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Clinical Trials |
Comprehensive Cancer Center, The University of Alabama at Birmingham, Birmingham, Alabama 35294-3300 [R. M. C., M. B. K., M. N. S., K. O. A., D. L. B., S. E. M., D. C., R. B. A., A. F. L.]; and Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892 [J. S.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% of breast cancers and 70% of non-small cell lung cancers (1)
. CEA is also expressed, to some extent, in normal colonic mucosa and fetal digestive organs (1)
. The cDNA for full-length CEA has been cloned from a human colon carcinoma cell library and inserted into the genome of a New York City attenuated strain of vaccinia virus (2)
. The recombinant virus (rV-CEA) is replication competent and directs cell surface expression of CEA. Immunization with rV-CEA has induced CEA-specific humoral and cellular immune responses in mice and nonhuman primates as well as protection against challenge with syngeneic CEA-expressing colon carcinoma cells in mice (3
, 4)
. However, it is important to emphasize that human CEA is a foreign gene in both mice and nonhuman primates, whereas humans appear to be immunologically tolerant to CEA: it is expressed in fetal and normal adult human tissues. The first clinical trial of rV-CEA was conducted in 1993 by the NCI-Navy Oncology Branch in 26 patients with metastatic adenocarcinoma (5) . The vaccine was administered by dermal scarification monthly for three exposures at doses up to 107 pfu. No severe toxicity occurred and no primary T-cell lymphoproliferative responses to soluble CEA protein were observed (5) . However, human class I HLA-A2-restricted cytolytic T-cell lines that are responsive to specific epitopes of CEA could be derived by prolonged in vitro culture of peripheral blood lymphocytes from patients after vaccination (6) .
An abbreviated trial of the same vaccine was conducted at our institution during 1994. Five patients who had surgically resected Dukes’ C colorectal carcinoma and who had completed adjuvant chemotherapy received two vaccinations with 107 pfu of rV-CEA 8 weeks apart by scarification (7)
. The trial was stopped prematurely due to identification of bovine viral diarrhea virus contamination within the rV-CEA vaccine. A new lot of rV-CEA was prepared for use in this study from a different producer cell line, which yielded a lower viral titer of 106 pfu/µl. The modest viral titer made it impossible to deliver the desired dose of 107–108 pfu by the traditional means of scarification, which uses a volume of only 1–2 µl suspended between the prongs of a bifurcated needle. Additional shortcomings of scarification include considerable variation in technique between clinicians and uncertainty regarding the actual dose that penetrates the cornified layer of the epidermis to contribute to productive infection, with resultant expression of the tumor-associated antigen. These problems are relevant to the administration of recombinant vaccinia virus vaccines for a variety of disorders, including cancer and HIV disease. A recognized advantage of scarification is that it delivers the virus to the skin, a highly immunogenic site that is rich in epidermal Langerhans cells, which function as potent antigen-presenting cells. Therefore, we elected to compare two alternative administration techniques that deliver some or all of the dose to the skin in a reproducible manner using injection volumes of 
100 µl. The techniques selected were intradermal needle injection versus s.c. jet injection using the Biojector. A limited dose escalation format was selected because little information was available regarding the toxicity of vaccinia virus when doses of 107–108 pfu are fully delivered beneath the cornified layer of the epidermis. We hypothesized that more effective delivery of the vaccine to dermal antigen-presenting cells by the administration techniques under investigation would elicit CEA-specific immune responses that are superior to those observed in the NCI Phase I trial of rV-CEA delivered by scarification.
Thus, the goals of this study were twofold: (a) to examine the safety and relative efficacy of intradermal needle injection versus Biojector administration of rV-CEA over a limited dose range and (b) to evaluate any CEA-specific immune responses or antitumor effects induced by rV-CEA immunization.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients were examined 3 and 7 days after immunization to assess the degree of inflammation at the inoculation site, regional adenopathy, or other signs of toxicity. A complete blood count, serum CEA level, and serum chemistries were obtained before each immunization as well as 7 and 28 days following each immunization. All patients were evaluated for evidence of objective antitumor response 8 weeks following the primary immunization.
Immunization Techniques.
The vaccine was stored in single-use vials at -70°C. On the day of dosing, the vaccine was thawed, vortexed to disperse viral clumps, and diluted as needed with sterile normal saline for injection. All patients received the rV-CEA (Mr 180,000) vaccine over the proximal lateral upper extremity. Patients in groups 1 and 3 received the vaccine by standard intradermal injection using a 0.1-ml volume of appropriately diluted viral stock in an insulin syringe. Those in groups 2 and 4 received the vaccine by s.c. jet injection using the Biojector 2000 needle-free injection device (Bioject, Inc., Raleigh, NC). Each dose consisted of a 0.5-ml volume of appropriately diluted viral stock drawn into a Biojector No. 2 syringe. The tip of the syringe was held firmly at the injection site at a 90° angle to the skin. Pressing the actuator lever results in release of compressed carbon dioxide (CO2) gas from a cartridge, which drives the syringe plunger to expel the vaccine through a micro-orifice at high velocity in a fraction of a second. Nothing pierces the skin except the fluid stream, and the CO2 never comes in contact with the vaccine. The Biojector delivers the vaccine in a conical distribution with the apex at the skin surface. Thus, the great majority is delivered to the s.c. tissue, although traces remain in the dermis.
Skin Test Technique and Scarification Challenge with Vaccinia Virus.
All patients received 10 µg of baculovirus recombinant CEA protein (MicroGeneSys, Meriden, CT) by intradermal injection on the ventral forearm as a test for delayed-type hypersensitivity to CEA 8 weeks after primary immunization. At the same time, they were challenged with 4 x 107 pfu of a different recombinant vaccinia virus encoding a truncated version of human CEA, rV-CEA (Mr 70,000), by scarification. The local inoculation site was examined for evidence of inflammation and/or pustule formation 3 and 7 days postscarification to determine the degree of immune-mediated attenuation of the skin reaction induced by the virus.
In a previous trial at our institution, the same dose of rV-CEA (Mr 70,000) administered by scarification induced local pustule formation in 10 of 12 patients, whose only prior exposure to the virus was childhood smallpox immunization. Repeat scarification with rV-CEA (Mr 70,000) 8 weeks later induced a local pustule in only 4 of 12 patients. These observations indicate that scarification boosts the immune response to vaccinia virus, thus attenuating the local skin reaction to subsequent viral challenge in the majority of patients. In this study, patients were vaccinia-primed by childhood smallpox vaccination and boosted with two doses of rV-CEA (Mr 180,000) 4 weeks apart by intradermal needle injection or s.c. jet injection. Viral challenge by scarification was undertaken 4 weeks later to determine whether these administration techniques provide sufficient viral delivery to antigen-presenting cells to boost the antivaccinia immune response with resultant attenuation of the local skin reaction to subsequent viral exposure.
Lymphoproliferative Assay.
PBMCs obtained by Ficoll-Hypaque density gradient centrifugation were resuspended in complete medium consisting of RPMI 1640 supplemented with 10% pooled normal human AB serum, 2 mML-glutamine, 50 µM 2-mercaptoethanol, and antibiotics. Cells were added at 1.5 x 105 per well to 96-well flat-bottomed plates. Stimulated cells were incubated in quadruplicate wells with UV light-inactivated vaccinia virus over a range of concentrations (2 x 106–2 x 107 pfu/ml), baculovirus recombinant human CEA (MicroGeneSys) over a range of concentrations (3–30 µg/ml), BSA (30 µg/ml) as a negative control antigen, tetanus toxoid (Wyeth-Ayerst Laboratories, Paoli, PA) as a positive control antigen, and PHA and pokeweed lectin as positive control mitogens. Control cells were cultured in complete medium alone. All cells were incubated in a humidified atmosphere of 5% CO2 in air for 6 days, followed by an overnight pulse with 1 µCi/well tritiated thymidine. Cells were harvested, and incorporated radioactivity was quantitated using a solid-phase ß-scintillation counter. The SI was calculated as the mean cpm of the stimulated cells divided by the mean cpm of the control cells. A positive response was defined as a postimmunization SI of >3 and at least 2-fold greater than the preimmunization SI for a given antigen.
IL-2 Release.
Mononuclear cells were cultured exactly as above with the same panel of antigens and mitogens over the same range of concentrations, with the exception that, after 3 days in culture, cell-free supernatants were harvested and assayed immediately or stored at -70°C. Supernatant IL-2 activity was quantitated based on the ability to support proliferation of CTLL-2 cells (9
, 10) . CTLL-2 cells were washed to remove exogenous cytokines and added at 5000 cells/well in 100 µl of complete RPMI-10% FCS. Each well also received 100 µl of cell culture supernatant to be assayed for IL-2 activity in triplicate. Following 24 h in culture, cells were pulsed with tritiated thymidine (1 µCi/well) for 8 h and harvested, and incorporated radioactivity was determined using a direct ß-scintillation counter. The mean cpm for triplicate cell cultures was converted to mIU/ml IL-2 in the culture supernatant by reading from a standard curve generated for each assay using recombinant human IL-2. A positive response was defined as 1 mIU/ml of IL-2 in the culture supernatant postimmunization and at least a 2-fold increase compared with the preimmunization sample.
Antibody Assays.
Anti-CEA and antivaccinia antibodies were quantitated by ELISA. To detect anti-CEA antibodies, we coated microtiter plates overnight with 200 ng/well native human CEA purified from human colon cancer liver metastases (Vitro Diagnostics, Littleton, CO). To detect antivaccinia antibodies, we coated microtiter plates overnight with 5 x 106 pfu per well of active vaccinia virus (Therion Biologicals Corporation) in PBS with 0.1 g/liter calcium and 0.1 g/liter of magnesium. The plates were blocked with 1% pig skin gelatin and 3% nonfat dry milk in PBS for 90 min at 37°C, followed by washing. The plates were then incubated with various dilutions of patient or normal donor sera in blocking buffer for 1 h at 37°C. Plates were washed and antibody binding was detected with horseradish peroxidase-conjugated goat antihuman IgG (heavy and light chain) antiserum (1:5000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). This antiserum binds human IgA, IgG, and IgM by virtue of light chain recognition. A positive anti-CEA antibody response was defined as an absorbance greater than the mean plus 2 SDs of 10 normal donor sera at the same dilution. Similarly, serum titers of antivaccinia antibody were calculated as the limiting dilution of serum producing an absorbance of >0.150 and greater than the mean plus 3 SDs of 10 vaccinia-naive normal donor sera at the same dilution.
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RESULTS |
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. The patients included 14 men and 6 women with a median age of 65 years (range, 42–77 years). All patients had metastatic disease, and 17 of 20 had elevated serum CEA levels at the time of study entry. Four of 20 patients had stable disease at 8 weeks of follow-up, whereas the remaining 16 patients demonstrated disease progression. None of the patients demonstrated a sustained decrease in serum CEA levels following vaccination, except for patient 15, who has medullary carcinoma of the thyroid, which often follows an indolent course.
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Toxicology.
Both techniques of inoculation produced local skin erythema, induration, and pustule formation similar to that reported following administration of vaccinia virus by scarification for smallpox immunization (11)
. Local inflammation and pustule formation have traditionally been regarded as desirable consequences of scarification with vaccinia virus, indicating an adequate inoculation with live virus. As shown in Table 2
, some degree of erythema and or induration was observed at the site of all initial inoculations with rV-CEA (Mr 180,000). Less local erythema and induration was seen after the second dose. Each technique of inoculation produced a pustule after the first dose in three of five patients at the initial dose level of 107 pfu. When the dose was increased to 108 pfu of rV-CEA, all five patients in the intradermal group and four of five patients in the Biojector group developed a pustule. Pustules were not observed following the second dose of rV-CEA in either group. Local pruritis at the inoculation site beginning 24 h after administration was noted in 14 of 20 patients, most commonly after the primary immunization. The incidence and severity were not dose related, but pruritis was more frequent and more intense following intradermal administration. Pruritis required no medications and resolved spontaneously within 3–5 days. A few patients reported mild inoculation site discomfort, fatigue, and/or fever, which were not dose related. No regional adenopathy or acute allergic reactions were observed after administration of rV-CEA.
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Granulocytes have been shown to express nonspecific cross-reacting antigen, raising the possibility that immunization against CEA could lead to autoimmune neutropenia (1 , 12) . Despite this theoretical concern, no consistent changes in WBC counts or neutrophil counts were observed. No deterioration in renal or hepatic function that could be attributable to immunization was observed.
Lymphoproliferative Responses.
The lymphoproliferative responses to vaccinia virus are provided in Table 3
. Table 3, A and B
, refers to patients from this study receiving rV-CEA (Mr 180,000) by intradermal needle injection and s.c. jet injection using the Biojector, respectively. Table 3C provides data from five additional patients with a history of colorectal carcinoma who received 107 pfu doses of rV-CEA (Mr 180,000) by scarification on weeks 0 and 8 as participants in a previous trial at our institution. Nine of 25 patients demonstrated lymphoproliferative responses to vaccinia virus prestudy, defined as a SI of >3. These patients were evenly distributed between the three groups. These responses are thought to represent persistent memory T cells induced by previous smallpox immunization (i.e., vaccinia priming), which was an inclusion criterion for each study. Patients 1, 2, and 15, who had robust lymphoproliferative responses to vaccinia virus prestudy, had received smallpox booster immunizations during adulthood as the result of military service. A positive lymphoproliferative response to vaccinia virus as the result of immunization with rV-CEA was defined as a postimmunization SI of >3 and at least 2-fold greater than the prestudy SI. By these criteria, 8 of 10 patients in the intradermal group, 9 of 10 patients in the Biojector group, and 5 of 5 patients immunized by scarification demonstrated a positive lymphoproliferative response to vaccinia virus following immunization with rV-CEA. The only patient in the Biojector group who did not achieve at least a 2-fold increase in SI was patient 1, who was strongly positive prestudy with a SI of 431. The booster immunization with rV-CEA increased the lymphoproliferative response to vaccinia virus in 8 of 10 patients in the intradermal group, 4 of 10 patients in the Biojector group, and 0 of 5 patients immunized by scarification. This is perhaps due to inability of the virus to generate a productive infection upon second exposure in the face of significant antivaccinia immune responses.
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No CEA-specific lymphoproliferative responses were observed in any of the 20 patients studied. Lymphoproliferative responses to tetanus toxoid as well as PHA and pokeweed mitogens were analyzed to determine the effect of progressive metastatic adenocarcinoma upon lymphocyte function in vitro. Proliferative responses to PHA and pokeweed mitogen did not change significantly over 8 weeks of follow-up, despite disease progression in 16 of 20 patients. The SIs (mean ± SE) in response to PHA prestudy and 8 weeks after primary immunization were 790 ± 120 and 940 ± 150, respectively. With regard to pokeweed responses, the SIs (mean ± SE) prestudy and 8 weeks after primary immunization were 420 ± 80 and 550 ± 80, respectively.
Ten of 20 patients demonstrated lymphoproliferative responses to tetanus toxoid prestudy, and 8 of these experienced disease progression during study participation. The SI in response to tetanus toxoid 8 weeks following primary immunization was lower than the prestudy SI in all eight of these patients. The tetanus toxoid-specific SI (mean ± SE) decreased by 54 ± 10% over 8 weeks of follow-up. The nonparametric Wilcoxon signed rank test used to perform a paired analysis of prestudy and week 8 SIs demonstrated that the decrease in SI was significant (P = 0.03).
IL-2 Release.
The mononuclear cell release of IL-2 in response to vaccinia virus is provided in Table 4
. Nine of 20 patients demonstrated IL-2 release in response to vaccinia virus prestudy, thought to represent persistent memory T cells induced by previous smallpox immunization. Patients 1, 2, and 15, who had robust IL-2 release in response to vaccinia virus prestudy, had received recent smallpox booster immunizations as a result of military service.
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1 mIU/ml and at least twice the patient’s prestudy level. By these criteria, 7 of 10 patients in the intradermal group and 10 of 10 patients in the Biojector group demonstrated a positive IL-2 response to vaccinia virus following immunization with rV-CEA. The booster immunization with rV-CEA increased the vaccinia virus-specific IL-2 response in only 4 of 10 patients in each group, perhaps due to inability of the virus to generate a productive infection upon second exposure. For the purpose of comparing vaccinia virus-specific IL-2 responses, patients with a prestudy IL-2 release of >20 mIU/ml were excluded, and calculations were based upon the maximum postimmunization IL-2 release for each patient. Prestudy vaccinia-specific IL-2 release (mean ± SE) was comparable between the two groups: 2.7 ± 1.4 and 1.8 ± 1.6 mIU/ml for the intradermal and Biojector groups, respectively. Postimmunization IL-2 release (mean ± SE) was likewise comparable: 66 ± 33 and 61 ± 20 mIU/ml for the intradermal and Biojector groups, respectively. Thus, each administration technique induced vaccinia-specific IL-2 release of comparable magnitude, although results were more consistently positive following Biojector delivery. No evidence of CEA-specific IL-2 release was observed among any of the 20 patients studied.
Antibody Responses to Vaccinia Virus and CEA.
The limiting dilution titers of antivaccinia antibody are provided in Table 5
. Seven of 20 patients demonstrated antivaccinia antibody titers ranging from 1:1000 to 1:10,000 prestudy. Nineteen of 20 patients demonstrated an increase in antivaccinia antibody titer within 4 weeks following primary immunization with rV-CEA. Booster immunization with rV-CEA did not have a consistent effect on the antivaccinia antibody titers. Analysis conducted with a one-sample t test confirmed that the antibody titers were significantly greater postimmunization compared with preimmunization (P = 0.002 and 0.018 for the intradermal and Biojector groups, respectively). Analysis was also carried out with a two-sample t test and Wilcoxon rank-sum test to examine the equality of prestudy versus postimmunization differences in antibody titer between the intradermal injection and Biojector groups. Results show that there is no significant difference between the two groups (P = 0.17 and P = 0.28 for the t test and Wilcoxon rank-sum test, respectively). Thus, both techniques of administering rV-CEA induce antivaccinia antibody responses of comparable magnitude. None of the patients demonstrated an antibody response to CEA as analyzed by ELISA with the native human protein.
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DISCUSSION |
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With regard to dose, we recommend the use of 108 pfu for subsequent clinical trials of recombinant vaccinia virus vaccines administered by intradermal needle injection or s.c. jet injection. This conclusion is based upon the favorable toxicity profile and the observation that this dose induced local pustule formation, which is indicative of an adequate inoculation of live virus, in 90% of patients, compared with 60% of patients receiving doses of 107 pfu. Furthermore, 108 pfu doses of rV-CEA (Mr 180,000) protected 90% of patients against pustule formation following scarification challenge with rV-CEA (Mr 70,000) on day 57 compared with 70% protection achieved with doses of 107 pfu. Vaccinia-specific lymphoproliferation, IL-2 release, and antibody response were not related to patient dose over the limited range studied.
No evidence of CEA-specific lymphoproliferation, IL-2 release, or delayed-type hypersensitivity was observed. The absence of demonstrable cellular immune response to CEA using fresh PBMCs is consistent with the observations of Hamilton et al. (5) in the initial trial of rV-CEA administered by scarification in the same patient population. However, Schlom and colleagues (6) have demonstrated that HLA-A2 restricted cytolytic T-cell lines responsive to specific epitopes of CEA could be derived by prolonged in vitro culture of postimmunization PBMCs from patients in the initial trial of rV-CEA. These data suggest that immunization with rV-CEA can evoke a cytolytic T-cell response to a specific epitope of the human CEA molecule but that it does so at a precursor frequency that is undetectable by current technology using PBMCs without prolonged in vitro culture in the presence of peptide antigen and IL-2. Such studies involving prolonged culture of PBMCs were not systematically undertaken in conjunction with this trial.
None of the patients demonstrated an antibody response to CEA, as determined by an ELISA assay with native human CEA protein. However, 85% of the patients had elevated circulating levels of CEA protein at the time of study entry. Therefore, anti-CEA antibodies in these patients would be expected to exist predominantly in the form of circulating immune complexes with CEA protein. The ELISA assay described herein may be inadequate to detect anti-CEA antibodies in the form of immune complexes. Therefore, we are continuing to analyze these sera by Western blot and modified ELISA techniques in an effort to detect CEA immune complexes. Detection of circulating immune complexes is a challenge shared by a variety of vaccine trials seeking to immunize against tumor associated antigens such as CEA and prostate-specific antigen, which are shed into the bloodstream (13) .
Evidence that vaccination is capable of breaking tolerance to CEA in humans has been provided by two sources: (a) Schlom and colleagues (6) have described the derivation of CEA-specific cytolytic T cell lines following rV-CEA immunization, as described above; and (b) Foon et al. (14) have reported CEA-specific antibody and lymphoproliferative responses following vaccination with an anti-idiotype monoclonal antibody to an anti-CEA monoclonal antibody in patients with colorectal carcinoma. Recent murine studies have demonstrated enhanced T-cell responses and antitumor effects when recombinant vaccinia viruses encoding tumor associated antigens were admixed with a recombinant vaccinia virus encoding the murine B7.1 costimulatory molecule (15 , 16) . Furthermore, priming with rV-CEA followed by multiple boosts with a nonreplicating canarypox recombinant encoding CEA (ALVAC-CEA) has been shown to enhance CEA-specific T cell immune responses and antitumor effects compared with use of either vector alone (17) . The use of diversified prime and boost strategies is thought to be particularly critical in vaccinating human populations who have previously received smallpox immunizations. Because vaccinia virus proteins are highly immunogenic, vaccinia recombinants can only be effectively administered once or twice due to the induction of high titers of antivaccinia antibody, which will neutralize subsequent doses of the virus, reducing recombinant gene expression (18 , 19) . This study confirms this shortcoming of recombinant vaccinia immunization. Pustules at the inoculation sites that are indicative of productive viral infection were observed after primary immunization in 90% of patients receiving 108 pfu of rV-CEA but were completely absent following booster immunization. Furthermore, the second inoculation with rV-CEA did not consistently boost vaccinia-specific lymphoproliferation, IL-2 release, or antibody responses. Therefore, the absence of demonstrable CEA-specific immune responses may be a consequence of inadequate booster immunizations. On the basis of these observations, we plan to carry out an additional clinical trial of vaccination against CEA using recombinant pox viruses. In this trial, patients will receive an admixture of rV-CEA and rV-human-B7.1 followed by multiple boosts with a canarypox recombinant encoding both CEA and human B7.1 to capitalize on the diversified prime and boost strategy as well as the costimulatory activity of B7.1. In this trial, we will use 108 pfu doses of the vaccinia recombinants administered by s.c. jet injection with the Biojector.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at The University of Alabama at Birmingham, 1824 Sixth Avenue, WTI 263, Birmingham, AL 35294-3300. Phone: (205) 934-7167; Fax: (205) 934-1608. 
2 The abbreviations used are: CEA, carcinoembryonic antigen; rV-CEA, recombinant vaccinia virus encoding carcinoembryonic antigen; NCI, National Cancer Institute; pfu, plaque-forming units; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; SI, stimulation index; IL-2, interleukin 2.
Received 9/29/98; revised 5/ 4/99; accepted 5/18/99.
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