Phase I/II clinical trial of a nonreplicative vaccinia virus expressing multiple HLA-A0201-restricted tumor-associated epitopes and costimulatory molecules in metastatic melanoma patients.

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens.

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients.

Identification and intracellular location of MAGE-3 gene product.

The human MAGE-3 gene encodes a melanoma antigenic epitope recognized by specific cytotoxic T lymphocytes, but its gene product has not been identified thus far. We produced a recombinant MAGE-3 gene product by expression cloning of the entire reading frame in the context of a fusion protein characterized by a 10-histidine tail, allowing purification by metal chelation on a nickel Sepharose column. The semipurified product was used to generate MAGE-3-specific monoclonal antibodies. One reagent could identify by immunoblotting the native MAGE-3 gene product as a M(r) 48,000 protein in lysates of cell lines showing evidence of MAGE-3 gene expression. No apparent cross-reactivity with recombinant or native MAGE-1 gene product was observed. Immunohistochemistry shows that, closely resembling the MAGE-1 gene product, MAGE-3 is a cytoplasmic protein.

MAGE-1 gene product is a cytoplasmic protein.

MAGE-1 gene encodes a human melanoma antigen, recognized by syngeneic cytotoxic T lymphocytes (CTL). MAGE-1 transcripts are also detectable in breast cancers, in non-small-cell lung carcinomas and in central nervous system tumors. In order to identify, in cellular preparations, the protein encompassing the antigenic peptide, we generated a panel of monoclonal antibodies (MAbs) against the MAGE-1 gene product by using, as immunogen, a full-length recombinant preparation (rMAGE-1), obtained through expression cloning of the relevant gene in E. coli. Four reagents were obtained recognizing both rMAGE-1 and the 46-kDa native protein in cell lines expressing MAGE-1 mRNA. No positivity could be detected in MAGE-1-mRNA-negative melanoma lines. No surface labelling of MAGE-1-positive cell lines could be observed. In contrast, on permeabilization of MZ2 melanoma cells, all 4 MAbs induced efficient staining, as detected by cytofluorography. Fluorescence microscopy shows that MAGE-1 gene product is a cytoplasmic protein clustered in paranuclear organelle-like structures. Thus, MAGE-1 protein location closely resembles that of P91A and P198 murine-tumor antigens.

On the relative roles of interleukin-2 and interleukin-10 in the generation of lymphokine-activated killer cell activity.

Induction of cytokine gene transcription by recombinant human IL-2 (rhIL-2) in peripheral blood mononuclear cells (PBMC) from healthy donors was studied by qualitative polymerase chain reaction. In all donors tested, optimal doses of rhIL-2-induced transcription of genes encoding for IL-5, GM-CSF, IFN-gamma, and TNF-alpha whereas transcription of IL-1-alpha, IL-3, IL-4, and IL-6 genes could only be detected in about half of the donors. Moreover, we observed that different doses of rhIL-2 were needed to induce transcription of different cytokine genes. In contrast, transcription of IL-2 and IL-10 genes was only observed in a minority of donors, irrespective of the concentration of rhIL-2 used. Since IL-10 displays a well-characterized inhibitory activity on the synthesis of cytokines possibly involved in the generation of lymphokine-activated killer (LAK) cells, we asked whether the absence of IL-10 gene transcription plays a role in the induction of LAK cells. Thus, we tested the effects of different doses of rhIL-10 on the rhIL-2-driven generation of LAK activity. Interestingly, rhIL-10 dose-dependently inhibited the production of IFN-gamma and TNF-alpha induced by IL-2, but had no effects on PBMC proliferation and generation of LAK activity. Similarly, purified CD3-/CD16+ lymphocytes, the precursors of LAK effector cells, could be optimally induced by low doses of rhIL-2 to proliferate and generate MHC-unrestricted cytotoxic activity against NK-resistant targets in the presence of rhIL-10. Altogether, our results indicate that rhIL-2 induces transcription of a preferential pattern of cytokine genes, with the IL-10 gene being infrequently transcribed. On the other hand, rhIL-10 shows diverse effects on rhIL-2-triggered PBMC activation, in that it inhibits IFN-gamma and TNF-alpha production but does not affect PBMC proliferation or generation of LAK activity.