Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

304 15 Bone Marrow Transplantation for Immune Therapy
15.7
The Suicide Gene Strategy
A suicide gene codes for a protein able to convert a non toxic pro-drug into a toxic
product. Therefore, cells expressing the suicide gene become selectively sensitive to
the pro-drug. The transfer of a suicide gene into donor lymphocytes could allow the
in vivo selective elimination of transduced lymphocytes and therefore switch off the
GvHD.
Different suicide genes have been investigated and described in recent years. At present,
the thymidine kinase of Herpes Simplex virus (HSV-tk) seems to be the most
effective, and it is the first one proposed and used in clinical trials [24, 25]. The HSVtk
protein converts the pro-drug gancyclovir (GCV) to its monophosphate intermediate
derivative. Cellular kinases phosphorylate it to a triphosphate (GCV-3P) compound,
which is the toxic form. GCV-3P can be incorporated into DNA, replacing
deoxyguanosine triphosphate, resulting in inhibition of DNA chain elongation.
The safety of gene transfer into peripheral blood lymphocytes (PBLs) has been evaluated
both in animal models and in previous clinical protocols of somatic gene therapy
[26±30].
The vectors that we used to modulate GvL in the context of allogeneic BMTand the
transduction procedure of human PBLs have been described previously [31]. For patient
1±8, we used the SFCMM-2 vector encoding for the truncated form of the lowaffinity
receptor for the nerve growth factor (DNGFR) and a bifunctional protein carrying
both the HSV-tk activity, conferring GCV sensitivity to transduced cells, and
neomycin phosphotransferase (neo R ) activity, conferring resistance to the antibiotic
neomycin and to its analogue G418. For patient 9±23, we removed the neo R gene in
order to reduce the immunogenicity of the transgene. The SFCMM-3 vector confers
higher GCV sensitivity to transduced cells: almost 100 % of cells are killed by GCV
concentrations reached in vivo. The cell surface molecule allows a rapid in vitro selection
of transduced cells by the use of magnetic beads conjugated with an antibody directed
to DNGFR; this selection requires a shorter culture time, an important variable
in order to preserve the immune repertoire. In addition, a surface marker allows
easy ex vivo detection and characterization of the transduced cells by FACS analysis.
Moreover, since NGFR is a human protein, it is not expected to be a target of a specific
immune response.
A phase I±II clinical study for the infusion of HSV-tk-transduced donor lymphocytes
to patients affected by severe complications after allogeneic BMT began in 1993 at
H. S. Raffaele (Milano). Two protocols were designed to evaluate the infusions of donor
lymphocytes transduced with a suicide retroviral vector in the context of allogeneic
BMT. In the Add-back protocol, 1 × 10 7 CD3 + HSV-tk transduced lymphocytes
were infused 6 weeks after transplant; the primary end-point of the Add-back protocol
was the immune reconstitution obtained by HSV-tk cells after T-depleted transplants.
In the Treatment protocol, patients received a mean dose of 4.4 × 10 7 CD3 +
HSV-tk cells for the treatment of disease relapse. More than 95% of transduced cells
infused were CD3 + and DNGFR + , and most were CD8 + with an activated phenotype.
The in vivo functional properties of transduced lymphocytes were evaluated by asses-