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Immunology |
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 [J. M. T., D. K. C., B. T., R. R., C. B. C., A. v. B., R. L.], and Vical Corporation, San Diego, California 92121 [G. S., G. H., P. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mouse immunoglobulin (MsIg) heavy- and light-chain constant regions chains, respectively. Patients in remission after chemotherapy received three monthly i.m. injections of the DNA in three dose escalation cohorts of four patients each (200, 600, and 1800 µg). After vaccination, 7 of 12 patients mounted either humoral (n = 4) or T-cell-proliferative (n = 4) responses to the MsIg component of the vaccine. In one patient, a T-cell response specific to autologous Id was also measured. Anti-Id antibodies were not detectable in any patient. A second series of vaccinations was then administered using a needle-free injection device (Biojector) to deliver 1800 µg both i.m. and intradermally (i.d.); 9 of 12 patients had humoral (n = 6) and/or T-cell (n = 4) responses to MsIg. Six of 12 patients exhibited humoral and/or T-cell anti-Id responses; yet, these were cross-reactive with Id proteins from other patient’s tumors. Subsequently, a third series of vaccinations was carried out using 500 µg of human granulocyte-macrophage colony-stimulating factor DNA mixed with 1800 µg of Id DNA. The proportion of patients responding to MsIg remained essentially unchanged (8 of 12), although humoral or T-cell responses were boosted in some cases. Throughout the study, no significant side effects or toxicities were observed. Despite the modest level of antitumor immune responses in this study, DNA vaccine technology retains potential advantages in developing anti-Id immunotherapies. Additional studies are warranted to optimize vaccine dose, routes of administration, vector designs, and prime-boost strategies. These results will help guide the design of such future DNA vaccine trials. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA vaccination may represent a more efficient route to anti-Id immunotherapy. Vaccination of mice with plasmid DNA expression vectors ("naked DNA vaccination") has been shown to induce protective immune responses against a variety of infectious diseases and tumors (8, 9, 10) . Plasmid DNA constructs are technically easier and less time consuming to prepare than recombinant or hybridoma-derived proteins. This is especially important in the case of a customized vaccine approach. In addition, bacterial plasmid DNA possesses inherent immune adjuvant activity based on its content of unmethylated CpG immunostimulatory sequences (11) that interact with toll-like receptors in mammalian immune cells (12) . Recent technological developments make a genetic approach to idiotypic vaccination possible (13) . PCR technology now allows for the rapid cloning of the variable (idiotypic) regions from immunoglobulin genes (14 , 15) . Genetic vaccination with Id-encoding plasmid DNA has shown efficacy in several murine lymphoma models (16, 17, 18, 19) . Syrengelas et al. found that vaccination of mice with plasmid DNA-encoding tumor Id developed strong humoral anti-Id immune responses and were protected against subsequent tumor challenge at a rate comparable with that after vaccination with Id-KLH protein. The protection depended on the inclusion of foreign constant region immunoglobulin sequences (human) linked to the mouse variable regions, which were felt to provide a "carrier" function analogous to that provided by the KLH in the case of protein vaccination. Anti-Id immune responses were further enhanced by coinjection of DNA encoding GM-CSF (13 , 16) . These findings, and the development of a bicistronic immunoglobulin expression vector (20) , formed the basis for the current study of DNA Id vaccination in humans.
We sought to explore the safety and immunogenicity of chimeric Id-encoding plasmid DNA vaccines with and without coinjected GM-CSF DNA in patients with lymphoma. Patient-specific plasmids were constructed to encode a chimeric immunoglobulin molecule consisting of tumor-specific variable (Id) regions linked to xenogeneic (murine) immunoglobulin constant regions, in a strategy analogous to that shown to be effective in our preclinical studies (16 , 20 , 21) . We found that with the doses and routes of injection used, the Id DNA vaccines were safe and that the vector was immunogenic. The majority of patients developed humoral or T-cell immune responses to the xenogeneic (murine immunoglobulin) component of the vaccine, and some patients made responses to their autologous tumor Id.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Production of Patient-specific Plasmid DNA Vaccines.
Tumor specimens were obtained before the initiation of chemotherapy. Total RNA was extracted from viable tumor cells obtained by Ficoll-Hypaque (Amersham Pharmacia, Uppsala, Sweden) sedimentation using RNAzol B (Tel-Test, Friendswood, TX) and was used to generate cDNA with oligo(dT) primers. Heavy- and light-chain variable regions were then PCR-amplified using family-specific leader and constant region primers as described previously (14
, 15)
, but incorporating SfiI restriction sites (20)
. The amplified, SfiI-digested fragments of the heavy and light chains were then ligated into plasmid VCL-1632 to yield plasmids VCL-1642.XXX (Fig. 1A)
. Plasmid VCL-1632 is a pUC18-derived kanamycin-selectable plasmid containing the CMV promoter followed by the murine constant region sequence, a translational enhancer, the murine IgG2a heavy-chain sequence, and a translation terminator. The completed plasmids, thus, expressed a bicistronic RNA encoding a chimeric human-murine immunoglobulin containing tumor-specific variable regions linked to murine constant regions. Proper insertion and orientation of the immunoglobulin domains was verified in individual clones by restriction digest mapping followed by DNA sequence analysis. To confirm functional expression of the encoded immunoglobulin, purified plasmids were used to transfect VM92 (murine melanoma) cells, and the supernatants were analyzed by ELISA and Western blot to detect full-length, functional immunoreactive chimeric immunoglobulin (data not shown). The best-producing clones were then chosen for vaccine production. Plasmid VCL-1723 encoding human GM-CSF (Fig. 1B)
has been described previously (23)
. The biological activity of plasmid VCL-1723 was confirmed by the ability of supernatants from VCL-1723-transfected VM92 cells to promote the growth of GM-CSF-dependent TF-1 cells (24)
. Large-scale production of clinical-grade plasmid DNAs, along with purification and quality assurance analyses, were performed as previously described (25
, 26)
. Vaccines were formulated to contain 200, 600, or 1800 µg of Id DNA or 500 µg of GM-CSF DNA in 1.0 ml of sterile PBS, and were stored frozen until use.
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Vaccine Treatments.
Patients received three series of vaccinations over the course of the study as summarized in Fig. 2
. Series 1 vaccinations represented a dose-escalation phase, in which three groups of four patients each received three monthly i.m. (deltoid) injections, by conventional needle and syringe, of 200, 600, or 1800 µg of the chimeric Id plasmid DNA. After the safety of the highest (1800 µg) dose level was demonstrated, these same patients were offered a repeat series of three monthly vaccinations (Series 2) at this dose level. For this series, patient 13 was substituted for patient 2, who had withdrawn from the study because of progressive disease. Series 2 injections were delivered using the Biojector needle-free jet injection device (Bioject Inc., Portland, OR). Eighty % of the dose was delivered i.m. (0.8-ml volume) and 20% was delivered to two adjacent i.d. sites in the skin of the contralateral posterior upper arm (0.1 ml each). Series 3 injections were given as in Series 2, but with the addition of 500 µg of human GM-CSF DNA admixed with the Id DNA just before injection.
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(S1C5; Ref. 27
) at 0.1, 1.0, 10, and 100 µg/ml. [3H]thymidine incorporation was measured after an overnight pulse on day 5. A response was interpreted as positive (+) when incorporation of more than twice background (medium alone) and prevaccine baseline was observed on two or more occasions. Responses were designated +/- when incorporation of more than twice background and prevaccine baseline was observed on only a single occasion postvaccine. Tumor-specific cytotoxicity and cytokine release by PBMCs cocultured with tumor cells were measured as described previously (6
, 7)
.
Humoral Immune Response Assessments.
Sera of all patients were analyzed for IgG anti-Id antibodies by ELISA. Tumor-derived Id proteins were of either IgM or IgG subclass depending on the type expressed by the tumor. Autologous IgM tumor Id proteins or irrelevant IgM Id proteins were captured onto microtiter plates coated with goat antihuman IgM (Biosource International, Camarillo, CA). When the tumor Id was an IgG, a recombinant Id protein was constructed containing the tumor Id linked to human IgG3 constant regions, and captured onto the plate with goat antihuman IgG3 (Biosource). The need to detect bound IgG antibodies necessitated isolation of the variable region/Id sequences from commonly used IgG constant region sequences to minimize background in the assay. The IgG3 isotype was chosen for target Id construction given its generally minor contribution to humoral responses. Prevaccine and postvaccine sera were serially diluted and allowed to bind to the target Id proteins. The reagents used to detect bound antibodies depended on the isotype of the target Id molecule. When the Id protein was an IgM, bound anti-Id antibodies of the IgG subclass were detected using polyclonal goat antihuman IgG (Biosource) coupled to HRP. When the target Id protein was an IgG3, bound anti-Id antibodies were detected with a cocktail of mouse monoclonal antihuman IgG1, IgG2, and IgG4 antibodies conjugated to HRP (Southern Biotech, Birmingham, AL). In each case, HRP-conjugated antibodies to the immunoglobulin light chain ( or 
) opposite to that of the tumor Id was also used to detect serum antibodies bound to the target Id. A response was considered positive (+) when a 4-fold increase in anti-Id titer was found compared with both the prevaccine serum and the isotype-matched irrelevant Id proteins used as specificity controls. Antibody responses to murine IgG2a/ were measured by coating microtiter plates with mouse antibody S1C5 followed by incubation with diluted sera as above and detection using polyclonal goat antihuman IgG-HRP. A response was considered positive (+) when a 4-fold increase in anti-MsIg titer was found compared with the prevaccine serum.
Safety Assessments.
Patients were observed for adverse effects for at least 1 h after each injection, and, thereafter, a diary was kept to record any local injections site reactions or other side effects. Serum was tested for rheumatoid factor and anti-dsDNA antibodies at baseline and 3 months after each vaccination series. For Series 3, evaluations also included serum creatine phosphokinase before, and 1 month after, each vaccination. Mouse IgG2a/ in serum was measured by ELISA (sensitivity of assay <0.1 µg/ml), using S1C5 as a standard. Serum GM-CSF was measured by ELISA (R&D Systems, Minneapolis, MN). Serum antibodies against GM-CSF were measured using a sandwich ELISA technique. Recombinant human GM-CSF (Immunex, Seattle, WA) was used to coat plates, to which serially diluted patient sera were added. Bound antibodies were detected using biotinylated GM-CSF followed by streptavidin-HRP (Vector Labs, Burlingame, CA). Sensitivity of the assay was <0.05 µg/ml.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The prevaccine tumor status, starting vaccine dosage levels, peripheral blood CD4+ T cell counts, and immune response data for each patient are shown in Table 1
. CD4+ T-cell counts were notably depressed in these patients postchemotherapy and remained so in most cases throughout the duration of the study (mean, 371 ± 119/µl; normal range, 540-1660/µl). At vaccine initiation, four patients had NED, four had MRD, and four had residual tumor in lymph nodes and/or bone marrow. The plasmid DNA was injected i.m. by needle and syringe into the deltoid region. No significant side effects or toxicities were noted other than those expected from routine i.m. injections. A minority of patients had mild local injection site tenderness lasting 1–2 days.
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and 2
). Immune responses were observed in a similar proportion of patients in each dose cohort; 2/4, 3/4, and 2/4 patients in the 200, 600, and 1800 µg dose cohorts, respectively (Table 1)
. An example of a T-cell response against MsIg observed in patient 5 is shown in Fig. 3
. In this case, the proliferation of PBMCs to MsIg was concentration dependent and was seen on multiple occasions after vaccination (data not shown). A more modest proliferative response to tumor Id was also observed at this particular time point. One other patient (patient 12), treated at the highest (1800 µg) dose level, developed a reproducible tumor Id-specific T-cell proliferative response after vaccination (Fig. 4)
.
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and 2
). Four of these nine immune responders had failed to respond after Series 1 vaccinations, whereas five of the six previously responding patients continued to display reactivity against mouse immunoglobulin. After Series 2 vaccinations, patient 12 no longer had T-cell reactivity to MsIg, yet did retain a specific T-cell proliferative response against autologous tumor Id. Four other patients had "nonspecific" T-cell anti-Id responses and two patients had nonspecific humoral anti-Id responses in which cross-reactivity to Id proteins from other patients’ tumors was observed.
Repeat Vaccinations Using Coinjected GM-CSF DNA.
Coinjection of plasmids encoding cytokines such as GM-CSF has been shown to augment protective immunity induced by DNA vaccines (9
, 13
, 16
, 30)
. We thus chose to administer a third round of vaccinations (Series 3) to these same patients using a coinjected plasmid encoding human GM-CSF (VCL-1723, Fig. 1B
) in an attempt to boost the immune response to tumor Id. Vaccines consisted of 1800 µg of the chimeric Id plasmid DNA admixed with 500 µg of GM-CSF DNA delivered via Biojector as in Series 2. Series 3 vaccinations were begun 14 months after the initiation of Series 2, after which time adequate safety data on Series 2 vaccinations was available and regulatory approval had been granted. The median interval between completion of Series 2 and the start of Series 3 vaccinations was 10 months (range, 4–12 months). By the time Series 3 vaccinations were initiated, 5 of the 12 patients had evidence of tumor progression (Table 1)
. The incidence and severity of i.m. injection site reactions in Series 3 were indistinguishable from that seen in Series 2, and no systemic toxicity was noted. Notably, no obvious local inflammatory reactions were observed at the sites of i.d. inoculation. After Series 3 vaccinations, there were a total of 11 immune responses against mouse immunoglobulin detected in eight patients (7 humoral, 4 T cell, 3 both humoral and T cell; Tables 1
and 2
). Seven of the nine previously responding patients continued to display humoral or T-cell reactivity toward mouse immunoglobulin, and there was one new responder. Patient 12, who had new evidence of tumor involving bowel and skin, no longer had a specific T-cell proliferative response against autologous tumor Id. No other patient showed humoral or T-cell reactivity toward Id that was tumor-specific, although five patients had nonspecific anti-Id responses.
In some instances, immune responses to MsIg were boosted by Series 3 injections. Fig. 5
shows the T-cell proliferative response for patient 13 at baseline and after Series 2 and Series 3 immunizations. The data indicate significant enhancement of the response after the repeat series of vaccinations. Boosting of the humoral immune response to MsIg with the sequential series of DNA vaccinations is shown in Fig. 6
. After Series 1, patient 5 showed no evidence of a humoral response to MsIg. However, after Series 2, a significant response was detectable, with further boosting of the response after Series 3 injections.
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and GM-CSF, yet no consistent tumor-specific pattern of cytokine release was observed (data not shown). We also investigated the cytotoxic activity of pre- and postvaccine PBMCs restimulated with autologous CD40 ligand-activated tumor cells toward cryopreserved tumor cells (6
, 7)
. No tumor-specific lysis was exhibited in any of the four cases tested (data not shown).
Safety of DNA Vaccinations.
No acute or long-term toxicities of the DNA vaccination maneuvers used have been identified in over 4 years of follow-up in this patient cohort. Five patients (patients 8, 9, 11, 12, and 13) developed modest, transient elevations of serum rheumatoid factor between Series 2 and 3 vaccinations (range, 21–58; normal level, <20). Patient 11 had a slightly elevated level of 26 at prevaccine baseline. This level peaked at 58 just prior to Series 2, subsided just prior to Series 3, and thereafter fell to within normal range. However, no patient developed clinical rheumatological manifestations or other symptoms or signs of autoimmune disease. Cases in which serum rheumatoid factors were elevated showed no obvious correlation to those in whom nonspecific anti-Id responses were detected (Table 1)
. Anti-dsDNA antibodies remained undetectable in all of the patients throughout the course of the study. Mouse IgG2a, human GM-CSF, or anti-GM-CSF autoantibodies were likewise undetectable in patient sera at all time points.
Long-Term Clinical Follow-Up.
Given that many patients had measurable tumor at different time points during this study, we had the opportunity to evaluate patients for tumor regression responses. Patient 4 had residual tumor in the bone marrow on initiation of Series 1 vaccinations. Bone marrow involvement was diminished after Series 2, and was not detected after Series 3 vaccinations, and this patient remains in complete clinical remission 49 months after completing chemotherapy. No other objective tumor regressions were observed. At a median follow-up of 44 months postchemotherapy, four patients remain without evidence of tumor, and four patients have stable RD. Five patients have developed progressive disease, and three of these (patients 2, 8, and 13) required additional therapies. Patient 8 died with progressive tumor because of complications after high-dose chemotherapy given in preparation for stem cell transplantation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The observed incidence of immune responses to the relevant Id tumor antigen was low. Only one patient had a tumor Id-specific immune response, as demonstrated by T-cell proliferation. Interestingly, this single positive anti-Id responder was in the highest-dose cohort during the initial dose escalation. This response persisted throughout Series 2, but was lost during Series 3, at which time new sites of tumor became evident. Six patients had nonspecific immune responses against tumor Id. In these cases, there was reproducible humoral or T-cell reactivity against tumor Id, but also cross-reactivity with Id proteins from other patients’ tumors. Such cross-reactivities have been observed with similar frequency in our previous trials (3
, 4
, 6)
, but have not been reported to date because their significance is unknown. It is possible that they represent antigen-specific reactivity against immunoglobulin determinants shared between different tumor Ids, although this has yet be proven. The T-cell proliferation assay used throughout our study detects primarily CD4+ T cell activities. It is possible that our assay technique is insufficiently sensitive to detect all immune priming against Id, particularly that of class I MHC-restricted CD8+ T cells. Although we found no tumor-specific secretion of tumor necrosis factor or GM-CSF by patient T cells after Series 3 vaccinations, the induction of T cells secreting other cytokines cannot be ruled out. The patients treated in this study had depressed CD4+ peripheral blood T-cell counts, presumably because of their prior chemotherapy, and this may have contributed to the observed low incidence of anti-Id immune responses. However, these low T-cell counts are not appreciably different from those of patients whom we have treated in other trials with protein Id vaccines and in whom anti-Id immune responses are readily measured.5
No obvious relationship between prevaccine CD4 counts and immune responses was evident, except that, in the three instances in which the CD4 counts were within the normal range (patients 4 and 11), both antibody and T-cell responses against MsIg were detected.
We used the same group of patients for the three phases of this study (vaccine Series 1–3), given the effort invested in the production of their customized vaccines and our principal interest in evoking therapeutic antitumor immunity. A greater proportion of patients had immune responses to MsIg or Id after the second series of vaccinations (Table 1)
, and several patients demonstrated further boosting of their responses after the third vaccination series. However, given our study design and the results achieved, we were unable to determine the relative contributions of the Biojector, combined i.m./i.d. injection route, and coinjected GM-CSF plasmid to the observed responses. We cannot exclude the possibility that these results are attributable solely to the repeated i.m. injections of 1800 µg of the chimeric Id DNA alone. With regards to clinical activity, only one patient (patient 4) had an objective improvement in tumor burden during the study. However, without an unvaccinated control group, we cannot exclude that the observed outcome of these patients was influenced by the DNA vaccination maneuvers.
It is important to view our results in the context of the limited data available on DNA vaccination in humans. In early trials of DNA vaccines encoding HIV or influenza antigens, the induction of antibodies, T-cell proliferation, or CTLs was reported, although most often in only a minority of patients (31) . More recently, i.m. injection of DNA encoding the malarial circumsporozoite protein has been shown to induce CD8+ CTLs in a majority of naïve, healthy subjects (32) . CTL responses were detectable with doses as low as 20 or 100 µg but were more frequent at higher (500 and 2500 µg) doses. Despite the induction of robust CTL responses, humoral responses to circumsporozoite protein were not detectable (33) . Particle-mediated delivery of a HBsAg DNA vaccine has now been shown to efficiently induce protective antibody titers and both CD4+ and CD8+ T-cell responses in healthy volunteers (34) . Thus, the immunogenicity of DNA encoding foreign proteins from infectious pathogens has been demonstrated repeatedly.
In contrast, eliciting antitumor immunity in cancer patients using DNA vaccines has proved more difficult. Hawkins et al. (15
, 35)
constructed plasmid DNAs encoding lymphoma Id as single-chain Fv fragments (heavy and light chain variable regions joined by a flexible linker) from 10 patients with low-grade B-cell lymphoma. However, at doses of 100–500 µg delivered i.m., they failed to find evidence of anti-Id immunity.6
Investigators at the National Cancer Institute Surgery Branch performed a study of DNA vaccination against the melanoma differentiation antigen gp100 (36)
. Twenty-three patients with metastatic melanoma were treated using doses of 100-1000 µg divided between two i.m. and i.d. sites, either with or without systemic interleukin 2. In this trial, no clear signs of anti-gp100 immunity or objective tumor regressions were noted. White and Conroy (31)
performed vaccination of 17 patients with metastatic colorectal carcinoma using a dual expression plasmid encoding the self tumor antigen carcinoembryonic antigen along with HBsAg as a control. Plasmid DNA was injected i.m. at doses of 100, 300, 1000, or 2000 µg. Although dose-dependent induction of anti-HbsAg antibodies was noted, no antibody or T- cell responses against carcinoembryonic antigen were detected. This latter result is analogous to the present study, in which immune responses against the foreign component of the vaccine were frequently induced, yet responses to the relevant tumor antigen were undetectable or weak (Figs. 3
and 4
). Thus, little evidence of antitumor immunity has been demonstrated using first generation tumor antigen DNA vaccines.
The results of tumor antigen DNA vaccine approaches might be improved by optimization of key variables such as dosage, route, vector design, and boosting strategies (37) . Most simply, elevated doses may prove to be more effective. Whereas the doses used in our study (200–1800 µg) were within the range of doses found to be immunogenic in humans using infectious disease antigens, on a per weight basis they are still 10–100 fold lower than those which provide optimum tumor protection in mice (9 , 16) . At the time our study was initiated, there were no published reports describing the safety or immunogenicity of plasmid DNA vaccines in humans; therefore, the appropriate dose range was not known, and the range of doses chosen reflected primarily safety concerns. Dose dependence of immune responses has now been demonstrated in numerous preclinical (37) and human studies (31 , 32 , 37) . Barouch et al. found that large doses of DNA (10 mg) were required to provide protection of primates against a simian immunodeficiency virus challenge.
The method and route of injection also greatly influences the efficacy of DNA vaccines. The i.m. needle injection appears to be the least efficient, whereas needle-free jet injection (Biojector) can improve the immunogenicity of a given DNA vaccine dose (28 , 29) . Delivery of DNA to the epidermis by gold particle bombardment also appears to greatly reduce the dose required to induce effective immunity (34 , 38) .
Improvements in vector design may also lead to greater efficacy of DNA vaccines in future studies. The first generation vector used in our study encoded a full-length, secreted tetrameric mouse-human chimeric immunoglobulin (Fig. 1A)
. The murine constant regions serve as a foreign carrier protein to enhance the immunogenicity of the Id, analogous to the function of KLH in traditional Id vaccines (4)
. Zhu et al. (39)
have now developed Id DNA vaccines as single-chain Fv fragments-tetanus toxin fusion proteins to provide a more potently immunogenic carrier protein. Using this approach, they have achieved excellent tumor protection in preclinical studies (18)
and are currently applying this strategy in a clinical study in lymphoma patients. Vectors incorporating genes encoding cytokines, chemokines, costimulatory molecules, apoptosis-inducing ligands, intracellular targeting sequences, or a higher content of immunostimulatory CpG sequences have also displayed enhanced immunogenicity in preclinical models (19
, 30
, 37
, 40
, 41)
.
Despite the unsatisfactory antitumor immune responses observed in the present study, DNA vaccination remains an attractive platform for the further development of Id vaccines. Promising clinical activity of protein and dendritic cell-based Id vaccines has already been demonstrated (3, 4, 5, 6, 7) , yet the broad application of patient-specific Id vaccines to the treatment of B-cell malignancies may necessitate more simplified vaccine production techniques. DNA vaccine technology has the potential to allow the streamlining of customized vaccine manufacturing, along with unique opportunities to manipulate the antigenic sequences to include additional immunogenic epitopes, cellular targeting sequences, and immunostimulatory motifs. Additional studies are warranted to optimize vaccine dosage, vector designs, routes of administration, and prime-boost sequences. Our results will help guide the design of such future Id DNA vaccine trials.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants HL57443 and CA33399. J. M. T. is the recipient of a Clinical Associate Physician award from the NIH (RR-00070-CAP). R. L. is an American Cancer Society Clinical Research Professor. 
2 Present address: Division of Hematology-Oncology, University of California, Los Angeles Medical Center, CHS 42-121, Los Angeles, CA 90095.
3 To whom requests for reprints should be addressed, at Department of Medicine, Division of Oncology, Stanford University Medical Center, CCSR Room 1105, Stanford, CA 94305. E-mail: levy{at}stanford.edu
4 The abbreviations used are: Id, idiotype; PBMC, peripheral blood mononuclear cell; MsIg, mouse immunoglobulin; CMV, cytomegalovirus; GM-CSF, granulocyte-macrophage colony-stimulating factor; KLH, keyhole limpet hemocyanin; dsDNA, double-stranded DNA; CT, computed tomography; NED, no (clinical) evidence of disease; RD, residual disease; MRD, minimal RD; i.d., intradermal; HRP, horseradish peroxidase; HbsAg, hepatitis B surface antigen.
5 J. Timmerman and R. Levy, unpublished observations.
6 F. Stevenson, personal communication.
Received 5/ 9/02. Accepted 8/ 9/02.
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