Christina J. Kim

Dendritic Cells Infected with Poxviruses Encoding MART-1/Melan A Sensitize T Lymphocytes In Vitro

Dendritic cells (DC) are potent professional antigen-presenting cells that can activate naive T lymphocytes and initiate cellular immune responses. As adjuvants, DC may be useful in enhancing the immunogenicity of tumor antigens and mediating tumor regression. Endogenous expression of antigen by DC offers the potential advantage of allowing prolonged constitutive presentation of endogenously processed epitopes and exploitation of multiple restriction elements for the presentation of the same antigen. In this report, we show that human DC are (a) capable of infection by recombinant poxviruses encoding melanoma-associated antigen (MAA) genes and (b) capable of efficiently processing and presenting these MAA to cytotoxic T cells. In 6/6 HLA A*0201-expressing melanoma patients tested, the virally driven expression of MART-1/Melan A MAA by DC was sufficient to generate CD8+ T lymphocytes that could recognize naturally processed epitopes on tumor cells. In most cases, specific anti-MART-1 reactivity could be detected after a single stimulation. Analysis of epitope dominance revealed that the amino acid sequence recognized by these cytotoxic T lymphocytes (CTL) corresponded to the MART-1(27-35) residues previously shown to be most commonly recognized by cytotoxic T lymphocytes expanded from metastatic melanoma lesions. These data show that the virally driven expression of MAA by DC can be exploited for the efficient induction of clinically relevant cytotoxic T-cell responses. This has clinical implications for active immunization therapy, and currently vaccine trials have been proposed for patients with metastatic melanoma.

Immunodominance across HLA polymorphism: Implications for cancer immunotherapy

Summary: Recent advances in the understanding of the mechanisms leading to tumor recognition by the immune system have shown that, at least in the case of human melanoma, the majority of cytotoxic T lymphocytes (CTL) identified in association with in vivo tumor regression after interleukin-2 therapy recognize nonmutated molecules expressed by most melanoma cells. For this reason, peptide-based or whole protein vaccination protocols against melanoma-associated antigens (MAA) are ongoing in several institutions, with the goal of inducing tumor regression by enhancing in vivo specific antitumor CTL reactivity. The rationale for the use of such vaccines is supported by: (a) preclinical evidence that vaccination with major histocompatibility complex class I restricted epitopes can enhance effectively cellular immunity, (b) evidence that potent antimelanoma CTL reactivity can be generated by repetitive in vitro stimulation of peripheral blood monocytes with MAA, and (c) evidence that the systemic administration of the same MAA can elicit antitumor CTL reactivity in vivo. As strategies are being developed for the development of sound vaccines, two basic approaches are investigated: one vaccination strategy is based on the administration of the specific amino acid sequence recognized by the CTL in association with a particular human leukocyte antigen (HLA) restriction element, and the other is based on the administration of the whole antigenic molecule, which relies on the organisms antigen-processing capabilities to render suitable the antigen for induction of HLA class I restricted CTL reactivity in vivo. Among the various factors complicating T-cell-based vaccination approaches stands the polymorphism of the HLA molecules. HLA are the most polymorphic of human genes, and because such polymorphism is clustered in the functional peptide-binding region, the binding of antigenic peptides is necessarily restricted to specific HLA alleles. This limits the interactions between CTL and antigen to specific sequences for each HLA allele. For this reason, the ability of an individual antigen to function as a T-cell immunogen in the context of different HLA allele restriction elements is an open question. It seems logical that whole- molecule vaccines have the potential advantage of broader use across patient populations. In particular, large antigenic molecules may contain multiple peptide sequences with putative binding properties for different HLA alleles, which in turn may elicit T-cell reactivity across the polymorphism of HLA. Such a concept, however, relies on the assumption that the same antigen may function with similar efficiency as an immunogen in association with different HLA alleles, independently from the epitopic sequence recognized in the various situations. This concept has been challenged recently by several practical observations and remains, in our opinion, an open question. This review will address the practical question of immunogenicity of molecules across the HLA polymorphism. We postulate that the complexity and success of the development of peptide-based vaccination strategies depend on the severity of this restriction, which is currently only incompletely studied and understood. Although no solutions are offered to the problem, emphasis is placed on the importance of this question, hopefully to stimulate the interest of other researchers, particularly in clinical settings, toward the investigation of this type of problem.

Use of recombinant poxviruses to stimulate anti-melanoma T cell reactivity

AbstractBackground: Dendritic cells (DC) are potent professional antigen-presenting cells that can activate naive T lymphocytes and initiate cellular immune responses. As adjuvants, DC may be useful for enhancing immunogenicity and mediating tumor regression. Endogenous expression of antigen by DC could offer the potential advantage of allowing prolonged constitutive presentation of endogenously processed epitopes and exploitation of multiple restriction elements for the presentation of the same antigen. Methods: DC were prepared from the peripheral blood of HLA A*0201 patients with metastatic melanoma in the presence of IL-4 (1000 IU/mL) and GMCSF (1000 IU/mL). Recombinant vaccinia and fowlpox viruses encoding the hMART-1 gene were constructed and used to infect DC. The efficiency of infection and expression of the MART-1 antigen were assessed by immunohistochemistry and intracellular FACS analyses. Cytotoxic lymphocytes (CTL) were generated by the stimulation of CD8+ T cells, with DC expressing the recombinant gene. Reactivity of the CTL was determined at weeks 1 and 2 by the amount of IFN-γ released. Results: DC were infected with recombinant poxviruses and demonstrated specific melanoma antigen expression by immunohistochemistry, immunofluorescence, and intracellular FACS analysis. The expression by DC of MART-1 MAA after viral infection was sufficient to generate CD8+ T lymphocytes that recognized naturally processed epitopes on tumor cells in 10 of 11 patients. Conclusions: Human DC are receptive to infection by recombinant poxviruses encoding MAA genes and are capable of efficiently processing and presenting these MAA to cytotoxic T cells. The potential advantage of this approach is the ability to present specific antigen independent of the identification of the epitope or the MHC restriction element. This strategy may be useful for the identification of relevant epitopes for a diverse number of HLA alleles and for active immunization in patients.

A prospective randomized evaluation of the prophylactic use of low-dose dopamine in cancer patients receiving interleukin-2

The administration of high-dose interleukin-2 (IL-2) causes tumor regression in 17-25% of patients with metastatic melanoma or renal cell carcinoma. Renal dysfunction is a common dose-limiting toxicity of IL-2 administration, limiting 26% of treatment cycles. We have conducted a prospective randomized trial to evaluate whether the prophylactic administration of low-dose dopamine (2 mg/kg/min) can minimize renal toxicity and thus affect the amount of IL-2 administered. Forty-two patients were randomly assigned to receive systemic high-dose IL-2 with standard supportive measures (group A = 21 patients) or with the addition of prophylactic dopamine (group B = 21 patients) at 2 mg/kg/min. For patients in group B, dopamine was instituted 1 h before the initiation of IL-2 administration and was discontinued 6-12 h after the maximum number of doses of IL-2 were given. There was no difference in the amount of IL-2 administered for each course of therapy for groups A and B. Despite differences in urine flow (milliliters per kilogram per day), fluid balance (liters per day), and overall weight gain, prophylactic low-dose dopamine did not significantly alter maximum plasma urea or creatinine levels in group B when compared with the control group (group A). The overall toxicity profile considering all grade 3 and 4 toxicities for patients in groups A and B was comparable. Thus, there is no evidence to support the routine use of prophylactic low-dose dopamine in patients receiving high-dose IL-2.

Combination therapy with interferon-γ and interleukin-2 for the treatment of metastatic melanoma

The toxicity and clinical response to treatment with the combination of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) in patients with metastatic melanoma was evaluated. From May 1993 through February 1994, 20 patients were treated with 24 courses of IFN-gamma with or without IL-2. A 7-day course of subcutaneous IFN-gamma alone was administered to cohorts of two or three patients each at doses of 0.1, 0.2, or 0.3 mg/m2. Thirteen patients received escalating doses of IFN-gamma between 0.2 and 0.5 mg/m2 followed by the intravenous (i.v.) administration of IL-2 (720,000 IU/kg) given three times a day. A treatment course consisted of two cycles (maximum of 15 doses of IL-2 per cycle) separated by a 10-day interval. Five additional patients were treated with five courses of IFN-gamma, IL-2, and tumor-infiltrating lymphocytes (TILs). All patients treated had the diagnosis of metastatic melanoma. The maximal tolerated dose of subcutaneous IFN-gamma was established at 0.3 mg/m2 with dose-limiting hepatotoxicity. Immunohistochemistry analyses showed detectable upregulation of MHC class I alleles in one (8%) of 12 patients. Two of 20 patients who received the combination of IFN-gamma and IL-2 had responses, one partial and one complete response. The duration of response was 7 months for the partial response and 12 months for the complete response. IFN-gamma was tolerated with minimal side effects of nausea, vomiting, malaise, and decreased hematopoiesis. No increased toxicities were found with the combination treatment, as compared with IL-2 alone. One death occurred on the third day of treatment with IFN-gamma alone from hemorrhage into brain metastases. There were no responders in the five patients who received the combination treatment of TIL, IL-2, and IFN-gamma. From these findings, we conclude that further studies looking at this combination treatment are not warranted.