Radiol Oncol 19%: .30: 260-7.
Current approaches to gene therapy in oncology: Construction of
tumor vaccines
Srdjan Novakovic
Institute of Oncology, Department of" Tumor Biology, Ljubljana Slovenia.
Current conventional treatment of malignancies is hasecl predominantly on the iise of radio- and chemotherapy. The mentioned therapies are not directed against cancer tissue only and have severe dose-limiting toxic side effecls accompanied with a suppressive effect on the patient's immune system. On the other hand, immunotherapy, and especially gene therapy, try to be more selective and less aggressive, having the purpose o( triggering a specific immune response against tumor cells. Therefore, different approaches to the creation and application of gene therapy in oncology have been formed in the pastfew years, yet the aim o( all of them is the same: to use extended knowledge ahout molecular mechanisms of the disease in order to devise a more specific mode of treatment. The major approach to present-day gene therapy of cancer is the generalion of tumor vaccines as ci possible future category of cancer treatmenl. The purpose of this article is to provide a brief overview on creation and potential applications of tiimor vaccines as well as of some modes of gene therapy in oncology.
Key words: neoplasms-therapy; gene therapy; tumor vaccines
Introduction
Owing to unspecific activities of conventional therapies against cancer (radiotherapy, chemotherapy) a treatment of this kind is quite often accompanied with unrecoverable damage of the normal tissue. The tremendous increase of knowledge in immunology as well as the exponential development of recombinant DNA technology conditioned the renewal of interest for creation of different immunotherapies that were supposed to be more effective, more specific for tumor cells, and cause no or negligible toxic side effects. The goal of each immunomodulatory treatment is to stimulate (enhance) immune response and in this way alter the dynamics of host-tumor relationship to therapeutic advantage. At the same time. this treatmenl modality has
Correspondence to: Srdjan Novakovic, Dr.Sci., Institute of Oncology. Department ofTumor Biology, Zaloska 2, 1105 Ljubljana. Slovenia. Fax: +386 61 131 41 80.
UDC: 6 l 6-006.6-097:615.37 I
to prevent the development of tumor celi resistance to such treatment, and cause no toxic deposition in the normal tissue. Therefore, for successful creation of immunotherapy, it is important first to understand the relationship between the host and specific tumor cells in order to choose the most appropriate approach. To induce tumor immunity more specifically and effectively, various methods of im-munotherapy have emerged using different biological agents such as monoclonal antibodies, cytokines. tumor antigens, hormones, activated killer cells. immune T cells. DNA and others.IJ1
Vaccination against cancer
The idea of vaccination against tumor cells has been a distant goal of immunologist for many years, ever since 1909, when Paul Ehrlich suggested that tumors might express antigens that coulcl be targets of immune system.'' Certainly, at that time there was hardly anything known about tumor-associat-
Current approaches to gene therapy in onco/ogy: Construction of tumor vaccines
261
ed antigens, B and T lymphocytes, antigen-specific receptors on lymphocytes, immunoregulatory cytokines etc. However, the observations that there is a' difference in the velocity of tumor growth, and that some tumors stagnate for a longer period of time (even some years), indicate that organisms possess powerful regulation mechanisms (i.e. immune system) for tumor growth control.' And stili, quite often tumor cells escape the control and do not trigger the immune response. Tumor vaccines were thus created with the inlention to rebuild or retrigger the immune system and induce systemic immunity against tumor cells. Por this purpose, irradiated autologous or allogeneic tumor cells, lysa-tes of tumor cells, and occasionally, virally infected tumor cells were used as tumor vaccines. To intensify additionally the immune response, nonspecific immunostimulators (e.g. Coryiiehacteriuiii paivwn or Bacillus Caliiielte-Giierin) were added to most of the above mentioned preparations.11-14 The basic working guide of all these experiments was to achieve an enhanced expression of MHC antigens on tumor cells and to increase the cytokine production.
Only the exponential development of molecular genetics and monoclonal antibody reagents, as well as the results of the latest investigations, provided enough information to allow speculation that diminished responsiveness or complete unresponsive-ness of the immune system could hc predominantly a consequencc of the changes of tumor cells at the molecular level. Among thc most important changes which enabled tumor cells to cscape immune control researchers classified the following: lx"'
•	inadequate expression of MHC (major histocompatibility complex) antigens,
•	prevention of tumor specific antigen presentation to T lymphocytes,
•	absence of adhesion molecules which are important for the activation of immune system, and
•	production of various factors (by tumor cells) which influence (change) thc host immune system.
Gene therapy and tumor vaccines
The "gene therapy" term has become a new paradigm, associated with any kind of disease where the origin can be connected with the defined genes. Gene therapy involves a variety of new techniques for gene transfer, gene replacement, gene repair or gene deletion.
Although the idea of vaccination against tumor cells dates in the beginning of 19th century, the modem tumor vaccines represent just one of the approaches (major) to gene therapy of cancer. In other words, modem tumor vaccines are a form of gene therapy where, by use of different vectors, genes of interest are transferred into the tumor cells or into immunocompetent cells. This can be achieved by direct DNA transfer or by using viral vectors. The most prevalent nonviral techniques used for gene transfer are calcium phosphate transfection, microinjection, electroporation, liposomal gene transfer, injection of naked DNA, and receptor mediated gene transfer. 1 7-22 Among the biological delivery systems for gene transfer the cardinal ones are retroviral vectors, adenoviral vectors, adeno-associated virus vectors and other viral vectors.2'
The first studies with genetically transformed tumor cells (that were used as tumor vaccines) confirmed that both classes of MHC antigens (MHC 1 and MHC II) play an important role in the process of triggering of the immune response and that the antitumor activity is predominantly a consequence of activation of cellular*immunity^ Class I MHC antigens are recognized by cytotoxic lymphocytes (CD8+) and their presence is obligatory for the activation of these cells. On the other hand, class II MHC antigens (they are presented by antigen-presenting cells in the form of endosomes or lyso-somes, respectively) take part in the activation of helper T lymphocytes (CD4+), cells which are classified as basic producers of different cytokines. Exactly, the defect in the helper arm (i.e. cytokine producing part) of the immune system is often the cause of inadequate immunogenicity of tumor cells: namely, the development of cellular immunity will fail in the case of inadequate cytokine production, regardless of the fact that MHC I antigens are normally expressed and active.2'
Considering those facts, tumor vaccines were created predominately to achieve:
•	enhanced production of various cytokines that participate in immune processes (1L-2, GM-CSF, IFN-u, TNF-u),
•	expression of allogeneic human leukocyte antigens (HLA antigens) or
•	enhanced concentration of products that are responsible for the expression or the activities, respectively, of oncogenes (e.g. of the product of p53 suppressor gene).
Therefore, depending on the manner chosen to fight tumor cells, quite a few different approaches
262
Novakovic S
to creation of gene therapy and tumor vaccines have been established. This review will deal with some of them, i.e. those that have been found most promising and attractive.
Preparation of twnor vaccines with insertion of genes coding for allogeneic leukocyte antigens into autologous minor cel/s
The purpose of such preparation of tumor vaccines is the transfer of gcnes encoding certain antigens (usually present on the surface of antigen-presenting cells) into tumor cells. B7 antigen is a molecule that normally functions as an activation molecule on antigcn-presenting cells (macrophages. B lymphocytes. dendritic cells). B7 antigen represents a ligand for two types of T lymphocyte receptors i.e. for CD28 (present on CD4+ and CDS+) and CTLA4 (present only on CDS+) receptors. Thc role of CTL4 has not been determined yet, while on the other hand CD28 is well known to be the cardinal receptor for activation of T lymphocytes and for stimulation of cytokine production."' These data led to formation of a hypothesis about the transfer of a gene coding for B7 antigen into the tumor cells and about the potentials of such tumor vaccine to trigger systemic antitumor immunity. So Chen et al..13 as well as Townsend and Allison,21 demonstrated that rejection of malignant melanoma cells expressing B7 ligand resulted from the aclivity of CDS+' T lymphocytes. Besides. in these experiments systemic immunity developed (in experimental animals) even against genetically unchanged melanoma cells (which were thus not expressing B7 ligand). On the basis of the cited studies we can conclude that tumor vaccines, created by transferring of B7 gene into autologous tumor cells, activate cytotoxic T lymphocytes and stimulate cytokine production in helper T lymphocytes, thus effectively triggering the development of systemic antitumor immunity. On the other hand. the best results with this kind of vaccines can be obtained (owing to the costimulatory mode of action of B7 on CDS+ and CD4+ T lymphocytes) only in the presence of MHC class I and class II antigens on tumor cells.
Vaccines created wilh insertion of genes coding for dijferenl cytokines inio aitlologoits tumor ce//.s
lnsertion of genes coding for different cytokines might play a role in "overcoming" Ihe unresponsiveness of immune system that derives from inability for normal cytokine production which is actually a consequence of complete absence or inade-
quate expression of MHC II antigens. In contrast to the activities of exogenous cytokines, the cytokines produced in genetically changed autologous tumor cells mimic the activities of natural endogenous cytokines (underlie to some extent the control mechanisms of the organism), which on one hand improves their effectiveness and on the other hand minimizes their toxic side effects. When preparing tumor vaccines, different researchers introduced genes for numerous cytokines or growth factors (IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-1O. IFN-a, TNF-a, GM-CSF and G-CSFJ, respectively, into tumor cells.2*-12 The effectiveness of vaccines tested on animal tumor models depended upon the type of cytokine produced by the cells. upon the abundance of cytokine synthesis and upon the type of tumor used in the study. Fearon et al. demonstrated that transfection of poorly immunogeneic mouse colon carcinoma cells with IL-2 gene results in reduction of tumorigenic potential of tumor cells and triggers the development of systemic immunity." They confirmed that the phenomenon of systemic immunity results from the influence of IL-2 on CD4+ and CDS+ T lymphocytes (activation of T lymphocytes). Similar conclusions were made by Gansbacher et al. alter the transduction of IL-2 gene into mouse fibrosarcoma cells (in syngeneic mice) and into human melanoma or renal carcinoma cells (in nude mice). 'u; The fact that IL-2 triggers the development of systemic immunity through its action upon T lymphocytes was also confirmed by Rusell et al. in experiments with rat tumor model.''' Namely, they transplanted transfected rat sarcoma cells either into syngeneic rats or into immunodeficient nude rats. The effect of vaccine in syngeneic rats with normal T lymphocyte production was highly superior to the one in nude rats. On the other hand, partly different results were obtained by Cavallo et al. '7 In agreement with other authors they demonstrated that vaccines prepared by IL-2 gene transduction are capable of challenging the immune response, which (according to Cavallo et al.) predominantly depends upon neutrophils activated with IL-2. Allione et al. created tumor vaccines with transfection of adenocarcinoma cells using genes encoding various interleukins (IL-2. IL-4, IL-7, IL-10), IFN-a, TNF-a or GM-CSF. The best antitumor protection was achieved with inoculation of tumor cells producing interleukins and IFN-, while tile treatment outcome alter application of tumor cells producing TNF-a was less favourable.'8 Quite interesting was also the comparison of the effectiveness
Current approaches to gene therapy in onco/ogy: Construction of tumor vaccines
263
of the therapy with genetically changed cclls, to therapy with tumor cclls admixed with Corynebac-leriwn parvwn. Namely, the authors established that the antitumor activity of ihe mixlure of tumor cells wilh Cory/?ebaclen//m pan>itm approximated in its degree ihe anlitumor activity of therapy with genetically modified cclls. Similar results were observed by Hock el al., who demonstrated that tumor vaccines prepared by mixing of tumor cells with nonspecific immunostimulalors exerl an antilumor effect which is comparable to lhe effect of tumor vaccines created of genelically transformed cells.1'' In contrast to lhe aulhors, who achieved relatively modest resulls with tumor vaccines containing gene for TNF-cí, Blankenslein presented encouraging oul-comes (his own and of other authors) using lhe very same vaccines.4" The anlilumor aclivities of such vaccines were supposed to be based predominantly on an indirecl effect mediated through slimulation of immune system and to a lesser extent on the direet antilumor effect of TNF-(,(. This kind of slimulation of immune system includes the activation of macrophages, as well as CD4+ and CD8+ T lymphocytes. Vaccines bearing TNF-(,( gene are also successful in the case of inhibited T lymphocyle production, bul anyway, the presence of these cells enhances the antitumor effect of such trealment. The best proteclion from challenge wilh wild type tumor cells, as well as the most pronounced antilumor activity against formed tumors, has been ascribed to vaccines created of tumor cells bearing gene for GM-CSF. Mulligan and Pardoll studied the effectiveness of vaccines bearing genes for various individual cylokines or for combination of cytokines.41 The most promising results were achieved with GM-CSF (in the group of vaccines bearing a gene for a single cytokine), while the most effective combination of genes for preparation of tumor vaccines comprised genes for IL-2 and GM-CSF. DranolT et al. quite early discovered thal tumor vaccines wilh GM-CSF gene are superior to vaccines prepared with genes encoding other cytokines in the case of slimulation of the antitumor immune response.10 However, the activation of CD4+ and CD8+ T lymphocytes was obligatory for the devel-opmenl of systemic immunity also with vaccines bearing GM-CSF gene, regardless of the MHC II antigen expression on tumor cells. The effectiveness of tumor vaccines with GM-CSF gene was finally confirmed by Golumbek et al.. since in their experiments not a single experimental animal immunized with lhe vaccine developed a tumor after
challenge with highly tumorigenic wild type tumor cells.'2 The effect of vaccines with enhanced expression of GM-CSF gene is being ascribed to the stimulation of differenliation of the precursor blood cells and dendrilic cells (imporlanl antigen-presenting cells for T lymphocytes).
Thus, the basic conclusions of these studies could be the following:
•	even low concentrations of cytokines produced by transformed cells are capable of stimulating the antitumor immune response (comparable results were achieved after systemic high dosage cytokine therapy which is often accompanied with numerous loxic side effecls);
•	imporlanl role of cytokines in the process of activation of nonspecific leukocytes e.g. granulocytes and macrophages;
•	cooperation belween granulocytes, macrophages, lymphocyles, fibroblasls and endolhelial cells represents the basis of immune reactions triggered by genelically lransformed cells;
•	degree of anlitumor activity depends upon the tumor type, the type of cytokine produced by tumor cells, and upon the abundance of cytokine produc-lion;
•	T lymphocyte aclivity is supposed to depend indireclly upon aclivation of macrophages and olh-er anligen-presenling cells, as well as upon secondarily induced cytokines (which play an importanl role in the aclivalion of T cells);
•	sublethally irradiated genetically changed cells are capable of challenging lhe immune response, yel a less pronounced one in comparison to lhe immune response triggered by proliferating cells, since sublethally irradiated cells produce cytokine only during a limited period of time and because lhe abundance of lumor-associalecl antigens is insu fficienl;
•	inserlion of GM-CSF gene inlo lumor cells does not change their tumorigenic polenlial, yet cells modified in this way and aflerwards sublethally irradiated, induce the development of a long lasting immune memory.
Application ( /«mor specific antigens as vaccines
The idea is to use specific antigens only, instead of intact tumor cells (as carriers of usually ill-defined tumor antigens), for the creation of tumor vaccines. In this case specific immunily can be enhanced (owing to the usage of specific antigens), and also whole work wilh gene lransfection becomes sur-
264
Novakovic S
plus. Thc basic condition for a successful application of vaccinc is that the choscn antigen has to be expressed exclusively on the .specific type of tumor cells and by no means on healthy normal cells. We are witnessing at present the identification of the first genes coding for human melanoma-associated antigens that are specifically recognized by autologous cytotoxic T lymphocytes. Mage-1 antigen represents an example of this kind. the antigen that cannot be found on normal cells of adults, but can be detected on approximately 50 % of human malignant melanoma cells.4-1
//we/'tio/i o/' genes coding for .viih.vta/ii.'e.v t/im make tumor cel/.v sltsce|lUh/e to che/iiothero/)««tic drugs
The use of tumor "suicide" genes offers an additional approach to ihe treatment of malignant disease. Thc idea is lo modify genetically lumor cells, and to render them vulnerable lo therapy with sys-temically delivered chemotherapeutic drugs. This kind of application of genetic engineering in cancer treatment represents gene iherapy in a classical sense. Moollen et al. quite early formed an idea of transferring the classically described "suicide" gene, herpes virus thymidine kinase (HSVTK) gene, into tumor cclls to make them sensitive to ganciclovir.44-45 Their starting point was ihe fact that normal mammalian cells are insensitive to ganciclovir owing to incapability of kinases (present in normal cells) to phosphorylate ganciclovir into toxic metabolites. On the other hand. HSVTK phosphor-ylates ganciclovir and its toxic metabolites inhibit DNA polymerase, thus impeding the elongation of DNA molecule. Therefore, the accumulation of toxic metabolites interferes with DNA synthesis, resulting in apoplosis and cell death. The mechanism of action in tumor cells may be analogous to the one in virally infected cells, yet lhe cffect of toxic metabolites spreads out also on genetically unchanged (not producing HSVTK) tumor cells - i.e. bystander effect. The exact mechanism of byslander effect remains queslionable, bul anyway, there is a hypothesis that toxic metabolites may be released from lhe cells (where they were produced) in form of lyposomes to enter genetically unchanged cells and affect them as described above. Besides, the antitumor activity also may be achieved through indirect mechanisms that include the activation of immune system. An affirmation derives from the observation that the effect of therapy with tumor vaccines (prepared with gene coding for HSVTK) followed by ganciclovir treatment is less pronounc-
ed in immunosuppressed animals (athymic nude mice).24 Short et al., as well as Culver et al., demonstrated the effectiveness of such system on in-tracranial tumors in experimental animals.46^7 Namely, they transferred in vivo HSVTK gene directly into tumors using vectors (fibroblasts) and afterwards treated the animals with ganciclovir. Even though they demonstrated that only a small number of tumor cells incorporated HSVTK gene, ganciclovir successfully destroyed both the transfected and the nontransfected cells.
Clinical trials and prospects
Preclinical studies have demonstrated that gene therapy represents a new and provocative mode of treatment with great therapeutic potentials. The insight into the mechanisms of growth and growth regulation of tumor cells has offered multiple potential methods for genetic intervention. Up till now, more than 100 trials with genetically altered tumor vaccines or gene therapy studies have received approval in humans. Most of them are using autologous tumor cells transfected with genes encoding different cytokines.
One of the first tumor vaccines applied in humans was Rosenberg's vaccine using tumor infiltrating lymphocytes stimulated in vitro with 1L-2 and infusing them to the patient with malignant melanoma, along with additional IL-2.4* In this case genetic manipulation was not included in the preparation of the vaccine, but exogenous biological response modifiers were applied to augment the immune response against tumor cells.
Another variant of creation of tumor vaccines was presented by Schirrmacher et al., who were employing a two-component human cancer vaccine. The purpose of such a vaccine was simply to challenge the immune system by inserting some viral antigens into tumor cells, thus rendering the cells much more immunogeneic. The idea was based on the analogy with virally induced tumors which are known to be the most immunogeneic tumors in humans. As the specific component (bearing specific antigens) they used the closest possible match to an individual cancer of a patient, namely autologous cancer cells from resected primary tumor or metastases. The non-lytic virus NDV (Newcastle Disease Virus) was applied as the second, nonspecific component for infection of tumor cells. In two clinical studies the vaccines were applied
Curre/// a/iproar//e.v lo gene ///erapy inn oncology: Ctms/ruction of tumor vw.ri/ie.s
265
postoperatively in patients with no macroscopic remnant of tumor, but with a high risk of developing recurrent disease (colorectal carcinoma and breast cancer), while in another three studies the vaccines were applied in combination with biological response modifiers to patients with remaining metastatic disease: renal carcinoma, metastatic breast carcinoma, and metastatic ovarian carcinoma.4''
As il was postulated before, presently there are many clinical trials with tumor vaccines going on and the studies of Rosenberg and Schirrmacher are the illustrations of only two different approaches to creation of tumor vaccines. Also it is worth mentioning that lately Rosenberg modified his concept for generation of tumor vaccines by introducing genes coding lor IL-2 or TNF-a into tumor-infiltrating lymphocytes.-"'
However, the transfer of preclinical knowledge and technology into clinical practice is accompanied with certain difficulties. For now the major concerns with tumor vaccines are inappropriate expression of the transferred gene, as well as frequent adverse immunological reactions of ihe organism against genetically transformed cells. Certainly, it would bc highly desirable if gene expression could be regulated in lime, quantity and place, yet with the current vectors this is impossible. Newer delivery systems should incorporate features that permit tissue/cell specific expression and allow liie level of gene expression to be regulated hy exogenous small molecules administered as a conventional pharmaceutical agent.
In addition, when autologous liimor vaccines are used, another group of questions, which have to be solved, comes to light. Namely, the basic term lor development of human autologous tumor vaccines is to establish primary celi cultures from patient's tumor specimens. Since this is a procedure, which is labour and time consuming, there was an idea to use allogeneic cells, stably transfected with cDNA of choice, instead of autologous tumor cells." Although the idea is attractive, conventional immunology stili dictates that autologous cells are far better for triggering an effective MHC-restricted immune response than allogeneic cells.
Finally, we also have to bear in mind that Hock et al. prepared a potent tumor vaccine without any kind of genetic manipulation to tumor cells.1'' Namely. in his experiments sublethally irradiated tumor cells admixed with Corynrh«c/ci'/u;;i had an immunogeneic activity by ali means comparable to the one of genetically transformed cells.
Conclusion
This article is dealing with a field of great importance, extremely last developing, and extremely wide - a fact that makes every general conclusion (become) obsolete in a very short period of time. Anyway. if we try to stress the major points, we have to admit that new biological approaches to treatment of cancer are of central importance not only I'or the treatment. but also for understanding of some basic rules governing antigen immune recognition, cancer metastasizing, bystander effect etc. Apart from some classical methodological problems that remain to be solved before final assessment of gene therapy and tumor vaccines validity will be given, there are also some social conventions that have to be changed. Namely, quite often are attractive ideas for biotherapy of cancer received with scepticism by established oncologists, and in the majority of cases, such therapy is acceptable only lor a patient who has failed every conventional treatment. Such patients are by no means the best candidates for establishing an active immune response, and studies of this kind can hardly prove the validity of immune therapy.
References
1.	Vieweg J. Boczkowski D. Roberson MK. e/ a/. Efficient gene transfer with adeno-associated virus-based plasmids complexed to cationic liposomes lor gene therapy of human prostate cancer. Cw/cer Re.. 1995: 55: 2366-72.
2.	Nakamura Y. Wakimoto H. Abe J. et o/. Adoptive immunotherapy with murine tumor-specific T lymphocytes engineered to secrete interleukin 2. Canccr Re.v 1994; 54": 5757-60,
3.	Tos GA. Cignetti A. Rovera G. Foa R. Retroviral vector-mediated transfer ofthe tumor necrosis factor gene into human cancer cells restores an apoptotic cell death program and induces a bystander-killing effect. /3/ood 1996; 87: 2486-95,
4.	Ehrke MJ. Verstovsek S. Krawczyk MC, e/ al. Cyclophosphamide plus tumor necrosis factor- chemoimnm-notherapy cured mice: life-long immunity and rejection of re-implanted primary lymphoma. hit J Canccr 1995; 63: 463-71.
5.	Kus B, Sersa G, Novakovic S, Urbancic J, Stalc A. Modification of TNF- pharmacokinetics in SA-1 tumor-hearing mice. //¡/ J Canccr 1993; 55: 1 10-4.
6.	Novakovic S. Fleischmann RWJr. Antitumor effect of interferon- administered by different routes of treatment. Radio/ Oncol 1993; 27: 286-92.
7.	Jezersek B. Novakovic S, Sersa G, Auersperg M, Fleischmann WR Jr. lnteractions of interl'eron and vinblast-
266
Novakovic S
ine on experimental tumor model melanoma B-16 in vitro. Anti-Cancer Drugs 1994: 5: 53-6.
8.	Novakovic' S, Boldogh l. /ii vitro TNF- production and in vivo alteration of TNF- RNA in niou.se peritoneal macrophages after treatment wilh different bacterial derived agents. Cancer tellers 1994: 81: 99-109.
9.	Ehrlich P. The collected papers of Paul Ehrlich. In: Himmelweit F, ed. /iimwnology and cancer research. London: Pergamon, 1957 (1909).
10.	Stevenson KF. Tumor vaccines. FASEB J 1991; 5: 2250-7,
11.	Hui K. Grosveld F. Festenstein H. Rejection of transplantable AKR leukaemia cells following MHC DNAmediated cell transformation. Nature 1984; 3ll: 7502.
12.	Wallich R, Bulbuc N, Hammerling G. Katzav S. Segal S, Feldman M. Abrogation of metastatic properties of tumor cells by de novo expresion of H-2K antigens following H-2 gene transfection. Nature 1985; 315: 301-5,
13.	Chen L. Ashe S. Brady W. et al. Costimulation of antitumor immunity by the B7 counlerreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell 1992: 71: 1093-102,
14.	Oettgen H, Old LJ. The history of cancer immunotherapy. In De Vita VT. Hellman S, Rosenberg SA eds. Biologic therapy of cancer. Philadelphia, Lippincott 1991: 53-66.
15.	Guo Y, Mengchao W, Chen H, et al. Effective tumor vaccine generated by fusion of hepatoma cells with activated^ cells. .Science 1994: 26.3: 518-20.
16.	Forni G. Giovarelli M, Cavallo F, e/ al. Cytokine-in-duced tumor immunogenicity: from exogenous cytokines to gene therapy. J /miiiuiiother 1993; 14: 2537.
17.	Perucho M, Hanahan D, Wigler M. Genetic and physical linkage of exogenous sequences in transformed cells. Cell 1980; 22:"309-17.
18.	Bosms SS. Targeted gene modification for gene therapy of stem cells. /»/ ./ Cell Cloiiiiig 1990: st80-96.
19.	Kubiniec RT, Liang H, Hui SW. Effects of pulse length and pulse strength on transfection by electroporation. Biofecluiiques 1990: 8: 16-20.
20.	Hug P, Sleight RG. Liposomes for the transfornationof eukaryotic cells. Biochiiii Bioplns Acta 1991: 1097: 1-17.
21.	Vitadcllo M, Schial'fino MV, Picard A. e/ al. Gene transfer in regenerating muscle. Hum Ueiie Ther 1994; 5: 11-8,
22.	Wagner E, Curiel D. Colten M. Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis. Adv Drug Del 1994; 14: 113-35.
23.	Afione AS, Conrad KC. Flotte RT. Gene Iherapy vectors as drug delivery systems. Cliii Pliarmacokiiiei 1995; 28: 181-9.
24.	Zwiebel AJ, Su N, MacPherson A, Davis T, Ojeifo OJ. The gene therapy of cancer: transgenic immunotherapy. Semii/ Hematol 1993: 30: 119-29.
25.	Berd D, Maguire HC, Mastrangelo MJ. lnduction of cell-mediated immunity to autologous melanoma cells and regression of metastases after treatment with a melanoma cell vaccine preceded by cyclophosphamide. Caiicer Res 1986; 46: 2572-8.
26.	Linsley PS, Brady W, Grosmaire L. Arulf'o A, Damle NK, Ledbetter JA. Binding of B cell activation antigen B7 to CD28 costimulates T cell proliferation and inter-leukin 2 mRNA accumulation. J Exp Med 1991: 173: 721-30.
27.	Townsend SE, Allison JP. Tumor rejection alter direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science 1993; 259: 368-70.
28.	Colombo MP, Ferrari G. Stoppacciaro A, et al. Granulocyte colony- stimulating factor gene transfer suppresses tumorigenicity of a murine adenocarcinoma iii vivo. J Erp Med 1991; 173: 889-97.
29.	Colombo MP, Lombardi L, Stoppacciaro A, et al. Gran-ulocyte-colony stimulating factor (G-CSF) gene transduction in murine adenocarcinoma drives neutrophil-mediated tumor inhibition /'» vivo. J /minimal 1992; 149: 113-9.
30.	Dranoff G. Jalfee E. Lazenby A. et al. Vaccination with irradiated tumor cells engineered to secrete murine GM-CSF stimulates potent, specific and long lasting anli-tumor immunity. Pmc Natl Acad Sci USA 1993: 90: 3539-43,
31.	Asher AL, Mule JJ, Kasid A, et al. Murine cells transduced with the gene I'or tumor necrosis factor . Evidence for paracrine immune effects of tumor necrosis factor against tumors. J /minima/ 1991; 146: 3227-34.
32.	Hock H, Dorsch M, Kunzendorf Uquin Z, Diamanstein T, Blankenstein T. Mechanisms of rejection induced by tumor cell-targeted gene transfer of interleukin 2. interleukin 4, interleukin 7, tumor necrosis factor, or interferon . Pmc Natl Acad Sri USA 1993; 90: 2774-8.
33.	Fearon ER, Pardoll DM, ltaya T, et al. lnterleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response. Cell 1990: 60: 397-403.
34.	Gansbacher B, Zier K, Daniels ß, e/ al. Interleukin-2 gene transferinto tumor cells abrogates tumorigenicity and induces protective immunity. J Exp Med 1990: 172: 1217-24.
35.	Gansbacher B. Zier K. Cronin K. et al. Retroviral gene transfer induced constitutive expression of interleukin-2 or interferon gamma in irradiated human melanoma cells. Blood 1992; 80: 2817-25.
36.	Russell SJ, Eccles SA, Flemining CL, et al. Decreased tumorigenicity of a transpantable rat sarcoma following transfer and expression of an IL-2 cDNA. /iit J Caiicer 1991; 47: 244-51.
37.	Cavallo F, Giovarelli M. Guliano A, et «l. Role of neutrophils and CD4+ T lymphocytes in the primary and memory response to noniinmunogenic murine mammary adenocarcinoma made imunogenic by IL-2 gene. J /iiiiiiwwl 1992; 149: 3627-35.
Current approaches to gene therapy in onco/ogy: Construction of tumor vaccines
267
38.	Allione A, Consalvo M, Nanni P, et a/. Immunizing and curative potential of replicating and nonreplicating murine mammary adenocarcinoma cells engineered with interleukin (IL)-2, IL-4, IL-6, IL-7, IL-10, tumor necrosis factor , granulocyte-macrophage colony-stim-ulaling factor, and -interferon gene or admixed with conventional adjuvants. CancerRes 1994; 54: 6022-6.
39.	Hock H, Dorsch M, Kunzendorf U, et al. Vaccinations with tumor cells genetically engineered to produce different cytokines: effectivity not superior to a classical adjuvant. Cancer Res 1993; 53: 1-3.
40.	Blankenstein T. Observations with tumor necrosis lac-tor gene-transfected tumours. Folia Bio/ - Prague 1994; 40: 19-28.
41.	Mulligan R. The basic science of gene therapy. Science 1993: 260: 926-32.
42.	Golumbek TP. Azhari R, Jaffee ME, ft al. Controlled release, biodegradable cytokine depots: a new approach in cancer vaccine design. Cancer Res 1993: 53: .5841-4.
43.	Van der Brugen P, Traversari C. Choniez P, et a/. Agene encoding an antigen recognized cytotoxic T lymphocytes on a human melanoma. Science 1991; 254: 1643-8.
44.	Moolten FL, Wells JM, Heyman RA, et al. Lymphoma regression induced by ganciclovir in mice bearing a herpes thymidine kinase transgene. Hum Geiie Ther 1990; 1: 125-34.
45.	Moolten FL, Wells JM. Curability of tumors bearing herpes thymidine kinase genes transferred ba retroviral vectors. JNCI 1990; 82: 297-300.
46.	.Short MP, Choi BC, Lee JK, et al. Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line. ./ Neu/osci Rev 1990; 27: 427-39.
47.	Culver KW, Ram Z, Wallbridge S, el al- In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 1992: 256: 1550-2.
48.	Rosenberg AS. Adoptive immunotherapy for cancer. Sci Am 1990; 262: 62-9.
49.	Schirnnacher V. Biotherapy of cancer. ./ Cancer Res C/in Onco/ 1995; 121: 443-51.
50.	Rosenberg AS, Anderson WF, Blaese M, et al. The development of gene therapy lor the treatment of cancer. Ann Surg 1993; 218: 455-64.
51.	Dalgleish A. The case lor therapeutic vaccines. Melanoma Res 1996: 6: 5-10.