Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma.

We report the development of a potent anti-CD38 immunotoxin capable of killing human myeloma and lymphoma cell lines. The immunotoxin is composed of an anti-CD38 antibody HB7 conjugated to a chemically modified ricin molecule wherein the binding sites of the B chain have been blocked by covalent attachment of affinity ligands (blocked ricin). Conjugation of blocked ricin to the HB7 antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Four to six logs of CD38+ tumor cell line kill was achieved at concentrations of HB7-blocked ricin in the range of 0.1 to 3 nmol/L. Low level of toxicity for normal bone marrow (BM) granulocyte-macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), colony-forming units-granulocyte/erythroid/monocyte/macrophage (CFU-GEMM) cells was observed. Greater than two logs of CD38+ multiple myeloma cells were depleted from a 10-fold excess of normal BM mononuclear cells (BMMCs) after an exposure to HB7-blocked ricin under conditions (0.3 nmol/L) that were not very toxic for the normal BM precursors. HB7-blocked ricin was tested for its ability to inhibit protein synthesis in fresh patients' multiple myeloma cells and in normal BMMCs isolated from two healthy volunteers; tumor cells from four of five patients were 100-fold to 500-fold more sensitive to the inhibitory effect of HB7-blocked ricin than the normal BM cells. HB7 antibody does not activate normal resting peripheral blood lymphocytes, and HB7-blocked ricin is not cytotoxic toward these cells at concentrations of up to 1 nmol/L. The potent killing of antigen-bearing tumor cells coupled with a lack of effects on peripheral blood T cells or on hematopoietic progenitor cells suggests that HB7-blocked ricin may have clinical utility for the in vivo or in vitro purging of human multiple myeloma cells.

Abrogation of secondary skin allograft rejection by veto-like cells in donor bone marrow.

To investigate whether a veto cell phenomenon is involved in the ability of donor strain bone marrow cells (BMC) to inactivate second-set skin graft rejection induced by the adoptive transfer of sensitized spleen cells (SSC), mice were grafted with ear skin allografts and injected on the following day with recipient strain SSC or SSC plus donor strain BMC. When skin and BMC donors from an F1 strain (B6AF1) were combined with a parental strain (C57BL/6) as recipient and SSC donors, the BMC were fully active in abrogating second-set rejection even though the SSC were naturally tolerant of the BMC. Thus, immunological recognition of the SSC by the BMC is not necessary for this abrogation. The suppressive activity of the BMC is specific in a manner similar to that reported for veto cells (i.e., they suppress an anti-self response). If BMC from various strains of mice were used to alter the rejection of C3H grafts by B6AF1 recipients, only BMC expressing the MHC antigens to which the SSC had been sensitized significantly suppressed the activity of SSC. Sharing of additional antigens between the graft and BMC (e.g., MHC antigens not recognized by the SSC or minor antigens) was neither necessary nor sufficient. Mitomycin C-treated BMC were unable to inhibit SSC-induced accelerated rejection. Overall, the characteristics of the SSC-suppressive activity of BMC are consistent with a veto cell nature of the BMC.

Abrogation of alloreactive spleen cell-induced second-set skin graft rejection in mice with donor-specific bone marrow cells.

To study the mechanism of induction of specific unresponsiveness to allografts in animals treated with antilymphocyte serum and donor bone marrow cells, we examined the effect of donor BMC on second-set graft rejection responses caused by antidonor-sensitized spleen cells (SSC) in mice bearing donor ear skin grafts. Anti-C3H SSC were obtained from skin-grafted, ALS-treated B6AF1 mice after rejection of their grafts. The second-set rejections of C3H skin grafts were assessed in B6AF1 mice following adoptive i.v. transfer of 30 x 10(6) SSC one day after grafting. Median survival time (MST) of C3H skin grafts in the group injected with SSC was 7 days, which is significantly lower than an MST of 11 days observed in the control group, which exhibited a first-set graft rejection response. Addition of 50 x 10(6) C3H BMC to 30 x 10(6) SSC abrogated the second-set rejection of C3H skin grafts (MST = 11 days). This abrogation effect of BMC is strain-specific, since BMC of a third-party strain (DBA/2) failed to abrogate the second-set rejection responses caused by anti-C3H SSC. Of the different C3H lymphoid cells tested for abrogation of the second-set graft rejection, BMC were the most effective. Splenocytes were more effective than thymocytes, which showed a partial effect. Lymph node cells had no effect. Our data suggest that unresponsiveness to allografts in animals treated with the ALS/bone marrow protocol may result from the inactivation of graft-reactive cells by donor BMC.

Evaluation of latex agglutination and microtube coagulase tests for detection of Staphylococcus aureus.

In a blind study, a latex agglutination test (Serostat Staphylococcus, Scott Laboratories) and a microtube coagulase test (Staphase, API) were evaluated for their ability to detect Staphylococcus aureus. Of 289 isolates of catalase-positive, gram-positive cocci, 122 were identified as S. aureus based on positive reactions in at least three of the following tests: tube coagulase, slide coagulase, DNase production, or anaerobic fermentation of mannitol. The latex agglutination test gave positive reactions for all S. aureus isolates and 10 (6%) non-S. aureus isolates. The slide coagulase test was positive for 121 S. aureus isolates and three (2%) non-S. aureus isolates. The microtube coagulase test detected 53, 90, and 98% of the S. aureus isolates after 2, 4, and 24 hr, respectively. In contrast, the conventional tube coagulase test detected 97% of the S. aureus isolates after 2 hr, and 98% after 4 and 24 hr. Two isolates of S. aureus gave negative tube coagulase reactions at 37 degrees C, but positive reactions at room temperature after 24 hr. The combination of tube and slide coagulase tests provided the most reliable results. The slide and tube coagulase tests gave more reliable results than the latex agglutination and microtube coagulase tests, respectively.