A major limitation to chemotherapy treatments for cancer is drug induced immune toxicity. One strategy to combat the severe toxicity is to genetically engineer blood or marrow cells by the introduction of retroviral vectors designed to express cDNA sequences that confer drug resistance. Introduction of drug resistant genes into hematopoietic stem cells (HSCs) results in transgene expression throughout the host hematopoietic system, including immunocompetent cells such as T cells and natural killer cells, after transplantation of gene-modified cells [20
]. We hypothesized that expression of a cDNA sequence that confers drug resistance within immunocompetent cells would allow for the combined use of chemotherapy and immune effector cell based immunotherapy.
Previously our laboratory evaluated the feasibility of using drug resistant immunotherapy in the context of drug-resistant hematopoietic cells [20
]. Mouse bone marrow cells were genetically engineered by retroviral mediated introduction of a cDNA encoding for a mutant form of DHFR, L22Y-DHFR, that confers resistance to trimetrexate (TMTX).
Mice were transplanted with gene-modified bone marrow cells, which resulted in transgene expression in all hematopoietic lineages, and treated with an immunotherapeutic agent, anti-CD137, or TMTX, or anti-CD137 and TMTX. In mice inoculated with AG104 sarcoma cells, TMTX chemotherapy reduced the efficacy of an anti-CD137 antibody in mice transplanted with non-modified cells. However, when mice were protected against chemotherapy-induced toxicity through transplantation of L22Y-DHFR-expressing bone marrow, the combined treatment of TMTX and anti-CD137 resulted in complete eradication of tumors in 100% of animals. In contrast to the protection of the entire hematopoietic system, in the present study we evaluated if genetically engineered human immunocompetent cells can be used in the context of drug resistance immunotherapy.
NK-92 and TALL-104 cells were selected as representative immune-effector cell lines since both of these cell types recognize and kill a wide range of malignant cells, including K562 cells [15
]. We found that P140KMGMT genetically engineered NK-92 and TALL-104 cells were resistant to TMZ and had cytotoxic activities similar to the non-modified cells. Additionally, the gene-modified cells showed cytolytic activities similar to non-transduced cells after drug selection. Therefore, genetic modification of these cells, and likely other antitumor immunocompetent cells, does not affect their cytotoxic activity.
The concept of drug resistant immunotherapy was then evaluated in a series of cytotoxic assays, in the presence and absence of drug. Importantly gene-modified immunocompetent cells displayed significant cytolytic activities toward drug resistant tumor cells in the presence of drug. In contrast, non-modified immunocompetent cells were ineffective at tumor killing when drug was administered. Taken together, these results demonstrate that in the presence of drug i) the effectiveness of non-modified effector cells is significantly diminished, ii) gene-modified effector cells remain active, and iii) greater killing is observed after treating gene-modified effector cells and non-modified target cells compared to non-modified effector cells and drug resistant target cells. Therefore, we conclude that genetically-modified drug resistant immunocompetent cells can be engineered to survive the toxic effects of chemotherapeutic agents and the effectiveness of tumor killing increases during a chemotherapy challenge.
Our in vitro proof-of-concept studies demonstrate that drug-resistant immunocompetent effector cells are superior cytotoxic effectors during a chemotherapy challenge. This is a significant finding which can potentially be combined with current cell-based and adoptive immunotherapies. Regression of large, vascularized tumors has been shown in patients with refractory metastatic melanoma. However, for maximum effectiveness a lympho-depleting regimen is necessary prior to autologous lymphocyte cell transfer [23
]. Generation and expansion of drug-resistant lymphocytes ex vivo
can allow, in this setting, for administration of immunocompetent cell-based therapy concurrently with chemotherapy, potentially improving tumor clearance while antitumor immunity is established and maintained. In this scenario, non-transduced lymphocytes can continually be depleted using a selective chemotherapy treatment, which could be repeatedly applied during the administration of adoptive immunotherapy. The co-administration of chemo- and immunotherapies could then lead to long-term tumor clearance. However, it has also been shown that the growth of CML cells in mice transplanted with bone marrow engineered to confer resistance to MTX can be exacerbated by the administration of chemotherapy [24
]. Thus chemotherapy treatment in the context of gene-modified whole bone marrow protection may induce secondary effects such as immune suppression that allow some cancers to survive a drug challenge. Based on our results, instead of transplanting drug resistant hematopoietic stem cells, a more effective strategy could involve transplantation of drug resistant immunocompetent lymphocytes.
It has recently been shown that melanoma and glioma cell lines are sensitive to the combination of TMZ and antifolates [25
]. Retroviral transfer of dual vectors that co-express multiple cDNAs that confer resistance to multiple chemotherapy agents can be used to improve tumor cell killing by the administration of a combination of chemotherapeutic agents. For example, expression of DHFR mutants provide resistance to antifolates, such as MTX, while MGMT expression provides resistance to monofunctional methylating agents, such as TMZ. Furthermore, the knowledge of drug resistant mechanisms for many chemotherapy agents can be employed to genetically engineer immunocompetent cells that are resistant to numerous anti-cancer agents. The engineered cells could then be tested in the context of drug resistance immunotherapy, which our results indicate should improve the effectiveness of tumor killing during a chemotherapy challenge.