title: Effects of anti-CD33 blocked ricin immunotoxin on the capacity of CD34+ human marrow cells to establish in vitro hematopoiesis in long-term marrow cultures.
abstract: Human marrow cells that express the CD34 antigen but lack CD33 are able to initiate sustained, multilineage in vitro hematopoiesis in long-term Dexter cultures and are believed to include the primitive stem cells responsible for effecting long-term hematopoietic reconstitution in vivo following marrow transplantation. In studies described in this report we investigated the effects of a novel anti-CD33 immunotoxin on the clonogenic potential of normal human CD34+ marrow cells and on the ability of these cells to initiate hematopoiesis in two-stage Dexter cultures (long-term marrow cultures, LTMC). This immunotoxin (anti-CD33-bR), shown previously to kill both clonogenic myelogenous leukemia cells and normal mature myeloid progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM), consists of an anti-CD33 monoclonal antibody conjugated to purified ricin that has been modified by blocking the carbohydrate binding domains of the ricin B-chain to eliminate nonspecific binding. For our studies, normal CD34+ human marrow cells were isolated from the light-density (less than 1.070 g/ml) cells of aspirated marrow by positive selection with immunomagnetic beads linked to the monoclonal antibody K6.1. These cell isolates were highly enriched with both multipotential and lineage-restricted clonogenic, hematopoietic progenitors (mixed lineage colony-forming units, CFU-Mix; CFU-GM; and erythroid burst-forming units, BFU-E) which constituted greater than or equal to 20% of the cells. Recovery of clonogenic progenitors from these CD34+ cell preparations, following treatment with anti-CD33-bR (10 nM), was reduced by greater than or equal to 85% for CFU-GM and 20%-40% for CFU-Mix and BFU-E. However, the capacity of these cells to initiate hematopoietic LTMC was preserved. Indeed, the production of high proliferative potential (HPP) CFU-GM, BFU-E, and CFU-Mix in cultures seeded with 10(5) anti-CD33-bR-treated CD34+ marrow cells was substantially greater than that observed in LTMC seeded with equivalent numbers of untreated CD34+ cells. Moreover, concentrations of long-term culture initiating cells in CD34+ cell isolates, quantified by a limiting dilution technique, were found to be increased following anti-CD33-bR treatment. These findings support the potential usefulness of anti-CD33-bR for in vitro marrow purging or in vivo treatment to eliminate CD33+ leukemic clones, while sparing normal CD34+/CD33- stem cells that support normal hematopoiesis and hematopoietic reconstitution in vivo.
authors.affiliation: Department of Hematology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
authors.forename: V F
authors.initials: VF
authors.lastname: La Russa
authors.forename: J D
authors.initials: JD
authors.lastname: Griffin
authors.forename: S W
authors.initials: SW
authors.lastname: Kessler
authors.forename: M A
authors.initials: MA
authors.lastname: Cutting
authors.forename: R D
authors.initials: RD
authors.lastname: Knight
authors.forename: W A
authors.initials: WA
authors.lastname: Blattler
authors.forename: J M
authors.initials: JM
authors.lastname: Lambert
authors.forename: D G
authors.initials: DG
authors.lastname: Wright
mesh_terms: D015703:Antigens, CD / Q000502:physiology*; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic / Q000502:physiology*; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D000922:Immunotoxins; D066298:In Vitro Techniques; D012276:Ricin / Q000008:administration & dosage; D063268:Sialic Acid Binding Ig-like Lectin 3; D013997:Time Factors
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D000922:Immunotoxins; D063268:Sialic Acid Binding Ig-like Lectin 3; D012276:Ricin