CONFERENCE REPORTS 303 CHIMIA 1999. 53. No.6

Derivation of the Immunosuppressive Macrolide Rapamycin: Chemical, Structural and Biological Aspects

Richard Sedrani, Novartis Pharma AG, Transplantation Research, S-507 .1.01, CH-4002 Basel

The macrolide rapamycin was discovered FKBP12. This complex formation involves ethers led to derivatives which were up to in 1975 at Ayers! in a screen for novel the 'binding domain'. The latter consists threefold more potent than rapamycin. antifungal agents. It has attracted interest mainly oftheC(1)-C(14) subunit, but also Methylation oftheC(28) hydroxy func- since the beginning of the nineties because includes the C(40) hydroxy group, which tion resulted in a thousandfold decrease in of its remarkable immunosuppressive makes a hydrogen bond with FKBPI2. activity, though binding to FKBP 12 was properties and is presently undergoing clin- Binding of rapamycin to FKBP12 is nec- not affected. The X -ray crystal structure of ical trials for the prevention of alIograft essary, but not sufficient, for immunosu- 28-0-methylrapamycin complexed with rejection. pressive activity. Indeed, in a second step, FKBP12 showed that the cyclohexyl ring the FKBP12/rapamycin complex binds to of this derivative has a completely differ- a protein termed FRAP, and it is this sec- ent orientation compared to the one found ond interaction which is ultimately re- for bound rapamycin. Structural evidence sponsible for immunosuppression. The in- indicated that the cyclohexyl ring in its teraction of the FKBPl2/rapamycin com- new position impedes the second complex- plex with FRAP is mediated by the 'effec- ation step with the target protein FRAP, tor domain' which comprises the region apparently mainly for steric reasons. These between C(14) and C(28) and, thus, in- results prompted us to further investigate cludes the conjugated triene subunit. the influence of the cyclohexyl moiety on Through chemical derivation of the the immunosuppressive activity. We found macrolide, we could show that the triene that cleavage of the C(39),C(40) bond, a moiety is indeed critical: partial or com- modification which does not add steric plete hydrogenation and dihydroxylation bulk to the molecule, abrogates the activ- drastically decreased or even abrogated ity. This finding indicated that part of the the immunosuppressive activity. Modifi- cyclohexyl subunit, which until now was cation of the allylic C(l6) methoxy group, thought to be only involved in binding to on the other hand, led to potent deriva- FKBP12, might also be important for in- Rapamycin exerts its immunosupres- tives. While replacement of this group by teraction with FRAP, and thus might also sive activity by forming, in a first step, a larger alkoxy moities resulted in loss of belong to the effector domain of rapamy- complex with its intracelIular receptor activity, the introduction of propargylic cm.

Somatic Cell and : Perspectives in

Roland Mertelsmann, Department of Medicine, University of Freiburg, Germany

The ex vivo expansion of primitive hae- haematopoietic stem cells could improve es for the biological safety and standardi- matopoietic cell harvests is of interest for the treatment of inherited diseases, viral sation of clinical gene transfer into hae- gene therapy and transplantation applica- infections and cancer, and aid the thera- matopoietic cells and have been shown to tions. The possibility of maintaining, ma- peutic evaluation of tumor-cell purging in allow greater cell-culture expansion than nipulating and expanding harvested hu- autologous stem cell transplantation. Suc- serum-containing conditions. Under these man haematopoietic stem cells in ex vivo cessful gene transfer into haematopoietic conditions, efficient purging (>3 logs) with culture could help to provide autologous repopulating cells has been reported at recombinant immunotoxins or dendritic and allogeneic transplants of improved low levels, but improvements are needed cell differentiation can readily be obtained. purity and performance. Such improved for clinical application. The same GMP medium (CellGro = AE) cell yields may be of clinical benefit to Serum-free GMP culture conditions also allows serum-free production of ret- poor-mobilising patients and those requir- have been developed for the expansion, roviral vector-containing medium using ing multiple transplantation, and enable differentiation, purging and gene transfer the gibbon ape leukaemia virus (GALV) access to gene therapy of the haematopoi- ofhaematopoietic stem cells. Such serum- pseudotyped packaging cell line PGT13/ etic system. Efficient gene transfer into free conditions have theoretical advantag- LN. The GAL V envelope binds to a spe- CONFERENCE REPORTS 304

CHIMIA 1999, 53, No.6 cific receptor expressed at high density on human cells but absent on producer cells, thus increasing biosafety. In chronic phase CML, Ph-stem cells Gene Transfer for Immune Therapy can be mobilised efficiently into the pe- ripheral blood by intensive chemothera- of Cancer py. In most CML patients, Ph+ cells have a CD34+/HLA-DR+ phenotype which can Claudio Bordignon, Director Gene Therapy and Cell Transplantation Pro- be depleted from the harvest by extended gram, Fondazione Centro S. Raffaele Del Monte Tabor, Milano, Italy long-term marrow culture. In a phase-I1II trial ofhaematopoietic stem cell autotrans- plantation for patients with CML, serum- free ex vivo gene marking has been per- The first clinical gene therapy protocol after treatment with ganciclovir, whereas formed after in vivo and in vitro purging of began in 1990 for the correction of adeno- one patient with chronic GvHD showed Ph+ cells. Retroviral gene transfer was sine-deaminase (ADA) deficiency. Since partial remission. Circulating PBLs ob- performed in committed and primitive then, retroviral, adenoviral, lentiviral, ad- tained from the patient before and after progenitor cells (long-term culture-initi- eno-associated and non-viral gene deliv- ganciclovir treatment showed no differ- ating cells, LTC-IC) for 7 days in suspen- ery techniques have been explored for ence in ganciclovir sensitivity in vitro and sion culture with serum-free vector-con- treatment of inborn diseases. An explo- high expression levels of the cell-surface taining media supplemented by stem-cell sive second phase began when the tech- marker LNGFR, suggesting that ganciclo- factor (SFC), interleukin-3 (IL-3) and flt- nology was exported to treatment of can- vir resistance of chronic GvHD is mainly 3 ligand, Gene-transfer efficiency was cer and AIDS. related to the cell-cycle dependent activity determined as the percentage of G418- In the context of T -cell-depleted allo- of the HSV -tk/ganciclovir system. resistant colonies relative to the total LTC- geneic bone-marrow transplantation (allo- Three patients treated with donor T- IC or CFU-C-derived colonies. BMT), delayed donor-lymphocytes infu- cells, which were genetically engineered After 7 days of transduction culture, sion plays a central therapeutic role in both to express HSV -tk, developed a specific the overall number of LTC-IC derived graft -versus-Ieukemia and immune recon- immune response against the tk protein, colonies in CD34+ and CD34+/HLA-Drlo stitution. However, the use of donor lym- which resulted in immediate elimination cells increased on average 3.9 ± 2.3 and phocytes is limited by the risk of severe of the infused genetically engineered cells. 2.7 ±2.4-fold, respectively. The transduc- graft-versus-host disease (GvHD) which This phenomenon was observed in pa- tion efficiency was 49 ± 1.3% and 57 is a frequent and life threatening compli- tients who received the first infusion of ± 8.4% for CFU-C and LTC-IC, respec- cation of BMT. Patients who relapsed or genetically modified donor cells late after tively, generated from CD34+ peripheral developed an Epstein-Barr-virus-induced BMT, documenting the role of the recipi- blood progenitor cells. The marker gene is after BMT were treated with ent immune constitution. To circumvent present in the peripheral blood for 200 donor lymphocytes transduced with the the intrinsic limitations of the HSV-tk days. Preliminary analysis suggests that thymidine kinase suicide gene of the Her- based strategy, such as immunogenicity, up to 10% of those cells from the ex vivo pes Simplex virus (HSV-tk). HSV-tk ex- partial ganciclovir resistance and cell-cy- culture that contribute to long-term hae- pression confers to donor lymphocytes a cle dependence, modified vectors for the matopoiesis contain the transgene. A new selective in vivo sensitivity to the drug new and promising suicide gene-strategy method of viral integration-site analysis ganciclovir allowing a specific treatment will be developed. demonstrates oligoclonal of GvHD, thus avoiding immunosuppres- with deferent clones of the marked popu- sive therapies. lation detectable at varying time points. The SFCMM -3 vectorthat encodes the While extended static culture (>7 days) gene for HSV -tk was generated under the leads to telomere shortening and decreased control of a viral LTR promoter as well as telomerase activity, shorter culture peri- the gene for a truncated and, therefore, ods maintain telomere length and increase non-functional version of the low-affinity telomerase activity. Telomere loss after receptor for nerve-growth factor (LNG- 12 days ex vivo expansion culture is not FR). This LNGFR gene served as a marker significantly greater than following stand- for successful transduction and was under ard-dose . Human periph- the control of the SV40 promoter. The eral blood-derived haematopoietic stem transduced cells survived for over 12 cells (HPSC) retain all biological in vitro months, in high proportions (up to 13.4% and in vivo properties for long-term hae- of circulating peripheral blood lym- matopoietic reconstitution. phocytes (PBL)) resulting in antitumor In conclusion, retroviral gene transfer activity in over 50% of patients. Complete into primitive haematopoietic progenitor remission could be achieved in patients cells can be achieved using long-term ex affected by EBV-lymphoma, chronic my- vivo transduction in serum-free culture elomonocytic , acute myeloid conditions supplemented by flt-3 ligand leukaemia, non-Hodgkin lymphoma, and and SCF, with or without IL-3, IL-6 or multiple myeloma. thrombopoietin. Preliminary data indicate Among three patients who developed a that efficient gene transfer into repopulat- GvHD after donor lymphocytes infusion, ing haematopoietic stem cells is possible. two patients showed complete remission