Generation of melanoma-specific, cytotoxic CD4(+) T helper 2 cells: requirement of both HLA-DR15 and Fas antigens on melanomas for their lysis by Th2 cells.

Recognition of melanoma antigens by HLA class-II-restricted CD4(+) T lymphocytes has been investigated. Two cytotoxic CD4(+) T cell lines were established by stimulating PBLs from a melanoma patient with either parental or IFN-gamma-transduced autologous tumor cells. These T cells secreted IL-4, but not IL-2, IFN-gamma, or TNF-beta, in response to the autologous melanoma cells, suggesting that they belong to the Th2 subtype. Their cytotoxicity was directed against the IFN-gamma-transduced melanoma cells and was HLA-DR-restricted. The autologous and two allogeneic IFN-gamma-modified melanoma cell lines shared melanoma antigen(s) presented in the context of HLA-DR15. HLA-DR15(+) nonmelanoma cells were resistant targets indicating that the shared antigen(s) is melanoma associated. Parental autologous and HLA-DR-matched allogeneic melanoma cell lines, displaying low levels of HLA-DR antigens, induced Th2 proliferation and cytokine release, but were insensitive to lysis prior to upregulation of HLA-DR and Fas antigens by IFN-gamma. Cytolysis was inhibited by anti-HLA-DR and by anti-Fas antibodies, suggesting that the cytolysis is mediated via the Fas pathway. While small amounts of HLA-DR15 molecules on melanoma cells are sufficient for Th2 proliferation and cytokine release, higher amounts of HLA-DR15 and the expression of Fas are required for CD4(+)-mediated lysis.

Dendritic cells infected with a vaccinia vector carrying the human gp100 gene simultaneously present multiple specificities and elicit high-affinity T cells reactive to multiple epitopes and restricted by HLA-A2 and -A3.

To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac-Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100-specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3-expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide-pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides.

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens.

Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.

Murine dendritic cells transfected with human GP100 elicit both antigen-specific CD8(+) and CD4(+) T-cell responses and are more effective than DNA vaccines at generating anti-tumor immunity.

Dendritic cells (DCs) are potent inducers of cytotoxic T lymphocytes (CTLs) when pulsed with an antigenic peptide or tumor lysate. In this report, we have used liposome-mediated gene transfer to examine the ability of plasmid DNA encoding the human melanoma-associated antigen gp100 to elicit CD8(+) and CD4(+) T-cell responses. We also compared the efficacy between gp100 gene-modified DCs and naked DNA (pCDNA3/gp100)-based vaccines at inducing anti-tumor immunity. DCs were generated from murine bone marrow and transfected in vitro with plasmid DNA containing the gp100 gene. These gp100-modified DCs (DC/gps) were used to stimulate syngeneic naive spleen T cells in vitro or to immunize mice in vivo. Antigen-specific, MHC-restricted CTLs were generated when DC/gps were used to prime T cells both in vitro and in vivo. Thus, these CTLs were cytolytic for gp100-transfected syngeneic (H-2(b)) tumor MCA106 (MCA/gp) and vaccinia-pMel17/gp100-infected syngeneic B16 and MCA106, but not parental tumor MCA106 and B16, or gp100-transfected allogeneic tumor P815 (H-2(d)). Immunization with DC/gp protected mice from subsequent challenge with MCA/gp but not parental MCA106. Antibody-mediated T-cell subset depletion experiments demonstrate that induction of CTLs in vivo is dependent on both CD4(+) and CD8(+) T cells. Furthermore, DC/gp immunization elicits an antigen-specific CD4(+) T-cell response, suggesting that DC/gps present MHC class II epitopes to CD4(+) T cells. In addition, our data show that gene-modified, DC-based vaccines are more effective than the naked DNA-based vaccines at eliciting anti-tumor immunity in both prophylactic and therapeutic models. These results suggest that the use of DCs transfected with plasmid DNA containing a gene for TAA may be superior to peptide-pulsed DCs and naked DNA-based vaccines for immunotherapy and could provide an alternative strategy for tumor vaccine design.

Tumor cells cotransduced with B7.1 and gamma-IFN induce effective rejection of established parental tumor.

Genetic modification of tumor cells with the gene for the B7.1 or with the genes for cytokines results in increased tumor cell immunogenicity. In the work reported here, immunization of naive animals with either B7.1 or gamma-IFN gene-modified MCA106 tumor cells effectively protects the host from subsequent challenge with parental tumor. The same treatment fails to induce regression of established tumors, although tumor-specific CTL are generated in the tumor-bearing animals. In contrast, a large tumor burden of the MCA106 fibrosarcoma can be successfully eliminated by treatment with MCA106 tumor cells cotransduced with the B7.1 and gamma-IFN genes. Antitumor immunity induced by the cotransductants is primarily dependent on CD8+ T cells and partly on CD4+ T cells and NK cells, and the enhanced therapeutic effect may be attributed to the in vivo increase of CTL precursors following treatment. The gamma-IFN and B7.1 genes must be expressed on the same tumor cell for optimal therapeutic effect. Our results suggest that tumor vaccines with a potent immunoprotective effect do not necessarily have therapeutic potential and that weakly immunogenic tumors may be rendered highly immunogenic by cotransfection with the genes for B7.1 and gamma-IFN.

Immunotherapeutic potential of tumor antigen-pulsed and unpulsed dendritic cells generated from murine bone marrow.

Dendritic cells (DC) are highly efficient antigen-presenting cells able to capture, process, and present antigens to naive and primed T-cells. In this study, we have investigated the ability of DC, derived from murine bone marrow and pulsed with tumor cell extracts, to induce regression of preexisting tumors. In an experimental model of B16 melanoma in B6 mice, a significant reduction in metastatic nodules in the lungs was observed in tumor-bearing animals treated with either DC alone or DC pulsed with tumor extracts. Kinetic studies demonstrate that the efficacy of these tumor vaccines is inversely related to tumor burden. In this model, tumor-specific cytotoxic T-cells (CTL) could also be induced in vitro from spleen cells derived from tumor-bearing animals treated with DC pulsed with tumor extracts. Untreated mice had no CTL. Furthermore, DC alone elicited tumor-specific CTL responses in tumor-bearing mice, but not in naive mice. Immune cell depletion experiments show that the therapeutic effects of DC are primarily mediated by CD8+ T-cells, while CD4+ T-cells and NK cells are involved in DC-mediated antitumor immunity to a limited extent. These results illustrate the potential use of DC and DC pulsed with tumor extracts as potent therapeutic reagents for cancer and provide a rationale for using DC in vivo to eliminate disseminated tumors or residual tumor deposits following surgery.

Generation of primary tumor-specific cytotoxic T lymphocytes from autologous and human lymphocyte antigen class I-matched allogeneic peripheral blood lymphocytes by B7 gene-modified melanoma cells.

Expression of B7.1 costimulatory molecules on tumor cells has been shown to elicit antitumor immunity in mice. In the present study, we have developed a human B7.1 retroviral vector system to effectively transduce human melanoma cell lines and investigated the potential role of B7.1 in the generation of tumor-specific CTLs from peripheral blood lymphocytes (PBLs) in vitro. We have demonstrated that B7.1-modified melanoma cells are able to induce primary CTL activity from autologous, human lymphocyte antigen (HLA) class I-matched allogeneic PBLs and purified CD8+ T cells in the absence of exogenous cytokines. CTLs generated by B7.1 are tumor specific and HLA class I restricted, and CD8+ T cells are primarily responsible for this specific cytotoxicity. Furthermore, CTLs generated from HLA class I-matched PBLs by B7.1 are cytolytic to tumor cells autologous to the stimulated PBLs. These data suggest that B7.1-modified tumor cells can be used as a potent tumor vaccine for both autologous and HLA class I-matched allogeneic patients.

Modulation of specific active immunization against murine melanoma using recombinant cytokines.

Specific active immunization with tumour cells and IL-1beta or IL-2 was examined in a murine model. Mice were treated with irradiated B16 melanoma, IL-1beta or IL-2 only, or with B16 plus cytokines prior to i.v. challenge with viable B16. Lung metastases were recorded after 28 days. Treatment with cytokine alone was not protective. Treatment with B16 alone afforded moderate protection. Treatment with B16 in combination with either cytokine resulted in a significant level of B16 specific protection which was dependent on the dose of cytokine used. Multiple immunizations with B16 provided limited protection which was significantly improved with IL-2. Immunization with B16 in combination with both cytokines at doses that alone failed to enhance immunity resulted in significant protection, suggesting that the two cytokines act at least additively. These studies demonstrate the significant benefit of specific active immunization with tumour cells in combination with low doses of IL-1beta or IL2.

Effect of irradiation on cytokine production, MHC antigen expression, and vaccine potential of interleukin-2 and interferon-gamma gene-modified melanoma cells.

Recent studies have shown that tumor cells transduced with interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) genes stimulated a potent and specific antitumor immunity in experimental animals. For use as a human vaccine, tumor cells must be inactivated by irradiation to ensure the arrest of their growth. This study was undertaken to examine the effects of irradiation (10,000 rad) on the growth characteristics and vaccine potential of IL-2 and IFN-gamma-modified human melanomas and B16 murine melanoma. Irradiation caused cessation of cell growth and gradual reduction of cell number. Irradiated melanoma cells displayed 1.5 to 10-fold increases in the surface expression of MHC class I and/or class II antigens. The increases in MHC antigens persisted for 7-14 days postirradiation and then declined thereafter. Furthermore, IL-2- and IFN-gamma-transduced melanoma cells showed enhanced expression of the cytokine mRNA and increased cytokine secretion after irradiation. The effect of irradiation on the vaccine potential of the transduced cells was examined in C57BL/ 6 mice by prophylactic immunization and immunotherapy, and in nude mice by mixed transplantation assays. The irradiated, cytokine-transduced B16 cell vaccine was as or more effective than the unirradiated vaccine. These irradiated vaccines protected the animals against a challenging tumorigenic dose of B16 parental cells and suppressed the growth of 4-day-established B16 lung metastases. The ability of the irradiated IL-2-transduced human melanomas to inhibit the growth of admixed parental melanoma cells was retained but was less efficacious than unirradiated cells. The results suggest that irradiation does not abrogate the vaccine potential of IL-2- and IFN-gamma-transduced melanomas. These findings have implications for designing specific active immunotherapy protocols utilizing cytokine gene-modified tumor cells.

Recognition and lysis of human melanoma by a CD3+, CD4+, CD8- T-cell clone restricted by HLA-A2.

The in vitro cytotoxic response to human melanoma is characterized by CD3+ CD8+ T-cells which recognize shared peptide antigens presented in the context of HLA class-I-encoded gene products. We report here studies of a CD3+, CD4+, CD8-, HLA-A2-restricted, melanoma-specific cytotoxic T-cell clone derived by limiting dilution from a T-cell line induced in PBLs from a melanoma patient following in vitro stimulation with an HLA-A2-matched melanoma cell line. The CD4+ cytotoxic T-cell clone is lytic only for melanomas which share the HLA-A2 allele, and the cytotoxicity is blocked by antibody to the T-cell receptor and by antibody to HLA class I. The clone proliferates only following stimulation with HLA-A2-matched melanoma tumor cells. The data suggest that cytotoxic CD4+ T-cells may play a significant role in immunity to melanoma, and HLA class-I-restricted recognition of melanoma may not necessarily require the CD8 molecule on the lytic T-cell.

Dual recognition of a human cytotoxic T-cell clone for melanoma antigens.

It is well known that tumor-specific CTLs have a crucial role in the elimination of tumors and that different CTL populations recognize tumor antigens in MHC-restricted and MHC-unrestricted manners. We have established two alpha beta CTL clones that recognize melanoma antigens in both human lymphocyte antigen (HLA)-A2-restricted and HLA-unrestricted manners. Flow cytometry analysis showed that these CTL clones carry CD3, CD8, and alpha beta T-cell receptor (TCR) and express low levels of CD56. In contrast, these CTL clones do not express CD16, indicating that they do not contain natural killer cells. TCR analysis of these CTL clones using an anchored PCR method revealed that each clone carries a single alpha beta TCR. Both CTL clones contained the same Valpha and Vbeta gene segments although they carried different Jalpha and Jbeta gene segments. Taken together, these results confirm that CTL clones that carry a single alpha beta TCR recognize melanoma antigens in both HLA-A2-restricted and HLA-unrestricted manners. It is strongly suggested that the dual recognition of these CTL clones for the melanoma antigens is mediated by TCRs. The novel mechanism for antitumor immunity by these CTLs may be important in the effective elimination of tumors in vivo.

Transduction of human melanoma cells with interleukin-2 gene reduces tumorigenicity and enhances host antitumor immunity: a nude mouse model.

Human melanoma tumor cells were genetically modified in vitro by transferring the interleukin-2 (IL-2) gene via a retroviral vector into established or fresh tumor cells. In addition, human melanoma cells were transduced in vivo by the direct injection of the IL-2/retroviral vector into melanoma xenografts in nude mice. The gene-modified melanoma cells expressed the IL-2 cytokine gene and secreted biologically active IL-2. Transduction of melanoma cells with the IL-2 gene did not affect the antigenic profile of the cells, but caused a strong abrogation of their tumorigenicity. One million parental cells formed subcutaneous tumors in nude mice. In contrast, various doses of up to 20 x 10(6) IL-2-transduced cells failed to form tumor in the mice. Coinjection of IL-2-producing cells with parental cells inhibited tumor formation even when highly tumorigenic doses of parental cells were used. Histochemical analysis of the injection sites of IL-2-modified cells showed an influx of host immune cells, predominantly macrophages, as early as the third day after inoculation. Neutrophils, mast cells, and eosinophils were also seen in the inflammatory exudate. Eventually, transduced cells showed signs of degeneration and necrosis and ultimately died in 4 weeks. Macrophages were seen in parental tumor sites only during the first few days after injection, and then parental tumors exhibited fast, progressive growth. The study suggests that melanoma cells transduced with the IL-2 cytokine gene may provide an effective vaccine for melanoma patients, whereas the in vivo transduction of tumors with cytokine genes is feasible and may represent a novel approach for the immunotherapy of cancer patients.

Transduction of human melanoma cells with the gamma interferon gene enhances cellular immunity.

Human tumor cells transduced with the gamma interferon (gamma IFN) gene are currently used in specific active immunotherapy protocols to enhance the antitumor immune responses of cancer patients. This in vitro study was undertaken to examine the initial events in the cellular immune response that may occur following the administration of the gamma IFN-transduced cell vaccine. Human melanoma tumor cell lines were transduced with a MoMLV-based retroviral vector carrying the human gamma IFN gene. The transduced cells expressed the cytokine gene, secreted biologically active gamma IFN, and exhibited enhanced expression of MHC class I and class II (HLA-DR), and ICAM-1 surface antigens. The gamma IFN-transduced and corresponding parental melanoma cells were used for the induction of short-term lymphocyte cultures. Peripheral blood lymphocytes or lymph node cells from 20 melanoma patients were stimulated for 5 to 15 days with autologous or MHC class I-matched allogeneic parental or gamma IFN-transduced melanoma cells. Seven of the 20 lymphocyte cultures showed substantial increases in lytic activity following stimulation with the transduced melanoma cells in comparison to control lymphocyte cultures stimulated with unmodified parental melanoma. The cytolytic activity stimulated with gamma IFN-modified melanomas was mediated partly by MHC-restricted cytotoxic T lymphocytes and partly by NK cells. Lymphocyte cultures that displayed increases in cytotoxicity after stimulation with the gamma IFN-transduced melanoma cells also exhibited enhanced expression or induction of one or more of the following lymphokines: IL-4, IL-1 alpha, IL-1 beta, gamma IFN, and TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines.

Of several thousand peptides presented by the major histocompatibility molecule HLA-A2.1, at least nine are recognized by melanoma-specific cytotoxic T lymphocytes (CTLs). Tandem mass spectrometry was used to identify and to sequence one of these peptide epitopes. Melanoma-specific CTLs had an exceptionally high affinity for this nine-residue peptide, which reconstituted an epitope for CTL lines from each of five different melanoma patients tested. Recognition by multiple CTL lines suggests that this may be a promising candidate for use in peptide-based melanoma vaccines.

Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy.

To study in vivo activated cytolytic T cells, CD8+ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8+ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8+ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell line, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8+ and 2 CD4+ CD8+ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) V alpha and V beta gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the V alpha 17/V beta 7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the V alpha 17/V beta 7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8+ CTL. CTL with a range of melanoma specificities and different TCR alpha beta dimers were encountered in this patient, perhaps as a result of hyperimmunization.(ABSTRACT TRUNCATED AT 400 WORDS)

Human melanoma-mediated inhibition of autologous CD4+ helper tumor-infiltrating lymphocyte growth in vitro.

Tumor-infiltrating lymphocytes (TIL) were isolated from a human melanoma metastatic to the abdomen. The TIL were 99% CD3+ and 99% CD4+ and CD8-. They were dependent on interleukin-2 (IL-2) for growth, as measured in a thymidine uptake assay, and were not cytotoxic to autologous or allogeneic melanoma or K562. When co-cultured with irradiated autologous tumor cells, or tumor cell supernatants, the TIL not only did not respond, but the IL-2-dependent growth was inhibited significantly. Inhibition occurred during the first 24 hours of co-culture and persisted as long as the tumor was present. After being washed free of inhibitory tumor cells, the TIL again were able to grow in the presence of IL-2, indicating that the inhibition was not caused by irreversible toxicity mediated by the tumor. Addition of excess IL-2 did not reverse the inhibitory effect, but addition of indomethacin, an inhibitor of cyclooxygenase and prostaglandin synthesis, partially blocked the inhibition. These data show melanoma-mediated inhibition of induction and expansion of human T-cells in vitro, which may reflect one of the mechanisms of inhibition of cellular responses in vivo. These results stress the need to examine the techniques for optimal in vitro expansion of tumor-specific TIL or cytotoxic T-cells for adoptive immunotherapy.

Inhibition of the growth of human melanoma metastases in nude mice by melanoma-specific murine monoclonal antibody.

The administration of anti-melanoma murine monoclonal antibody (MAB) 16.C8 (IgG2a) to nude mice bearing established human melanoma lung or liver metastases resulted in a significant inhibition of tumour growth. A total dose of 2 mg of affinity purified 16.C8 caused complete inhibition of tumour growth in 89 and 100% of animals in the liver and lung model, respectively. In contrast, a significant tumour growth was found in most control animals which received an irrelevant IgG2a MAB or 2% human serum albumin in Hanks Balanced Salt Solution (HBSS). The MAB was most effective when treatment was started on day 1 or 4 following tumour inoculation. When the 16.C8 MAB treatment was delayed 7 or 14 days, 33 and 67% of 16.C8 treated animals, respectively, developed tumours. The MAB-mediated anti-tumour activity appeared to be dose dependent, and the effect of a suboptimal dose was potentiated by the concomitant administration of recombinant interleukin 2 (rIL-2). Recombinant IL-2 alone in a similar dose did not elicit comparable anti-tumour activity. Moreover, the MAB 16.C8 inhibited tumour growth in irradiated animals which may suggest the involvement of host-radioresistant cellular elements in the 16.C8 antibody-mediated anti-tumour activities in nude mice. These results suggest that MAB 16.C8 alone or combined with rIL-2 may prove useful in the immunotherapy of metastatic melanoma.

Generation of human IgG, IgA, and IgM anti-melanoma monoclonal antibodies utilizing lymphocytes of an actively immunized melanoma patient.

Active specific immunotherapy with irradiated allogeneic melanoma cells has been shown to enhance the humoral immune response in melanoma patients. An increased titer of melanoma-binding antibodies was demonstrated in sera of immunized patients. Lymph node cells and splenocytes isolated from an actively immunized melanoma patient were fused with the human-murine heteromyeloma cell lines SHMD-33, SPM4-0, and SBC-H20. A group of human anti-melanoma monoclonal antibodies (MABs) were generated from the SHMD-33 fusion. Isolated MABs (one IgG2, one IgA, and two IgM) have been stable in cultures for more than 12 months and have produced human immunoglobulins at 0.2-0.9 Ug/ml/day. As shown by solid phase radioimmunoassays, the MABs react with autologous tumor cells and allogeneic melanoma tumors, including the cell line that was used for immunotherapy. In immunocytochemical assays, all four MABs react with a number of melanoma tumor cell lines. The IgG2 and IgA MABs stained preferentially melanoma tumor cells. In contrast, the IgM MABs cross-reacted with a broad panel of tumor cells from colon, prostate, pancreas, lung, and other human tumors. The MABs appear to be directed to intracellular rather than membrane-associated antigens as shown by immunofluorescence assays on live and permeabilized cells. The IgG2 antibody recognizes a 70 kDa antigen in melanoma cell lysates by Western immunoblotting. The target antigens for the other MABs have not yet been defined. Stability in culture and strong binding to melanoma tumor cells provide the basis for evaluating the potential of these human MABs. The IgG2 MAB, in particular, may prove useful for diagnostic and therapeutic applications in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

MHC-restricted recognition of autologous melanoma by tumor-specific cytotoxic T cells. Evidence for restriction by a dominant HLA-A allele.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.