Modification of the Immune Response

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The documentation of the graft vs leukemia (GVL) effect in decreasing relapse after allogeneic transplantation and the success of donor lymphocyte infusion (DLI) in producing sustained remission after leukemic relapse are clear indicators of the power of the immune system to control leukemia. Malignant cells may evade immune surveillance through several mechanisms, including actively suppressing the immune response (171-174), alteration of the CD95/Fas system (175) and downregulation of major histocompatibility complex (MHC) class I or II expression (176). The concept of exploiting the immunologic response to provide therapy for leukemia is not new. Mathé in 1965 treated children with ALL in remission with vaccinations consisting of leukemic blasts and BCG, documenting prolonged DFS in 8 of 20 patients, while all 10 controls relapsed (177). Our current ability to transfer and express genes has led to myriad possibilities to manipulate the immune system as a means of therapy. Of these, two primary approaches can be defined: (1) genetic modification of the malignant cell, or a cell to be used with it as a vaccine to augment the immunogenicity of the leukemia, and (2) to engineer an increased capacity of an immunologically active cell population (effector cells) to eradicate cancerous cells otherwise inefficiently identified or killed. These approaches have the common goal of eliminating residual leukemic cells using an immunologic approach, but are distinct and are discussed independently.

3.2.1. Engineering of Leukemia Cells to Increase an Immunologic Response

These applications have generally had the goal of designing a more effective tumor vaccine than was previously possible, and, with few exceptions, this is performed by modification of cells ex vivo. More than a decade ago it was shown that the endogenous expression of immunologically active cytokines can decrease tumorigenicity and enhance immunogenicity in several murine malignancies (178-181). Since that time, numerous cytokines, human leukocyte antigen (HLA) molecules, costimulatory proteins, and integrins have been tested and shown to have value in this role. A detailed description of these approaches is beyond the scope of this discussion, and previous reviews have well documented the issues involved (1,182-185). Several investigators have described increased survival of mice vaccinated with leukemia or lymphoma cells engineered to express cytokine genes, such as interleukin (IL)-2 and granulocyte macrophage colony stimulating factor (GM-CSF) (186-190). It is hypothesized that the production of high levels of a cytokine directly stimulatory to cytotoxic T cells or antigen-presenting cells in the immediate environment surrounding the malignant cell may prove more effective in initiating a generalized immune response than systemic administration, which can only hope to achieve modest serum levels. The transfer of the human IL-2 gene stimulates an increased immune response to murine myeloma (189). Clinically, an adenoviral vector encoding the IL-2 gene was used to transduce an intracranial plasmacytoma in a patient with myeloma. Although gene transfer was documented, no apparent clinical response was observed (191). Brenner has tested the combination of human CD40 ligand (hCD40L) to increase the expression of costimulatory factors on the malignant cells and enhance immune recognition and IL-2 in CLL blasts and lymphoma, and has shown that the expression of both molecules provides increased antitumor immunity than either alone (114,118). Vaccination of animals with AML blasts engineered to express IL-12, another cytokine stimulatory to T cells, provides increased survival in mice with previously established leukemia (192,193).

The use of agents that can enhance the processing and presentation of antigens, most notably GM-CSF, may also be effective at establishing specific cytotoxic T cells directed against the malignant cell. In a murine model of AML, vaccinations of leukemic cells engineered to produce GM-CSF by retro-viral-mediated gene transfer were superior to vaccine strategies in which the B7.2, IL-4, or TNF-a genes were used (194). In a murine Philadelphia chromosome positive (Ph+) ALL model, GM-CSF expression was effective in vaccination experiments but CD80 (B7.1) and GM-CSF coexpression provided the greatest protection, against the administration of wildtype leukemia (195). An interesting approach to GM-CSF production in the local environment for the purposes of leukemia vaccinations uses the K562 cell line engineered to express high GM-CSF levels (196). The development of a cell line producing GM-CSF that can be used instead of transducing autologous cells has the potential to make vaccinations much less complex. In addition, because the K562 line does not express class I or II MHC molecules, the likelihood that the cells will be readily rejected is diminished.

A means of providing increased immunologic recognition of B-cell malignancies, which express immunoglobulin idiotype molecules on the cell membrane, is the development of a T-cell response to idiotype antigens using vaccinations. This concept has merit without the use of gene transfer techniques, because vaccinations of idiotype-specific proteins and keyhole limpet hemocyanin (KLH) administered to 16 patients with follicular non-Hodgkin's lymphoma (NHL) resulted in an increase in the cytotoxic T-cell precursor (CTLp) frequency all patients in vitro, and eight sustained responses were noted (197). However, B-cell neoplasms do not present antigens well, which limits the potency of a T-cell response (113). These cells can be engineered to express costimulatory molecules such as B7.1 and B7.2 or CD40L (CD154) by gene transfer, making them much more likely to generate a T-cell response. This strategy was employed in a clinical trial in which an adenoviral vector was used to express CD154 in CLL cells ex vivo (117). The infusion of engineered cells was in general well tolerated, although fevers, elevated transaminases, and arthralgias were observed. The elevation of plasma cytokine levels, including TNF and IL-12, was observed, and an increase in the number of leukemia-specific T-cells was reported, suggesting that this line of intervention may prove important for B-cell malignancies.

An alternative mechanism by which an enhanced antitumor response may be obtained is by increasing the processing and presentation of tumor-associated antigens by cells other than the malignant population. The primary cell target of this approach is the dendritic cell, which was first described in murine tissues by Steinman in the early 1970s (198,199). T-cells can be "educated" to respond to peptide fragments derived from antigens bound to MHC class I (intracellular or endogenous antigens) or class II (exogenous antigens) molecules (200). The dendritic cell has a greater density of MHC, adhesion, and costimulatory molecules on the cell surface than other cells capable of presenting antigen, making them well suited for interacting with T-cells toward the generation of specific cellular responses (201). As techniques for the generation of dendritic cells from peripheral blood have been developed, they have become a primary focus in the engineering of T-cell responses (38,202). To facilitate antigen presentation and increase antileukemia or myeloma T-cell responses, several potential antigen sources can be used, including synthetic peptides, proteins derived from tumor cell extracts, or RNA derived from tumor cells (203-209). The use of a BCR/ABL-derived peptide and dendritic cells markedly increases the number of T-cells directed against murine CML, and vaccination with peptide-pulsed dendritic cells had protective effects against the administration of wildtype leukemia (210). It is possible to obtain dendritic cells directly from myeloid leukemia cells, which appear to present antigens of leukemic origin and increase the specific T-cell response (206). Westermann reported similar findings for dendritic cells derived from patients with CML and that the transduction of dendritic cells with the IL-7 gene increased the T-cell response to CML cells (205). To increase antigen specificity, the desired antigen can be produced within the dendritic cell using viral vectors. The engineering of dendritic cells to express LMP2A antigen with an adenoviral vector results in an enhanced T-cell response, which may prove a viable means of increasing the response to Hodgkin's lymphoma (168).

3.2.2. Genetic Modification of Effector Cells

An alternative approach to the manipulation of malignant cells or antigen-presenting cells is the direct modification of the effector cell population to increase the recognition, function, or killing of effector cells. Clinical trials were initiated in which TIL were genetically modified to express the TNF gene, with the intention of killing the malignant cells through the local production of providing TNF at the tumor site, resulting in increased control of the malignancy (211,212). The endogenous expression of IL-2 in a human natural killer (NK) cell line or donor-derived human NK cells increases the ability to kill tumor targets (213,214). This approach was also tested by transducing murine T-cells with a retroviral vector containing the IL-2 cDNA. T-cells expressing IL-2 grew independently and maintained antigen specificity (215). Similarly, transduction of hematopoietic precursors has also been reported with IL-2 vectors, and animals receiving transduced cells have improved survival after a leukemia challenge (216). A drawback to this approach, however, is the potential for autocrine stimulation and uncontrolled proliferation of cells transduced with a stimulatory cytokine, which would likely require coupling the expression of the gene designed to enhance function with a "suicide gene" such as the herpes simplex virus thymidine kinase (HSV-tk) gene. Another means of responding to this concern uses constructs designed with alternative cytokine genes to stimulate T-cells; Minamoto designed a fusion gene in which the extracellular moiety of the erythropoietin receptor was combined with genes encoding signaling domains of the IL-2 receptor. The presence of erythropoietin in the environment was sufficient to drive T-cell proliferation (217).

In contrast to providing a nonspecific stimulus to T-cells, the capacity to generate effector cell specificity through gene transfer has also been explored. Dem-bic et al. documented that it is possible to transfer the a and P T-cell receptor genes from a T-cell with the desired specificity into an otherwise naive T-cell and thereby generate specificity (218). However, the ability to recognize antigen after the transfer of T-cell receptors is MHC restricted, because the antigen is presented in association with the MHC molecule, which is a significant drawback to this approach (219). Chimeric genes have been described in which the variable regions of antibodies were fused to T-cell receptors to generate specificity, because this would not require antigen to be expressed in association with MHC molecules and, therefore, cells could be engineered in a non-MHC-

Fig. 4. Engineering of T-cell specificity. The variable regions of the light and heavy chain (VL and HV) antibody genes are combined with a flexible linker, and joined to the transmembrane and cytoplasmic portions of a gene that can provide signaling within a T-cell, such as the zeta chain of the T-cell receptor. This construct can be introduced into the effector cell by various gene transfer methods. The protein is anchored in the cell membrane with the single chain Fv region (scFv) external to the cell where it can bind the desired antigen of leukemia or lymphoma cells. Binding results in killing of the cell expressing the antigen and activation of the effector cell through signaling pathways.

Fig. 4. Engineering of T-cell specificity. The variable regions of the light and heavy chain (VL and HV) antibody genes are combined with a flexible linker, and joined to the transmembrane and cytoplasmic portions of a gene that can provide signaling within a T-cell, such as the zeta chain of the T-cell receptor. This construct can be introduced into the effector cell by various gene transfer methods. The protein is anchored in the cell membrane with the single chain Fv region (scFv) external to the cell where it can bind the desired antigen of leukemia or lymphoma cells. Binding results in killing of the cell expressing the antigen and activation of the effector cell through signaling pathways.

restricted fashion (219,220). The ability to isolate genes encoding the variable regions of antibodies and combine them into a single gene with maintained antigen specificity allowed further modification of this strategy (221,222). These single-chain Fv (scFv) genes can be combined with genes encoding the transmembrane and intracytoplasmic domains of a protein that signal T-cells to proliferate and become cytotoxic, such as the Z chain of the T-cell receptor or y chain of the Fc receptor (223,224) (Fig. 4). The expression of these chimeric genes have been shown to markedly increase the killing of several malignant cells bearing a target antigen and signaling through the intracytoplasmic Z or y chain results in activation and cytokine expression (223-228). A clinical trial is underway in which an anti-CD20 scFv/Z plasmid is transfected into autologous T-cells expanded in the presence of antigen before infusion (229). This method for producing specific effector cells has great promise, but issues such as prolonged function of expanded cells and the potential for rejection of cells with chimeric antibody/T-cell receptor proteins remain to be addressed.

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