenting cells after the uptake of vaccine cells or their antigens; or it can be achieved by direct antigen presentation of vaccine cell-antigens to the recipient's T cells, provided at least one MHC allele is matched between the vaccine cell and the patient. A clinically effective, cell-based vaccine furthermore requires that the tumor-specific antigens present in the vaccine cells are shared by the tumor of the patient. There is limited information available about shared antigens in NSCLC;^29,30 however, there is evidence for expression of identical tumor-associated antigens in melanomas^31,32,33,34 and other lung tumors.²³ Our study suggests that shared lung tumor antigens may be present and that they can be presented both by the direct and indirect antigen presentation pathway.

The answer to the primary aim of this study — is it possible to generate specific CD8 T-cell responses in patients with advanced disease? — is clearly affirmative; the cell-based vaccine-induced CD8 CTL responses in all but one patient with advanced stage lung cancers. Even though NSCLC is nonimmunogenic and potentially immuno suppressive,¹ and although NSCLC tumors are infiltrated with T-regulatory cells that can inhibit autologous T-cell activation,¹ our study shows that a CD8 response could be induced by a whole-cell vaccine. The CD8 cells recognized the NSCLC line AD100 in vitro, while K562 did not elicit Ifn- responses suggesting specificity. Whether the immune response generated against AD100 also results in immune reactivity towards the patient's own tumor could not be answered in this study, because autologous tumor material was not available for testing. Future studies will address this question in more detail. Three patients were examined for NSCLC reactivity of their CD8 cells 20–54 weeks after the final immunization. All three showed considerable CD8 frequencies and two of the three showed an increase in the frequencies of Ifn--producing cells after cessation of immunization, suggesting that the initial vaccine-induced immune response was perpetuated by the patients' own tumor.

An important question in this trial related to the effect of "autologizing" the allogeneic vaccine cells by HLA A1 or A2 transfection and determining the CD8 response of HLA A-matched or unmatched patients. It is clear that both patient groups generated a significant response and that there is no statistical difference between the two groups.

The second question related to the effectiveness of an immune response for the rejection of lung tumors. Lung tumors do not appear to generate an immune response normally; therefore, lung carcinoma cells have not been subjected to immune attack and to selective pressure to evade effector responses. Thus, CTL response to vaccination may be clinically effective. Although the number of vaccinated patients is small, this study supports this hypothesis. Prolongation of survival of the patients suggests clinical benefit and was accompanied by a good quality of life with minimum side effects when compared to the option of continued palliative care or more toxic standard chemotherapy with limited success in these advanced situations. Our findings arise from a nonrandomized single-institution study. Despite this limitation, we believe that the results are sufficiently compelling to justify further, larger studies of whole-cell vaccines for lung cancer.

While all but one of the 14 evaluated patients in this trial had a good to strong CD8 response to the vaccination regimen, eight patients, nonetheless, resumed progressive disease even while they generated an immune response. Analysis of survival time in relation to MHC matching of the vaccine revealed a trend to increased survival in the HLA unmatched group. This finding, if confirmed in larger studies, suggests that the allo-response due to the transfected A1 or A2 allele may be beneficial for the clinical outcome, although it was not reflected in further increased CD8 responses. Several explanations including an increased NK response, increased inflammatory reaction or effects on T-regulatory cells may be offered for this observation. Systematic investigations on the effect of syngeneic and allogeneic MHC molecules on whole-cell vaccines are clearly needed to resolve these questions.

A critical component in the design of our study was the use of a whole-cell vaccine. This choice was necessary because lung tumor antigens have not been identified. The use of simple or complex adjuvants such as mycobacterial antigens had only limited success.³⁵ However, and more importantly, the use of whole-cell vaccines allows the generation of immune responses to the whole plethora of putative lung tumor-specific or -associated antigens. A poly-epitope T-cell response is likely to be more efficient in blocking tumor evasion than T-cell responses to single epitopes.

In summary, the study suggests that favorable clinical responses to immunization are associated with a strong CD8 response, and that CD8 responses may be associated with increased survival. Measuring the CD8 titer of Ifn-spot-forming cells is a valuable surrogate marker for antitumor activity.

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Acknowledgements

We gratefully acknowledge support for this work from the family and friends of Iris Zeitler and the University of Miami Sylvester Comprehensive Cancer Center. LR is supported by a Clinical Career Development Award and SP by a Young Investigator Award from ASCO. Further support came from NIH Grant RO1-CA39201-14.