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[Cancer Research 59, 5106-5111, October 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 5106-5111, October 15, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Comparative Studies of a Retrovirus versus a Poxvirus Vector in Whole Tumor-Cell Vaccines

James W. Hodge and Jeffrey Schlom1

Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
A number of experimental and clinical studies have used retroviral vectors to express transgenes in whole tumor-cell vaccines. Recently, poxvirus vectors such as vaccinia or avipox have been used toward this goal. The studies reported here compare for the first time the use of a retroviral vector versus a poxvirus vector (vaccinia) in whole tumor-cell vaccines. The transgene used was the T-cell costimulatory molecule B7-1, and the tumor was the weakly or nonimmunogenic MC38 murine colon adenocarcinoma. Recombinant retrovirus (R-B7) and the recombinant vaccinia (V-B7) induced equivalent expression of B7 on the surface of the carcinoma cell. Using live whole-tumor cells as vaccine, cells transduced via recombinant retrovirus (MC38/R-B7) and recombinant vaccinia (MC38/V-B7) equally induced protection against challenge by native MC38 cells 14 days later. Upon rechallenge with native MC38 cells 40 days later, however, the MC38/R-B7 vaccine was shown to be less effective than the MC38/V-B7 vaccine. Similar results were obtained when the tumor cells were irradiated prior to administration. When comparative studies were conducted in which X-irradiated tumor-cell vaccines were given to mice bearing experimental lung metastases, the MC38/V-B7 vaccine was shown to be significantly (P = 0.0351) more effective than the MC38/R-B7 vaccine. Additional studies were carried out in mice that had received vaccinia virus previously. Again, the X-irradiated MC38/V-B7 vaccine was statistically (P = 0.024) more effective than the MC38/R-B7 vaccine in the elimination of metastases. When the naïve and vaccinia-immune mice for each vaccination group were combined for meta-analysis (n = 16), the MC38/V-B7 was significantly more effective than the MC38/R-B7 in the treatment of pulmonary metastases (P = 0.0014) in this model. These studies thus demonstrate for the first time that a whole tumor-cell vaccine (either live or X-irradiated) containing a vaccinia transgene is at least as efficient, and sometimes more efficient, in inducing antitumor effects compared with the same vaccine using a retrovirus to express the transgene. The implications for the clinical applications of such approaches are discussed.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Numerous studies have now demonstrated the antitumor efficacy of whole tumor-cell vaccines in experimental models. In some cases, the vaccines consisted of live or X-irradiated tumor cells that were highly to moderately immunogenic. In many cases, rodent tumors were induced by carcinogens and were shown to express tumor-specific transplantation antigens; in other cases, the tumor-associated antigens were shown to be retroviral env or gag proteins. In some studies, the tumor cells were shown to be weakly or nonimmunogenic (1) ; for example, the tumor cells would readily grow in the host, and vaccines consisting of X-irradiated tumor cells would not induce antitumor immunity.

In cases of poor tumor immunogenicity, tumor cells have been rendered more immunogenic by the insertion and expression of transgenes. Three basic modalities have been used: (a) insertion of cytokine genes such as IFN-{gamma} (2) , interleukin-2 (3 , 4) , granulocyte/macrophage-colony stimulating factor (5, 6, 7) , and interleukin-12 (5 , 8 , 9) to enhance T-cell function or the migration of antigen-presenting cells (dendritic cells); (b) insertion of "neo-antigens" to enhance immunogenicity (e.g., histocompatibility antigens; Ref. 10 ); or (c) insertion of T-cell costimulatory molecules to enhance antitumor T-cell activation (1 , 10, 11, 12, 13) . This latter methodology is particularly attractive because it has now been shown that, with the exception of hematopoietic malignancies, the vast majority of tumors (both murine and human) do not express T-cell costimulatory molecules (14) . Furthermore, it has now been demonstrated that T-cell activation requires two signals: signal 1, via peptide/MHC interaction with the T-cell receptor; and signal 2, via a costimulatory molecule(s) interacting with a ligand on the T cell.

The most studied of the costimulatory molecules is B7-1, which has been shown to activate T cells via its ligands (CD28 to up-regulate and CTLA-4 to down-regulate). Other costimulatory molecules such as intercellular adhesion molecule-1, leukocyte function-associated antigen-3 and CD70 have been shown to interact with their own T-cell ligands. Numerous studies have now shown that one can use a retroviral vector to introduce a costimulatory molecule into a murine tumor cell and, in some cases, enhance immunogenicity for use as an antitumor vaccine (15 , 16) . Recent studies have now shown that one can also use poxvirus vectors such as vaccinia, fowlpox, or canarypox (ALVAC) to express a costimulatory molecule transgene in a tumor cell to induce antitumor immunity (17) .

One question that now arises concerns the relative merits of a poxvirus vector, such as vaccinia, versus a retroviral vector for use in modified whole tumor-cell vaccines. Points to be considered are as follows: (a) ease of vector preparation; (b) whether the vector infects tumor cells efficiently; (c) the need for tumor-cell division if a transgene is to be expressed; (d) ability of the vaccine (either live or X-irradiated cells) to induce antitumor activity; (e) safety; and (f) the potential for using vectors that can express multiple transgenes.

In the studies reported here, we have compared for the first time the use of a poxvirus vector versus a retroviral vector to express the murine B7-1 costimulatory molecule transgene in both live and X-irradiated whole tumor-cell vaccines. Relative degrees of effort in the generation of these vaccines are noted, along with comparative studies using both vectors in experiments involving: (a) protection against tumor challenge; and (b) vaccination of mice that previously received experimental lung metastases.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines.
The amphotropic packaging cell line PA317 was obtained from Dr. Robert Bassin (National Cancer Institute, NIH, Bethesda, MD). The MC38 murine colonic adenocarcinoma cell line has been described previously (18) . These cell lines were maintained in DMEM with 10% fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, 0.1 mM sodium pyruvate, and 50 µg/ml gentamicin sulfate (Life Technologies, Inc., Gaithersburg, MD).

Recombinant Vaccinia Virus.
The recombinant vaccinia virus expressing the murine costimulatory molecule B7-1 (designated V-B7) was described and characterized previously (12) . Wild-type vaccinia virus was designated V-WT.

Recombinant Retrovirus.
Cloning of the murine B7-1 gene has been described (12) . The DNA fragment coding for B7-1 was ligated into the HindIII/CLA-1 restriction enzyme sites of the retroviral transfer vector pLNCX (a kind gift of Dr. Dusty Miller, Fred Hutchinson Cancer Research Center, Seattle, WA). The pLNCX-B7–1 plasmid was transfected into the PA317 packaging cell line by Lipofectin (Life Technologies, Inc.) according to the manufacturer’s instructions. Cells were harvested, plated onto 60-mm dishes, and incubated with 200–500 µg/ml G418 for four weeks. Clones of PA317 cells containing the B7-1 gene were identified by flow cytometric analysis using a FITC conjugated anti-CD80 (B7-1) MAb.2 Analysis was performed with a FACScan (Becton Dickinson, Mountain View, CA).

The retroviral supernatants of pLNCX-B7-transduced PA317 cells were collected, concentrated, and used to transfect MC38 cells in the presence of Polybrene. After transfection, MC38 cells were selected by cloning G418-resistant colonies and screened by FACS analysis using the B7-1 antibody. The resultant B7-1-positive cell line was designated MC38/R-B7. MC38 cells were also transfected with R-WT in an identical manner and designated MC38/R-WT.

Preparation-modified Whole Tumor-Cell Vaccines.
Parental MC38 cells were infected at a multiplicity of infection (MOI; pfu/cell) of 10 with V-WT or V-B7 for 5 h and designated MC38/V-WT and MC38/V-B7, respectively. For retrovirus-modified tumor cells, MC38/R-WT and MC38/R-B7 were used. Cell surface expression of B7-1 on MC38 after infection with V-B7 or transfection with R-B7 cells was confirmed by flow cytometric analysis. Virus-modified tumor cells were administered immediately or, for certain experiments, irradiated (50 Gy) prior to administration.

Prevention of s.c. Tumors by Modified Whole Tumor-Cell Vaccines.
MC38/V-WT, MC38/V-B7, MC38/R-WT, or MC38/R-B7 were washed and suspended in HBSS at a concentration of 1 x 107 cells/ml. Female C57BL/6 mice (Taconic Farms, Germantown, NY) were given a s.c. injection of 100 µl (1 x 106 cells) in the right flank. Mice remaining tumor free for 40 days were challenged on the opposite flank with 1 x 106 uninfected cells. In all experiments, tumors were measured by caliper in two dimensions, and volumes were calculated as described previously (12) . Animals were sacrificed in all experiments when any tumor measurement (length or width) exceeded 20 mm and/or when mice appeared moribund or cachectic.

Therapy of Established Positive Pulmonary Metastases.
C57BL/6 mice were challenged i.v. with 1 x 106 parental MC38 tumor cells. After 3 days, mice were randomized and inoculated s.c. with either 1 x 106 MC38/V-WT, MC38/V-B7, MC38/R-WT, or MC38/R-B7. Identical inoculations were performed on days 10 and 17. Mice were sacrificed 28 days after tumor transplant, and experimental pulmonary metastatic nodules as defined by Wexler (19) were stained. These metastatic nodules were enumerated in a blind fashion. Lungs with more nodules than could be counted were assigned an arbitrary count of >200. For certain experiments, mice were immunized with 1 x 107 pfu V-WT via scarification 14 days before tumor challenge.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
It took ~18 weeks for preparation of the recombinant retrovirus pLNCX containing the murine B7-1 costimulatory molecule gene, transfection of the MC38 tumor cells, drug selection, and selection and expansion of a high-expression clone (Fig. 1A)Citation . Approximately 9 weeks were necessary to generate the recombinant vaccinia expressing B7 and to infect the MC38 cells. The methods used are described in "Materials and Methods." In terms of application (i.e., once the vectors were produced), it took ~24–40 days to transfect MC38 cells with R-B7 or R-WT and for drug selection and expansion of a high-expression clone (Fig. 1B)Citation . In contrast, it took 3–5 h to infect MC38 cells with V-B7 and have them express B7 on the cell surface.



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  		Fig. 1. Comparative time and effort requirements to prepare retroviral and vaccinia recombinant vectors and vaccines (A) and to prepare vaccines once vectors have been made (B). C, cell surface expression of B7-1 of MC38 colon carcinoma cells after infection with R-B7 or V-B7. The numbers in panels MC38/R-B7 and MC38/V-B7 are the percentage of B7-positive cells and mean fluorescent intensity (in parentheses). The dashed lines depict staining by the isotype control antibody.

 
The level of murine B7-1 transgene expression on the surface of MC38 cells was defined. Parental MC38 and MC38 infected with R-B7 or V-B7 (designated MC38/R-B7 and MC38/V-B7, respectively) were all negative for B7 surface expression by FACS analysis (Fig. 1C)Citation . The high-expression clone of MC38/R-B7 was shown to express B7 on 97% of cells. MC38/V-B7 analyzed 5 h after infection also showed B7 expression on 98% of cells. The cell surface density of B7-1 on the MC38/V-B7 and MC38/R-B7 was similar as determined by mean fluorescent intensity.

The first comparative studies involved the efficacy of live whole tumor-cell vaccines to express the B7-1 transgene via either the use of a retrovirus or recombinant vaccinia. MC38 carcinoma cells were infected with V-B7, V-WT, R-B7, or R-WT. Because the R-B7 cells were cloned and drug selected, >98% of the cells expressed B7 (Fig. 1C)Citation . The V-B7 and V-WT were used at an MOI of 10 to infect MC38 cells. This MOI was used because previous studies have shown that it would lead to efficient levels of expression of transgenes on the majority of cells within 5 h. Moreover, this MOI led to expression levels similar to those observed with the drug-selected MC38/R-B7 cells used in these studies (Fig. 1C)Citation .

All four groups of MC38 tumor-cell vaccines were injected s.c. into C57BL/6 mice using 1 x 106 live cells/mouse. Fourteen days later, mice were challenged on the opposite flanks with 5 x 105 parental MC38 tumor cells injected s.c. Tumor growth was monitored weekly. In mice vaccinated with MC38/V-WT, parental MC38 cells grew at levels comparable with those in unvaccinated mice (Fig. 2A)Citation . In mice vaccinated with MC38/R-WT, parental MC38 tumors grew at a slightly accelerated rate (Fig. 2C)Citation . This is most likely due to the fact that the MC38/R-WT vaccine cells grew and gave rise to tumors in the opposite flank. Mice vaccinated previously with MC38/V-B7 or MC38/R-B7 were both protected against challenge with parental MC38 cells in that none of the five mice in each group developed tumors upon challenge (Fig. 2, B and DCitation , respectively). To determine whether long-term immunological memory was achieved, mice that were originally vaccinated with MC38/V-B7 or MC38/R-B7 and then challenged with MC38 cells 14 days later were challenged again with 1 x 106 native MC38 tumor cells 40 days later (54 days after the original vaccination). Mice that originally received the MC38/V-B7 vaccine remained tumor free (Fig. 3B)Citation , whereas two of five mice in the group receiving the MC38/R-B7 vaccine developed tumors (Fig. 3C)Citation . The growth rate of control mice that originally received HBSS buffer as control and were later challenged with MC38 cells is shown in Fig. 3ACitation .



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  		Fig. 2. The ability of whole tumor-cell vaccines to protect against tumor challenge. Nonirradiated viable MC38 cells (A–D) or irradiated MC38 cells (E–H) were used as vaccines. Mice were vaccinated on day 0 with 1 x 106 cells in the right flank. Vaccines were MC38 cells infected with V-WT (A and E), V-B7 (B and F), R-WT (C and G), and R-B7 (D and H). Fourteen days later, mice were challenged on the opposite flank with 1 x 106 viable native MC38 cells. The numbers at the bottom right of each panel refer to the number of mice in the group of five that were tumor free up to 35 days after challenge.

 


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  		Fig. 3. Rechallenge of mice after vaccination with whole tumor-cell vaccines. Mice that received live MC38 vaccine infected with V-B7 or R-B7 or X-irradiated MC38 vaccine infected with V-B7 or R-B7 were challenged 14 days later with 1 x 106 viable uninfected MC38 tumor cells (results are seen in Fig. 2Citation ). Those mice depicted in Fig. 2Citation that remained tumor free were rechallenged with 1 x 106 viable tumor cells 40 days later (54 days after vaccination). The growth of these tumors is seen in B–E, along with the growth of MC38 cells in unvaccinated mice (A). Numbers at the bottom right of each panel represent tumor-free mice/total mice.

 
The experiments were repeated as described above, except that the MC38 tumor-cell vaccines were X-irradiated prior to administration. When X-irradiated MC38/V-WT or MC38/R-WT were used as vaccine and mice were challenged 14 days later with native MC38, tumors grew in all mice (Fig. 2, E and G)Citation . All mice vaccinated with X-irradiated MC38/V-B7 were protected against tumor challenge (Fig. 2F)Citation . Of mice receiving X-irradiated MC38/R-B7 vaccine, two of five developed tumors upon rechallenge with native MC38 cells (Fig. 2H)Citation . All mice that received these X-irradiated vaccines and remained tumor-free were then rechallenged with 5 x 105 native MC38 tumor cells 40 days later (54 days after the original vaccination). Again, all of the mice originally receiving the X-irradiated MC38/V-B7 vaccine displayed memory and were resistant to tumor challenge (Fig. 3D)Citation . As indicated in Fig. 2HCitation , three of five mice originally receiving the X-irradiated MC38/R-B7 vaccine were resistant to tumor challenge at 14 days. However, when these mice were then rechallenged with native MC38 tumor cells 40 days later, all three developed tumors (Fig. 3E)Citation . These studies thus indicate that when using either live or X-irradiated whole tumor-cell vaccines, the vaccinia-infected cell vaccines appeared more efficient than those using retrovirus-infected cells. These experiments were repeated one additional time with similar results.

Experiments were then conducted to determine the relative therapeutic efficacies of X-irradiated MC38 tumor-cell vaccines infected with V-B7 versus R-B7 in an experimental lung metastasis model (Fig. 4)Citation . On day 0, vaccinia-naïve mice were injected i.v. with 1 x 106 live native MC38 tumor cells. On days 3, 10, and 17, groups of 16 mice received one of the following vaccines s.c.: X-irradiated MC38 (Fig. 4A)Citation or X-irradiated MC38 cells infected with V-WT (Fig. 4B)Citation , V-B7 (Fig. 4C)Citation , R-WT (Fig. 4D)Citation , or R-B7 (Fig. 4E)Citation . All mice were sacrificed 28 days later, and tumor nodules in lungs were quantitated. Half of the mice in each group were preimmunized with V-WT via tail scarification. As can be seen in Fig. 4ACitation , all vaccinia-naïve mice receiving the X-irradiated MC38 tumor-cell vaccine developed >200 metastatic nodules/lung and were similar to mice receiving no vaccine (Fig. 4F)Citation . All mice receiving vaccine consisting of X-irradiated MC38 cells infected with V-WT (Fig. 4B)Citation or R-WT (Fig. 4D)Citation also developed lung metastases, with 3 of 10 mice developing a slight [but not statistically significant (P = 0.3)] reduction in the number of lung metastases as compared with the control groups. Mice receiving the X-irradiated MC38/R-B7 vaccine displayed a statistically significant (P < 0.001) reduction in lung metastases as compared with mice receiving X-irradiated MC38/R-WT (Fig. 4, D and E)Citation . Mice receiving the X-irradiated MC38/V-B7 vaccine similarly displayed a statistically significant (P < 0.001) reduction in the development of lung metastases as compared with mice receiving X-irradiated MC38/V-WT vaccine (Fig. 4, B and C)Citation . Mice receiving the X-irradiated MC38/V-B7 vaccine, however, also displayed a statistically significant (P = 0.0351) reduction in lung metastases compared with those mice receiving the X-irradiated MC38/R-B7 vaccine (Fig. 4, C and E)Citation .



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  		Fig. 4. Treatment of lung metastases using X-irradiated whole tumor-cell vaccines. Groups of eight vaccinia-naïve (•, A–F) or vaccinia-immune ({blacksquare}, A–F) mice were inoculated i.v. with 1 x 106 native MC38 cells on day 0. On days 3, 10, and 17, mice received as vaccine either X-irradiated MC38 tumor cells (A); X-irradiated tumor cells infected previously with V-WT (B), V-B7 (C), R-WT (D), or R-B7 (E); or no tumor cells (HBSS buffer; F). On day 28, mice were sacrificed, and lung metastases were counted. G, average numbers of pulmonary metastases for vaccinia-naïve mouse groups; H, average metastases for vaccinia-immune mouse groups. Bars, SD.

 
One of the concerns inherent in using a vaccine containing a vaccinia vector is that previous immunity via the smallpox vaccine would inhibit its effectiveness. However, for reasons explained below, this most likely would not be the situation in the use of whole-cell vaccines (see "Discussion"). Nevertheless, to determine whether prior exposure to vaccinia virus would inhibit the antitumor efficacy of the X-irradiated whole tumor-cell vaccine with the V-B7 vector, the studies described above (Fig. 4)Citation were performed again. In this case, however, mice received 107 pfu V-WT 14 days before receiving the live MC38 cells i.v. and 17 days before the first vaccination. We demonstrated previously that this dose and time interval led to the development of substantial levels of anti-vaccinia immunoglobulin (20) . Results of these studies are shown in Fig. 4Citation . The use of X-irradiated MC38 (Fig. 4A)Citation , MC38/V-WT (Fig. 4B)Citation , or MC38/R-WT (Fig. 4D)Citation as vaccine led to little, if any, reduction in the number of lung metastases. The X-irradiated MC38/R-B7 vaccine significantly (P < 0.001) reduced the number of metastases compared with the MC38/R-WT vaccine (Fig. 4, D and E)Citation . The x-irradiated MC38/V-B7 vaccine significantly (P < 0.001) reduced lung metastases compared with the MC38/V-WT vaccine (Fig. 4, B and C)Citation . Moreover, there was still a statistically significant (P = 0.024) antitumor efficacy of the MC38/V-B7 vaccine compared with the MC38/R-B7 vaccine. Thus, the fact that mice had been exposed previously to live V-WT virus did not inhibit the efficacy of the MC38/V-B7 vaccine to induce antitumor immunity over that of the MC38/R-B7 vaccine. The results of these experiments involving an experimental lung metastasis model were also analyzed as "the average number of lung metastases" for the different vaccine groups. The fact that mice were either vaccinia-naive or were previously administered V-WT apparently had no effect on the antitumor efficacy of these vaccines (Fig. 4G)Citation . When the naive and vaccinia-immune mice for each vaccination group were combined for meta-analysis (n = 16), the MC38/V-B7 was significantly more effective (P = 0.0014) than the MC38/R-B7 in the treatment of pulmonary metastases in this model.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Numerous preclinical studies using retroviral vectors in whole tumor-cell vaccines have been reported. Principally, cytokine genes and costimulatory molecule genes have been used as transgenes to enhance immunogenicity. Several studies have recently used replication-competent vaccinia and replication-defective fowlpox and avipox vectors in whole tumor-cell vaccines. To date, retrovirus delivery of transgenes (either cytokines or B7) in whole tumor-cell vaccines has been used in 36 clinical trials, some completed, and others ongoing (21) . Two trials have begun recently in which pox vectors were used to express B7-1 in tumor cells. To our knowledge, no one has conducted studies to compare the effectiveness of whole tumor-cell vaccines using a retrovirus with those using vaccinia virus as the vector. It should be pointed out that studies using anti-CTLA-4 MAb to inhibit tumor growth in murine models have been reported (22 , 23) . This MAb has been described as having antitumor activity for some tumors but not for others that are poorly immunogenic. The use of anti-CTLA-4 was shown to be ineffectual in the MC38 model reported here.

There are obviously pros and cons in the use of any vector. Advantages in the use of retroviral vectors include: (a) stable integration, thus prolonging expression; (b) transgene expression by all cells (if the cells are drug selected and cloned); and (c) proposed lack of immunogenicity.

Several potential disadvantages in the use of retroviral vectors also exist. One is that infected cells require a drug-selection and cloning process, although some of the newer vectors have reported a higher efficiency of infection and require little or no selection (24, 25, 26) . In addition, actively dividing cells are required for viral integration. Although this is quite easy using established murine tumor-cell lines, this is not always true with cells derived from human tumor biopsies. For example, in the case of carcinomas such as breast and colorectal cancer, it is extremely difficult to propagate tumor cells from biopsies. This is most probably also the case for direct injection of vectors into the tumor in situ. Recently, it has been reported that a new HIV-derived lentivirus vector can express transgenes in cells that are not dividing (27) . The use of this vector awaits further analysis. The real or proposed recombination of retroviral vector genes with endogenous or exogenous human retrovirus also remains an issue. Finally, one is limited by the number of genes that can be inserted into a retroviral vector.

Poxviruses also have their potential advantages and disadvantages. Potential disadvantages include pre-existing or induced immunity to poxvirus proteins, although this was not the case in the studies reported here (Fig. 4)Citation . Moreover, it may well not be the case in other experimental or clinical studies using whole tumor-cell vaccines. In these instances, the virus is intracellular, and there is no need for virus replication. The fact that mice were either vaccinia-naive or were previously administered V-WT apparently had no effect on the antitumor efficacy of these vaccines (Fig. 4)Citation . When the naive and vaccinia-immune mice for each vaccination group were combined for meta-analysis (n = 16), the MC38/V-B7 was significantly more effective (P = 0.0014) than the MC38/R-B7 in the treatment of pulmonary metastases in this model. Pox vector proteins expressed intracellularly may actually act as adjuvant. This observation was the basis for the use of oncolysates as vaccines, such as using extracts of vaccinia virus-infected cells (28) . Without costimulation, however, these types of vaccines may not have fulfilled their potential. Moreover, other types of poxviruses, such as fowlpox and canarypox (ALVAC), are replication defective and less immunogenic. Preclinical studies (20) and clinical studies have now shown that repeated administrations of these recombinant avipox vectors continue to enhance the immune response to the inserted transgene (29) . Because vaccinia virus is a live vector, the potential to induce viremia in an immunosuppressed individual is also a consideration. Hundreds of patients with advanced cancer have received vaccinia virus recombinant vaccines without adverse effects. All of these patients had previously shown positive skin test reactions to standard recall antigens. Of course, this is not an issue with replication-defective avipox viruses. Moreover, the strains of vaccinia virus used in the worldwide eradication of smallpox have since been attenuated (30) , as in the case of the vaccinia virus vector used in the studies reported here.

The potential advantages in using pox vectors in whole tumor-cell vaccines include: (a) a wide host range of infectability (most mammalian cells, including most human cell types, are readily infected); (b) the lack of a need for cell division and drug selection; and (c) a highly efficient rate of infection. Moreover, there is a short interval between infection and expression. As shown in this study, one can infect >95% of tumor cells within 5 h using a poxvirus vector. There is also the fact that poxvirus vectors such as vaccinia virus, fowlpox, and canarypox allow the insertion of multiple genes. Although retroviruses can accept only 10 kb of genetic material and, at most, two genes (11 , 31, 32, 33) , poxviruses have already been shown to faithfully express up to seven transgenes (Ref. 34 ; up to 25 kb of genetic information). This may prove to be extremely important in the case of whole tumor-cell vaccines, which are weakly immunogenic by nature. One may wish to express multiple transgenes in X-irradiated tumor-cell vaccines, such as cytokine or chemokine genes, as well as multiple costimulatory molecule genes. To that end, it has been demonstrated recently that by inserting three costimulatory molecule genes (B7-1, intercellular adhesion molecule-1, and leukocyte function-associated antigen -3) into either vaccinia virus or fowlpox vectors, and then infecting tumor cells with these vectors, one can activate T cells to far greater levels than is possible with the use of any one of the costimulatory molecules alone.3 Moreover, the effects on T-cell activation were shown to be synergistic when using the three costimulatory (i.e., TRICOM) vectors. For instance, T-cell activation far exceeded the additive effects of the use of single costimulatory molecules.

The studies reported here thus reveal some potential advantages in the use of a poxvirus versus a retrovirus vector in several different modalities involving whole tumor-cell vaccines expressing the B7-1 transgene. These advantages include: (a) time required and ease of cellular infection (Fig. 1)Citation ; (b) use in a live whole tumor-cell vaccine to prevent tumor development (Fig. 2, B and D)Citation ; (c) use of an X-irradiated whole tumor-cell vaccine in a primary challenge of tumor cells (Fig. 2, F and H)Citation , and in rechallenge with tumor after an extended period of time (Fig. 3, B and D)Citation ; (d) use in a therapy model against experimental lung metastases in vaccinia-naive mice (Fig. 4)Citation ; and (e) use in mice that previously received vaccinia virus (Fig. 4)Citation . In the above studies, care was taken to drug-select a retrovirus high-expression clone to avoid influencing results toward the use of vaccinia. Indeed, both types of vectors expressed the transgene at comparable levels (Fig. 1C)Citation . These observations, taken with the fact that one can insert multiple costimulatory molecule transgenes into pox vectors to enhance the immunogenicity of whole tumor-cell vaccines, should lend credibility to the use of these vectors in clinical applications.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, 10 Center Drive, Room 8B07, Bethesda, MD 20892. Phone: (301) 496-4343; Fax: (301) 496-2756; E-mail: js141c{at}nih.gov Back

2 The abbreviations used are: MAb, monoclonal antibody; FACS, fluorescence-activated cell sorter; R-WT, retrovirus-wild type; MOI, multiplicity of infection; pfu, plaque-forming unit(s). Back

3 J. W. Hodge, H. Sabsevari, M. G. O. Lorenz, A. G. Gomez, L. Gritz, and J. Schlom. A triad of costimulatory molecules synergize to amplify T-cell activation, Cancer Res., submitted for publication, 1999. Back

Received 7/ 8/99. Accepted 9/ 2/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Chen L., McGowan P., Ashe S., Johnston J., Li Y., Hellstrom I., Hellstrom K. E. Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity. J. Exp. Med., 179: 523-532, 1994.[Abstract/Free Full Text]
  2. Nemunaitis J., Bohart C., Fong T., Meyer W., Edelman G., Paulson R. S., Orr D., Jain V., O’Brien J., Kuhn J., Kowal K. J., Burkeholder S., Bruce J., Ognoskie N., Wynne D., Martineau D., Ando D. Phase I trial of retroviral vector-mediated interferon (IFN)-{gamma} gene transfer into autologous tumor cells in patients with metastatic melanoma. Cancer Gene Ther., 5: 292-300, 1998.[Medline]
  3. Gunji Y., Tasaki K., Tagawa M., Matsubara H., Takenaga K., Suzuki T., Asano T., Ochiai T., Isono K., Sakiyama S. Inhibition of peritoneal dissemination of murine colon carcinoma cells by administrating retrovirus harboring IL-2 gene. Cancer Gene Ther., 5: 339-343, 1998.[Medline]
  4. Tasaki K., Tagawa M., Gunji Y., Matsubara H., Takenaga K., Muramatsu M., Fujimura S., Suzuki T., Asano T., Ochiai T., Isono K., Kouzu T., Sakiyama S. Inhibition of experimental lung metastasis of murine colon carcinoma cells depends on the amount of interleukin-2 secreted from the transduced cells. Anticancer Res., 18: 813-817, 1998.[Medline]
  5. Aruga A., Tanigawa K., Aruga E., Yu H., Chang A. E. Enhanced adjuvant effect of granulocyte-macrophage colony-stimulating factor plus interleukin-12 compared with either alone in vaccine-induced tumor immunity. Cancer Gene Ther., 6: 89-95, 1999.[Medline]
  6. Hsieh C. L., Pang V. F., Chen D. S., Hwang L. H. Regression of established mouse leukemia by GM-CSF-transduced tumor vaccine: implications for cytotoxic T lymphocyte responses and tumor burdens. Hum. Gene Ther., 8: 1843-1854, 1997.[Medline]
  7. McLaughlin J. P., Abrams S., Kantor J., Dobrzanski M. J., Greenbaum J., Schlom J., Greiner J. W. Immunization with a syngeneic tumor infected with recombinant vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM- CSF) induces tumor regression and long-lasting systemic immunity. J. Immunother., 20: 449-459, 1997.
  8. Lotze M. T., Zitvogel L., Campbell R., Robbins P. D., Elder E., Haluszczak C., Martin D., Whiteside T. L., Storkus W. J., Tahara H. Cytokine gene therapy of cancer using interleukin-12: murine and clinical trials. Ann. NY Acad. Sci., 795: 440-454, 1996.[Medline]
  9. Tahara H., Zitvogel L., Storkus W. J., Zeh H. J., III, McKinney T. G., Schreiber R. D., Gubler U., Robbins P. D., Lotze M. T. Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector. J. Immunol., 154: 6466-6474, 1995.[Abstract]
  10. Marti W. R., Oertli D., Meko J. B., Norton J. A., Tsung K. Induction of antigen-presenting capacity in tumor cells upon infection with non-replicating recombinant vaccinia virus encoding murine MHC class II and costimulatory molecules. J. Immunol. Methods, 200: 191-198, 1997.[Medline]
  11. Cavallo F., Martin-Fontecha A., Bellone M., Heltai S., Gatti E., Tornaghi P., Freschi M., Forni G., Dellabona P., Casorati G. Co-expression of B7–1 and ICAM-1 on tumors is required for rejection and the establishment of a memory response. Eur. J. Immunol., 25: 1154-1162, 1995.[Medline]
  12. Hodge J. W., Abrams S., Schlom J., Kantor J. A. Induction of antitumor immunity by recombinant vaccinia viruses expressing B7-1 or B7-2 costimulatory molecules. Cancer Res., 54: 5552-5555, 1994.[Abstract/Free Full Text]
  13. Uzendoski K., Kantor J. A., Abrams S. I., Schlom J., Hodge J. W. Construction and characterization of a recombinant vaccinia virus expressing murine intercellular adhesion molecule-1: induction and potentiation of antitumor responses. Hum. Gene Ther., 8: 851-860, 1997.[Medline]
  14. Hellstrom K. E., Gladstone P., Hellstrom I. Cancer vaccines: challenges and potential solutions. Mol. Med. Today, 3: 286-290, 1997.[Medline]
  15. Dunussi-Joannopoulos K., Weinstein H. J., Nickerson P. W., Strom T. B., Burakoff S. J., Croop J. M., Arceci R. J. Irradiated B7-1 transduced primary acute myelogenous leukemia (AML) cells can be used as therapeutic vaccines in murine AML. Blood, 87: 2938-2946, 1996.[Abstract/Free Full Text]
  16. Townsend S. E., Su F. W., Atherton J. M., Allison J. P. Specificity and longevity of antitumor immune responses induced by B7-transfected tumors. Cancer Res., 54: 6477-6483, 1994.[Abstract/Free Full Text]
  17. Kawakita M., Rao G. S., Ritchey J. K., Ornstein D. K., Hudson M. A., Tartaglia J., Paoletti E., Humphrey P. A., Harmon T. J., Ratliff T. L. Effect of canarypox virus (ALVAC)-mediated cytokine expression on murine prostate tumor growth [see comments]. J. Natl. Cancer Inst., 89: 428-436, 1997.[Abstract/Free Full Text]
  18. Fox B. A., Spiess P. J., Kasid A., Puri R., Mule J. J., Weber J. S., Rosenberg S. A. In vitro and in vivo antitumor properties of a T-cell clone generated from murine tumor-infiltrating lymphocytes. J. Biol. Response Modif., 9: 499-511, 1990.[Medline]
  19. Wexler H. Accurate identification of experimental pulmonary metastases. J. Natl. Cancer Inst., 36: 641-645, 1966.
  20. Hodge J. W., McLaughlin J. P., Kantor J. A., Schlom J. Diversified prime and boost protocols using recombinant vaccinia virus and recombinant non-replicating avian pox virus to enhance T-cell immunity and antitumor responses. Vaccine, 15: 759-768, 1997.[Medline]
  21. Human Gene Therapy Protocols. Hum. Gene Ther., 9: 928-976, 1998.
  22. Leach D. R., Krummel M. F., Allison J. P. Enhancement of antitumor immunity by CTLA-4 blockade. Science (Washington DC), 271: 1734-1736, 1996.[Abstract]
  23. Kwon E. D., Hurwitz A. A., Foster B. A., Madias C., Feldhaus A. L., Greenberg N. M., Burg M. B., Allison J. P. Manipulation of T cell costimulatory and inhibitory signals for immunotherapy of prostate cancer. Proc. Natl. Acad. Sci. USA, 94: 8099-8103, 1997.[Abstract/Free Full Text]
  24. Grignani F., Kinsella T., Mencarelli A., Valtieri M., Riganelli D., Lanfrancone L., Peschle C., Nolan G. P., Pelicci P. G. High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein. Cancer Res., 58: 14-19, 1998.[Abstract/Free Full Text]
  25. Dachs G. U., Dougherty G. J., Stratford I. J., Chaplin D. J. Targeting gene therapy to cancer: a review. Oncol. Res., 9: 313-325, 1997.[Medline]
  26. Kim S. H., Yu S. S., Park J. S., Robbins P. D., An C. S., Kim S. Construction of retroviral vectors with improved safety, gene expression, and versatility. J. Virol., 72: 994-1004, 1998.[Abstract/Free Full Text]
  27. Naldini L. Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr. Opin. Biotechnol., 9: 457-463, 1998.[Medline]
  28. Wallack M. K., Sivanandham M., Ditaranto K., Shaw P., Balch C. M., Urist M. M., Bland K. I., Murray D., Robinson W. A., Flaherty L., Richards J. M., Rosen L., Bartolucci A. A. Increased survival of patients treated with a vaccinia melanoma oncolysate vaccine: second interim analysis of data from a Phase III, multi-institutional trial. Ann. Surg., 226: 198-206, 1997.[Medline]
  29. Marshall J. L., Richmond E., Pedicano J., Tsang A., Schlom J. Phase I/II trial of Vaccinia-CEA and ALVAC-CEA in patients with advanced CEA-bearing tumors. Proc. Am. Soc. Clin. Oncol. Annu. Meet., 18: 609a 1999.
  30. Moss B. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc. Natl. Acad. Sci. USA, 93: 11341-11348, 1996.[Abstract/Free Full Text]
  31. He X. S., Rivkina M., Robinson W. S. Construction of adenoviral and retroviral vectors coexpressing the genes encoding the hepatitis B surface antigen and B7-1 protein. Gene (Amst.), 175: 121-125, 1996.[Medline]
  32. Zitvogel L., Tahara H., Cai Q., Storkus W. J., Muller G., Wolf S. F., Gately M., Robbins P. D., Lotze M. T. Construction and characterization of retroviral vectors expressing biologically active human interleukin-12. Hum. Gene Ther., 5: 1493-1506, 1994.[Medline]
  33. Dubey C., Croft M., Swain S. L. Costimulatory requirements of naive CD4+ T cells. ICAM-1 or B7-1 can costimulate naive CD4 T cell activation but both are required for optimum response. J. Immunol., 155: 45-57, 1995.[Abstract]
  34. Ockenhouse C. F., Sun P. F., Lanar D. E., Wellde B. T., Hall B. T., Kester K., Stoute J. A., Magill A., Krzych U., Farley L., Wirtz R. A., Sadoff J. C., Kaslow D. C., Kumar S., Church L. W., Crutcher J. M., Wizel B., Hoffman S., Lalvani A., Hill A. V., Tine J. A., Guito K. P., de Taisne C., Anders R., Horii T., Paoletti E., Ballou W. R. Phase I/IIa safety, immunogenicity, and efficacy trial of NYVAC-Pf7, a pox-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria. J. Infect. Dis., 177: 1664-1673, 1998.[Medline]



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