Michael L. Salgaller

Evaluation of phase I/II clinical trials in prostate cancer with dendritic cells and PSMA peptides.

A phase I trial involving patients with advanced prostate cancer was conducted to assess the safe administration of dendritic cells (DC) and HLA‐A0201‐specific prostate‐specific membrane antigen (PSMA) peptides (PSM‐P1 or ‐P2). Thirty‐three of the phase I participants were subsequently enrolled in a phase II trial, which involved six infusions of DC pulsed with PSM‐P1 and ‐P2 peptides.

Analysis of expression of the melanoma-associated antigens MART-1 and gp100 in metastatic melanoma cell lines and in in situ lesions

MART-1 and gp100 melanoma associated antigens (MAA) are expressed by cells of the melanocytic lineage and are recognized by the majority of HLA-A2 restricted tumor-infiltrating lymphocytes. Heterogeneity of expression of MAA in tumor deposits may affect the natural history or response to therapy of patients with melanoma. In this study, we evaluated the expression of these MAA with a new monoclonal antibody (mAb) directed against MART-1 (M2-7C10) and the commercially available HMB45 mAb directed against gp100. Expression was tested in vitro by intracellular fluorescence analysis and in vivo by immunophenotyping of tissue specimens. Nine melanoma cell lines and 25 tissue specimens from metastatic melanoma were analyzed. One cell line did not express MART-1 or gp100. The expression of both antigens was more heterogeneous and significantly reduced (p < 0.01) in melanoma cell lines compared with melanocytes, suggesting progressive loss of expression of MAA by neoplastic cells. None of the nonmelanoma cancer lines tested stained for MART-1 or gp100. Analysis of melanoma lesions by immunohistochemistry showed significant heterogeneity of expression of both MART-1 and gp100 MAA either as a percentage of cells expressing MAA or as intensity of expression. Ten of 25 frozen sections expressed MART-1 in < 50% of the cells. In 6 of 25 lesions, immunoreactivity for MART-1 was totally absent. Fine needle aspiration of metastatic lesions seemed to yield information accurately about amount and heterogeneity of expression of MAA in tumor lesions in vivo. Heterogeneity of expression of MAA may be one of several mechanisms leading to tumor escape from immune recognition, and pretreatment evaluation of tumor lesion for expression of these antigens may help in selecting patients best suited to antigen-specific vaccine therapies.

Differential anti-MART-1/MelanA CTL activity in peripheral blood of HLA-A2 melanoma patients in comparison to healthy donors: evidence of in vivo priming by tumor cells.

MART-1 is expressed ir both normal and neoplastic cells of melanocytic origin. Peripheral blood mononuclear cells (PBMC) from melanoma patients recognize and lyse tumor cells after repetitive in vitro stimulation with the immunodominant peptide MART-1(27-35). In this study, we compared the characteristics of the cytotoxic T lymphocyte (CTL) response to MART-1 in PBMC from 13 HLA-A2 melanoma patients with PBMC from 9 normal healthy donors stimulated in vitro with MART-1(27-35) (AAGIGILTV) or FluM1(58-66) (GILGFVFTL) peptides. The expansion rate among CTLs from different patients was variable and did not correlate with the development of specificity against the MART-1(27-35) or FluM1(58-66) peptides. Specific anti-MART-1(27-35) cytotoxicity could be generated in 13 of 13 melanoma patients but only in 5 of 9 healthy donors (p < 0.001). Anti-FluM1(58-66) activity could be generated in six of seven melanoma patients and six of seven healthy donors. Specific activity against MART-1(27-35), but not FluM1(58-66), was detectable significantly earlier after repetitive in vitro stimulation in melanoma patients (22.7 +/- 2.0 days compared with 32.7 +/- 1.7 days for healthy donors, p < 0.01). This report provides the first evidence of an enhanced level of sensitization of tumor-bearing hosts compared with normal individuals against a differentiation antigen shared by tumor and normal cells of the same lineage. These findings may have important implications for delineating events involved in the biology of tumor rejection naturally or in response to active specific immunotherapy.

Report of immune monitoring of prostate cancer patients undergoing T-cell therapy using dendritic cells pulsed with HLA-A2-specific peptides from prostate-specific membrane antigen (PSMA)

In this paper we describe our program for the immune monitoring of phase II participants given dendritic cell (DC)/prostate‐specific membrane antigen (PSMA)‐based immunotherapy, and we also present some initial findings.

Current methods for loading dendritic cells with tumor antigen for the induction of antitumor immunity

The immunotherapy of cancer is predicated on the belief that it is possible to generate a clinically meaningful antitumor response that provides patient benefit, such as improvement in the time to progression or survival. Indeed, immunotherapeutics with dendritic cells (DC) as antigen-presenting delivery vehicles for cell-based vaccines have already improved patient outcome against a wide range of tumor types (1–9). This approach stimulates the patients own antitumor immunity through the induction or enhancement of T-cell immunity. It is generally believed that the activity of cytotoxic T lymphocytes (CTL), the cells directly responsible for killing the tumor cells in vivo, are directed by DC. Therefore, the goal of many current designs for DC-based vaccines is to induce strong tumor-specific CTL responses in patients with cancer. In practice, most studies for DC-based cancer vaccine development have focused on the development of methods that can effectively deliver exogenous tumor antigens to DC for cross-priming of CD8+ T cells through the endogenous MHC class I processing and presentation pathway (10). To date, many methods have been developed or evaluated for the delivery of defined and undefined tumor antigens to DC. This review provides a brief summary on these methods, the techniques used in these methods, as well as the advantages and disadvantages of each method.

Generation of tumor-specific cytolytic T lymphocytes from peripheral blood of cervical cancer patients by in vitro stimulation with a synthetic human papillomavirus type 16 E7 epitope.

OBJECTIVE Approximately 90% of squamous carcinomas of the cervix harbor the human papillomavirus and type 16 has been detected in nearly 50% of cases. Recent studies in mice have shown that the human papillomavirus type 16 E7 oncoprotein contains peptide epitopes that are processed and presented in association with a major histocompatibility antigen for recognition by cytolytic T lymphocytes. We investigated whether an epitope from human papillomavirus type 16 E7 could be used to generate specific human cytolytic T lymphocytes in patients with cervical carcinoma. STUDY DESIGN After radiation therapy, three patients with antigen HLA-A2 and with locally advanced cervical cancer underwent leukapheresis. Epitope-specific cytolytic T lymphocytes were generated from the peripheral blood mononuclear cells by in vitro stimulation with autologous peripheral blood mononuclear cells pulsed with a human papillomavirus type 16 E7, HLA-A2-restricted, synthetic peptide, E7(11-20) (YMLDLQPETT). RESULTS In two patients cytolytic T lymphocytes were capable of E7(11-20)-specific, HLA-A2-restricted cytolysis of the peptide-pulsed, HLA-matched, T2 target cell line. Cytolytic T lymphocytes from one of these patients also demonstrated specific cytolysis against the HLA-A2+, HPV-16+ CaSki cervical cancer cell line but did not lyse either HLA-A2+, HPV-16- MS-751 cells or HLA-A2-, HPV-16- HT-3 cells. CONCLUSIONS These experiments demonstrate that novel cytolytic T lymphocytes that recognize a human papillomavirus type 16 E7 epitope can be generated by using the peripheral blood mononuclear cells from irradiated patients with cervical cancer. In addition, because CaSki cells were specifically lysed by the cytolytic T lymphocytes, these data indicate that the peptide E7(11-20) is endogenously processed and presented on the cell surface of the CaSki cells. The demonstration of epitope-specific lysis of cytolytic T lymphocytes of HPV-16+ cervical cancer cells supports further efforts to develop human papillomavirus peptide-based vaccines or antigen-specific adoptive immunotherapy for the prevention and treatment of cervical carcinoma.

Use of cellular and cytokine adjuvants in the immunotherapy of cancer

Cellular and cytokine adjuvants, often immune effector cells and soluble factors, respectively, are supplemental and/or follow‐up treatments of human origin for cancer patients who have unsatisfactory clinical responses to conventional chemotherapy, radiotherapy, and surgery. Since many human studies with these reagents are in their infancy, extensive data collection is only now being performed to determine which strategy provides the greatest therapeutic benefit. Research published in the literature since the genesis of this approach to cancer treatment is summarized in this report. Methodologies attempting to generate anticancer responses by provoking or enhancing the patients own immune system are new compared with the other standard types of cancer treatment. Although a few encouraging human studies can be discussed, many of the most promising techniques are only now being transferred from the laboratory to the clinic. The administration of immune effector cells in combination with immunomodulators, such as interferons or interleukins, often enhances clinical outcome. The literature cited in this report indicate that immune‐cell‐ and cytokine‐based therapies hold promise in our attempts to improve the quality and duration of life in those with cancer. With each report reaching the literature, more effective clinical trials are being designed and implemented. J. Surg. Oncol. 1998;68:122–138.

Generation of specific anti-melanoma reactivity by stimulation of human tumor-infiltrating lymphocytes with MAGE-1 synthetic peptide

The MAGE-1 gene encodes a tumor-specific antigen, MZ2-E, which is recognized by cloned, specific cytolytic T cells (CTL) derived from the peripheral blood of a patient with melanoma. We have produced a MAGE-1-specific CTL line derived from the tumor-infiltrating lymphocytes (TIL) of a melanoma patient by weekly restimulation with autologous EBV-B cells pulsed with the synthetic HLA-A1-restricted MAGE-1 epitope nonapeptide EADPTGHSY. The 1277. A TIL line grew in long-term culture in low-dose interleukin-2 (IL-2) and IL-4, and exhibited antigen-specific, MHC-class-I-restricted lysis of HLA-A1-bearing MAGE-1+ cell lines. Cytolysis of target cells pulsed with the synthetic MAGE-1 decapeptide KEADPTGHSY was superior to that of cells pulsed with the immunodominant nonapeptide. Single amino-acid or even side-chain substitutions in the immunodominant nonamer abrogated cytolysis. 1277. A TIL specifically secreted tumor necrosis factor α after co-incubation with HLA-A1-expressing MAGE-1+ cell lines or fresh tumor. These data suggest that tumor-antigen-specific, MHC-restricted CTL may be grown from TIL in the presence of synthetic epitope peptides and expanded for adoptive immunotherapy in melanoma patients.

HLA associations in the antitumor response against malignant melanoma

In this study we analyzed the human leukocyte antigen (HLA) pattern of North American Caucasian patients with metastatic melanoma as compared with the North American Caucasian (NAC) population. We also investigated whether the HLA type of melanoma patients had an effect on their tolerance and response to interleukin-2 (IL-2)-based therapy. Four hundred twelve serologic phenotypes of Caucasian melanoma patients referred to the National Cancer Institute, National Institutes of Health, from February 1989 through December 1993 were collected by typing the patients peripheral blood lymphocytes. Furthermore, 74 melanoma patients were typed for HLA class II by high-resolution sequence specific primer-polymerase chain reaction. Response rate and treatment-related toxicity in those patients receiving IL-2-based treatment (N = 272) were compared with HLA serologic types. The frequency of four HLA-B alleles was significantly different in the melanoma compared with the NAC population: Of these, HLA-B5, -B8, and -B15 had a frequency falling between the NAC and the Northern European population. No other significant differences between melanoma patients and NAC population were noted for other HLA loci. A correlation was noted between HLA-DR3 and -DR4 alleles and decreased tolerance to IL-2, whereas homozygosity for HLA-DR decreased the chance of response. There were no significant associations between HLA type and response. It is unlikely that the associations noted between some HLA-B alleles and melanoma bear significantly on the etiology of the disease. The differences seen between American melanoma patients and the NAC population are probably best explained by geographical ancestry. The association between HLA-DR and tolerance to IL-2 therapy noted in this study may offer insight toward the understanding of mechanisms regulating the cascade of events after the systemic administration of IL-2.

Expression of MAGE genes in ocular melanoma during progression from primary to metastatic disease

Primary melanomas that form within the eye have a unique pattern of disease progression as compared with melanomas that form within the skin. A high percentage of patients (approximately 50%) develop metastatic tumors that occur predominately in the liver. An unusual characteristic of ocular melanomas is the prolonged disease-free interval that extends for many years between the development of primary and metastatic tumors. It is estimated that the shortest interval between dissemination of tumor cells from the eye and the appearance of clinically detectable metastases is 6 years. A recent report indicated that fresh uveal melanoma tissue and metastatic tumor biopsies failed to express melanoma antigen gene (MAGE)-1, MAGE-2, or MAGE-3. In the present study, we examined the expression of MAGE genes on fresh and cultured tumor cells obtained from an ocular melanoma patient during different stages of progressive disease. MAGE gene expression was determined by reverse transcription-polymerase chain reaction using MAGE-1, MAGE-2 and MAGE-3 specific primers. Our results demonstrate that primary ocular tumor tissue and cultured tumor cells both express significant levels of MAGE-1, 2, and 3 at the time of enucleation. A high percentage of tumor cells within the primary tumor appear to express MAGE as demonstrated by consistent MAGE expression in 16 tumor cell clones. Metastatic liver tumors that developed 3 years after enucleation and 18 years after the initial formation of the primary tumor also expressed high levels of MAGE-1, -2, and -3. MAGE was expressed on fresh tumor tissue from a single biopsy and cultured tumor cells obtained from three of four different metastatic tumor nodules. When the MAGE-negative metastatic tumor cells were treated with the demethylating agent 5-Aza-2-Deoxycytidine (5-Aza-dC), transcription of MAGE-1 was restored, indicating the MAGE genes were not deleted. Our results demonstrate that in some patients, MAGE genes are expressed on primary and metastatic ocular melanomas.