Induction of anti-tumor immunity by intrasplenic administration of a carcinoembryonic antigen DNA vaccine.

BACKGROUND: We have previously reported that intramuscular, intradermal or epidermal gene gun administration of a plasmid encoding carcinoembryonic antigen (CEA) under transcriptional regulatory control of the cytomegalovirus (CMV) early promoter/enhancer elicits CEA-specific humoral and cellular immune responses in mice with resultant immunoprotection against challenge with syngeneic, CEA-expressing colon adenocarcinoma cells. METHODS: In the present work, we examine the ability of this DNA vaccine construct (pCEA) to elicit CEA-specific immunity following intrasplenic administration. Groups of mice were immunized with pCEA by intrasplenic or intramuscular injection. Six weeks later, mice were evaluated for the presence of anti-CEA humoral responses and were challenged with syngeneic, CEA-expressing colon carcinoma cells. RESULTS: Intrasplenic administration of pCEA produced a frequency of CEA-specific antibody responses comparable to that elicited by intramuscular pCEA inoculation. Both intrasplenic and intramuscular administration of pCEA generated IgG2a antibody responses to CEA, consistent with the induction of T helper-1-biased immune responses. In addition, partial immunoprotection against tumor challenge was observed after a single plasmid DNA dose by either route of administration. Subsequent studies revealed that antibody responses to intrasplenic DNA vaccination are dose and schedule dependent. CONCLUSION: These findings support future investigations of DNA vaccination strategies that specifically promote the uptake of plasmid by splenocytes.

Phase I trial of a recombinant vaccinia virus encoding carcinoembryonic antigen in metastatic adenocarcinoma: comparison of intradermal versus subcutaneous administration.

The principal objectives of this trial were twofold: (a) to examine the safety and relative efficacy of intradermal needle injection versus s.c. jet administration of a carcinoembryonic antigen (CEA)-encoding recombinant vaccinia virus (rV-CEA) over a limited dose range and (b) to evaluate CEA-specific immune responses or antitumor effects induced by rV-CEA vaccination. Patients were randomly assigned to one of two groups, depending upon the technique of vaccine administration. All 20 patients received two doses of 10(7) or 10(8) pfu of rV-CEA at a 4-week interval. Toxicity was limited to modest local inflammation at the inoculation site as well as low-grade fever and increased fatigue, each affecting fewer than 20% of the patients. No evidence of CEA-specific lymphoproliferation, interleukin 2 release, delayed-type hypersensitivity, or antibody response was observed. Thus, the efficacy comparison between the two administration techniques was based upon the induction of immune responses to the vaccinia virus vector. Both techniques induced vaccinia-specific lymphoproliferation, interleukin 2 release, and antibody responses of comparable magnitude and frequency as well as protected 80% of patients against pustule formation following vaccinia scarification. Thus, there is no compelling reason to recommend one administration technique over the other based upon toxicity or efficacy. We have selected s.c. jet injection for subsequent trials of rV-CEA based on the ability to accommodate larger injection volumes, enhanced standardization between clinicians, and avoidance of needles that could transmit the vaccine or blood-borne pathogens to health care workers. We recommend use of 10(8) pfu doses for subsequent trials of recombinant vaccinia virus vaccines based upon the favorable toxicity profile and more consistent local pustule formation indicative of an adequate inoculation of live virus. No objective clinical responses to the rV-CEA vaccine were observed among this population of patients with widely metastatic adenocarcinoma.

Polynucleotide immunization of nonhuman primates against carcinoembryonic antigen.

In preparation for a Phase I trial of DNA immunization against carcinoembryonic antigen (CEA) in patients with colorectal carcinoma, we have produced a single plasmid DNA encoding CEA and hepatitis B surface antigen (HBsAg) under transcriptional regulatory control of two separate cytomegalovirus promoters within separate eukaryotic expression cassettes, designated pCEA/HBsAg. Hepatitis B surface antigen was included to provide an internal positive control for the efficacy of this immunization strategy without regard to the issue of breaking tolerance to a self-antigen. In the present work, we sought to examine the immunogenicity of this plasmid in a nonhuman primate model with close phylogenetic relationship to humans. Groups of pig-tailed macaques were immunized with pCEA/ HBsAg by i.m. injection or particle bombardment of the skin according to a dose and schedule thought to be optimal for the respective technique of DNA immunization. Both administration techniques produced humoral and lympho-proliferative responses of comparable magnitude. However, delayed type hypersensitivity to CEA and CEA-specific interleukin-2 release were observed only in the i.m. group, suggesting a qualitative difference in the character of the immune response elicited by the two techniques of DNA immunization. The antibody responses to CEA and HBsAg were surprisingly persistent in that all immunized animals maintained moderate antibody titers against both antigens for more than 15 months after the last boost. No toxicity was observed during 2 years of follow-up, including no measurable levels of anti-DNA antibody. This antitumor immunization strategy is presently being examined in patients with metastatic colorectal carcinoma using pCEA/HBsAg administered by i.m. injection.

Effects of a novel purine nucleoside phosphorylase inhibitor, BCX-34, on activation and proliferation of normal human lymphoid cells.

The antiproliferative effect of BCX-34 was tested in normal human peripheral blood mononuclear cells (PBMCs) induced to proliferate with OKT3, tetanus toxoid, the mixed lymphocyte reaction, or IL-2. In the case of OKT3, tetanus toxoid, or the MLR the IC50s ranged between 0.7 and 4 microM. With IL-2, the IC50 was 14.6 microM. In T-cells purified by rosetting the IC50 with IL-2 was 0.62 microM. In CD4 or CD8 cells obtained by magnetic activated cell sorting the IC50s with IL-2 were 0.24 and 0.62 microM, respectively. BCX-34 inhibition of proliferation in human PBMCs may not depend entirely upon the accumulation of intracellular dGTP because tetanus toxoid-induced proliferation was inhibited in the absence of deoxyguanosine and was not reversed by deoxycytidine. BCX-34 did not inhibit IL-2 release from PBMCs and did not alter PBMC viability. The results of these studies show that BCX-34 is a potent inhibitor of normal human T-cell proliferation induced by antigenic or IL-2 stimulation. BCX-34 in normal human T-cells has a deoxyguanosine-independent mechanism to suppress in vitro proliferation. BCX-34 appears to have little effect on T-cell viability. The data suggest that BCX-34 may be useful in the treatment of T-cell proliferative disorders.

Selected strategies to augment polynucleotide immunization.

We sought to amplify the immune response to polynucleotide immunization through co-delivery of complementary DNA (cDNA) encoding a cytokine or co-stimulatory molecule to enhance antigen presentation. In the context of intramuscular immunization, we examined co-delivery of cDNAs for B7-1 and human carcinoembryonic antigen (CEA) within separate plasmids or a dual plasmid with two independent expression cassettes. Intramuscular delivery of the dual expression plasmid produced anti-CEA antibody responses and antitumor effects superior to those generated by plasmid DNA encoding CEA alone. However, co-delivery of cDNAs encoding B7-1 and CEA in the form of two separate plasmids produced no augmentation. The importance of single plasmid delivery suggests the effectiveness of this strategy is contingent upon co-expression of B7-1 and CEA within the same cell. The success of cutaneous polynucleotide immunization by particle bombardment is thought to derive largely from the presence of Langerhans cells within the skin. We hypothesized that co-delivery of plasmid DNA encoding granulocyte-macrophage colony stimulating factor (GM-CSF) by particle bombardment would enhance the antigen presenting capacity of Langerhans cells at the inoculation site similar to its effects in vitro. Augmentation of CEA-specific lymphoblastic transformation and antibody response was observed when plasmid GM-CSF (pGM-CSF) was administered 3 days prior to each dose of plasmid DNA encoding CEA. These strategies for augmentation of immune response to polynucleotide immunization should be applicable to a wide variety of antigenic targets including infectious agents and other tumor-associated antigens.

A carcinoembryonic antigen polynucleotide vaccine for human clinical use.

We have constructed a plasmid DNA encoding the full-length complementary DNA for human carcinoembryonic antigen (CEA) under transcriptional regulatory control of the cytomegalovirus early promoter/enhancer (pCEA) and demonstrated that this plasmid can function as a polynucleotide vaccine to elicit a CEA-specific immune response. This immune response protects against tumor challenge with syngeneic CEA-transduced colon carcinoma cells in mice. In the present work, the pCEA construct and purification method were modified to eliminate nonessential viral sequences, the ampicillin selectable marker, mutagens, and endotoxin to produce a reagent suitable for human clinical trials. The human use plasmid (pGT37) directs CEA expression at levels comparable with the original pCEA plasmid and can be propagated to yield large quantities of plasmid DNA based on kanamycin selection. A simple extraction technique greatly reduces contamination by endotoxin. Six weekly intramuscular injections of pGT37 elicited CEA-specific lymphoblastic transformation and antibody response in five of five mice and fully protected 10 of 10 mice against tumor challenge with syngeneic CEA-expressing colon cancer cells 42 days from the first plasmid injection. Thus, pGT37 encoding a tumor-associated antigen (CEA) has been shown to elicit cellular and humoral immune responses and mediate antitumor effects in vivo. This plasmid is suitable for human use and can be easily propagated in the laboratory.

A carcinoembryonic antigen polynucleotide vaccine has in vivo antitumor activity.

We have constructed a plasmid DNA encoding the full-length cDNA for human carcinoembryonic antigen (CEA) driven by the cytomegalovirus early promoter/enhancer and demonstrated that this plasmid can function as a polynucleotide vaccine. The immune response elicited by the CEA polynucleotide vaccine is dose and schedule dependent. There appears to be a threshold dose of 50 micrograms capable of inducing CEA-specific lymphoblastic transformation, lymphokine release, and antibody response. Doses of 10 micrograms were significantly less effective. When 50-micrograms doses are employed, thrice weekly or weekly vaccination schedules more reliably elicit CEA-specific immune responses by day 43 than does an every-3-weeks schedule. Furthermore the CEA polynucleotide vaccine can immunoprotect against challenge with syngeneic CEA-transduced colon carcinoma cells as early as 3 weeks after the first vaccination. Studies are ongoing to demonstrate the ability of CEA polynucleotide vaccination to treat pre-existing syngeneic mouse colon and breast carcinomas expressing human CEA.

Immune response to a carcinoembryonic antigen polynucleotide vaccine.

We have constructed a DNA plasmid encoding the full length complementary DNA for human carcinoembryonic antigen (CEA) driven by the cytomegalovirus early promoter/enhancer (plasmid DNA encoding human CEA) and demonstrated that this plasmid can function as a polynucleotide vaccine. This polynucleotide vaccine induced humoral and/or cellular immune responses specific for human CEA in all 5 immunized mice. Lymphoblastic transformation data with the use of enriched T-cell populations detected the presence of CEA-specific memory T-cells in 3 of 5 mice. Lymphocytes from 2 of 5 mice had interleukin 2/interleukin 4 release in response to CEA. CEA specificity was confirmed by the absence of reactivity to a control antigen and lack of CEA reactivity among mice vaccinated with a control plasmid encoding chloramphenicol acetyltransferase. Four of 5 mice vaccinated with plasmid DNA encoding human CEA demonstrated anti-CEA antibody responses. This immune response compared favorably with a positive control group of mice immunized with vaccinia-CEA by a dose and schedule previously shown to induce immunoprotection and therapy against a human CEA expressing syngeneic murine colon carcinoma model. Studies are ongoing to establish the construct, dose, and schedule to elicit optimal CEA-specific immune response as well as immunoprotection and therapy against human CEA expressing syngeneic murine adenocarcinoma models.