Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines.

Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA-A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. E75-pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA-A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens.

Melanoma differentiation-associated gene-7 (mda-7)/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model.

Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3(+)CD8(+) but not the CD3(+)CD4(+) cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.

Increased sensitivity of adriamycin-selected tumor lines to CTL-mediated lysis results in enhanced drug sensitivity.

The emergence of drug resistance to chemotherapeutic agents is a major cause of treatment failure in cancer therapy. Therefore, much effort has been aimed at circumventing or reversing this undesired effect. Recently, we found that tumor cell lines selected for their multidrug-resistant phenotype can also exhibit increased levels of TAP mRNA and MHC class I proteins. This raised the question of whether drug-resistant tumors are more readily recognized by MHC-restricted CTLs. In this report, we show that five of five MHC class I+ tumor cell lines grown in medium containing Adriamycin developed into variants that expressed higher levels of MHC class I than did their corresponding parental cell lines. This was not observed with a MHC class I- cell line. No similar association was noted for changes in the expression of either HER-2 or intercellular adhesion molecule 1 protein. We also found that MHC class I+ drug-selected variants were more readily lysed by MHC-restricted, tumor-associated CTLs than were the drug-sensitive parental cell lines. When the drug-selected variants were cocultured with the same CTLs to eliminate tumor cells expressing higher levels of MHC-I (MHC-Ihi), the CTL-resistant tumor cells exhibited a drug sensitivity profile similar to that of the parental cell lines that were not exposed to Adriamycin. These findings suggest that certain chemotherapeutic drugs may increase the immunogenicity of some tumors, and that CTL immunotherapy may help reverse drug resistance.

Tumor cytolysis by lymphocytes infiltrating ovarian malignant ascites.

Tumor-associated lymphocytes (TAL) were isolated from the ascitic fluid of patients with adenocarcinoma of the ovary. These cells proliferated and expanded by 100-600-fold as either CD3+ CD4+ or CD3+ CD8+ cultures in the presence of moderate concentrations (50-200 cetus units/ml) of recombinant interleukin 2 and reached high numbers (5 x 10(8)-1 x 10(9)). After expansion of 16 TAL samples from 15 patients, 5 of the 7 isolated ovarian cytotoxic T-lymphocyte cell lines of T-cell receptor (TCR) (alpha beta)+ CD3+ CD8+ CD4- phenotype exhibited preferential cytolytic activity against autologous tumor targets and significantly lower cytolytic activity against allogeneic tumor targets and the natural killer-sensitive cell line K562. The cytolytic activity of the CD8+ TAL was inhibited by operationally anti-TCR (alpha beta) monoclonal antibody and monoclonal antibody specific for the CD3 differentiation antigen, indicating that the TCR and CD3 are involved in the cytolytic process. The other TAL cultures demonstrated similar cytolytic activity against both autologous and allogeneic tumors. The phenotype of these TAL was predominantly TCR (alpha beta)+ CD3+ CD4+ CD8-. Certain CD3+ CD8+ T-cell clones isolated from representative TAL exhibited preferential autologous tumor-specific cytotoxicity that may be major histocompatibility complex restricted. Other CD3+ CD8+ and CD3+ CD4+ clones exhibited nonmajor histocompatibility complex restricted cytotoxicity. These results demonstrate that CD3+ CD4+ and CD3+ CD8+ T-cells present in the ovarian malignant ascites can be propagated in large numbers and for long time intervals as T-cell lines in vitro. This finding may be significant for further investigation of ovarian tumor-specific cytotoxic T-lymphocytes and future adoptive specific immunotherapy studies.

Identification of naturally processed human ovarian peptides recognized by tumor-associated CD8+ cytotoxic T lymphocytes.

Identification of naturally processed peptides recognized by tumorspecific CTLs may lead to epitope-specific tumor vaccines. Because these epitopes may be expressed differently on epithelial tumors and may differ in their ability to induce CTL in vivo, we have isolated the HLA-A2-peptide complexes by immunoaffinity from an established ovarian tumor line transfected with and expressing HLA-A2 gene. High-performance liquid chromatography-fractionated peptides were used to reconstitute epitopes recognized on HLA-A2 by three HLA-A2+ CD8+ CTL lines. These lines recognized at least three of the same groups of fractions (designated SKOV3.A, -B, and -C) but showed differences in the pattern of recognition of other fractions. To gain insight in the epitope distribution by freshly isolated ovarian tumors, we compared the recognition of peaks SKOV3.B and -C with the corresponding peaks from an ovarian tumor (OVA-6) that expressed similar levels of HLA-A2, using one of these lines (CTL-OVA-5) as indicator. CTL-OVA-5 recognized a large number of epitopes from peaks B and C rechromatographed on more resolving high-performance liquid chromatography gradient. Although a number of peaks appeared to be coincident on both SKOV3 and OVA-6, an even higher number appeared either not to overlap or to overlap only partially. These findings, which represent the first analysis of the epitopes presented by a patient tumor, suggest that the use of tumor line-derived peptides for vaccination may require selection of the epitopes corresponding to the ones presented by freshly isolated human tumors.

Peptide priming of cytolytic activity to HER-2 epitope 369-377 in healthy individuals.

The presence of tumor-reactive CTLs in tumor infiltrates and in the peripheral blood of cancer patients demonstrates an immune response against tumors that apparently cannot control disease spread. This raises concerns as to whether amplification of this response may be useful during disease progression. Induction of tumor-reactive CTLs in healthy donors at risk, as well as in patients free of disease, may be therapeutically important, based on the hypothesis that CTLs that recognize tumors early may be more effective in containing their progression than CTLs that expand only when the disease progresses. To address the feasibility of priming cytolytic activity in healthy donors, we used the HER-2 peptide E75 (369-377) as an immunogen and autologous peripheral blood mononuclear cell-derived dendritic cells as antigen-presenting cells. We found that of 10 healthy donors tested, two responded at priming with E75 presented on autologous dendritic cells by induction of E75-specific CTL activity. Three other responders were identified after two additional restimulations. Of these five responders, three recognized E75 presented on the ovarian tumor line SKOV3.A2, as demonstrated by cold-target inhibition experiments. Induction of cytolytic activity at priming was enhanced in responders by tumor necrosis factor-alpha and interleukin 12 but not in the nonresponders. AlphaB7.1 monoclonal antibody added at priming enhanced induction of lytic activity in only one of the four nonresponding donors tested, suggesting that in the majority of donors, E75-precursor CTLs were not tolerized. Because of the possibility that disease may develop in nonresponders, strategies to improve the immunogenicity of tumor antigens for healthy donors may be required for development of cancer vaccines.

Vaccine implications of folate binding protein, a novel cytotoxic T lymphocyte-recognized antigen system in epithelial cancers.

The immune system can be efficiently stimulated and targeted to specific antigens expressed exclusively or preferentially by experimental cancers. The foremost limitations to extending this vaccine technology to the prevalent epithelial-derived cancers are the lack of: (a) identified tumor-associated antigens recognized by cellular immunity; (b) antigens expressed on the majority of tumor cells during disease progression; and (c) immunogenic CTL epitopes. To date, only HER-2/neu has been shown to be the source of naturally occurring, MHC-restricted, CTL-recognized peptides in epithelial tumors. In this study, we demonstrate that the human high-affinity folate binding protein (FBP), which is a source of antigenic peptides recognized in ovarian cancer, is also recognized in breast cancer. Both immunodominant E39 (FBP, 191-199) and subdominant E41 (FBP, 245-253) epitopes are presented by HLA-A2 in these cancers. These peptides are efficient at amplifying the response of tumor-associated lymphocyte populations in terms of lytic function, enhanced proliferation, and specific IFN-gamma release. On a per cell basis, tumor-associated lymphocytes stimulated with the FBP peptides exhibit enhanced cytotoxicity not only against peptide-loaded targets but also against FBP-expressing epithelial tumors of different histologies. Furthermore, FBP peptides induced E39-specific CTLs and E39- and E41-specific IFN-gamma and IP-10 secretion in certain healthy donors. The broad distribution of FBP among >90% of ovarian and endometrial carcinomas, as well as 20-50% of breast, lung, colorectal, and renal cell carcinomas, along with pronounced differential overexpression in malignant tissues compared with the extremely limited expression in normal epithelium, suggests the exciting potential of a widely applicable FBP-based vaccine in epithelial cancers.

Proliferative and cytokine responses to class II HER-2/neu-associated peptides in breast cancer patients.

Previous studies have characterized the reactivity of CD8+ CTLs with ovarian and breast cancer. There is little information about the antigens and epitopes recognized by CD4+ T cells in these patients. In this study, we analyzed the ability of T cells from peripheral blood mononuclear cells of breast cancer patients to recognize HER-2/neu (HER-2) peptides. We found that 13 of 18 patients responded by proliferation to at least one of the HER-2 peptides tested. Of these peptides, one designated G89 (HER-2: 777-789) was recognized by T cells from 10 patients. Seven of nine responding patients were HLA-DR4+, suggesting that this peptide is recognized preferentially in association with HLA-DR4. Analysis of the specificity and restriction of the cytokine responses to G89 by G89-stimulated T cells revealed that these cells secreted significantly higher levels of IFN-gamma than interleukin 4 and interleukin 10, suggesting priming for a Th0-T helper 1 response. The same pattern of cytokine responses was observed to the intracellular domain of HER-2 protein, suggesting that G89-stimulated T cells recognized epitopes of the HER-2 protein in association with HLA-DR4. Because HLA-DR4 is present in 25% of humans, characterization of MHC class II-restricted epitopes inducing Th0-T helper 1 responses may provide a basis for the development of multivalent HER-2-based vaccines against breast and ovarian cancer.

Ovarian and breast cytotoxic T lymphocytes can recognize peptides from the amino enhancer of split protein of the Notch complex.

In this study we investigated recognition by ovarian tumor associated lymphocyte (OVTAL), and breast tumor associated lymphocytes (BRTAL), of peptides corresponding to the sequence 125-135 of the Aminoenhancer of split (AES) protein. Three of these peptides designated as G75:AES1/2 (128-135), G60: AES1/2 (127-137) and G61: AES1/2 (125-133) correspond to the wildtype AES sequence, while the fourth G76:GPLTPLPV, AES1/2 (128-135) corresponds to a variant sequence of the peptide G75 with the N-terminal Leu substituted to glycine. These sequences were chosen for study because mass-spectrometric analysis (MS) of a CTL active HPLC peptide fraction eluted from immunoaffinity precipitated HLA-A2 molecule, revealed: (a) the presence of an ion with a mass-to-charge ratio (m/z) of 793 which was more abundant than other ions of similar masses; (b) the tentatively reconstituted sequence of the ion 793 matched the sequence of peptide G76. We found that AES peptides G75 (128-135) and G76 (128-135) (L128G) reconstituted CTL recognition at concentrations ranging between 200-500 nM. These concentrations are lower than concentrations reported to activate effector function of CTL recognizing other epithelial tumor Ag. Furthermore, analysis with cloned CD8+ T cells indicated that G75 and G76 were not cross-reactive specificities, suggesting a key role for the N-terminal residues of the variant peptide in dictating specificities. Since the AES proteins are part of a set of transcriptional repressors encoded by the Enhancer of split [E(spl)] genes, and since these repressors are activated to suppress cell differentiation in response to Notch receptors signalling, the AES peptides may represent a novel class of self-antigens that deserve further consideration as tumor Ag in epithelial cancers.

Secretion of CXC chemokine IP-10 by peripheral blood mononuclear cells from healthy donors and breast cancer patients stimulated with HER-2 peptides.

CXC chemokines play an important role in recruitment of T cells to the site of activation and regulation of angiogenesis. CXC chemokines are secreted by T cells stimulated with cytokines or by established cytotoxic T lymphocyte (CTL) lines at recognition of conventional antigen (Ag), but the activation requirements and the relationship of interferon-gamma (IFN-gamma) inducible protein (IP-10) secretion with IFN-gamma induction in lymphocytes are still unclear. We studied the induction of IP-10 from nonadherent peripheral blood mononuclear cells (PBMC) by IFN-gamma, interleukin-12 (IL-12), and the HER-2 peptide E75, which forms a CTL-defined antigen. We found that IFN-gamma alone was a weak inducer of IP-10 in these cells, whereas IL-12 was a significantly stronger inducer of IP-10. In the presence of IL-12, the tumor peptide E75 (HER-2, 369-377) was a stronger inducer of IP-10 than was IL-12 alone. E75 and its variants mutated at position 5 could also induce IP-10 in the absence of exogenous IL-12 or IFN-gamma. IP-10 induction by E75 required HLA-A2 presentation and B7-CD28 interactions and was partially inhibited by blocking of CD40-CD40L interactions. These results indicate that presentation of tumor peptides to peripheral T cells can induce a fast chemokine response, which in its early phase may be higher than the IFN-gamma response. This shows that the IP-10 response was independent of any early-phase IFN-gamma response in peripheral T cells. This may be important for understanding the regulation of the balance between chemoattractant chemokines (CC) and CXC chemokines by tumor Ag and may have implications for understanding the mechanisms of polarization of T cells and conditioning of antigen-presenting cells (APC) by tumor antigens.

Growth and antigen recognition of tumor-infiltrating lymphocytes from human breast cancer.

In the present study, we isolated tumor-infiltrating lymphocytes (TIL) from 21 primary solid tumors and tumor-associated lymphocytes (TAL) from 9 malignant effusions, respectively, of breast cancer patients. Significant proliferation and expansion of T cells was observed in 23 of 30 distinct samples. TIL were isolated from primary tumors by either enzymatic digestion or mechanical disruption. The TIL cultures were initiated using OKT3 mAb in the presence of moderate concentrations (25-50 U/ml) of IL-2, followed by 100 U/ml of tumor necrosis factor (TNF)-alpha. TAL were not stimulated with OKT3 mAb, but all were successfully expanded in culture in the presence of IL-2 alone or together with TNF-alpha. Seven of nine distinct TAL grew in culture as predominantly CD4+ lines. In contrast, only 14 of 21 (66%) of primary breast TIL expanded in culture and were predominantly of CD8+ phenotype. Autologous tumor lysis was observed in seven of eight cases tested. Only one of the four TIL tested and one of the four TAL tested preferentially lysed autologous tumor. HER-2 peptide E75 (369-377) was recognized by two TIL lines of the five primary TIL tested and three of the four TAL tested. This suggests that E75 may be recognized by primary breast tumors. This may be of interest in developing vaccine strategies for therapeutic management of breast cancer.

Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumoral injection of an adenovirus vector expressing wild-type. P53 (Ad-p53).

The immune responses of 10 patients with advanced non-small cell lung cancer receiving monthly intratumoral injections of a recombinant adenovirus containing human wild-type p53 (Ad-p53) to adenovirus and transgene antigens were studied. The predominate cellular and humoral immune responses as measured by lymphocyte proliferation and neutralizing antibody (Ab) formation were to adenovirus serotype 5 vector antigens, with increased responses in posttreatment samples. Consistent alterations in posttreatment cellular and humoral immune responses to p53 epitopes were not observed, and cytotoxic Abs to human lung cancer cells were not generated. Patients in this study had evidence of an antitumoral effect of this treatment with prolonged tumor stability or regression; however, neither Abs to p53 protein nor increased lymphocyte proliferative responses to wild-type or mutant p53 peptides have been consistently detected.

Clinical trials of HER-2/neu-specific vaccines.

Recently there has been a renewed interest in developing vaccines for use in cancer treatment. Part of this interest stems from a better understanding of the immune system, the identification of a number of T-cell-specific tumor antigens, more effective adjuvants, and the ability to construct more immunogenic molecules using recombinant DNA techniques. Studies from several laboratories have shown that breast cancer patients have preexisting immunity to the HER-2/neu oncoprotein receptor (HER-2) in the form of elevated antibody titers and T-cell immunity. Preclinical studies showed enhanced delayed-type hypersensitivity and cytotoxic T-lymphocyte precursors in spleens from animals immunized with several human leukocyte antigen class I and class II peptides derived from the HER-2 protein. Phase I trials of these peptides combined with the cytokine granulocyte-macrophage colony-stimulating factor as a part of therapy in patients with HER-2-positive cancers have shown minimal local toxicity, along with enhanced helper T-cell activity and antibody production in patients with minimal disease. Increases in cytotoxic T-lymphocyte precursor activity were less frequent, but in some cases could be enhanced when patient lymphocytes were incubated ex vivo with the proinflammatory cytokine interleukin-12. Other preclinical studies designed to enhance HER-2 peptide immunogenicity are in progress. Additional current and future clinical trials using HER-2-derived vaccines will be discussed.

T cell responses to ovarian tumor vaccines: identification and significance for future immunotherapy.

The cellular immune responses to ovarian cancer patients treated with viral oncolysates (VO) ovarian tumor vaccines to vaccines are described. CD3+ cells proliferated after stimulation with the tumor vaccines in a dose-dependent manner. The proliferation of CD3+ cells stimulated with the tumor vaccine was blocked by anti-HLA-DR monoclonal antibody and anti-CD4 mAb indicating that CD3+ CD4+ cells from the blood of the patients treated with VO recognize tumor derived determinants in conjunction with MHC class II antigens. The regulatory activity of the T cells collected after VO treatment was assayed by co-cultivation with PBMC collected before VO treatment. These cells demonstrated increased helper activity for immunoglobin production by cells collected before vaccination and secreted IL-2 in response to stimulation by vaccine. Finally, when biochemical fractionation of the components of VO was attempted, PBMC from VO treated patients responded by proliferation to several fractions suggesting that they recognize multiple epitopes in the ovarian tumor vaccine. Therefore, these data provide novel evidence for the involvement of the T cells in response to ovarian tumor vaccines.

Identification of activated tumor antigen-reactive CD8+ cells in healthy individuals.

We investigated the ability of HER-2 peptide E75, which maps an immunodominant CTL epitope for ovarian and breast tumor-associated lymphocytes (TAL), to activate effector functions in freshly isolated CD8+ cells from healthy individuals. IFN-gamma was rapidly induced by E75 within 20-24 h, in five of six healthy donors, in the presence of IL-12 and was detectable as early as 6 h. The IFN-gamma levels were Ag-concentration dependent. Similar results were obtained with peptides mapping CTL epitopes from two other tumor Ag: folate binding protein (FBP) and amino-enhancer of split of Notch (AES). IFN-gamma was also detected, from freshly isolated, unstimulated PBMC in response to HLA-A2 matched tumors + IL-12 but not of IL-12 alone. The major source of IFN-gamma were CD45RO+ CD8+ cells. Induction of IFN-gamma and IL-2 from CD8+ cells and of IL-12 from dendritic cells (DC) by CD8+ cells reactive with E75 mirrored their induction by the influenza matrix peptide (M1: 58-66) in the same individual. Responses to M1 are used to define the presence of activated memory cells in healthy individuals. Compared to M1 responses E75 recognition induced 2-4-fold lower levels of IL-12 from the same APC and IFN-gamma and IL-2 from the same CD8+ cells. At lower Ag concentrations the endogenous IL-12 induced by E75-reactive CD8+ cells did not reach the threshold required to co-stimulate for IFN-gamma. alphaB7.1 synergized with E75 in increasing the overall levels of IL-2 induced within 24 h. The presence of tumor Ag-reactive activated CD8+ cells in healthy individuals may improve our understanding of the mechanisms of immunosurveillance and regulation of immune responses by tumors.

Selective usage of TCR V beta in tumor-specific CTL lines isolated from ovarian tumor-associated lymphocytes.

We have developed a series of six CTL lines exhibiting preferential killing of autologous tumour cells from tumour-associated lymphocytes (TALs) infiltrating malignant ovarian ascites. Five out of six CTL lines showed increased percentages of V beta 8.1+ cells, four showed increased percentages of V beta 5.3+ cells and one showed increased percentages of V beta 6.7+ cells. On the contrary, fresh isolated TALs or autologous PBMCs, when stimulated by OKT3 mAb or other TAL cultures with nonspecific cytolytic function, did not show preferential usage of any of these families. Most important, V beta 8.1+ and V beta 6.7+ T cells within the CTL-TALs mediated cytoxicity against autologous targets. This represents the first demonstration of preferential usage of TCR V beta in ovarian tumor-specific CTLs and suggests possible association between tumor surveillance and TCR V beta genes.

Increased ovarian tumor cell surface reacting antibodies in patients with ovarian adenocarcinoma after viral oncolysate treatment.

Antibodies reacting with the cell surface of ovarian tumor cell lines were detected in the sera of untreated patients with ovarian cancer using cell surface immunofluorescence and FACS analysis. Vaccination of these patients with viral oncolysates increased the antibody production against ovarian cell surface antigens. These antibodies cross-reacted with a melanoma cell line (WM-75), and a lymphoblastoid cell line (Daudi). Immunoprecipitation and SDS-PAGE analysis of 125I-iodine labelled cell surface antigens of ovarian cells revealed a response to private and common antigens expressed on the cell surface of the tumor cells.

T-cell functions in ovarian cancer patients treated with viral oncolysates: I. Increased helper activity to immunoglobulins production.

We investigated the humoral and cellular responses of ovarian cancer patients treated with viral oncolysates (VO) tumor vaccines. All patients treated with VO developed tumor cell surface-reacting antibodies and anti-hemagglutinin antibodies. Furthermore, PBMC from these patients developed cellular proliferative responses to the cellular oncolysates (CO). The in vitro proliferating population consisted mainly of CD4+ cells, as demonstrated by depletion experiments using the corresponding monoclonal antibodies and complement and by examination of the phenotype of the cells stimulated in culture with VO or cellular oncolysates. Furthermore, in the patients treated with VO we observed an increased helper activity to immunoglobulin production by PBMC collected post-VO treatment in the PWM-driven differentiation system. The helper activity was much higher in patients treated with IL-2 plus VO than IL-2 alone.

Viral oncolysates in cancer treatment: immunological mechanisms of action (review).

Viral oncolysates (VO) are homogenates of virus-infected tumor cells. VO have been demonstrated in experimental animals to induce protection against transplanted autologous tumors, when administered prior to the challenge with the tumor. VO were first introduced into clinical trials as a treatment adjunct to chemotherapy and nonspecific immunomodulators (BCG). In recent years, beneficial clinical and biological activity responses have been observed in melanoma and gynecologic cancer (cervix, ovary), after administration of VO alone. Our group has continued clinical trials with PR8 strain influenza A. We have demonstrated that PR8 VO augments delayed type hypersensitivity in coded skin testing and have emphasized regional active immunization in treatment protocols. The efficacy of active immunization strategies have yet to be defined, but recent findings in ovarian and uterine cervix cancer highlight the importance of route and careful integration with other therapeutic modalities. The VO are postulated to trigger immunological mechanisms in the host that may ultimately lead to the control of the metastatic spread and elimination of the tumor. Humoral immune responses induced by VO have been described, while cellular immune responses associated with VO administration have been investigated to a lesser extent. We present here what is known and we discuss hypothesized mechanisms of humoral and T cell mediated immunity induced by VO now under investigation. The elucidation of these mechanisms may provide conceptual advances in the field of human tumor immunity as well as a rationale for the development of tumor vaccines.

Regulation of natural killer cytotoxicity by recombinant alpha interferons. Augmentation by IFN-alpha 7, an interferon similar to IFN-alpha J.

The structure-function relationship of several recombinant human alpha interferons (IFN-alpha) (IFN-alpha 1, IFN-alpha 2, IFN-alpha 4, IFN-alpha 7, IFN-alpha 2/alpha 1 and IFN-delta 4 alpha 1) was investigated with respect to their ability to augment natural killer (NK) cytotoxicity of human peripheral blood mononuclear cells (PBMC) against hemopoietic tumor cell lines. Although all these IFNs significantly augmented NK cytotoxicity against the K562, Daudi and U937 targets, significant quantitave differences were observed in their ability to augment NK. INF-alpha 4, IFN-alpha 2 and IFN-alpha 2/alpha 1 were able to augment NK at low concentrations (less than 0.1 ng/ml), whereas IFN-alpha 7, IFN-alpha 1 and IFN-delta 4 alpha 1 required significantly higher concentrations (3 ng/ml or higher). The cumulative rank order of INFs on the basis of NK augmenting ability was found to be: IFN-alpha 4 approximately IFN-alpha 2 approximately IFN-alpha 2/alpha 1 greater than IFN-alpha 7 greater than IFN-alpha 1 approximately IFN-delta 4 alpha 1. To determine synergism or potentiation in the ability of IFNs to augment NK cytotoxicity, we investigated the effect of simultaneous, sequential and reversed order of treatment of human PBMC by these IFNs. Such potentiation or synergism was not observed. In addition, all these IFNs were able to augment NK cytotoxicity against targets from malignant melanoma cell lines. IFN-alpha 7 augmented regularly and reproducibly NK cytotoxicity in 15 of 19 normal donors examined (79%). This augmentation was blocked by an anti-IFN-alpha antibody. Concentrations of IFN-alpha 7 as low as 0.06 ng/ml were able significantly to augment NK cytotoxicity of PBMC after incubation for one hour at 37 degrees C. In contrast to these findings, IFN-alpha J, an interferon similar to IFN-alpha 7, has been report to be incapable of augmenting NK cytotoxicity and also of interfering with augmentation of NK by other IFNs. Sequential treatment of PBMC first with IFN-alpha 7 and then with other interferons did not prevent the augmentation of NK. Similarly, simultaneous treatment with IFN-alpha 7 and other interferons did not prevent augmentation of NK. In both treatments IFN-alpha J has been reported to prevent augmentation of NK. IFN alpha J and IFN-alpha 7 differ only by one amino acid, at position 107, where a lysine in IFN-alpha J has been replaced by a glutamic acid in the IFN-alpha 7.(ABSTRACT TRUNCATED AT 250 WORDS)