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Nov 30, 2011

Engineered Blood Stem Cells Form Cancer-Killing Lymphocytes In Vivo

  • Scientists say human blood stem cells can be engineered to develop in vivo into fully functional melanoma-killing lymphocytes. A multidisciplinary University of California Los Angeles (UCLA)-led team engineered human hematopoietic stem cells (hHSCs) to express a melanoma antigen, and then reimplanted these cells back into a humanized mouse model carrying human thymus and other organs. The animals subsequently generated melanoma-specific naive CD8+ T cells, which were able to clear melanoma tumors without any additional manipulation. Encouragingly, the transduced hHSCs established long-term bone marrow engraftment.

    Jerome A. Zack M.D., and colleagues report on their studies in PNAS in a paper titled “Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells.”

    The feasibility of using autologous T cells engineered to express a natural T-cell receptor (TCR) specific for the melanoma-associated antigen recognized by T cells 1 (MART-1) has already been demonstrated in patients, the authors write. However, the approach does have some drawbacks, including the potential generation of autoreactive clones when introducing a TCR into peripheral T cells, and the need to extensively manipulate T cells prior to transduction, which can lead to loss of potency and ability to become long-term memory cells. Also, while peripheral blood T cells used in the procedure can trigger strong immune responses, they are generally not long lived.

    Circumventing these problems could be accomplished by using genetically modified hHSCs to generate functional antigen-specific T cells, the authors continue. Indeed, some work using murine progenitors has been undertaken, but the use of human progenitors to generate human transgenic T cells has to date been restricted mainly to in vitro studies. The UCLA team has previously used severe combined immunodeficient (SCID)-hu mice bearing human thymus/liver (thy/liv) implants to generate HIV-specific cytotoxic T lymphocytes (CTLs) from transduced hHSCs. They have now expanded their research by using a modified version of the bone marrow/liver/thymus (BLT) humanized mouse model, which contains a human thymus, enables long-term peripheral immune reconstitution, mimics the human immune function, and is an effective model to test transgenic T-cell functionality in vivo. The aim was to generate transgenic CTLs carrying MART-1, and test the antitumor effects in mice carrying both matched and nonmatched human melanoma tumors.

    The team transduced human hematopoietic progenitors with an HLA-A*0201–restricted, melanoma-specific TCR, and reconstituted the thy/liv implant with a mix of transduced and non-transduced progenitors, followed by an intravenous injection of autologous transduced progenitors. In multiple experiments using this approach, high levels of mature circulating CD8+ T cells that expressed the MART-1–specific TCR were observed 4–6 weeks after injection. The majority of circulating CD8+MART-1+ T cells displayed a naïve T-cell phenotype. Importantly, the authors add, only rarely was the MART-1-specific TCR expressed on CD4 T cells, providing a strong indication that the transduced cells underwent proper T-cell lineage-commitment processes. In addition, there was no expression of the MART-1 TCR on mature CD8+ cells in mice that had thy/liv implants that did not express the HLA-A*0201 molecule.

    Animals were then challenged with one of two types of melanoma tumor in each shoulder. One tumor, M202, could serve as a target for the transgenic CTL, and the other, M207, couldn’t. Encouragingly, the M202 tumors regressed over time when compared with the nontarget M207 tumors. “More specifically, when comparing the M202 and M207 tumors in mice receiving transduced progenitors (treated mice), M202 tumors were targeted in seven of the nine treated mice, and four of nine mice cleared the M202 tumors,” the authors write.

    The authors then used flow cytometric analysis and PET imaging to detect the presence of reporter-tagged transgenic tumor-inflitrating lymphocytes (TILs) in both the HLA-matched and nonmatched tumors. These analyses confirmed that at six weeks after tumor injection (before the tumors had started to regress), there were statistically significantly more transduced peripheral blood cells in the M202 tumors than in the control M207 tumors.

    Ex vivo stimulation of transgenic CTLs taken from the M202- and M207-bearing humanized mice indicated that the cells maintained their antitumor activity, and a direct cell killing assay demonstrated that the MART-1-specific CTLS were also able to kill the specific HLA-A*0201+ targets effectively. Phenotypic characteristics of CD45+CD3+CD8+MART-1+ cells in the spleens and blood of transgenic animals supported the high level of CTL activity exhibited by the MART-1–specific CTLs, the authors continue. Compared with control animals, those receiving TCR-transduced HSCs had significantly higher levels of CD25-expressing cells receiving, suggesting the presence of activated T cells. There were also naive, central, and effector memory cells, as well as terminally differentiated cells in the spleen. In contrast, the CD8 T cell population in control mice was predominantly naive.

    Further supporting the notion that transgenic CTLs were responsible for the anticancer effects, there was a correlation between tumor metabolic activity and levels of reconstitution or ex vivo cytolytic activity. Higher ex vivo CTL activity correlated strongly with decreased tumor growth and clearance of the tumors, and animals demonstrating a higher percentage of CD8/MART-1+/TCR+ cells in the spleen more efficiently tackled tumors than those exhibiting less reconstitution of transgenic cells.

    Finally, the researchers determined that the transduced CD34 cells stably repopulated the bone marrow of irradiated recipient mice. Indeed bone marrow samples from the treated mice exhibited significant levels of integrated vector and expressed the reporter gene on stimulation.

    “We have demonstrated as a proof of principle the therapeutic efficacy of a stem cell-based genetically enhanced immune response against melanoma and established a viable and successful model for testing new, alternative therapeutic approaches for chronic diseases such as cancer,” they conclude.

     


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