Edward D. Ball

Centrifugal enhancement of retroviral mediated gene transfer

Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.

U.S.-Canadian Consensus Recommendations on the Immunophenotypic Analysis of Hematologic Neoplasia by Flow Cytometry: Medical Indications

Bruce H. Davis,1* Kathy Foucar,2 Wlodek Szczarkowski,3 Edward Ball,4 Tom Witzig,5 Kenneth A. Foon,6 Denise Wells,7 Pat Kotylo,8 Rebecca Johnson,9 Curtis Hanson,5 and David Bessman10 1William Beaumont Hospital, Royal Oak, Michigan 2University of New Mexico, Albuquerque, New Mexico 3Cytometry Associates, Brentwood, Tennessee 4University of Pittsburgh, Pittsburgh, Pennsylvania 5Mayo Clinic, Rochester, Minnesota 6Markey Cancer Center, Lexington, Kentucky 7Hematologics, Seattle, Washington 8Indiana University, Indianapolis, Indiana 9Berkshire Medical Center, Pittsfield, Massachusetts 10University of Texas, Galveston, Texas

Posttransplant adoptive immunotherapy with activated natural killer cells in patients with metastatic breast cancer.

Relapse after high-dose chemotherapy is the main cause of therapeutic failure in patients with metastatic breast cancer. Adoptive immunotherapy with interleukin-2 (IL-2) plus activated natural killer cells may eliminate residual disease without excessive toxicity. The authors sought to determine if immunotherapy immediately after transplantation would affect engraftment and the toxicity associated with transplantation. Fifteen consecutive patients with metastatic breast cancer were allocated to three cohorts. Cohort 1 (five patients) received high-dose cyclophosphamide, thiotepa, and carboplatin (CTCb) followed by peripheral blood stem cell infusion and granulocyte colony-stimulating factor at 10 micrograms/kg. Cohort 2 (five patients) received in addition rhIL-2 (2 x 10(6) IU/m2/day) for 4 days intravenously via continuous infusion after peripheral blood stem cell infusion. In cohort 3 (five patients), peripheral blood stem cell transplant was followed by infusion of autologous activated NK cells and rhIL-2 (2 x 10(6) IU/m2/day) for 4 days (via continuous intravenous infusion). Generation of activated NK cells was possible in all patients in cohort 3. All patients has successful engraftment. Median time to absolute neutrophil count more than 0.5 x 10(9)/L was 8 days (range, 8 to 11 days) in cohort 1, 9 days (range, 7 to 11 days) in cohort 2, and 9 days (range, 8 to 9 days) in cohort 3. Median time until the platelet count was more than 20 x 10(9)/L was 14 days (range, 9 to 22 days) in cohort 1, 11 days (range, 6 to 14 days) in cohort 2, and 12 days (range, 11 to 21 days) in cohort 3. All patients developed neutropenic fevers, but the overall toxicity associated with the infusion of IL-2 (cohort 2) or IL-2 plus activated NK cells (cohort 3) did not differ from that observed in cohort 1. Complete responses were achieved in one patient in cohort 1, in two patients in cohort 2, and in one patient in cohort 3. In conclusion, post-transplant adoptive immunotherapy with activated NK cells plus IL-2 is feasible, well tolerated, and does not adversely affect engraftment.

Targeting gastrin-releasing peptide receptors for cancer treatment.

Growth factor receptors play critical roles in cancer cell proliferation and progression. A number of such receptors have been targeted for cancer treatment by either a monoclonal antibody or a specifically designed small molecule to inhibit the receptor function. Bombesin/gastrin-releasing peptide receptors (BN/GRP-Rs) are expressed in a variety of cancer cells and have limited distribution in normal human tissue. Inhibition of BN/GRP-Rs has been shown to block small cell lung cancer growth in vitro. Early phase clinical trials targeting human GRP-R showed anti-cancer activity. This review will focus on the study of the distribution of BN/GRP-Rs in normal and malignant tissues, and various approaches to targeting BN-GRP-Rs for cancer diagnosis and treatment.

Busulfan and cyclophosphamide (BU/CY2) as preparative regimen for patients with lymphoma.

The combination of busulfan and cyclophosphamide has seldom been employed as a conditioning regimen for patients with lymphoma. Twenty patients with relapsed or refractory lymphoma were treated with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (BU/CY) followed by peripheral blood stem cell rescue in 19 patients or autologous bone marrow in one patient. There were 12 females and eight males, with a median age of 48 years (range 30–65). Four patients had Hodgkin’s disease, and 16 patients had non-Hodgkin’s lymphoma. Disease status at the time of BU/CY was: first relapse in 10 patients (four patients with chemosensitive disease and six patients with chemoresistant disease), primary refractory disease in six patients, and more advanced disease in four patients. Excessive treatment-related toxicity was not noted. There were no cases of interstitial pneumonitis, but three cases of veno-occlusive disease occurred. At 2 years, the estimated overall survival and event-free survival are 50% and 33%. We concluded that BU/CY seems to have sufficient anti- lymphoma activity, is devoid of excessive toxicity and warrants further investigation in this patient population.

Hematopoietic progenitor cell content of vertebral body marrow used for combined solid organ and bone marrow transplantation.

While cadaveric vertebral bodies (VB) have long been proposed as a suitable source of bone marrow (BM) for transplantation (BMT), they have rarely been used for this purpose. We have infused VB BM immediately following whole organ (WO) transplantation to augment donor cell chimerism. We quantified the hematopoietic progenitor cell (HPC) content of VB BM as well as BM obtained from the iliac crests (IC) of normal allogenic donors (ALLO) and from patients with malignancy undergoing autologous marrow harvest (AUTO). Patients undergoing WO/BM transplantation also had AUTO BM harvested in the event that subsequent lymphohematopoietic reconstitution was required. Twenty-four VB BM, 24 IC BM-ALLO, 31 IC AUTO, and 24 IC WO-AUTO were harvested. VB BM was tested 12 to 72 hr after procurement and infused after completion of WO grafting. IC BM was tested and then used or cryopreserved immediately. HPC were quantified by clonal assay measuring CFU-GM, BFU-E, and CFU-GEMM, and by flow cytometry for CD34+ progenitor cells. On an average, 9 VB were processed during each harvest, and despite an extended processing time the number of viable nucleated cells obtained was significantly higher than that from IC. Furthermore, by HPC content, VB BM was equivalent to IC BM, which is routinely used for BMT. We conclude that VB BM is a clinically valuable source of BM for allogeneic transplantation.

9 Gaucher's disease: studies of gene transfer to haematopoietic cells

Transfer of the gene coding for glucocerebrosidase (GC) via a retroviral vector (MFG-GC) to haematopoietic progenitors results in engraftment and life-long expression of the human protein at high levels in transplanted mice. Studies of human CD34 cells were carried out to evaluate their potential use in a gene therapy approach to Gauchers disease. High transduction efficiency and correction of the enzyme deficiency was possible in CD34 cells obtained from patients with Gauchers disease. Based on these results, a clinical trial of gene therapy was designed and initiated. Preliminary results of this study indicate the persistence or engraftment of genetically corrected cells in the transplanted patients.

Autologous bone marrow transplantation for acute myeloid leukemia in remission or first relapse using monoclonal antibody-purged marrow: results of phase II studies with long-term follow-up

One hundred and thirty-eight patients with AML underwent ABMT with monoclonal antibody plus complement-purged marrow between August 1984 and March 1997. One hundred and ten patients were in CR (CR1: 23; CR2/3: 87) and 28 were in first relapse (R1) at ABMT. Preparative regimens included busulfan (16u2009mg/kg) and CY (120u2009mg/kg) (nu2009=u200993), CY (120u2009mg/kg over 2 days) with TBI (1200u2009cGy) (nu2009=u2009 35), and busulfan (16u2009mg/kg) plus etoposide (60u2009mg/kg) (nu2009=u200910). CR1 patients treated with CY/TBI (nu2009=u20097) had 3- and 5-year disease-free survival (DFS) rates of 71% and 57%. CR1 patients treated with BU/CY (nu2009=u200912), had 3- and 5-year DFS rates of 45%. Three and 5-year DFS for CR2/3 patients treated with CY/TBI (nu2009=u200926) was 23%. Three- and 5-year DFS for patients in CR2/3 treated with BU/CY (nu2009=u200955) was 31 and 28%. Three- and 5-year DFS for patients in R1 treated with BU/CY (nu2009=u200926) was 37%. In multivariate analysis, increased age was associated with greater risk of death and relapse. For CR2/3 patients, the length of CR1 was a significant predictor of DFS. ABMT performed in CR or R1 results in excellent 5-year DFS and OS. The contribution of purging may require a randomized trial comparing purged vs unpurged stem cell infusions. Bone Marrow Transplantation (2000) 25, 823–829.

Induction of graft-versus-leukemia effect in a patient with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is incurable with conventional therapy. Recent data report favorable results with allogeneic transplant. We report a patient with CLL with persistent leukemia post-transplant who obtained remission after discontinuing immune suppression.

Targeting Gastrin-Releasing Peptide Receptors on Small Cell Lung Cancer Cells with a Bispecific Molecule that Activates Polyclonal T Lymphocytes

Purpose: Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. Experimental Design: We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). Results: The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNγ production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule–armed human T cells significantly reduced SCLC tumor burdens in a mouse model. Conclusion: The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor–positive tumors, such as SCLC, are warranted.