Novel approaches to current cellular therapies continue progress toward disease prevention

December 08, 2003

(San Diego, Calif., December 8, 2003) - Over the past few decades, the medical community has made great strides in the search for effective treatments and cures for some of society's most debilitating diseases. One field that has seen a great deal of advancement over the past decade is cellular therapy. Through the use of stem cells, bone marrow transplants, and therapeutic cloning, researchers explore ways to replace diseased or dysfunctional cells with healthy, functioning ones. Although advances in cellular therapy have been limited by complications such as donor availability, immune rejection, and graft versus host disease (GVHD), several studies presented during the 45th Annual Meeting of the American Society of Hematology explore novel approaches that show promise for future medical benefits.

"There are a number of serious diseases about which very little is known," said Janis Abkowitz, M.D., Head of the Hematology Section, University of Washington Medical Center, and Professor of Medicine, University of Washington, Seattle. "Through innovative techniques and continual medical advancements, the scientific community provides us with the hope that we will one day find effective therapies for even the most elusive diseases."

A Bovine Model of Long-Term Hematopoietic Engraftment with Stem Cells Generated by Nuclear Transplantation (Abstract 259)

Therapeutic cloning, also known as somatic cell nuclear transfer, which is used in experimental tissue and organ generation, has recently been explored as a possible treatment for a wide range of degenerative diseases. A study led by Malcom A.S. Moore, D. Phil., Memorial Sloan-Kettering Cancer Center, New York, used the therapeutic cloning technique to examine its potential for hematopoietic stem cell transplantation therapy. Results showed that this technique appears to have the potential to generate cloned, compatible hematopoietic stem cells that are capable of long-term, multi-lineage engraftment in a large animal model.

"These results are very encouraging and pave the way for further studies in this area," said Dr. Moore. "Although the level of engraftment was not particularly high, the fact that we observed engraftment at all is extraordinarily interesting."

Researchers obtained skin fibroblasts from several old (10 to 13 years) donor cows and selectively grew them in culture for the neor genetic marker, a genetic "tag" which aids in the identification of successfully engrafted cells after transplantation. They then removed the nucleus from these neor positive fibroblast cells and transplanted it into an oocyte (immature egg) that was devoid of all genetic material and came from another unrelated donor cow. This therapeutically cloned embryo was grown in vitro to the blastocyst stage and implanted in the uterus of yet another unrelated donor cow. Embryos were removed from the maternal donor cow's uterus at 100 to 110 days gestation and harvested for fetal liver cells, which are known to be a rich source of hematopoietic stem cells at that stage of development.

These fetal liver cells, still positive for the neor genetic marker, were transplanted into several of the original old donor cows by means of intravenous injection. To make room for the new cells and aid full stem cell engraftment, two of the animals were treated with busulfan (an alkylating agent that decreases the number of stem cells in bone marrow). This process, known as "conditioning," helps achieve a state of myelosuppression, in which the bone marrow's production of blood cells and platelets slows or stops altogether. One animal remained untreated. Stem cell engraftment was monitored by multiple analysis procedures, including progenitor (CFC) assay.

Upon observation after 414 and 488 days, there appeared to be a percentage of peripheral blood leukocytes that originated from cloned donor cells, peaking at nine to 17 percent in the granulocytes, and at lower levels in lymphocyte subsets (0.0 to 0.01 percent). Circulating progenitor cells appeared to have an even higher incidence of cloned cells that were positive for the neo(r) genetic marker.

Surprisingly, clonally derived stem cells appeared to differentiate into endothelial cells and were found to be present in the aorta. Another unexpected outcome was a lack of immune rejection after the transplant. Due to the need for multiple, unrelated donor cows during the cloning process, there are slight differences in the mitochondrial proteins of transplanted stem cells from those of the recipient cow. This difference in mitochondrial proteins has the potential to trigger the body's immune response. However, persisting engraftment suggested the absence of immune rejection.

Transferring Donor Immunity to Pathogens Across HLA Barriers (Abstract 129)

Bone marrow transplants are inherently dangerous and are associated with many potentially fatal health risks. In order to make room for donor cells in a recipient's bones, conditioning of the patient prior to a transplant requires harsh radiation and chemotherapy that significantly weakens a recipient's immune system, effectively rendering the patient defenseless and very susceptible to opportunistic infection, GVHD, or immune rejection. Re-building immunity to pathogens after a bone marrow transplant is important for the continued health of a transplant recipient. A study led by Andrea Velardi, M.D., University of Perugia, Italy, examines the possibility of speeding up the development of pathogen immunity after transplant by transferring immune system T-cells from the donor to the recipient.

Cytomegalovirus (CMV), a herpes virus, and Aspergillus, a fungus that includes many common molds, act as opportunistic infectious agents in immunosuppressed patients. Researchers identified CMV and Aspergillus, potentially allo-reactive pathogens, in the donors' system, then created allo-deleted T-cell clones specific to these pathogens (>2000 CMV-specific CD4+ clones from 21 CMV positive donors and >1,000 Aspergillus-specific CD4+ clones from 10 donors). Results from this study were positive and appeared to identify a safe and effective way to re-build immunity to pathogens after bone marrow transplantation.

"Many patients who undergo bone marrow transplants experience unnecessarily high rates of GVHD and frequently suffer from opportunistic infections as their bodies re-build immunity to pathogens," said Dr. Velardi. "This study shows great promise for preventing these complications in future transplants and significantly improving patient outcomes."

Thirty-one recipients underwent transplants and on day +20 were given a single dose of adoptive therapy containing T-cell clones specific to the donor's CMV and Aspergillus pathogens (21 received CMV-specific T-cells and 10 received Aspergillus-specific T-cells). Twenty-three recipients in the control group underwent transplants, but did not receive adoptive therapy. In the 31 patients receiving adoptive therapy, antigen specific T-cells were present at near normal levels as early as two to three weeks after infusion and T-cell counts remained stable over time (monitored for approximately one year). Nine to 12 months post transplant, antigen-specific T-cells were detected at very low frequency in 23 patients of the control group.

In the 21 transplant recipients who received anti-CMV T-cell clones, CMV antigen activity became negative from two to three weeks after the infusion onward. Two patients who had CMV disease at the time of infusion appeared to be cleared of the disease and continued to be well at six and 12 months. None of the other 19 patients who received adoptive therapy developed CMV disease. No deaths due to CMV were observed in this group.

In the 10 transplant recipients who received adoptive therapy with anti-Aspergillus T-cell clones, nine had Aspergillus antigen activity immediately following the transplant, and five of the nine had Aspergillus pneumonia. After adoptive therapy, antigen activity appeared to decrease, and all nine recipients eventually cleared the disease.

In the 23 recipients of the control group who did not receive adoptive therapy following the transplant, 22 experienced repeated CMV reactivation approximately 18 weeks after transplant. Six patients developed pneumonia from the CMV disease, and five of the six subjects died. Thirteen out of 23 recipients in the control group displayed Aspergillus antigen activity, which persisted for several weeks after transplant. All 13 had pneumonia and seven of the 13 died.

Transplant patients were at risk for GVHD, due to the study's design and inherent risk in bone marrow transplants. The only case of GVHD occurred in a patient who received 3x106/kg cells of adoptive therapy. Doses of antigen specific T-cells ranged from 105 to 3x106/kg cells.

Lastly, two transplants from CMV negative donors into CMV positive recipients used pre-transplant recipient cells to clone CMV-specific, non-donor reactive T-cells. Infusion with adoptive therapy did not cause rejection and it was followed by prompt recovery of CMV specific T-cell counts that were of recipient origin, unlike the other T-cells present in the body that appeared to be from the donor.
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The American Society of Hematology is the world's largest professional society concerned with the causes and treatment of blood disorders. Its mission is to further the understanding, diagnosis, treatment, and prevention of disorders affecting blood, bone marrow, and the immunologic, hemostatic, and vascular systems, by promoting research, clinical care, education, training, and advocacy in hematology.

American Society of Hematology

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