Christoph Noppen

Phase I study in melanoma patients of a vaccine with peptide‐pulsed dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells

Dendritic cells (DCs) are professional antigen‐presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T‐cell response in vivo against melanoma‐associated antigens. We have shown that the sequential use of early‐acting hematopoietic growth factors, stem cell factor, IL‐3 and IL‐6, followed by differentiation with IL‐4 and granulocyte‐macrophage colony‐stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34+ peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF‐α. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide‐pulsed DCs. Fourteen HLA‐A1+ or HLA‐A2+ patients received at least 4 i.v. infusions of 5 × 106 to 5 × 107 DCs pulsed with a pool of peptides including either MAGE‐1, MAGE‐3 (HLA‐A1) or Melan‐A, gp100, tyrosinase (HLA‐A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low‐grade fever (WHO grade I–II). Clinical and immunological responses consisted of anti‐tumor responses in 2 patients, increased melanoma peptide‐specific delayed‐type hypersensitivity reactions in 4 patients, significant expansion of Melan‐A‐ and gp100‐specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA‐A2+ patient. Our data indicate that the vaccination of peptide‐pulsed DCs is capable of inducing clinical and systemic tumor‐specific immune responses without provoking major side effects. Int. J. Cancer 86:385–392, 2000.

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.

Culture-Independent Molecular Subtyping of Mycoplasma pneumoniae in Clinical Samples

ABSTRACT A new molecular subtyping approach was developed which is based on the amplification and sequencing of a repetitive region of the P1 gene of Mycoplasma pneumoniae. It allows the differentiation of all known subtypes and variants of M. pneumoniae as well as the identification of new subtypes directly in clinical samples to characterize endemic and epidemic M. pneumoniae infections.

Rapid induction of specific cytotoxic T lymphocytes against melanoma-associated antigens by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo.

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.

Naturally processed and concealed HLA-A2.1-restricted epitopes from tumor-associated antigen tyrosinase-related protein-2.

In this study, a computer‐assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP‐2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA‐A2.1‐binding motifs, two induced specific CD8+ cytotoxic T lymphocytes. HLA‐A2.1+ melanoma cells expressing TRP‐2 were lysed by clones specific for TRP‐2360–368 (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T‐cell clones directed against TRP‐2476–484 (VMGTLVALV) were unable to lyse HLA‐matched TRP‐2+ cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini‐genes encoding the TRP‐2476–484 peptide alone or carrying N‐ or C‐terminal extensions. Specific T‐cell clones recognized target cells expressing the cytotoxic T‐lymphocyte (CTL)‐defined epitope or its C‐terminally extended precursor, but failed to recognize cells expressing the N‐terminally extended TRP‐2476–484 peptide, suggesting the presence of a negative processing signal (NPS). Regarding C‐terminus‐flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP‐2476–484 epitope. Interestingly, proteasome inhibitors preventing the generation of the MART‐1/Melan‐A27–35 immunodominant melanoma tumor‐associated antigen (TAA) promoted detectable presentation of TRP‐2476–484 epitope in HLA‐A2.1+ and TRP‐2+ tumor lines, as witnessed by cytokine release by specific T‐cell clones. Int. J. Cancer 87:241–246, 2000.

C-type lectin-like receptors in peptide-specific HLA class I-restricted cytotoxic T lymphocytes: differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope specificity

C‐type lectin‐like inhibitory receptors are heterodimers consisting of CD94 and NKG2‐A‐B molecules expressed on NK cells and on a subset of activated T lymphocytes. Their inhibitory effects on NK cytotoxicity and on the NK‐like activity of T cell clones have been demonstrated, but no data are currently available on antigen‐specific class I‐restricted cytotoxic T lymphocytes (CTL). We have generated a panel of HLA‐A2.1‐restricted CTL clones directed against a nonapeptide derived from a melanoma‐associated antigen, dopachrome tautomerase (TRP‐2). All clones were CD8+ and TCR α β+. About half of them expressed a CD94bright phenotype, whereas the remaining were CD94dim. Only the CD94bright CTL expressed the NKG2‐A‐B gene, consistent with the expression of a C‐type, lectin‐like, inhibitory CD94/NKG2‐A‐B heterodimer. Both CD94bright and CD94dim clones appeared to require similar amounts of synthetic epitope sensitizing target cells. Addition of anti‐CD94 mAb resulted in a significant increase of specific killing by CD94bright, but not by CD94dim clones in the presence of suboptimal concentrations of peptide, whereas, when optimal amounts were used, the mAb did not induce a significant modulation of the cytotoxicity. Antigen‐induced inward [Ca2+]i fluxes were unaffected, but an enhancement of TCR down‐modulation could be observed in the presence of anti‐CD94 mAb at high concentration of antigenic peptide. The analysis of the TCR‐Vβ repertoire of the CTL clones by RT‐PCR and immunofluorescence revealed that all clones regardless of CD94 phenotype shared Vβ22 expression. Most importantly, sequence analysis showed that they all expressed identical Vβ22 TCR rearranged with Jβ2.1 and Cβ2. Taken together, these data indicate that different expression of functionally active lectin‐like inhibitory receptors can be detected in CTL clones sharing identical TCR sequence and peptide specificity.

Modulation of dendritic cell phenotype and mobility by tumor cells in vitro

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro. Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15(+) cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.

FLT3 ligand gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens.

Dendritic cells (DC) are professional antigen presenting cells (APC) whose proliferation and functional differentiation can be induced by hematopoietic growth factors including GM‐CSF and FLT3 ligand (FL). Colorectal cancers are known to be infiltrated by dendritic cells (DC) and neoplastic cells have been shown to produce GM‐CSF. In this work we investigated FLT3 ligand (FL) gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. Using reverse transcription polymerase chain reaction (RT‐PCR), 6 out of 6 established tumor lines were found to express to variable extents FL gene. In 1 of them, SW480, FL immunoreactivity could be observed by taking advantage of specific antibodies. In contrast, soluble FL could not be detected in any culture supernatant. FLT3 receptor (FR) gene was not expressed and exogenous addition to the cultures of recombinant FL (rFL) did not affect the proliferation of the tumor lines. FL gene expression was investigated using a densitometry‐assisted, semiquantitative RT‐PCR in clinical tumor specimens. Specific FL gene transcripts were amplified from 12 of 12 surgical samples. In these cases, FL gene expression of significantly lower intensity was also detected in healthy mucosa sampled in the vicinity (2 cm) or at a distance (10 cm) from neoplastic outgrowth. Immunohistochemical studies identified FL‐positive cancer cells in 5 of 5 cases tested. No positivity was detected in healthy mucosa epithelia at a distance from the tumor or in stromal cells. FL content in preoperative sera from colorectal cancer patients (n = 13) did not exceed the levels detected in healthy donors (≤ 100 pg/ml). Int. J. Cancer 86:238–243, 2000.

The tumour-associated antigen MAGE-1 is detectable in formalin-fixed paraffin sections of malignant melanoma

The MAGE-1 gene encodes a protein encompassing a HLA-A1-restricted target epitope for cytolytic T lymphocytes. Monoclonal antibodies directed against the MAGE-1 protein were tested for usage in immunohistology of routine pathology material. Seven formalinfixed, paraffin-embedded malignant melanomas were studied by the Avidin-Biotin complex (ABC) method with or without different antigen retrieval methods. Native, frozen tissues from the same tumours were used to validate the results by immunohistochemistry on frozen sections, by PCR for mRNA and by protein demonstration in tissue extracts using western blotting. Of 4 monoclonal antibodies tested, mAB 34B and mAB 77B were highly efficient in detecting MAGE-1 protein in deparaffinised sections with the regular ABC method after microwave pretreatment. In a series of an additional 28 patients 75% expressed MAGE-1, 50% in a substantial proportion. Follow-up studies in 6 patients indicate that the expression pattern remains stable but may change substantially within a short range. Immunohistology is thus a rapid and well-established method that might be used to select and monitor HLA-A1 positive patients with malignant melanoma and other candidate tumours for MAGE-1-directed immuno-therapy.