Cytotoxic T lymphocyte-mediated immunotherapy

#The present invention is directed to methods for stimulating primary and secondary effector cell responses for cellular immunotherapy. Cellular immunotherapy can successfully prevent or treat various viral infections and tumors, such as posttransplant EBV lymphoma. The present invention relates to methods for effecting cellular immunotherapy by providing for presentation of viral particles or specific antigens to effector cells, by the most effective antigen presenting cells without the need to develop an active viral infection in the antigen presenting cells. Furthermore, the present invention relates to generating effector cells specific for more than one opportunistic pathogen, e.g., Epstein-Barr virus and adenovirus.

#Immunocompromised patients lack a fully active and effective immune system, and are vulnerable to infection by a host of opportunistic organisms that are effectively controlled in a healthy individual. Cancer patients and transplant recipients are especially vulnerable to these infections since their therapeutic regimen often includes radiation and chemotherapeutic agents, which compromise the immune system. Immunodeficient patients, such as AIDS and SCID patients, are also at high risk from these opportunistic pathogens. In particular, patients undergoing bone marrow transplantation (BMT) are severely immunocompromised until their immune systems reconstitute. During the period prior to reconstitution, these patients are susceptible to serious, and sometimes fatal, virus infections caused by normally benign viruses such as adenovirus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV).

#In a normal individual, recognition and destruction of virally infected cells is performed principally by CD8.sup.+ cytotoxic T lymphocytes (CTLs). The mounting of a CTL immune response requires that the viral proteins undergo intracellular processing to peptide fragments. Selected peptides of defined length are subsequently presented at the cell surface in conjunction with MHC class I molecules. This complex provides the first stimulatory signal recognized by the specific cytotoxic T lymphocyte.

#FIG. 1. Proliferation of mononuclear cells (MNCs) in response to irradiated dendritic cells (Den) pulsed with nothing, wild-type adenovirus type 5 (Ad), or dl312 (E1A-), an Ad5 mutant deleted for the E1A region at effector to target ratios of 10:1 (dense diagonal), 2:1 (bold diagonal), and 0.4:1 (open diagonal). The numbers represent counts per minute of .sup.3 H-thymidine incorporated into DNA. The effector cell population was characterized as follows: 59% CD4; 32% CD8; 84% .alpha./.beta.; 3% .gamma./.delta.; 70% HLA-DR (class II); and 8% CD56.

#FIGS. 2A-2H. Cytotoxicity and cell-surface phenotype of T cells that grow in response to virus-pulsed dendritic cells. T cell lines were prepared from donors LW (A, C, E, G) and CR (B, D, F, H) and assayed against mock-(A,B), Ad5 (WT)-(C,D), dl312 (E1A-)-(E,F), or dl7001 (E3-)-(G,H) infected fibroblasts. Both autologous (AUTO, solid circle) and HLA-mismatched (ALLO, solid square) fibroblasts were used. CTLs from LW were 52% CD4, 25% CD8, 13% CD56, 87% HLA-DR, 83% .alpha./.beta., and 2% .gamma./.delta.; CTLs from CR were 67% CD4, 27% CD8, 6% CD56, 76% HLA-DR, 91% .alpha./.beta., and 4% .gamma./.delta..

#FIG. 3. Cytotoxicity of CD56.sup.+ -depleted versus non-depleted CTLs. CD56.sup.+ -depleted (solid bars) and non-depleted (cross-hatched bars) CTLs were prepared from LW and assayed against either uninfected K562 cells or against autologous (AUTO) or allogeneic (ALLO) fibroblasts either mock infected or infected with the indicated viruses. The relative populations before and after depletion are listed in the figure.

#As noted above, the present invention is directed to methods for stimulating primary and secondary effector cell responses for immunotherapy. In principle, immunotherapy can prevent or treat opportunistic pathogenic viral infections or tumors (including viral tumors) in immunodeficient subjects. However, cellular immunotherapy with ultrafractionated donor derived population of T cells can lead to graft-versus host (GVH) disease when a subset of administered T cells cross-react with host antigens. The ability to generate pathogen-specific effector cell populations without engendering GVH disease is critical for successful CTL immunotherapy.

#CTL immunotherapy has great promise for treating Epstein-Barr virus (EBV) lymphoproliferative disease and EBV-associated Hodgkin disease following bone marrow transplant (BMT). EBV lymphoproliferative disease occurs in about 3-30% of BMT recipients who receive marrow from unrelated donors. Adenovirus infections are common in patients receiving chemotherapy, organ transplantation rejection therapy, and in 10-30% of BMT patients. CTL immunotherapy has significant potential for treating these infections and for treating other viruses associated with malignancies such as adult nasopharyngeal carcinoma (NPC), and for recipients of organ transplants (who are also at risk for cytomegalovirus, CMV, infection), papillomavirus-induced laryngeal papillomatosis and certain cancers, and in immunocompromised patients, such as acquired immunodeficiency syndrome (AIDS) or severe combined immunodeficiency disease (SCID) patients.

#Prior efforts to generate virus-specific CTLs depended on using antigen presenting cells actively infected with virus as stimulators. For example, Riddell et al. ›Science 257:238 (1992)! described generating human CMV-specific CTL clones in vitro with CMV-infected fibroblasts as the stimulating cells. Riddel used CD8.sup.+ clones (no CD4 cells were present), which persist only for 6-12 weeks ›as described in Walter et al., NEJM 333:1038 (1995)!. The present invention uses CD8.sup.+ and CD4.sup.+ cells (which maintain survival of CD8.sup.+ cells), and thereby permitting CTLs to persist for many months. Rooney et al. ›Lancet 345:9-13 (1995); see also Heslop et al., Nature Med. 2:551 (1996)! describe stimulating EBV-specific CTLs with EBV-transformed B cell lymphoblastoid lines. As shown in the Examples, infra, the present invention provides a number of advantages over the prior art by providing for presentation of viral particles or specific antigens to effector cells by the most effective antigen presenting cells, without the need to develop an active viral infection in the antigen presenting cells.