'1-8 interferon inducible gene family': putative colon carcinoma-associated antigens.

D(b-/-)xbeta2 microglobulin (beta2m) null mice transgenic for a chimeric HLA-A2.1/D(b)-beta2m single chain (HHD mice) are an effective biological tool to evaluate the antitumour cytotoxic T-lymphocyte response of known major histocompatibility-restricted peptide tumour-associated antigens, and to screen for putative unknown novel peptides. We utilised HHD lymphocytes to identify immunodominant epitopes of colon carcinoma overexpressed genes. We screened with HHD-derived lymphocytes over 500 HLA-A2.1-restricted peptides derived from colon carcinoma overexpressed genes. This procedure culminated in the identification of seven immunogenic peptides, three of these were derived from the 'human 1-8D gene from interferon inducible gene' (1-8D). The 1-8D gene was shown to be overexpressed in fresh tumour samples. The three 1-8D peptides were both antigenic and immunogenic in the HHD mice. The peptides induce cytotoxic T lymphocytes that were able to kill a colon carcinoma cell line HCT/HHD, in vitro and retard its growth in vivo. One of the peptides shared by all the 1-8 gene family primed efficiently normal human cytotoxic T lymphocyte precursors. These results highlight the 1-8D gene and its homologues as putative immunodominant tumour-associated antigens of colon carcinoma.

O-glycosylated versus non-glycosylated MUC1-derived peptides as potential targets for cytotoxic immunotherapy of carcinoma.

Due to the fact that many cellular proteins are extensively glycosylated, processing and presentation mechanisms are expected to produce a pool of major histocompatibility complex (MHC) class I-bound protein-derived peptides, part of which retain sugar moieties. The immunogenic properties of the presented glycosylated peptides in comparison to their non-glycosylated counterparts have not been determined clearly. We assessed the cellular immunogenicity of MUC1 (mucin)-derived peptides O-glycosylated with a Tn epitope (GalNAc) using HLA-A*0201 single chain (HHD)-transfected cell lines and transgenic mice. For part of the compounds Tn moiety did not interfere with the HLA-A*0201 binding. Moreover, part of the glycopeptides elicited effective cytotoxic responses, indicating recognition of the glycopeptide-HLA-A*0201 complex by the T cell receptor (TCR) and subsequent cytotoxic T lymphocyte (CTL) activation. The CTLs exhibited a substantial degree of cross-reactivity against target cells loaded with glycosylated and non-glycosylated forms of the same peptide. The studied (glyco)peptides showed cellular immunogenicity in both MUC1-HHD and HHD mice and induced effective lysis of (glyco)peptide-loaded target cells in CTL assays. However, the elicited CTLs did not induce selective lysis of human MUC1-expressing murine cell lines. Moreover, immunization with (glyco)peptide-loaded dendritic cells (DCs) did not induce significant immunotherapeutic effects. We conclude that Tn glycosylated MUC1-derived peptides can be presented by MHC class I molecules, and may be recognized by specific TCR molecules resulting in cytotoxic immune responses. However, the studied glycopeptides did not offer significant benefit as targets for cytotoxic immune response due apparently to (a) cross-reactivity of the elicited CTLs against the glycosylated and non-glycosylated forms of the same peptide and (b) low abundance of glycopeptides on tumour target cells.

MAGE-A8 overexpression in transitional cell carcinoma of the bladder: identification of two tumour-associated antigen peptides.

Bladder carcinoma is the fourth most common cancer in men and the eighth most common cancer among women. Our study is aimed to characterise tumour-associated antigen peptides of transitional cell carcinoma of the bladder (TCC). A DNA micro-array-based differential display analysis of 10 000 genes was carried out, and MAGE-A8 gene expression was detected in the tumour, and not in the normal bladder. High occurrence of MAGE-A8 expression was observed in fresh tumour samples (17 out of 23) and TCC lines (four of eight). The MAGE-A8 protein sequence was screened for HLA-A2.1-binding motifs, six potential peptides were synthesised, and peptides binding to HLA-A2.1 were assured. Immunogenicity and antigenicity of the MAGE-A8 peptides were examined in the HHD system, murine class I MHC knockout mice, transgenic for HLA-A2.1. The MAGE-A8 peptide immunogenicity was examined in three modes of vaccination, delivered intranasally with cholera toxin, injected into the tail base with complete Freund's adjuvant (CFA), or presented directly as loaded onto cell surface HLA-A2.1 molecules. Two peptides, 8.1 and 8.3, induce CTL that kills the T24 TCC line in vitro, and prime human lymphocyte response of healthy donors. These results demonstrate the potential use of the MAGE-A8 peptides for specific immunotherapy of TCC.

Anti-tumor vaccination in heterozygous congenic F1 mice: presentation of tumor-associated antigen by the two parental class I alleles.

Peptide vaccination of homozygous mice against syngeneic tumors using single major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) epitopes elicits effective immune responses against metastatic growth. So far, single-peptide vaccination of patients against their autologous tumors seems to elicit less satisfactory results. In this study, the authors tried to determine whether effective anti-metastatic immunity requires the presentation of peptides restricted by the two parental class I major histocompatibility complex alleles in heterozygous hosts. The immune response against the H-2b-derived 3LL Lewis lung carcinoma was evaluated in heterozygous recombinant congenic F1 mice (Kk x K(b)) and (Kd x K(b)). Vaccination of such heterozygous animals with dendritic cells expressing the two parental H-2K alleles, pulsed with total tumor extract, elicited a potent anti-metastatic response. A comparable response was obtained after vaccination with tumor cells genetically modified to express the two class I alleles. In contrast, vaccination of the heterozygous mice with dendritic cells expressing only one of the parental F1 H-2K alleles or with tumors expressing only one H-2K allele failed to elicit effective immunity against tumor metastasis in recombinant congenic F1 mice. It appears, therefore, that to achieve effective anti-metastatic immunotherapy in heterozygous organisms, presentation of cytotoxic T lymphocyte epitopes restricted by the two parental class I alleles is required.

Induction of antitumor immunity by proteasome-inhibited syngeneic fibroblasts pulsed with a modified TAA peptide.

CTLs specific for tumor antigens play a major role in immunity against cancer. Improved binding affinity of putative TAA peptides could enhance the in vivo immunogenicity of these self-altered self- tumor antigens. We examined here the efficacy of tumor vaccines composed of an altered peptide ligand of MUT-1, designated MUT-D, which exhibited significantly higher class-I allele K(b) binding affinity than its native counterpart MUT-1. The peptide was loaded on antigen presenting cells composed of the C57BL/6-syngeneic fibroblast cell line BLK.CL4. These cells were treated with proteasome inhibitor in order to shut off the degradation of proteins and the subsequent loading of endogenous peptides onto MHC class-I molecules, thus allowing for the pulsing of these cells with the modified peptide MUT-D. Proteasome-inhibited and modified peptide-loaded fibroblasts induced a peptide-specific CTL that significantly delayed primary tumor progression and protected the pre-immunized mice against the development of lung metastasis following the surgical removal of the primary tumor. Genetic modification of the fibroblasts to express the immunostimulatory cytokine IL-2 did not improve the APC function of the modified cells, nor did it result in augmentation of the potency of the vaccine. Our results suggest that the proteasome-inhibited fibroblasts pulsed with modified, high binder tumor-associated antigen peptide are good antigen-presenting cells and represent an effective form of tumor vaccine.

Antigenicity and immunogenicity of an intracellular delivery system of major histocompatibility complex class I epitopes that bypasses proteasome processing.

The development of a cell-free synthetic vaccine to induce an effective cytotoxic T lymphocyte response is an important challenge in T-cell--mediated immunity. Because standard vaccinations with nominal epitopes were found to be only partially effective in vivo, the authors suggest an alternative strategy: the delivery of epitopes directly to the cell cytosol in a proteasome bypass mechanism of processing. Two model peptides, the presentation level on the cell surface of which can be directly assessed, were conjugated via a cross-linker to an internalization peptide derived from an antennapedia homeobox protein. The linker was designed to undergo spontaneous hydrolysis, after which the epitope is subsequently released. The conjugates were shown to enter RMA and P815 cells, where the epitopes were released mainly in cytosol and endogenously loaded on the major histocompatibility complex class I molecules to be presented on the cell surface. Concomitant inhibition of proteasome activity by MG132 significantly increased the presentation level of both model peptides, indicating proteasome-independent processing. This phenomenon was exploited to enhance the immunogenicity of the conjugates. Conjugates were emulsified with MG132 in incomplete Freund's adjuvant and injected into mouse footpads. Analysis of the draining lymph nodes indicated an increase in the percentage of both CD4+ and CD8+ lymphocytes. In vitro cytolytic assays implied significant, albeit moderate, priming only when the proteasome inhibitor was administered with the conjugate. This approach may be useful for the development of efficient synthetic cell-free vaccines.

Immunogenicity of H-2Kb-low affinity, high affinity, and covalently-bound peptides in anti-tumor vaccination.

CTL induction by immunization with synthetic peptide epitopes has been shown to inhibit tumor growth and its metastatic spread. Ex vivo pulsing of peptides on MHC class I-bearing cells such as RMA-S cells or professional APCs elicits an effective CTL response. Since the stability of the MHC-peptide complex is strongly correlated with the overall immunogenecity, we compared the effect of immunization with low affinity, high affinity, and irreversibly bound MHC peptides in the context of immunotherapy of metastasis. MUT1, a tumor-associated antigen peptide that was isolated from 3LL Lewis lung carcinoma, is a low H-2Kb binder. MUT1 was modified into a high binder by changing positions 3, 5, and 8 to the favorable anchor residues. In addition, we introduced a photo-active chemical moiety, which can bind irreversibly to MHC upon illumination. These peptides, loaded onto RMA-S, were used to immunize mice against the 3LL tumor. Vaccination via the covalent conjugation of the low binder peptide was found to increase the CTL response measured against MUT1 loaded cells and against H-2Kb transfected D122 cells relative to the native MUT1 peptide. However, the photo cross-linking of the high affinity peptide to the MHC did not significantly improve the induction of specific CTL. The level of CTL activity was elevated to the same extent by either cross-linking the peptide to the MHC or by modifying it into a high-binder peptide. The protective capacity of all the peptide-based vaccines against D122 metastatic spread to the lungs was found to be comparable. These results indicate that augmentation of the affinity of a TAA peptide to the RMA-S surface MHC molecules, by conversion to a high-affinity mimotope or by photo-conjugation, can significantly enhance the immune response. There seems to be, however, a ceiling beyond which increase in the peptide-binding affinity does not lead to a corresponding enhancement of the overall immunogenicity of the peptide.

MHC class I-restricted epitope spreading in the context of tumor rejection following vaccination with a single immunodominant CTL epitope.

Epitope spreading is a process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become targets of an evolving immune response. This phenomenon has been associated most notably with the progression of naturally occurring or experimentally induced chronic autoimmune diseases. We have investigated the potential occurrence of epitope spreading in the context of antitumor cytotoxic T cell (CTL) responses using chicken ovalbumin (OVA) as a model antigen. Our results indicate that following rejection of OVA-expressing EG.7 tumor cells effectuated by a CTL response which is induced against the MHC class I-restricted immunodominant epitope OVA257-264, there occurs intramolecular diversification of the CTL response to two additional OVA-derived epitopes, OVA176-183 and OVA55-62, as well as intermolecular spreading to other endogenous tumor-derived determinants. It seems that CTL-mediated tumor cell destruction in vivo favors cross-presentation of additional epitopes with the consequent activation of additional tumor-reactive lymphocytes. The process of epitope spreading in that context has obvious important implications for the design of antigen-specific antitumor immunotherapies.

Induction of antitumor immunity with modified autologous cells expressing membrane-bound murine cytokines.

Development of cytokine gene-modified autologous tumor vaccines must take into account the strictly paracrine physiology of cytokines whose expression at the tumor microenvironment is important for the successful induction of tumor-specific immunity. In this study, we investigated the efficacy of a tumor vaccine composed of inactivated autologous cells transfected with two plasmid vectors encoding a mutant membrane-bound murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and murine interferon-gamma (MuIFN-gamma). Expression of both cytokines as cell surface ligands on the highly metastatic D122 clone of Lewis lung carcinoma led to abrogation of their tumorigenicity and metastatic phenotype. More importantly, vaccination with irradiated tumor cells expressing the membrane-bound GM-CSF and IFN-gamma induced a cytotoxic T lymphocyte (CTL) response that protected syngeneic mice against a subsequent challenge with D122 cells as a primary tumor in preimmunized mice as well as against lung metastasis developing after surgical removal of the primary tumor in naive mice. Autologous cells expressing the membrane-bound GM-CSF and IFN-gamma exhibited comparable efficacy as an antimetastatic vaccine to a vaccine composed of transfectants expressing wild-type secreted cytokine molecules. These results indicate that membrane-bound cytokines can cause enhanced immunogenicity when transfected into tumor cells for the induction of antitumor immunity.

Antimetastatic vaccination against Lewis lung carcinoma with autologous tumor cells modified to express murine interleukin 12.

Interleukin 12 (IL-12) is a disulfide-linked heterodimer molecule produced predominantly by professional antigen presenting cells. It promotes the induction of sundry biological effects with significant relevance to antitumor immunity, such as enhancing a T(H)1 helper response, an in vivo antiangiogenic effect, induction of adhesion molecules that assist in lymphocyte homing to sites of tumor growth, and a direct stimulatory effect on both T-cells and NK cells. We tested the efficacy of an antimetastatic vaccine composed of autologous murine D122 cells transfected with both subunits of IL-12 cDNA to express biologically-active IL-12 molecule. Expression of IL-12 by D122 cells significantly reduced their tumorigenicity and metastatic potential in immunocompetent syngeneic hosts. Furthermore, vaccination of mice with 2 x 10(6) irradiated IL-12-transfected D122 cells engendered a protective CTL response which rejected a subsequent challenge with parental D122 cells and eradicated lung micrometastasis in animals whose primary tumors have been surgically removed. The antitumor effects of IL-12 were mediated primarily by its ability to induce gammaIFN expression in vivo. CD8+ T-cells as well as NK cells were crucial in the execution of the antitumor effects of IL-12. These results suggest that autologous tumor cells expressing IL-12 by gene transfer are a potent antitumor vaccine able to induce a systemic immune response against poorly immunogenic and spontaneously metastatic tumors.

DAP kinase links the control of apoptosis to metastasis.

DAP kinase is a new type of calcium/calmodulin-dependent enzyme that phosphorylates serine/threonine residues on proteins. Its structure contains ankyrin repeats and the 'death' domain, and it is associated with the cell cytoskeleton. The gene encoding DAP kinase was initially isolated as a positive mediator of apoptosis induced by interferon-gamma, by using a strategy of functional cloning. We have now tested whether this gene has tumour-suppressive activity. We found that lung carcinoma clones, characterized by their highly aggressive metastatic behaviour and originating from two independent murine lung tumours, did not express DAP kinase, in contrast to their low-metastatic counterparts. Restoration of DAP kinase to physiological levels in high-metastatic Lewis carcinoma cells suppressed their ability to form lung metastases after intravenous injection into syngeneic mice, and delayed local tumour growth in a foreign 'microenvironment' Conversely, in vivo selection of rare lung lesions following injection into syngeneic mice of low-metastatic Lewis carcinoma cells or of DAP kinase transfectants, was associated with loss of DAP kinase expression. In situ TUNEL staining of tumour sections revealed that DAP kinase expression from the transgene raised the incidence of apoptosis in vivo. DAP-kinase transfectants also showed increased sensitivity in vitro to apoptotic stimuli, of the sort encountered by metastasizing cells at different stages of malignancy. We propose that loss of DAP kinase expression provides a unique mechanism that links suppression of apoptosis to metastasis.

Modification of PDGFalpha receptor expression or function alters the metastatic phenotype of 3LL cells.

Functional PDGFalpha receptors are selectively expressed on highly lung-metastasizing clones of the 3LL Lewis lung carcinoma, but not on low-mestastatic clones. The highly metastatic clones are also growth induced in vitro by PDGF and lung conditioned medium. To investigate whether modification of PDGFalpha receptor expression or function can affect metastatic capability, we transfected cells of a low-metastatic 3LL clone with a full length PDGFalpha receptor gene and cells of a highly-metastatic clone with a truncated kinase domain PDGFalpha receptor gene. Introduction of the full length PDGFalpha receptor conferred upon low-metastatic cells the ability to grow in vitro in the presence of PDGF-AA and to colonize the lung in experimental and spontaneous metastases assays. Conversely, introduction of a truncated version of the PDGFalpha receptor into highly metastatic cells reduced their metastatic load to control levels. Accordingly, their responses to PDGF-AA, including growth stimulation and receptor autophosphorylation, were reduced. These results demonstrate that PDGFalpha receptor expression and function can control the capacity of tumor cells to generate metastases in the lung. The response of this receptor to lung-derived PDGF-like factors may define a paracrine mode of metastatic cell growth in the target organ.

Tuftsin-THF-gamma 2 chimeric peptides: potential novel immunomodulators.

Synthesis of two chimeric peptides composed of tuftsin and thymic humoral factor-gamma 2 (THF-gamma 2) conjugates was accomplished. Our goal was the generation of novel immunomodulators. Initially, we demonstrate an IL-6 inducing activity of the phagocytic cells stimulant, tuftsin, on murine macrophages. This activity was documented only in the presence of antigen, either KLH or lysozyme. The augmentation was dose dependent, with optimal activity at a concentration of 200 and 20 nM, respectively. The chimeric peptides, either H2N-tuftsin-THF-gamma 2-OH or H2N-THF-gamma 2-tuftsin-OH, were also evaluated in the IL-6 system in the presence of the more potent antigen, KLH. The IL-6 inducing effect was maintained, although maximal activity appeared only at a concentration an order of magnitude greater than that of tuftsin. The chimeric peptides were further tested in an assay evaluating enhancement in murine bone marrow myeloid colony formation, a system in which THF-gamma 2, a T cell stimulant, has an established beneficial effect. The compounds were found to be inactive at the 25-200 ng/ml (14-112 nM) concentration range evaluated. Finally, the chimeric peptides were tested in a combined macrophages-T cells assay, i.e. antigen presentation, in which H2N-tuftsin-THF-gamma 2-OH was found to be more active than either parent peptide, thus representing a possible therapeutic agent.

Identification of shared tumor-associated antigen peptides between two spontaneous lung carcinomas.

CTLs recognize antigenic peptides bound to MHC class I Ags on the cell surface of tumor cells. Tumor-associated Ag (TAA) peptides are 8 to 10 amino acids long and can be derived from normal, mutated, or viral proteins. The majority of T cell-defined Ags have been identified in human melanoma cells. These were shown to be commonly expressed by different allogeneic melanomas that share the same MHC molecule. We have recently isolated Kb-restricted TAA peptides, which are mutations of the gap junction protein connexin 37, from the spontaneous C57BL/6 Lewis lung carcinoma (3LL). These peptides, named MUT 1 and MUT 2, serve as CTL epitopes and can induce CTL activity in vivo. Using CTL cross-reaction assays, peptide extraction, HPLC fractionation, and reverse transcriptase-PCR amplification, we show that clones of another spontaneous C57BL/6 lung carcinoma, CMT 64, share TAA peptides with the 3LL carcinoma. Vaccination with synthetic MUT 1 or MUT 2 induces CTLs that efficiently lyse CMT 64-derived clones, protects mice from CMT 64 metastasis, and affords therapy of established CMT 64 metastases. Hence, shared CTL epitopes exist between two spontaneous murine lung carcinomas.

Expression of two H-2K genes, syngeneic and allogeneic, as a strategy for potentiating immune recognition of tumor cells.

Metastatic clones of some tumors manifest an impaired expression of class I major histocompatibility complex (MHC) antigens. High metastatic, low immunogenic Lewis lung carcinoma clones (C57BL-H-2b origin) express low levels of the H-2Kb MHC antigen. These cells metastasize spontaneously in C57BL/6J mice. Transfection of syngeneic or allogeneic H-2K genes converted such cells to the nonmetastatic state, but did not prevent the growth of the local tumors. Transfection of two H-2K genes, syngeneic and allogeneic, into the highly metastatic clone D122, resulted in reduction of the growth rates of the transfectants and protected the mice from D122 metastases. In contrast, cells transfected with a single class I gene (syngeneic or allogeneic) gave partial protection, or did not protect the mice at all from D122 metastases. The combination of syngeneic and allogeneic genes in the same tumor cell elevated the immunogenic properties of the expressing cells and potentiated the immune response as was demonstrated by in vitro cytotoxicity analysis and by limiting dilution cytotoxicity analysis. Increased immunogenicity by double transfection may constitute an effective therapeutic modality.

Regression of established murine carcinoma metastases following vaccination with tumour-associated antigen peptides.

The cure of micrometastases following surgery is the major goal of cancer immunotherapy. We have recently isolated tumour-associated antigen (TAA) peptides, MUT 1 and MUT 2, derived from a mutated connexin 37 gap-junction protein, from the malignant 3LL-D122 murine lung carcinoma. We now report that synthetic MUT 1 or MUT 2 induces effective antitumour cytoxic T lymphocytes. Peptide vaccines protect mice from spontaneous metastases of 3LL-D122 tumours. Moreover, peptide vaccines reduce metastatic loads in mice carrying pre-established micrometastases. Tumour-specific immunity was primarily mediated by CD8+ T cells. This is the first evidence that peptide therapy may be effective in treatment of residual tumours and provides a rationale for the development of peptide vaccines as a modality for cancer therapy.

Combined vaccination with major histocompatibility class I and interleukin 2 gene-transduced melanoma cells synergizes the cure of postsurgical established lung metastases.

We have analyzed and compared in detail the malignant phenotypes of, the immune mechanisms induced by, and the immunotherapeutic potentials of B16-F10.9 melanoma cells manipulated by gene transfer to express syngeneic H-2Kb molecules or to secrete the cytokines interleukin 2 (IL-2) or IL-6. Local tumor growth in the footpad of transduced cells is mainly retarded by expression of H-2Kb and IL-2 genes and less by expression of IL-6. Mice given injections intrafootpad of tumorigenic doses of transduced clones manifested significantly reduced postsurgical spontaneous metastasis. After i.v. inoculation, mice given injections of F10.9-Kb expressors did not develop experimental lung metastases; mice given injections of F10.9-IL-6 secretors developed reduced metastatic loads; whereas mice given injections of F10.9-IL-2 secretors developed high loads of lung metastases. On the basis of injections into nude mice, in vivo depletions of CD4+, CD8+, and NK1.1+ cells, and in vitro CTL and natural killer (NK) assays, we show that all F10.9-modified cells induce CD8+ tumor-specific CTL activity and that F10.9-IL-2 secretors also induce nonspecific NK/lymphokine-activated killer cell activity. Vaccinations with F10.9-modified cells were capable of significantly reducing metastatic spread from small established F10.9 footpad tumors. However, in mice carrying preestablished lung metastases, a highly therapeutic effect was achieved only when H-2Kb expressors and IL-2 secretors were combined in vaccination, whereas individual vaccines or other combinations had marginal effects. This higher efficiency of the combined vaccine is due to the combined effect of efficient CTL induction and NK/lymphokine-activated killer cell activity as concluded from depletion of CD8+ and NK1.1 cells during immunotherapy. Thus, the cure of established metastasis can be achieved by the synergistic effects of vaccination with class I and IL-2-transduced tumor cells.

Effective anti-metastatic melanoma vaccination with tumor cells transfected with MHC genes and/or infected with Newcastle disease virus (NDV).

The therapeutic efficacy of active immunization with B16-F10.9 melanoma cells transfected with syngeneic major histocompatibility complex (MHC) class-I genes, modified by infection with Newcastle Disease virus (NDV) or modified by both treatments, was compared. B16-F10.9 tumor-bearing mice were treated at various stages of tumor growth and metastasis with irradiated, modified tumor-cell vaccines. Irradiated tumor cells and H-2Db transfectants did not stimulate anti-tumor immunity while H-2Kb transfectants and NDV-modified F10.9 cells showing low and high expression of MHC class-I genes efficiently prevented metastasis of small established tumors. NDV-modified parental-cell vaccines functioned optimally and improved overall survival by about 60%, also at early stages of metastasis establishment. A synergistic effect of H-2Kb expression and virus modification on rejection of micrometastases was observed in mice bearing advanced tumors. Postoperative vaccination of mice carrying multiple metastases with NDV-modified vaccines caused significant, but incomplete, reduction of metastatic tumor load. The therapeutic effect of NDV-modified tumor vaccines was dependent on multiple immune mechanisms. Depletion of CD8, CD4 or NK cells by in vivo treatment with monoclonal antibodies reversed the immunotherapeutic effects of the vaccine. Thus, tumor xenogenization and gene modification may act synergistically to vaccinate against advanced tumors, while single modalities can effectively vaccinate against metastasis at early stages of tumor growth.

Immunotherapy via gene therapy: comparison of the effects of tumor cells transduced with the interleukin-2, interleukin-6, or interferon-gamma genes.

We have comparatively analyzed the immune mechanisms induced by and the immunotherapeutic potentials of a highly metastatic clone of the Lewis lung carcinoma, D122, transduced with the interleukin-2 (IL-2), IL-6, or interferon-gamma (IFN-gamma) genes. All of the D122 cytokine gene-transduced cells induced antitumor CD8+ cytotoxic T lymphocytes (CTLs), as can be judged from in vivo depletion of CD8+ cells and in vitro CTL assays. In vivo depletion of CD4+ cells did not affect the malignant phenotypes of the different D122 gene-modified cells, but in vivo depletion of natural killer (NK) cells resulted in increased malignancy of both D122 cells and D122 gene-modified cells. In accordance with the effects of in vivo NK depletion, D122 as well as D122 derivative cells were sensitive to lysis by polyinosinic-polycytidylic acid (poly I:C)-induced activity. We discuss the immune responses generated by the different D122 gene-modified cells in view of their in vivo behavior in syngeneic and nude mice. We also performed comparative analysis of the capacity of vaccinations with irradiated D122 gene-modified cells to cure established micrometastases of parental D122 cells in tumor-operated mice. Vaccinations with D122-IL-2 or -IL-6 secretors did not generate a significant effect. Also, vaccinations with D122-IFN-gamma cells, which showed increased major histocompatibility complex class I expression but did not secrete detectable levels of IFN-gamma, did not cure established micrometastases. Only vaccination with D122-IFN-gamma high secretors efficiently cured postoperated mice carrying established micrometastases. We discuss the relevance of these results to the application of immunotherapy via cytokine gene therapy of human malignancy.

Interleukin 6 gene transfection into Lewis lung carcinoma tumor cells suppresses the malignant phenotype and confers immunotherapeutic competence against parental metastatic cells.

To investigate the influence of interleukin 6 (IL-6) production on malignancy of tumor cells we transfected cells of the high-metastatic, low-immunogenic D122 clone of the Lewis lung carcinoma with a mammalian expression vector containing the human IL-6 complementary DNA. In vitro, IL-6 positive transfectants showed growth inhibition that was directly correlated with the levels of IL-6 production. The in vitro growth arrest did not seem to be a function of an autocrine system mediated via the secreted human IL-6 acting on the tumor cell surface receptors since neutralizing antibodies to human IL-6 did not prevent the growth inhibition. Neither did exogenous human recombinant IL-6 affect the growth of D122 cells. In vivo, IL-6 positive transfectants showed reduction of tumorigenicity and significant suppression of metastatic competence in syngeneic, immunocompetent mice. In mature T-cell deficient nude mice, the IL-6 transfectants showed some arrest of local growth but no suppression of lung metastasis. It seems therefore that the reduction of metastatic competence of IL-6 transfectants is primarily a function of stimulation by the transfectants of host T-cell immune responses. Immunization with inactivated high-positive IL-6 transfectants induced high levels of anti-tumor cytotoxic T-lymphocytes and protected mice against metastatic growth of a subsequent graft of parental tumor cells. Moreover, reduction of metastatic growth of parental highly metastatic D122 cells was also achieved when immunization of mice was begun after establishment of the primary parental tumors. Thus, inactivated IL-6 transfectants were effective when used as a cellular vaccine for experimental immunotherapy of metastasis.