Susan K. Fautsch

Good manufacturing practices production of natural killer cells for immunotherapy: A six-year single-institution experience

BACKGROUND: Natural killer (NK) cells, a subset of lymphocytes and part of the innate immune system, play a crucial role in defense against cancer and viral infection. Herein is a report on the experience of clinical‐scale, good manufacturing practices (GMPs) production of NK cells to treat advanced cancer.

Successful “in-flight” activation of natural killer cells during long-distance shipping

BACKGROUND: Natural killer (NK) cells have shown promise in the treatment of malignancy. However, the widespread use of these cells may be limited by both the lack of resources and the expertise needed to manufacture them and the apparent need to use only fresh cells. The NHLBI‐sponsored Production Assistance for Cellular Therapies group was established to provide the resources and expertise to carry out cell therapy research, including support of clinical trials. Here we describe the qualification of in transit activation of an NK‐cell therapy product in preparation for a Phase I clinical trial at a distant medical center.

Concentration of citrate anticoagulant in peripheral blood progenitor cell collections

Background: Peripheral blood progenitor cell (PBPC) collection by hemapheresis has become widely used in recent years. For anticoagulation during cytapheresis, citrate solutions, commonly ACD‐A, are used, at a recommended anticoagulant‐to‐whole blood ratio of 1:11 to 1:12. Although the apheresis procedure is generally well tolerated, the most common patient complaints are attributable to transient hypocalcemia, which is a side effect of the citrate anticoagulant. Patients experiencing discomfort due to hypocalcemia are sometimes managed by a decrease in the flow rate of the anticoagulant.

Mislabeling of elutriation medium: a demonstration of the need to improve the quality control of reagents produced for cell processing.

BACKGROUND: The processing of hematopoietic cells to remove red cells, T‐lymphocytes, or malignant cells has become common. However, few of the reagents used are produced specifically for marrow processing and approved by the Food and Drug Administration. CASE REPORTS: A labeling error is reported in a commercially manufactured, research‐grade reagent used in an elutriation procedure to deplete T cells from mononuclear cells obtained from bone marrow for allogeneic transplantation. The elutriation medium used in the procedure had been labeled as containing 0.9‐percent sodium chloride, 0.1‐percent dextrose, and 0.3 mM EDTA, but it actually contained 3.0 mM EDTA. When this medium was not available from the manufacturer, it was prepared in this hospital, according to the stated formulation, with 0.3 mM EDTA. When marrows from two unrelated donors were processed with this 0.3 mM EDTA‐containing medium, cell loss due to aggregation occurred, and, in one recipient, the marrow did not engraft. After the error in the medium composition was discovered, marrow was collected a second time from the same donor. The marrow was processed with medium containing 3.0 mM EDTA, and no cell aggregation was observed.

Monitoring the Response to Neo-Antigen in Patients Vaccinated with Keyhole Limpet Hemocyanin (KLH) after Hematopoietic Cell Transplantation (HCT)

Monitoring the Response to Neo-Antigen in Patients Vaccinated with Keyhole Limpet Hemocyanin (KLH) after Hematopoietic Cell Transplantation (HCT) Angela Panoskaltsis-Mortari, Melinda Berthold, Julie Curtsinger, Kirsten Malvey, Susan Fautsch, Bruce Blazar, Jeffrey Miller. Hematology, Oncology, Blood and Marrow Transplant Program, University of Minnesota Cancer Center, Minneapolis, MN. Although HCT is used in the treatment of cancer patients, obstacles still remain in achieving complete immune reconstitution and effective immune function, even after autologous HCT. To test the integrity of the immune response, we vaccinated 12 cancer patients after autologous HCT and 27 normal volunteers with a single injection of KLH, a neo-antigen. KLH (Intracel, Rockville, MD or Biosyn Corp, Carlsbad, CA) was given as a single 1 mg subcutaneous injection. Immune response was measured by ELISA, proliferation or ELISPOT assays on blood 1 mo after vaccination compared to preimmunization. In normals (n 1⁄4 17), Intracel KLH (containing the intact KLH molecule, 6000–8000 kDA) induced potent IgM, IgG1 and IgG2 responses as well as a cellular response. Because of sporadic availability, Biosyn KLH, produced as 350/390 kDA subunits, was tested. Biosyn KLH alone failed to induce either a humoral or cellular response. This was not due to loss of antigenic epitopes since anti-KLH Ab levels in sera of Intracel KLHvaccinated subjects were similar when measured against Biosyn KLH. We reasoned that lack of response to Biosyn KLH was due to product purity and decreased complexity or size resulting in loss of adjuvant properties. Biosyn KLH was then mixed with the oil/surfactant Montanide ISA-51, that completely restored immunogenicity to a single 1 mg KLH test vaccine in normals (n 1⁄4 10) similar to the Intracel KLH. In marked contrast to the normal response, humoral responses were significantly impaired in 11 patients 58– 144 [median 103] days after HCT and in 1 as late as 16 months post-HCT (the only patient with a cellular response to KLH). The decreased KLH-specific IgG2 response was partially explained by decreased total IgG2 levels. Total IgG1 and IgM levels were not significantly different between groups. The defect in isotype switching correlated with decreased absolute CD4 counts between the normal (891 6 58/mm) and HCT (290 6 119/mm) cohorts. Although some of the post-HCT patients had protective levels of antibodies to Tetanus toxoid, these patients demonstrated a diminished memory response to TT. We conclude that patients after autologous HCT have immune defects significantly impairing neo-antigen and memory responses. This has important implications for tumor vaccine strategies in these settings. KLH vaccines are safe, and require only a single injection to test for normal cellular and humoral neo-antigen responses including isotype switching, an optimal platform for definitive testing of strategies to promote immune reconstitution after HCT.